Rozprawy doktorskie na temat „RUNX2”

RUNX2 belongs to the RUNT domain family of transcription factors of which three have been identified in humans (RUNX1, RUNX2 and RUNX3). RUNX proteins are vital for metazoan development and participate in the regulation of cellular differentiation and cell cycle progression (Coffman, 2003). RUNX2 is required for proper bone formation by driving the differentiation of osteoblasts from mesenchymal progenitors during development (Ducy et al, 1997; Komori et al, 1997; Otto et al, 1997). RUNX2 is also vital for chondrocyte maturation by promoting the differentiation of chondrocytes to the hypertrophic phenotype (Enomoto et al, 2000). The consequences of completely disrupting the RUNX2 locus in mice provided compelling and conclusive evidence for the biological importance of RUNX2 where knockout mice died shortly after birth with a complete lack of bone formation (Komori et al, 1997; Otto et al, 1997). A further indication of the requisite role of RUNX2 in skeletal development was the discovery that RUNX2 haploinsufficiency in humans and mice caused the skeletal syndrome Cleidocranial Dysplasia (CCD) (Mundlos et al, 1997; Lee et al, 1997). A unique feature of RUNX2 is the consecutive polyglutamine and polyalanine tracts (Q/A domain). Mutations causing CCD have been observed in the Q/A domain of RUNX2 (Mundlos et al, 1997). The Q/A domain is an essential part of RUNX2 and participates in transactivation function (Thirunavukkarasu et al, 1998). Previous genotyping studies conducted in our laboratory identified several rare RUNX2 Q/A variants in addition to a frequently occurring 18 base pair deletion of the polyalanine tract termed the 11Ala allele. Analysis of serum parameters in 78 Osteoarthritis patients revealed the 11Ala allele was associated with significantly decreased osteocalcin. Furthermore, analysis of 11Ala allele frequencies within a Geelong Osteoporosis Study (GOS) fracture cohort and an appropriate age matched control group revealed the 11Ala allele was significantly overrepresented in fracture cases indicating an association with increased fracture risk. To further investigate the 11Ala allele and rare Q/A variants, 747 DNA samples from the Southeast Queensland bone study were genotyped using PCR and PAGE. The experiment served two purposes: 1) to detect additional rare Q/A variants to enrich the population of already identified mutants and 2) have an independent assessment of the effect of the 11Ala allele on fracture to either support or refute our previous observation which indicated the 11Ala allele was associated with an increased risk of fracture in the GOS. From the 747 samples genotyped, 665 were WT, 76 were heterozygous for the 11Ala allele, 5 were homozygous for the 11Ala allele and 1 was heterozygous for a rare 21 bp deletion of the polyglutamine tract. Chi-square analysis of RUNX2 genotype distributions within fracture and non-fracture groups in the Southeast Queensland bone study revealed that individuals that carried at least one copy of the 11Ala allele were enriched in the fracture group (p = 0.16, OR = 1.712). The OR of 1.712 was of similar magnitude to the OR observed in the GOS case-control investigation (OR = 1.9) providing support for the original study. Monte-Carlo simulations were used to combine the results from the GOS and the Southeast Queensland bone study. The simulations were conducted with 10000 iterations and demonstrated that the maximum probability of obtaining both study results by chance was less than 5 times in two hundred (p < 0.025) suggesting that the 11Ala allele of RUNX2 was associated with an increased fracture risk. The second element of the research involved the analysis of rare RUNX2 Q/A variants identified from multiple epidemiological studies of bone. Q/A repeat variants were derived from four populations: the GOS, an Aberdeen cohort, CAIFOS and a Sydney twin study. Collectively, a total of 20 rare glutamine and one alanine variants were identified from 4361 subjects. All RUNX2 Q/A variants were heterozygous for a mutant allele and a wild type allele. Analysis of incident fracture during a five year follow up period in the CAIFOS revealed that Q-variants (n = 8) were significantly more likely to have fractured compared to non-carriers (p = 0.026, OR 4.932 95% CI 1.2 to 20.1). Bone density data as measured by quantitative ultrasound was available for CAIFOS. Analysis of BUA and SOS Z-scores revealed that Q-repeat variants had significantly lower BUA (p = 0.031, mean Z-score of -0.79) and a trend for lower SOS (p = 0.190, mean Z-score of -0.69). BMD data was available for all four populations. To normalize the data across the four studies, FN BMD data was converted into Z-scores and the effect of the Q/A variants on BMD was analysed using a one sample approach. The analysis revealed Q/A variants had significantly lower FN BMD (p = 0.0003) presenting with a 0.65 SD decrease. Quantitative transactivation analysis was conducted on RUNX2 proteins harbouring rare glutamine mutations and the 11Ala allele. RUNX2 proteins containing a glutamine deletion (16Q), a glutamine insertion (30Q) and the 11Ala allele were overexpressed in NIH3T3 and HEK293 cells and their ability to transactivate a known target promoter was assessed. The 16Q and 30Q had significantly decreased reporter activity compared to WT in NIH3T3 cells (p = 0.002 and 0.016, for 16Q and 30Q, respectively). In contrast 11Ala RUNX2 did not show significantly different promoter activation potential (p = 0.54). Similar results were obtained in HEK293 cells where both the 16Q and 30Q RUNX2 displayed decreased reporter activity (p=0.007 and 0.066 for 16Q and 30Q respectively) whereas the 11Ala allele had no material effect on RUNX2 function (p = 0.20). The RUNX2 gene target reporter assay provided evidence to suggest that variation within the glutamine tract of RUNX2 was capable of altering the ability of RUNX2 to activate a known target promoter. In contrast, the 11Ala allele showed no variation in RUNX2 activity. The third feature of the research served the purpose of identifying potential RUNX2 gene targets with particular emphasis on discovering genes cooperatively regulated by RUNX2 and the powerful bone promoting agent BMP2. The experiment was conducted by creating stably transfected NIH3T3 cells lines overexpressing RUNX2 or BMP2 or both RUNX2 and BMP2. Microarray analysis revealed very few genes were differentially regulated between standard NIH3T3 cells and cells overexpressing RUNX2. The results were confirmed via RT-PCR analysis which demonstrated that the known RUNX2 gene targets Osteocalcin and Matrix Metalloproteinase-13 were modestly induced 2.5 fold (p = 0.00017) and 2.1 fold (p = 0.002) respectively in addition to identifying only two genes (IGF-II and SCYA11) that were differentially regulated greater than 10 fold. IGF-II and SYCA11 were significantly down-regulated 27.6 fold (p = 1.95 x 10-6) and 10.1 fold (p = 0.0002) respectively. The results provided support for the notion that RUNX2 on its own was not sufficient for optimal gene expression and required the presence of additional factors. To discover genes cooperatively regulated by RUNX2 and BMP2, microarray gene expression analysis was performed on standard NIH3T3 cells and NIH3T3 cells stably transfected with both RUNX2 and BMP2. Comparison of the gene expression profiles revealed the presence of a large number of differentially regulated genes. Four genes EHOX, CCL9, CSF2 and OSF-1 were chosen to be further characterized via RT-PCR. Sequential RT-PCR analysis on cDNA derived from control cells and cells stably transfected with either RUNX2, BMP2 or both RUNX2/BMP2 revealed that EHOX and CSF2 were cooperatively induced by RUNX2 and BMP2 whereas CCL9 and OSF-1 were suppressed by BMP2. The overexpression of both RUNX2 and BMP2 in NIH3T3 fibroblasts provided a powerful model upon which to discover potential RUNX2 gene targets and also identify genes synergistically regulated by BMP2 and RUNX2. The fourth element of the research investigated the role of RUNX2 in the ascorbic acid mediated induction of MMP-13 mRNA. The study was carried out using NIH3T3 cell lines stably transfected with BMP2, RUNX2 and both BMP2 and RUNX2. The cell lines were grown to confluence and subsequently cultured for a further 12 days in standard media or in media supplemented with AA. RT-PCR analysis was used to assess MMP-13 mRNA expression. The RT-PCR results demonstrated that AA was not sufficient for inducing MMP-13 mRNA in NIH3T3 cells. In contrast RUNX2 significantly induced MMP-13 levels 85 fold in the absence of AA (p = 0.0055) and upregulated MMP-13 mRNA levels 254 fold in the presence of AA (p = 0.0017). The results demonstrated that RUNX2 was essential for the AA mediated induction of MMP-13 mRNA in NIH3T3 cells. The effect of BMP2 on MMP-13 expression was also investigated. BMP2 induced MMP-13 mRNA transcripts a modest 3.8 fold in the presence of AA (p = 0.0027). When both RUNX2 and BMP2 were overexpressed in the presence of AA, MMP-13 mRNA levels were induced a massive 4026 fold (p = 8.7 x 10-4) compared to control cells. The investigation revealed that RUNX2 was an essential factor for the AA mediated induction of MMP-13 and that RUNX2 and BMP2 functionally cooperated to regulate MMP-13 mRNA levels.

RUNX2 belongs to the RUNT domain family of transcription factors of which three have been identified in humans (RUNX1, RUNX2 and RUNX3). RUNX proteins are vital for metazoan development and participate in the regulation of cellular differentiation and cell cycle progression (Coffman, 2003). RUNX2 is required for proper bone formation by driving the differentiation of osteoblasts from mesenchymal progenitors during development (Ducy et al, 1997; Komori et al, 1997; Otto et al, 1997). RUNX2 is also vital for chondrocyte maturation by promoting the differentiation of chondrocytes to the hypertrophic phenotype (Enomoto et al, 2000). The consequences of completely disrupting the RUNX2 locus in mice provided compelling and conclusive evidence for the biological importance of RUNX2 where knockout mice died shortly after birth with a complete lack of bone formation (Komori et al, 1997; Otto et al, 1997). A further indication of the requisite role of RUNX2 in skeletal development was the discovery that RUNX2 haploinsufficiency in humans and mice caused the skeletal syndrome Cleidocranial Dysplasia (CCD) (Mundlos et al, 1997; Lee et al, 1997). A unique feature of RUNX2 is the consecutive polyglutamine and polyalanine tracts (Q/A domain). Mutations causing CCD have been observed in the Q/A domain of RUNX2 (Mundlos et al, 1997). The Q/A domain is an essential part of RUNX2 and participates in transactivation function (Thirunavukkarasu et al, 1998). Previous genotyping studies conducted in our laboratory identified several rare RUNX2 Q/A variants in addition to a frequently occurring 18 base pair deletion of the polyalanine tract termed the 11Ala allele. Analysis of serum parameters in 78 Osteoarthritis patients revealed the 11Ala allele was associated with significantly decreased osteocalcin. Furthermore, analysis of 11Ala allele frequencies within a Geelong Osteoporosis Study (GOS) fracture cohort and an appropriate age matched control group revealed the 11Ala allele was significantly overrepresented in fracture cases indicating an association with increased fracture risk. To further investigate the 11Ala allele and rare Q/A variants, 747 DNA samples from the Southeast Queensland bone study were genotyped using PCR and PAGE. The experiment served two purposes: 1) to detect additional rare Q/A variants to enrich the population of already identified mutants and 2) have an independent assessment of the effect of the 11Ala allele on fracture to either support or refute our previous observation which indicated the 11Ala allele was associated with an increased risk of fracture in the GOS. From the 747 samples genotyped, 665 were WT, 76 were heterozygous for the 11Ala allele, 5 were homozygous for the 11Ala allele and 1 was heterozygous for a rare 21 bp deletion of the polyglutamine tract. Chi-square analysis of RUNX2 genotype distributions within fracture and non-fracture groups in the Southeast Queensland bone study revealed that individuals that carried at least one copy of the 11Ala allele were enriched in the fracture group (p = 0.16, OR = 1.712). The OR of 1.712 was of similar magnitude to the OR observed in the GOS case-control investigation (OR = 1.9) providing support for the original study. Monte-Carlo simulations were used to combine the results from the GOS and the Southeast Queensland bone study. The simulations were conducted with 10000 iterations and demonstrated that the maximum probability of obtaining both study results by chance was less than 5 times in two hundred (p < 0.025) suggesting that the 11Ala allele of RUNX2 was associated with an increased fracture risk. The second element of the research involved the analysis of rare RUNX2 Q/A variants identified from multiple epidemiological studies of bone. Q/A repeat variants were derived from four populations: the GOS, an Aberdeen cohort, CAIFOS and a Sydney twin study. Collectively, a total of 20 rare glutamine and one alanine variants were identified from 4361 subjects. All RUNX2 Q/A variants were heterozygous for a mutant allele and a wild type allele. Analysis of incident fracture during a five year follow up period in the CAIFOS revealed that Q-variants (n = 8) were significantly more likely to have fractured compared to non-carriers (p = 0.026, OR 4.932 95% CI 1.2 to 20.1). Bone density data as measured by quantitative ultrasound was available for CAIFOS. Analysis of BUA and SOS Z-scores revealed that Q-repeat variants had significantly lower BUA (p = 0.031, mean Z-score of -0.79) and a trend for lower SOS (p = 0.190, mean Z-score of -0.69). BMD data was available for all four populations. To normalize the data across the four studies, FN BMD data was converted into Z-scores and the effect of the Q/A variants on BMD was analysed using a one sample approach. The analysis revealed Q/A variants had significantly lower FN BMD (p = 0.0003) presenting with a 0.65 SD decrease. Quantitative transactivation analysis was conducted on RUNX2 proteins harbouring rare glutamine mutations and the 11Ala allele. RUNX2 proteins containing a glutamine deletion (16Q), a glutamine insertion (30Q) and the 11Ala allele were overexpressed in NIH3T3 and HEK293 cells and their ability to transactivate a known target promoter was assessed. The 16Q and 30Q had significantly decreased reporter activity compared to WT in NIH3T3 cells (p = 0.002 and 0.016, for 16Q and 30Q, respectively). In contrast 11Ala RUNX2 did not show significantly different promoter activation potential (p = 0.54). Similar results were obtained in HEK293 cells where both the 16Q and 30Q RUNX2 displayed decreased reporter activity (p=0.007 and 0.066 for 16Q and 30Q respectively) whereas the 11Ala allele had no material effect on RUNX2 function (p = 0.20). The RUNX2 gene target reporter assay provided evidence to suggest that variation within the glutamine tract of RUNX2 was capable of altering the ability of RUNX2 to activate a known target promoter. In contrast, the 11Ala allele showed no variation in RUNX2 activity. The third feature of the research served the purpose of identifying potential RUNX2 gene targets with particular emphasis on discovering genes cooperatively regulated by RUNX2 and the powerful bone promoting agent BMP2. The experiment was conducted by creating stably transfected NIH3T3 cells lines overexpressing RUNX2 or BMP2 or both RUNX2 and BMP2. Microarray analysis revealed very few genes were differentially regulated between standard NIH3T3 cells and cells overexpressing RUNX2. The results were confirmed via RT-PCR analysis which demonstrated that the known RUNX2 gene targets Osteocalcin and Matrix Metalloproteinase-13 were modestly induced 2.5 fold (p = 0.00017) and 2.1 fold (p = 0.002) respectively in addition to identifying only two genes (IGF-II and SCYA11) that were differentially regulated greater than 10 fold. IGF-II and SYCA11 were significantly down-regulated 27.6 fold (p = 1.95 x 10-6) and 10.1 fold (p = 0.0002) respectively. The results provided support for the notion that RUNX2 on its own was not sufficient for optimal gene expression and required the presence of additional factors. To discover genes cooperatively regulated by RUNX2 and BMP2, microarray gene expression analysis was performed on standard NIH3T3 cells and NIH3T3 cells stably transfected with both RUNX2 and BMP2. Comparison of the gene expression profiles revealed the presence of a large number of differentially regulated genes. Four genes EHOX, CCL9, CSF2 and OSF-1 were chosen to be further characterized via RT-PCR. Sequential RT-PCR analysis on cDNA derived from control cells and cells stably transfected with either RUNX2, BMP2 or both RUNX2/BMP2 revealed that EHOX and CSF2 were cooperatively induced by RUNX2 and BMP2 whereas CCL9 and OSF-1 were suppressed by BMP2. The overexpression of both RUNX2 and BMP2 in NIH3T3 fibroblasts provided a powerful model upon which to discover potential RUNX2 gene targets and also identify genes synergistically regulated by BMP2 and RUNX2. The fourth element of the research investigated the role of RUNX2 in the ascorbic acid mediated induction of MMP-13 mRNA. The study was carried out using NIH3T3 cell lines stably transfected with BMP2, RUNX2 and both BMP2 and RUNX2. The cell lines were grown to confluence and subsequently cultured for a further 12 days in standard media or in media supplemented with AA. RT-PCR analysis was used to assess MMP-13 mRNA expression. The RT-PCR results demonstrated that AA was not sufficient for inducing MMP-13 mRNA in NIH3T3 cells. In contrast RUNX2 significantly induced MMP-13 levels 85 fold in the absence of AA (p = 0.0055) and upregulated MMP-13 mRNA levels 254 fold in the presence of AA (p = 0.0017). The results demonstrated that RUNX2 was essential for the AA mediated induction of MMP-13 mRNA in NIH3T3 cells. The effect of BMP2 on MMP-13 expression was also investigated. BMP2 induced MMP-13 mRNA transcripts a modest 3.8 fold in the presence of AA (p = 0.0027). When both RUNX2 and BMP2 were overexpressed in the presence of AA, MMP-13 mRNA levels were induced a massive 4026 fold (p = 8.7 x 10-4) compared to control cells. The investigation revealed that RUNX2 was an essential factor for the AA mediated induction of MMP-13 and that RUNX2 and BMP2 functionally cooperated to regulate MMP-13 mRNA levels.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Faculty of Health
Full Text

In the United States, metastatic breast cancer kills approximately 40,000 women and 400 men annually, and approximately 200,000 new cases of breast cancer are diagnosed each year. Worldwide, breast cancer is the leading cause of cancer deaths among women. Despite advances in the detection and treatment of metastatic breast cancer, mortality rates from this disease remain high because the fact is that once metastatic, it is virtually incurable. It is widely accepted that a major reason breast cancer continues to exhibit recurrence after remission is that current therapies are insufficient for targeting and eliminating therapy-resistant cancer cells. Emerging research has demonstrated that these therapy-resistant cells possess stem cell-like properties and are therefore commonly referred to as breast cancer stem cells (BCSCs). A major hallmark of BCSCs is the cell surface expression of CD44 and lack of expression of CD24, the so-called CD24-/CD44+ phenotype. Research indicates that this dangerous and rare subpopulation of BCSCs may be responsible for cancer onset, recurrence, and ultimately metastasis that leads to death. Two different model systems were utilized in this research. The first was the MCF7 cell line, a luminal A tumor subtype representative of a mildly invasive breast ductal carcinoma with an ER+/PR+/-/HER2- immunoprofile. The second was the MCF10A breast cancer progression model, which consists of three cell lines: MCF10A, MCF10AT1, and MCF10CA1a. In this system, spontaneously immortalized, non-malignant MCF10A cells were transfected with constitutively active H-Ras to form pre-malignant MCF10AT1 cells, which were then subcutaneously injected into mice and allowed to metastasize in order to form the oncogenic MCF10ACA1a cell line. This thesis presents evidence of a CD24low/-/CD44+ BCSC subpopulation within the MCF10A breast cancer progression model system. Findings indicate that RUNX1 and RUNX2 expression levels are involved in maintaining the BCSC phenotype. Across two different model systems, qRT-PCR analysis revealed that decreased levels of RUNX1 expression and increased levels of RUNX2 expression are essential for the maintenance of the BCSC subpopulation. It was also shown that low expression levels of RUNX1 and high expression levels of RUNX2 are present in CD24low/-/CD44+ BCSCs as compared to CD24+/CD44+ non-BCSCs. Furthermore, shRNA knockdown of RUNX1 was shown to enhance tumorigenicity, while shRNA knockdown of RUNX2 repressed tumorigenicity in BCSCs, as measured by the tumorsphere-formation assay. This research lays the groundwork for future investigations into the roles of RUNX1 and RUNX2 in regulating stemness in breast cancer.

Magíster en ciencias médicas, mención biología celular
El Osteosarcoma (OS) es el tumor sólido maligno más frecuente en la infancia y la adolescencia, corresponde al 20% de todos los tumores óseos y al 5% de los cánceres pediátricos. Los tratamientos actuales logran tasas de sobrevida a 5 años de 75% en pacientes sin metástasis, mientras que el 80% de los pacientes con enfermedad metastásica recaen a pesar del tratamiento efectuado. Runx2 es un factor de transcripción maestro que regula el proceso de diferenciación osteogénica, aunque este factor también promueve progresión tumoral en células de cáncer de próstata y de mama. Recientemente, se demostró la amplificación del gen Runx2 en pacientes con OS, así como la expresión aumentada de la proteína Runx2 en líneas celulares de OS. En esta tesis se propuso la hipótesis que el factor de transcripción Runx2 promueve migración e invasión en líneas celulares de osteosarcoma. Así, el objetivo general del proyecto fue definir el rol de Runx2 en los procesos de progresión tumoral en osteosarcoma. Para estudiar el rol de Runx2 sobre parámetros de progresión tumoral en líneas celulares de osteosarcoma, se moduló la expresión de Runx2 y se estudió su efecto en migración e invasividad. Nuestros resultados muestran que la sobreexpresión de Runx2 mediante adenovirus produjo un aumento en la capacidad de migración e invasión de líneas celulares de OS con bajos niveles de Runx2. El silenciamiento de Runx2 mediante siRNA disminuyó la capacidad de migración e invasión en líneas celulares de OS. De esta forma, hemos demostrado por primera vez que Runx2 promueve progresión tumoral en líneas celulares de OS.
Osteosarcoma (OS) is the most common malignant solid tumor in childhood and adolescence and represents 20% of all bone tumors and 5% of childhood cancers. Survival rates after five years are at 75% in patients without metastases, while 80% of patients with metastatic disease relapse despite the treatment. Runx2 is a master transcription factor that regulates the osteogenic differentiation process. However, this factor also functions as tumor promoter in prostate cancer cells and breast cancer. Runx2 gene amplification in patients with OS, as well as increased expression of Runx2 protein in OS cell line have recently been demonstrated. To this project, we hypothesized that the transcription factor Runx2 promotes migration and invasion in OS cell lines. Thus, the general aim was to define the role of Runx2 in osteosarcoma tumor progression processes. To study the role of Runx2 on parameters involved in tumor progression of OS cell lines, we modulated the expression of Runx2 and studied the outcome of this modifications on cell migration and invasiveness. Our results demonstrated that overexpression of Runx2 by adenovirus in OS cell lines with low levels of Runx2 increased cell migration and invasion. Runx2 silencing by siRNA in OS cell lines decreased their ability to migrate and invade. Thus, we have demonstrated for the first time that Runx2 promotes tumor progression in OS cell lines.

Bone tissue engineering is a promising approach to address the limitations of currently used bone tissue substitutes. However, an optimal cell source for the production of osteoblastic matrix proteins and mineral deposition has yet to be defined. In response to this deficiency, ex vivo gene therapy of easily accessible non-osteogenic cells, such as skeletal myoblasts, has become a prevalent strategy for inducing an osteoblastic phenotype. The majority of these approaches focus on constitutive overexpression of soluble osteogenic growth factors such as bone morphogenetic proteins (BMPs). In order to avoid aberrant effects of unregulated growth factor secretion, this work focuses on delivery of the osteoblastic transcription factor Runx2 as an autocrine osteogenic signal under the control of an inducible expression system. The overall objective of this research was to engineer an inducible cell source for bone tissue engineering that addresses the limitations of current cell-based approaches to orthopedic regeneration. Our central hypothesis was that inducible Runx2 overexpression in skeletal myoblasts would stimulate differentiation into a regulated osteoblastic phenotype. We have demonstrated that Runx2 overexpression stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype. Furthermore, we have established Runx2-engineered skeletal myoblasts as a potent cell source for bone tissue engineering applications in vitro and in vivo, similar to BMP-2-overexpressing controls. Finally, we exogenously regulated osteoblastic differentiation by myoblasts engineered to express a tetracycline-inducible Runx2 transgene. This conversion into an osteoblastic phenotype was inducible, repressible, recoverable after suppression, and dose-dependent with tetracycline concentration. This work is significant because it addresses cell sourcing limitations of bone tissue engineering, develops controlled and effective gene therapy methods for orthopedic regeneration, and establishes a novel strategy for regulating the magnitude and kinetics of osteoblastic differentiation.

Tissue engineering has emerged as a promising alternative to conventional orthopaedic grafting therapies. The general paradigm for this approach, in which phenotype-specific cells and/or bioactive growth factors are integrated into polymeric matrices, has been successfully applied in recent years toward the development of bone, ligament, and cartilage tissues in vitro and in vivo. Despite these advances, an optimal cell source for skeletal tissue repair and regeneration has not been identified. Furthermore, the lack of robust, functional orthopaedic tissue interfaces, such as the bone-ligament enthesis, severely limits the integration and biological performance of engineered tissue substitutes. This works aims to address these limitations by spatially controlling the genetic modification and differentiation of fibroblasts into a mineralizing osteoblastic phenotype within three-dimensional polymeric matrices. The overall objective of this project was to investigate transcription factor-based gene therapy strategies for the differentiation of fibroblasts into a mineralizing cell source for orthopaedic tissue engineering applications. Our central hypothesis was that fibroblasts genetically engineered to express Runx2 via conventional and biomaterial-mediated ex vivo gene transfer approaches will differentiate into a mineralizing osteoblastic phenotype. We have demonstrated that a combination of retroviral Runx2 overexpression and glucocorticoid hormone treatment synergistically induces osteoblastic differentiation and biological mineral deposition in primary dermal fibroblasts cultured in monolayer. We report for the first time that glucocorticoids induce osteoblastic differentiation in this model system by modulating the phosphorylation state of a negative regulatory serine residue (Ser125) on Runx2 through an MKP-1-dependent mechanism. Furthermore, we utilized these Runx2-genetically engineered fibroblasts to create mineralized templates for bone repair in vitro and in vivo. Finally, we engineered a heterogeneous bone-soft tissue interface with a novel biomaterial-mediated gene transfer approach. Overall, these results are significant toward the ultimate goal of regenerating complex, higher-order orthopaedic grafting templates which mimic the cellular and microstructural characteristics of native tissue. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with a compromised ability to recruit progenitors to the sight of injury as result of traumatic injury, radiation treatment, or osteodegenerative disease.

The main hypotheses tested within this research focussed on the identification of polymorphisms within Runx2, and the classification of these with respect to bone mineral density (BMD). A separate set of hypotheses focussed upon the effects of calcium treatment in an elderly population, to identify any differences in BMD, bone mineral content (BMC) and bone area between the specific genotypes identified. The initial research strategy included taking subjects’ from the extremes of a population to identify alleles specifically related to the bone mineral density trait. The idea was tested using Runx2, the well known osteoblast transcription factor. From a population of 1300 subjects (from the Geelong Osteoporosis Study: the GOS) the age-weight adjusted femoral neck BMD was ranked and the upper and lower deciles taken to represent the adjusted extremes. After adjusting and ranking, the two groups (n=130 each) were not significantly different for age or weight. In these 260 subjects, we identified 16 allelic variations within the Runx2 gene and gene promoters (P1 and P2), and characterized these novel variations with respect to BMD strata by genotype using DHPLC.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Breast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction. Effective treatment of bone metastasis remains a considerable clinical challenge. In the UK around 70% of the 12,000 patients that die from breast cancer annually have bone metastasis. Whilst existing therapies provide some pain relief, by limiting the tumour-mediated bone degradation, bone metastases are presently incurable. There is therefore an urgent need to develop therapies to prevent bone metastatic breast cancer. The transcription factor complex Runx2/CBFβ is a key regulator of bone development and is aberrantly expressed in breast cancer, leading to up-regulation of bone metastasis-associated genes. Previous work has demonstrated that Runx2/CBFβ determines the invasive phenotype of metastatic breast cancer cells and is required for the expression of metastatic genes. The Runx2/CBFβ complex also has a role in normal breast gene expression, activating expression of the milk protein β-casein in response to hormones. However, little is known about the normal role of Runx2/CBFβ in breast cells. The overarching aim of this project was to determine the role of Runx2/CBFβ in metastasis and identify the target genes that determine the metastatic phenotype. In order to understand the role of Runx2/CBFβ in breast cancer, initial experiments were performed to determine the role of Runx2/CBFβ in normal breast cells. A 3D culture system was established to examine the role of Runx2/CBFβ in regulating gene expression in non-cancerous differentiated epithelial breast cells. Attempts were also made to determine the Runx2/CBFβ target genes after lactogenic hormone stimulation. Unfortunately siRNA knockdown of Runx2 was incompatible with hormonal stimulation. However, 3D cell culture of normal mammary gland cell line HC11 showed Runx2 was expressed throughout the development of mammary acini structures. In addition the expression of CBFβ was confirmed in these cells. Having established the 3D culture system, experiments were subsequently performed to examine the role of CBFβ in the metastatic breast cancer cell line MDA-MB-231. These experiments demonstrated that depletion of CBF has a remarkable effect on the phenotype of the cells, leading to the development of mammary acini structures normally formed by non-cancerous breast cancer cell lines. Thus, depletion of CBF results in a reversion to an epithelial phenotype, suggesting that CBF is required to maintain the epithelial to mesenchymal transition (EMT). RT-PCR analysis also revealed changes in the expression of EMT marker genes. We also demonstrated that the EMT reversion could be rescued by re-expressing an inducible form of CBFβ. These data suggest that CBFβ is required to maintain the mesenchymal phenotype of metastatic breast cancer cells. Finally, a microarray analysis of MDA-MB-231 cells was performed to identify Runx2/CBFβ target genes that might contribute to the mesenchymal phenotype. Cells depleted of CBFβ and grown in 3D revealed reduced expression of IL11. This is known to be involved in bone remodelling. Inspection of the IL11 promoter revealed potential DNA binding sites which confirmed binding to Runx2 using EMSA.

L’hypertension artérielle pulmonaire (HTAP) est une pathologie vasculaire résultant de l’obstruction des artères pulmonaires distales. L’obstruction de ces artères conduit à une augmentation drastique de la pression artérielle pulmonaire au-delà du niveau physiologique (≥ 25 mmHg), entrainant une adaptation pathologique du cœur droit. En effet, le cœur droit deviendra incapable de compenser cette augmentation de pression, ce qui conduira à l’arrêt cardiaque et la mort du patient. Jusqu’à présent, les traitements ont permis une amélioration de la qualité de vie des patients, mais aucun ne permet de soigner cette maladie. L’obstruction des artères pulmonaires observées chez les patients atteints d’hypertension artérielle pulmonaire s’explique, notamment, par l’acquisition d’un phénotype prolifératif des cellules musculaires lisses (CML) de ces artères. Précédemment, le laboratoire a démontré le rôle majeur de la diminution du micro-ARN 204 dans l’acquisition de ce phénotype des CML. Cette même diminution est observée dans les maladies vasculaires systémiques où elle conduit à l’acquisition d’un phénotype calcifiant des CML en permettant l’expression du facteur de transcription ostéogénique RUNX2. Durant ma thèse, j’ai investigué le rôle de la surexpression du facteur de transcription RUNX2 régulé par le micro-ARN 204 dans les CML des artères pulmonaires de patients atteints d’HTAP. Ce projet a permis de démontrer le rôle central de RUNX2 dans l’HTAP non seulement en participant à l’acquisition du phénotype prolifératif des CML, mais également en favorisant la transition phénotypique des CML vers un phénotype calcifiant conduisant à la formation de lésions calcifiantes. De plus, la diminution de l’expression de RUNX2 dans un modèle murin sugen/hypoxie d’HTAP permet l’amélioration des paramètres histologiques et hémodynamiques de l’HTAP, faisant de RUNX2 une cible thérapeutique prometteuse.
Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease that drives the narrowing of distal pulmonary arteries. Pulmonary artery narrowing leads to an increase in the pulmonary arterial pressure above 25 mmHg. In order to compensate this increased pressure, the right ventricle undergoes a pathological adaptation. Shortly, the right ventricle becomes unable to compensate this increase leading to right heart failure and patient’s death. So far, PAH treatment has allowed significant improvement for patient’s life quality but none of them can cure the disease. Distal pulmonary artery narrowing is due, at least in part, to the acquisition of a proliferative phenotype of the smooth muscle cell (SMC). Previously, the laboratory has demonstrated the down-regulation of the micro-RNA 204 in the SMC and its major role in the acquisition of this proliferative phenotype. Interestingly, the same down-regulation is observed in systemic vascular disease where it leads to the acquisition of a calcifying phenotype of the SMC by allowing the expression of the osteogenic transcription factor RUNX2. During my thesis, I investigated the role of RUNX2 up-regulation in pulmonary artery SMC allowed by the down-regulation of the micro-RNA 204. This project uncovered a critical role for RUNX2 in PAH, not only by participating to the acquisition of SMC proliferative phenotype but also by allowing the acquisition of a calcifying phenotype of the SMC. Furthermore, targeting RUNX2 in a Sugen/hypoxia rat model of PAH reverse histologic and heomodynamic PAH phenotype, making RUNX2 an attractive therapeutic target.

O objetivo deste estudo foi avaliar a resposta dos tecidos perirradiculares após perfuração de furca intencional e selamento com Biodentine (BD), agregado trióxido mineral (MTA) ou guta percha. Foram utilizados pré-molares de 3 cães, num total de 30 dentes, distribuídos em 3 grupos: experimental BD (n= 14), controle negativo (MTA) (n= 10) e controle positivo (guta percha) (n= 6), por um período de 120 dias. Radiograficamente foi analisada a área correspondente à perfuração de furca. Na análise histopatológica qualitativa foi avaliada a presença, ou não de tecido mineralizado no local da perfuração de furca e adjacências. Na análise histopatológica semi-quantitativa foram atribuídos escores para os parâmetros: presença ou ausência de tecido mineralizado, intensidade do processo inflamatório e reabsorção dos tecidos mineralizados. Na análise histopatológica quantitativa foi medida a espessura de tecido mineralizado na área de perfuração de furca. Foram realizados ensaios de imuno-histoquímica para os marcadores de mineralização: RANKL e osteoprotegerina (OPG). Ensaio de imunofluorescência indireta avaliou a expressão Runx-2 para a síntese de proteínas de mineralização. Os dados foram avaliados pelos testes qui-quadrado, teste exato de Fisher e teste de Mann Whitney, utilizando o programa estatístico Graph Pad Prism 6.0. Os grupos foram comparados entre sí pelo Teste de Kruskal Wallis com pós-teste de Dunn. O nível de significância adotado para todas as análises foi de 5%. Na análise radiográfica a (BD) apresentou melhor desempenho em relação ao MTA, em todos os aspectos analisados. Histologicamente, tanto o MTA quanto a BD induziram a formação de tecido mineralizado, quando comparado à guta percha, que não induziu a formação de tecido mineralizado (p<.001). O selamento completo das perfurações de furca foi mais frequente com o MTA, que induziu a deposição de tecido mineralizado com área e espessura maiores. Tanto as amostras seladas com BD, quanto com MTA, não apresentaram reabsorção óssea em área de furca, apresentaram poucas células inflamatórias e maior intensidade do imunomarcador RUNX2 quando comparadas com a guta percha. A OPG esteve presente em amostras seladas com BD e com MTA. Embora o MTA tenha apresentado maior frequência de selamento completo e maior área e espessura de tecido mineralizado recém-formado, a BD também apresentou bons resultados histopatológicos e pode ser considerada como um adequado material de selamento de perfuração de furca.
The objective of this study was to evaluate the periradicular tissue response after intentional furcation and sealing with Biodentine (BD), mineral trioxide aggregate (MTA) or gutta percha. Pre-molars of 3 dogs were used, in a total of 30 teeth, distributed in 3 groups: experimental BD (n = 14), negative control (MTA) (n = 10) and positive control (gutta percha), for a period of 120 days. The area corresponding to furcation was analyzed radiographically. In the qualitative histopathological analysis, the presence or not of mineralized tissue at the furcation site and adjacent areas was evaluated. In the semi-quantitative histopathological analysis, scores were assigned to the parameters: presence or absence of mineralized tissue, intensity of the inflammatory process and reabsorption of mineralized tissues. In the quantitative histopathological analysis the thickness of mineralized tissue in the furcation area was measured. Immunohistochemical assays were performed for the mineralization markers: RANKL and osteoprotegerin (OPG). Indirect immunofluorescence assay evaluated RUNX-2 expression for the synthesis of mineralization proteins. Data were evaluated by chi-square test, Fisher exact test and Mann Whitney test using the Graph Pad Prism 6.0 statistical software. The groups were compared by the Kruskal Wallis test with Dunn\'s post-test. The level of significance adopted for all analyzes was 5%. In the radiographic analysis the (BD) presented better performance in relation to the MTA, in all aspects analyzed. Histologically, both MTA and BD induced the formation of mineralized tissue when compared to gutta percha, which did not induce the formation of mineralized tissue (p <.001). The complete sealing of furcation holes was more frequent with the MTA, which induced the deposition of mineralized tissue with a larger and thickness area. Both the BD and MTA sealed samples did not show bone resorption in the furcation area, showed few inflammatory cells and a greater intensity of the RUNX2 immunomarker when compared to the gutta percha. OPG was present in samples sealed with BD and with MTA. Although the MTA presented higher frequency of complete sealing and greater area and thickness of newly formed mineralized tissue, BD also presented good histopathological results and can be considered as a suitable furcation perforation sealing material.

Runx2 is a transcription factor required for bone formation and osteoblastic differentiation during normal development and is implicated in metastatic disease during breast cancer progression. Runx2 is highly expressed in many metastatic breast cancers and breast cancer cell lines Knockdown of Runx2 in various breast cancer cell lines restores epithelial characteristics and reduces proliferation, migration, and invasion. However, the role of Runx2 in breast cancer progression from early to late stages is not well understood. The MCF10A derived breast cancer progression model provides the opportunity to study the role of Runx2 in a series of cell lines that progress from nearly normal, with low Runx2 levels, to highly metastatic and aggressive, with much higher Runx2 levels. To address if removal of Runx2 affects gene expression and what pathways it may influence, specifically focused on breast cancer progression, we knocked down Runx2 using an shRNA lentivirus. Depletion of Runx2 inhibits the expression of mesenchymal markers including N-cadherin, Fibronectin, and Vimentin. Despite this finding, functional characteristics including proliferation, migration, and invasion were minimally affected. Possible reasons for the difference in results compared to other cell systems are discussed. As an alternative approach, we have generated stable, inducible cell lines using CRISPRi dCas9-KRAB to target Runx2 and in the future will investigate the effects of Runx2 knockdown in these cells.

Filamin A is a well-characterised cytoskeletal protein which regulates cell shape and migration by cross-linking with actin. Filamin A mutations cause a number of human developmental disorders, many of which exhibit skeletal dysplasia. However, the molecular mechanisms by which Filamin A affects skeletal development are unknown. The transcription factor Runx2 is a master regulator of osteoblast and chondrocyte differentiation. Data presented in this thesis show that Filamin A forms a complex with Runx2 in osteoblastic cell lines. Moreover, it is demonstrated that Filamin A is present in the nucleus in several cell lines, including those of osteoblastic origin. The data presented show that the Filamin A/Runx2 complex suppresses the expression of the gene encoding the matrix-degrading enzyme, matrix metalloproteinase-13 (MMP-13), which is an important osteoblastic differentiation marker. ChIP assays were employed to demonstrate that endogenously expressed Filamin A associates with the promoter of the MMP-13 gene. In addition, Filamin A is not only located in the nucleus but also in the nucleolus, an important nuclear compartment involved in ribosomal RNA (rRNA) transcription. Ribosomal DNA promoter-driven reporter assays, Filamin A-knockdown experiments and exogenous Filamin A transfections demonstrated that Filamin A and Runx2 can repress ribosomal gene expression activity. Importantly, Filamin A is recruited to the human ribosomal DNA promoter, suggesting its direct involvement in the regulation of rRNA transcription. These findings reveal a novel role of Filamin A in the direct regulation of ribosomal gene expression. Finally, by using microarray technology, changes in gene expression were identified when Filamin A was downregulated. Some of the differentially expressed genes were known orchestrators of bone development. The data presented in this thesis strengthen the link between Filamin A and bone development and provide a molecular rationale for how Filamin A, acting as a regulator of gene expression, might influence osteoblastic differentiation.

Runx2 is a transcription factor that has a crucial role in the development of bone; haploinsufficiency of Runx2 leads to the autosomal-dominant disorder, cleidocranial dysplasia (CCD) characterized by various skeletal abnormalities. Zinc finger protein 521 (Zfp521) is a transcription factor that is expressed in several cell types including bone. Recent studies demonstrated that Zfp521 interacts with Runx2 and regulates osteoblast maturation at least in part by repressing the transcriptional activity of Runx2; furthermore, it was demonstrated that the repression of Runx2 by Zfp521 involves the recruitment of HDAC3. The interaction of Runx2 with HDAC3 is strongly enhanced by Zfp521. Zfp521 may regulate osteoblast commitment and differentiation by modulating the Runx2 transcriptional activity by decreasing the levels of Runx2 acetylation. Objective: Runx2 is a key regulator of osteoblast differentiation; Zfp521 may regulate osteoblast commitment and differentiation, at least in part by decreasing the levels of Runx2 acetylation. We investigated 1) Effect of Zfp521 on Runx2 acetylation in HEK293 cells 2) The endogenous Runx2 acetylation levels in MC3T3-E1 cells during osteoblast differentiation. Results: These studies demonstrated that Runx2 acetylation is decreased when Zfp521 is stable expressed in HEK293 cells. Runx2 acetylation levels were detected using immunoprecitation analyses. Sodium butyrate (NaB) prevents protein deacetylation by inhibiting HDACs. The treatment of cells with NaB increased global protein acetylation levels. Importantly, stable expression of Zfp521 did not change global protein acetylation. Therefore, this study suggested that Zfp521 specifically influences Runx2 acetylation. In addition, our findings suggest that Zfp521 impairs Runx2 acetylation by HDACs. Preliminary results show that transient transfection of P300 in HEK293 increased Runx2 acetylation levels. However, stable expression of Zfp521 can still partially decrease Runx2 acetylation levels. This study suggests that Zfp521 function can be linked to P300. Runx2 acetylation levels were then assessed in MC3T3-E1 cells during osteoblast differentiation. While Runx2 protein levels increase by 7 days in culture, and gradually decreases by days 14, Runx2 acetylation was undetectable. Conclusion: Runx2 is the transcription factor that has essential role in osteoblast commitment and differentiation. Zfp521 represses the transcriptional activity of Runx2 by recruiting HDAC3. These studies suggest that Zfp521 modulates Runx2 activity by decreasing Runx2 acetylation level. These studies have extended our knowledge of the mechanisms by which Zfp521 regulates osteoblast differentiation and bone formation, which could have important implications for on the development of future osteo-anabolic treatments.

Members of the Runx family of transcription factors play essential roles in the differentiation and development of several organ systems. Here we address the contribution of the osteoblast-related Runx gene, Runx2, to the osteogenic and chondrogenic differentiation of mesenchymal stem cells. Using a transgenic mouse model, we observe Runx2 transcription through one of its two known promoters (designated P1 in pre-cartilaginous mesenchymal condensations as early as E9.5. Runx2 gene activity is later repressed at the onset of cartilage formation, both in vivo and in vitro, necessitating examination of the regulation and function of Runx2 in mesenchymal stem cells. We demonstrate that Runx2 gene activity is repressed by the direct interaction of the homeodomain transcription factor Nkx3.2 with the proximal Runx2 P1 promoter. This repression was found to be required for the progression of BMP-induced chondrogenesis, thereby identifying Runx2 as a modulator of BMP activity in the chondrogenic as well as osteogenic differentiation program. To further understand the regulation of the Runx2 P1 promoter and to determine the contribution of P1-derived gene product, Runx2 Type II, to the formation of mineralized tissue, we have generated a Runx2 Type II-LacZ gene replacement mouse model in which the initial coding sequences and splice donor sites of the Type II isoform are replaced with the LacZ reporter gene. Activity of the endogenous P1 promoter can therefore be monitored by β-galactosidase production. Analysis of Runx2 Type II-LacZ mice demonstrates that the P1 promoter is transcriptionally most active in mature osteoblasts, but its product, Runx2 Type II is dispensable for embryonic skeletal formation. Lastly, we examine the link between growth control and osteogenic differentiation by tissue-specific deletion of the Mdm2 proto-oncogene in developing skeletal tissues of the mouse embryo. Loss of Mdm2 results in impaired bone formation, with skeletal elements exhibiting lower bone mineral content and higher porosity. Ex vivo cultures of calvarial osteoprogenitor cells exhibit severely decreased osteoblastogenesis and bone nodule formation accompanied by a failure to activate Runx2 gene activity. These findings suggest that Mdm2 is required for inhibition of p53 activity that ultimately allows for post-confluent proliferation and induction of Runx2 during maturation of the osteogenic phenotype. Taken together, our findings suggest that Runx2 modulates the commitment of progenitor cells to the osteogenic and chondrogenic lineages, and that Runx2 activity is inextricably linked to mechanisms that control cellular proliferation.

Thyroid Tumorigenesis is typically a well understood process, with well delineated oncogenic factors. Follicular and papillary thyroid cancers are typically survivable, with 5-year survival rates being >95% for Stage I-III of both cancer types. Anaplastic thyroid cancer, in contrast, lacks this prognosis, and is the most lethal of all endocrine-related cancers. The median survival time after a diagnosis is generally between 6-8 months, with a 5-year survival rate of <10%. Current treatment for anaplastic thyroid cancers routinely meet roadblocks, as resistance is quickly developed. Even non-discriminatory kinase inactivators, such as sorafenib, which are generally considered a drug of last resort, are unable to effect survival rates. As such, there is a clear need for further investigation of the causes of anaplastic thyroid cancer mechanisms. Previous work in the Carr lab revealed a novel regulatory pathway of an oncogene that is associated with several other endocrine-related cancers, as well as other non-endocrine-related cancers. Specifically, the Runt-related transcription factor 2 (Runx2) was found to be suppressed via direct binding of the thyroid hormone receptor beta 1 isoform (TRß1) to its proximal promotor. Runx2 was previously shown to be associated with increasing malignancy, with Runx2 occurring at low-levels in indolent cell lines, whilst occurring at high-levels in more malignant cell lines. TRß1, conversely, exhibited the opposite relationship. Endogenous levels of TRß1 were found to be high in indolent cell lines and were depleted in malignant cell lines. These findings were further confirmed via tissue microarrays. Restoration of TRß1 in malignant cell lines diminished Runx2 mRNA and protein levels, which was corroborated by evidence from electrophoretic mobility-shift assays, and chromatin immunoprecipitations that TRß1 was able to directly bind Runx2 promotor 1. Current studies have investigated the nuclear protein profile that associates with TRß1 to alter Runx2 transcription. Through EMSA-to-Mass Spectrometry methodologies, as well as novel DNA pulldown techniques, binding partners have been elucidated. Findings have also been confirmed via classical immunoprecipitations. Specifically, our findings show that TRß1 complexes with the brahma-related gene 1 (BRG1) protein, the nuclear co-repressor (NCOR), and BRG1-associated protein 60 (BAF60). BRG1 functions by preferentially recruiting histone deacetylases (HDAC), with BRG1 and the HDAC’s acting to alter chromatin, and thus transcription. Future studies aim at examining whether other proteins complex with TRß1 to alter Runx2 transcription, and whether these complexes are altered in aggressive cell lines.

Os morcegos da família Phyllostomidae, endêmicos da região Neotropical, apresentam notáveis padrões de diversificação nas formas craniofaciais que são associadas às especializações alimentares. Entretanto, as bases genéticas e do desenvolvimento responsáveis pela geração e manutenção de tais especializações adaptativas são amplamente desconhecidas; não apenas nesta linhagem evolutiva, mas para vertebrados de forma geral. Dentre os diversos genes já investigados e associados à morfologia craniofacial, o RUNX2 apresenta amplo potencial de envolvimento causal na diversificação de formas observadas em morcegos filostomídeos. Repetições in tandem na taxa de aminoácidos Glutamina por Alanina (taxa Q/A), relacionadas à morfologia craniana, foram verificadas em carnívoros, porém, o padrão não se manteve quando analisadas outras linhagens de mamíferos como um todo. Neste contexto, o presente estudo analisa, de forma pioneira, o papel da taxa Q/A na diversificação morfológica craniofacial em uma linhagem específica de mamíferos (morcegos filostomídeos) com particular adaptação (distintas dietas, e.g. carnivoria, frugivoria, insetivoria, nectarivoria, onivoria, sanguinivoria). Utilizou-se a abordagem de associação genótipo-fenótipo através de diferentes métodos estatísticos (e.g. correlação de Pearson e inferência Bayesiana), incluindo ainda a relação entre os hábitos alimentares e os traços genotípicos e fenotípicos como variável latente. As análises foram controladas para a não-independência filogenética dos dados, empregando métodos filogenéticos comparativos. Foram verificadas correlações significativas entre a taxa Q/A e diferentes medidas craniométricas que descrevem as mudanças na morfologia integrativa do crânio, em especial a largura e comprimento da maxila superior. Uma correlação positiva entre o aumento da taxa Q/A e os hábitos de frugivoria e onivoria foi verificada, sugerindo também a existência de interação entre o hábito alimentar e as medidas de comprimento e largura da maxila superior, especialmente para animais frugívoros e onívoros.
Phyllostomidae bats, a family endemic to the Neotropics, show remarkable diversification in craniofacial forms that are associated with feed specializations. However, the genetic and developmental bases responsible for the generation and maintenance of such morphological adaptation are largely unknown, not only on this evolutionary lineage, but for vertebrates in general. Among the several genes associated with craniofacial morphology so far, RUNX2 is a putative candidate to underlie diversification of forms found in phyllostomids. Tandem repeats in the rate of Glutamine by Alanine amino acids (Q/A ratio), related to cranial morphology, have been demonstrated in carnivores. However, such pattern was not clear when other mammalian lineages were analyzed as a whole. In this context, the present study examines, for the first time, the role of rate Q/A on craniofacial variation in a specific lineage of mammals (phyllostomids) with marked feed specializations (e.g. carnivorous, frugivorous, insectivorous, nectarivorous, omnivorous, hematophagous). The correlation between genotypes and phenotypic traits were made with different statistical methods (e.g. Pearson correlation and Bayesian inference), including the latent variable of diet as well. Such analysis were controlled for the non-independence phylogenetic effect using phylogenetic comparative methods. Significant correlations were observed between different Q/A rates and cranial measurements, which describes changes in the integrative morphology of the skull, especially the length and width of the upper jaw. In addition, a positive correlation between the increase in Q/A rate and frugivorous/omnivorous diet habits was observed, also suggesting the interaction between feed specializations, length and width of the upper jaw, especially for fruit-eating and omnivores animals.

Cell Biology
Ph.D.
Connective Tissue Growth Factor (CTGF) is a matricellular protein that has been shown to mediate cell adhesion, and as a consequence, it regulates cell proliferation, migration, differentiation and gene transcription. Although previous in vivo and in vitro studies supported the anabolic role of CTGF in skeletogenesis, to date mechanisms of this effect remain unknown. So far, no specific receptor has been identified for CTGF, although previous studies have shown that integrins can serve as functional signaling receptors for CTGF. The CTGF-integrin interaction initiates intracellular signaling cascades that ultimately regulate cell cytoskeleton reorganization, gene transcription and cell function. To study the effect of CTGF on osteoblasts, we first conducted adhesion assays using the MC3T3-E1 osteoblastic cell line. We confirmed that osteoblasts adhere to rCTGF in a concentration-dependent manner and we showed this adhesion has characteristics of integrin mediated adhesions. Next, we used an array of blocking antibodies directed against the individual alpha and beta; integrin subunits that are known to be expressed in osteoblasts. Significant decreases in cell adhesion were observed upon treatment with anti-alpha-v or anti-beta1 blocking antibodies. Subsequent coimmunoprecipitation analyses demonstrated that CTGF interacts with alpha-v and beta1 integrins in osteoblasts. Furthermore, we showed that the specificity of this CTGF-integrin interaction occurs in the C-terminal domain (fourth module) of CTGF. The immunefluorescence staining of cells cultured on substrates of rCTGF, fibronectin (positive control) or BSA (negative control) demonstrated that osteoblast adhesion to rCTGF results in actin cytoskeleton reorganization, focal adhesion formation, enhanced cell spreading and Rac activation. These series of events are necessary for proper cell-matrix interaction and integrins' downstream signaling initiation. Next, through alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining, we demonstrated that osteoblast attachment to CTGF matrix enhances cell maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves activation of specific signaling molecules, we performed Western blot and chromatin immunoprecipitation (ChIP) assays. Osteoblasts cultured on rCTGF expressed higher levels of both total and phosphorylated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) compared to the cells cultured on BSA. In addition, these osteoblasts showed an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter compared to the negative control. These experiments confirmed CTGF's effect on enhancing osteoblast differentiation through regulation of important signaling molecules. In another series of experiments, we used primary osteoblasts isolated from CTGF KO mice, their WT littermates, or WT cells infected to overexpress (OE) CTGF to study the effect of different levels of endogenous CTGF on osteoblast cytoskeleton reorganization and motility. Our assays showed enhanced cell adhesion, spreading and Rac expression in CTGF OE osteoblasts, while in CTGF KO osteoblasts, cell adhesion, spreading and Rac expression were significantly decreased. In contrast, CTGF OE osteoblasts that showed high adhesion and spreading, exhibited diminished cell motility and low levels of RhoA expression, while KO cells migrated quickly and expressed high levels of RhoA. Together, these experiments establish CTGF as an adhesion protein for osteoblasts; they demonstrate that the alpha-v beta1 integrin is a functional signaling receptor for CTGF; they confirm that osteoblast differentiation is enhanced when cultured on CTGF matrix through activation of regulatory signaling molecules; and finally, these experiments establish a role for CTGF in the regulation of small RhoGTPases expression, which in turn implies a significant role for CTGF in cell cytoskeleton reorganization and motility.
Temple University--Theses

Runx2 is a transcription factor widely known for its roles in skeletal development. It is recognised that its regulation and transcriptional activity are also linked to increased growth, invasion, and metastasis in various tumour types. Apoptosis resistance is one of the major hallmarks of cancer, and Bcl-2 is one of the major proteins involved in controlling apoptosis. Recent studies suggest that Runx2 is involved in apoptosis resistance. Previous work carried out in our laboratory showed an association between hypoxia and Runx2 and Bcl-2 expression in prostate tumours, and a consensus DNA binding sequence for Runx2 was found within the Bcl-2 promoter. Results from these studies formed the basis of the hypotheses of this study: The Runx2 transcription factor binds to the Bcl-2 promoter resulting in increased expression of Bcl-2 and resistance to apoptosis and that hypoxia affects the levels of Runx2 thus potentiating apoptosis resistance. Cell proliferation and apoptosis assays have shown that an increased level of Runx2 confers a survival advantage and increased resistance to apoptosis in breast and prostate cells. Protein- DNA interaction assays have suggested that Runx2 binds to and interacts with the Bcl-2 promoter. Runx2-associated apoptosis resistance has been shown to be mediated, at least in part, by increased Bcl-2 expression. mRNA expression analysis carried out has highlighted other potential targets of Runx2 which may be involved in the increased apoptosis resistance demonstrated. Investigation of the effects of acute hypoxia on Runx2 expression have established that Runx2 expression can be increased by hypoxia, and that the associated increase in survival can be diminished by silencing Runx2. However, further work is needed to substantiate these findings. Overall, the work in this thesis adds to the body of knowledge on Runx2 in cancer development and progression, demonstrating its importance in mediating apoptosis resistance in breast and prostate cancer cells. As well as presenting data to show the role of Runx2-associated increased Bcl-2 expression in this process, it also reveals novel Runx2-responsive genes potentially involved in mediating the apoptosis resistance observed. Finally, this work shows that hypoxia may be an important upstream regulator of Runx2 expression in hormone sensitive prostate cancer cells.

Ingeniera en Biotecnología Molecular
Anualmente se producen cerca de 9 millones de fracturas por fragilidad ósea en el mundo. El hueso es el segundo tejido con mayor demanda de trasplantes en el sistema de salud, en donde se emplean injertos autógenos y alógenos. Ante este escenario, el desarrollo de injertos óseos biocompatibles utilizando células troncales adultas para la optimización de la regeneración ósea es una solución con enorme potencial biomédico. En este trabajo se propuso aumentar el potencial osteoblástico de células madre mesenquimáticas derivadas de la gelatina de Wharton (WJ-MSC), a través de la pérdida de función de JARID1B, represor epigenético del gen maestro RUNX2. En investigaciones antecedentes al seminario de titulo, se demostró que el silenciamiento de JARID1B en células C2C12 es capaz de incrementar la expresión de RUNX2-P57. Además, se logró establecer que el estado epigenético de los promotores de RUNX2 y SP7, en cuya regulación participa JARID1B como demetilasa de histonas, posee un rol fundamental en la mantención del limitado potencial osteoblástico de WJ-MSC. En este seminario de título se establecieron las condiciones experimentales para el tratamiento de WJ-MSC con el fármaco PBIT, un inhibidor de la actividad de JARID1B, durante la diferenciación osteoblástica. El tratamiento con PBIT resultó en un aumento significativo de la expresión de RUNX2-P57, en conjunto con el enriquecimiento de la marca epigenética H3K4me3 en el promotor P1 de RUNX2. Sin embargo, no se observaron diferencias con respecto a la mineralización por deposición de sales de calcio en etapas tardías de la diferenciación en respuesta al tratamiento farmacológico. El silenciamiento de JARID1B resultó en un aumento de la expresión de RUNX2-P57, además del enriquecimiento de las marcas epigenéticas H3K4me3 y H3K27Ac en su promotor. xiii En concordancia con estudios previos realizados en el laboratorio, se concluye que las WJ-MSC poseen un potencial osteoblástico restringido, evidenciado por la activación de genes osteoblásticos tempranos y no de marcadores tardíos, frente a la inducción a diferenciación osteoblástica. Se reafirma así la propuesta de una barrera epigenética en RUNX2, que restringe el compromiso a linaje osteoblástico en WJ-MSC, dada por el origen ontogénico temprano de estas MSC, directamente relacionado con su potencial de desarrollo. La modulación epigenética en WJ-MSC, por medio de JARID1B, es capaz de incrementar significativamente la expresión de genes osteoblásticos tempranos, no así de potenciar la formación de osteoblastos y el proceso de mineralización. Proponemos el co-cultivo de WJ-MSC tratadas con PBIT sobre matrices biocompatibles, además de la adición de otros inductores de diferenciación osteoblástica, como BMP2, al medio de cultivo. Asimismo, se sugiere ensayar la adición de las moléculas de la vía de señalización Wnt, WISP1 y sFRP4, con el objetivo de incrementar el potencial osteoblástico de cultivos primarios de WJ-MSC. En definitiva, a pesar del enorme potencial terapéutico de las WJ-MSC, es necesario un mayor entendimiento de los mecanismos celulares que limitan el compromiso a linaje óseo, particularmente en relación a la barrera a nivel epigenético propuesta a raíz de nuestro trabajo con WJ-MSC, para respaldar sólidamente su uso en ingeniería de tejidos óseos y terapia regenerativa.
Worldwide, over 9 million bone fragility fractures are estimated to occur per year. Bone is the second most demanded tissue for transplantation in the health system, where autologous and allogenic grafts are used. In this scenario, the development of biocompatible bone grafts using adult stem cells for the optimization of bone regeneration is a solution with enormous potential in biomedicine. In this work, we aimed to increase the osteoblastic potential of mesenchymal stem cells derived from the Wharton’s Jelly of the umbilical cord (WJ-MSC), through the loss of function of JARID1B, an epigenetic repressor of the master gene RUNX2. In previous work, it was demonstrated that knockdown of JARID1B in the cell line C2C12 is capable of increasing RUNX2-P57 expression. Also, it was established that the epigenetic state of the promoters of RUNX2 and SP7 has a fundamental role in the maintenance of the limited osteoblastic potential of WJ-MSC. In the present thesis, we set the experimental conditions for the treatment of WJ-MSC in osteoblastic differentiation with the drug PBIT, an inhibitor of the activity of JARID1B. PBIT treatment resulted in a significant increase in RUNX2-P57 expression, along with the enrichment of the epigenetic mark H3K4me3 at RUNX2 P1 promoter. However, there were no differences in mineralization due to calcium salts deposition in late stages of osteoblastic differentiation, in response to pharmacological treatment. Knockdown of JARID1B resulted in an increase in RUNX2-P57 expression and in the enrichment of the epigenetic marks H3K4me3 and H3K27Ac at RUNX2 promoter. In concordance with previous research from our laboratory, we conclude that WJ-MSC have a restricted osteoblastic potential, as evidenced by the activation of early osteoblastic genes, but not late osteoblatic markers, when induced to osteoblastic differentiation. The proposal of an epigenetic barrier in RUNX2 that restricts the xv commitment to the osteoblastic lineage in WJ-MSC is then reaffirmed. It is given by the early ontogenetic origin of this MSC and directly related with its developmental potential. Epigenetic modulation in WJ-MSC through JARID1B is capable of significantly increasing the expression of early osteoblastic genes, but it is not capable of enhancing osteoblasts formation and mineralization. We propose the culture of PBIT-treated WJ-MSC over biocompatible matrix and the addition of other osteoblastic differentiation inducers such as BMP2. Similarly, we suggest testing the adittion of molecules from the Wnt signaling pathway, like WISP and sFRP4, to increase the osteoblastic potential in primary cultures of WJ-MSC. In summary, and despite their extensive therapeutic potential, a deeper understanding of the cellular mechanisms underlying the limitation to the commitment of WJ-MSC to the bone lineage is needed to strongly support its application in bone tissue engineering and regenerative therapy, particularly in respect to the epigenetic barrier proposed in the light of our work with WJ-MSC primary cultures.
Proyecto FONDEF D09E1047 de la Dra. Verónica Palma por el financiamiento de la investigación.

Bone tissue engineering strategies are currently being developed as alternative mechanisms to address the clinical demand for bioactive and biomechanical graft material. To date, these efforts have been largely restricted by inadequate supply of committed osteoprogenitor cells and loss of osteoblastic phenotype expression following in vitro culture and expansion. The objective of this thesis research was to address the cell sourcing limitations of tissue-engineered bone grafts through constitutive and sustained overexpression of the osteoblast-specific transcriptional activator Runx2/Cbfa1 in osteogenic marrow-derived stromal cells using retroviral gene delivery. Runx2 overexpression enhanced expression of multiple osteoblastic genes proteins and, more importantly, significantly up-regulated matrix mineralization in both monolayer culture and following cell seeding in 3-D polymeric scaffolds. To evaluate in vivo performance, Runx2-expressing cells were seeded into 3-D constructs and implanted both subcutaneously and in a critical size craniotomy bone defect model. Notably, in vitro pre-culture of Runx2-transduced cell-seeded constructs prior to implantation significantly enhanced their capacity to form mineralized tissue in the subcutaneous space and induce new bone formation in the critical size defect model compared to control cells. The described series of analyses provided a novel combination of tissue and genetic engineering techniques toward the development of a Runx2-modified stromal cell/polymeric scaffold composite tissue-engineered bone graft substitute.

The Runx family of transcription factors performs an essential role in animal development by controlling gene expression programs that mediate cell proliferation, growth and differentiation. The work described in this thesis is concerned with understanding mechanisms by which Runx proteins support this program of gene expression within the architectural context of the mammalian cell nucleus. Multiple aspects of nuclear architecture are influenced by Runx2 proteins including sequence-specific DNA binding at gene regulatory regions, organization of promoter chromatin structure, and higher-order compartmentalization of proteins in nuclear foci. This work provides evidence for several functional activities of Runx2 in relation to architectural parameters of gene. expression for the control of cell growth and differentiation. First, the coordination of SWI/SNF mediated chromatin alterations by Runx2 proteins is found to be a critical component of osteoblast differentiation for skeletal development. Several chromatin modifying enzymes and signaling factors interact with the developmentally essential Runx2 C-terminus. A patent-pending microscopic image analysis strategy invented as part of this thesis work - called intranuclear informatics - has contributed to defining the C-terminal portion of Runx2 as a molecular determinant for the nuclear organization of Runx2 foci and directly links Runx2 function with its organization in the nucleus. Intranuclear informatics also led to the discovery that nuclear organization of Runx2 foci is equivalently restored in progeny cells following mitotic division - a natural perturbation in nuclear structure and function. Additional microscopic studies revealed the sequential and selective reorganization of transcriptional regulators and RNA processing factors during progression of cell division to render progeny cells equivalently competent to support Runx2 mediated gene expression. Molecular studies provide evidence that the Runx proteins have an active role in retaining phenotype by interacting with target gene promoters through sequence-specific DNA binding during cell division to support lineage-specific control of transcriptional programs in progeny cells. Immunolocalization of Runx2 foci on mitotic chromosome spreads revealed several large foci with pairwise symmetry on sister chromatids; these foci co-localize with the RNA polymerase I transcription factor, Upstream Binding Factor (UBFl) at nucleolar organizing regions. A series of experiments were carried out to reveal that Runx2 interacts directly with ribosomal DNA loci in a cell cycle dependent manner; that Runx2 is localized to UBF foci within nucleoli during interphase; that Runx2 attenuates rRNA synthesis; and that this repression of ribosomal gene expression by Runx2 is associated with cell growth inhibition and induction of osteoblast-specific gene expression. This thesis has identified multiple novel mechanisms by which Runx2 proteins function within the hierarchy of nuclear architecture to control cell proliferation, growth and differentiation.

La parathormone (PTH) ou son fragment 1-34 (rh 1-34 PTH) administrée de façon intermittente à faible dosse conduit chez le rongeur, le primate et l'Homme provoque une augmentation de la densité et de la masse osseuse. La rh 1-34 PTH utilisée chez les patientes ayant une ostéoporose avérée réduit significativement la perte osseuse et prévient l'apparition de nouvelles fractures vertébrales. Les mécanismes cellulaires et moléculaires participant cet l'effet anabolique de la PTH sont encore à l'heure actuelle mal connus. Dans notre étude, nous avons exploré in vivo deux voies possibles qui permettrait de mieux comprendre le mode d'action de la PTH : les métalloprotéases matricielles (MMPs) et le facteur de transcription et de différenciation ostéoblastique Runx2. Des études ont par ailleurs montré que c'est par l'intermédiaire du facteur de transcription Runx2 que la parathormone pourrait agir sur le niveau d'expression de la collagénase-3 synthétisée par l'ostéoblaste. Dans une première approche, nous avons traité des souris femelles adultes sur exprimant un inhibiteur des métalloprotéases matricielles, le TIMP-1, dans les ostéoblastes ainsi que leur contrôle de même âge par la PTH à la dose de 40 μg/kg/j pendant 6 semaines. Cette étude indique que l'inhibition des MMPs ostéoblastiques qui conduit à une diminution de la résorption osseuse in vivo induit une augmentation des effets positifs d'un traitement anabolique par la PTH. Nous montrons que l'augmentation de la résorption osseuse par la PTH est absente chez les souris sur exprimant le TIMP-1 mais que l'augmentation de la formation osseuse est maintenue. Dans la seconde partie de notre étude, des souris femelles de 1 mois sur exprimant le facteur de transcription Runx2 dans les ostéoblastes et leur contrôle de même âge ont reçu de la PTH à la dose de 100 μg/kg/j pendant 6 semaines. Les résultats de cette étude montrent qu'une augmentation du niveau d'expression de facteur Runx2 réduit la réponse des ostéoblastes à la PTH. Notre analyse moléculaire et cellulaire démontre que c'est probablement par le biais d'une modification de l'état de différenciation des ostéoblastes que Runx2 affecte la réponse à la PTH in vivo
Parathyroid hormone (PTH) and its 1-34 fragment (rh 1-34 PTH) increase bone density and bone mass in rodents, primates and humans when administrated intermittently at low dosage. Rh 1-34 PTH administration to post-menopausal women with severe osteoporosis leads to a decrease in bone loss and a reduction in the number of new vertebral fractures. The cellular and molecular mechanisms underling this effect are not well understood. In our study, we have explored two pathways that could possibly be involved in die anabolic action of PTH : Matrix metalloproteinase (MMPs) and the transcription factor Runx2. Other studies have indeed presented evidence that Runx2 could be a key mediator of the enhancement of collagenase-3 expression induced by PTH. In the first part of the study, we treated adult female mice which over express TIMP-1 in osteoblasts and their wild type littermates with PTH at a dose of 40μg/kg/d during 6 weeks. Our study indicates that inhibition of osteoblastic MMPs that reduces bone resorption in vivo has a positive effect on the anabolic treatment with PTH. We showed that the increase in bone resorption induced by PTH is abolished in transgenic mice but also that bone formation is maintained. In the second part of this study, we treated young female mice which over express the transcription factor Runx2 in osteoblast and their wild type littermate with PTH at a dose of 100μg/kg/d during 6 weeks. Our results showed that increasing the level of expression of the Runx2 transcription factor reduced the osteoblastic response to PTH. Our molecular and cellular analysis moreover demonstrated that Runx2 blunt the anabolic effect of PTH probably through a mechanism affecting osteoblastic differentiation. These two independent studies helped us to decipher the molecular mechanisms involving Runx2 and MMPs in the action of PTH. We suggest that Runx2 action in the PTH anabolism is independent from those involving MMPs

Epithelial-mesenchymal transition (EMT) transforms developing embryonic endothelial cells of the heart into mesenchymal cells in the process of activation of downstream regulators of the TGFβ pathway, a major component in EMT signaling, such as the positive transcription factor, Runx2. EMT has been shown to occur in pathogenic, cardiac diseases in the adult as well as in developmental embryonic situations. Here, we explore the Runx2 isoforms and their role in each of these processes, as well as the roles of Runx2’s inhibition on downstream transcription and intracellular (stem-cell possessing) factors, Zeb2 and ALDH1, respectively. We concluded that Runx2 isoforms show a relationship in the calcification of the heart, and that Runx2-I expression is also in the embryonic stages. Also, TGFβ-2 induces Runx2 expression, while silencing RNA against Runx2 reduces its prominence, and essentially induces expression of Zeb2 and ALDH1 through a limited regulation. By deciphering the roles of these fundamental yet intricate players and mechanisms in EMT, a gateway to deciphering potential target therapeutics will also be prominent in future developments for patients with congenital cardiac disease.

京都大学
0048
新制・課程博士
博士(医学)
甲第22646号
医博第4629号
新制||医||1044(附属図書館)
京都大学大学院医学研究科医学専攻
(主査)教授 篠原 隆司, 教授 松田 秀一, 教授 安達 泰治
学位規則第4条第1項該当
Doctor of Medical Science
Kyoto University
DFAM

Osteoporosis is a general skeletal disorder. It is characterized by a low bone mass and a microarchitectural deterioration of bone tissue, which increase bone fragility and susceptibility to fracture. LRP5 is a widely expressed member of the low-density lipoprotein receptor superfamily, which acts as a co-receptor for Wnt. Wnt/β-catenin signaling plays an important role in the development and maintenance of many organs and tissues, among others bone. In bone, LRP5 is expressed by osteoblasts and not by osteoclasts; however, little is known about its regulation. Genetic studies show that LRP5 has a major influence on BMD. Loss-of-function mutations in the LRP5 gene cause osteoporosis pseudoglioma syndrome (OPPG) and Gain-of-function mutations in the LRP5 gene cause high bone mass phenotype, an autosomal dominant condition of increased BMD. RUNX2 is a member of the Runt family of transcription factors with a major role in the control of osteoblast commitment and differentiation, whose expression is necessary for the regulation of skeletal genes. Mutations in the RUNX2 locus in human cause cleidocranial dysplasia (CCD), which is an autosomal-dominant condition. Skeletons from homozygous Runx2 -/- (knock out) mice showed complete lack of Ossification. The DNA-binding sites of Runx2 in major bone matrix protein genes, including Col1a1; Col1a2; Spp1; Ibsp/BSP; Bglap2; Fn1/fibronectin; Mmp13, and Tnfrsf11b/Opg, have been identified, and Runx2 induced the expression of these genes or activated their promoters in vitro. Until recently, RUNX2 and LRP5 had not been directly connected. Studies have revealed the presence of five RUNX2 binding sites within a 2.9 kb region upstream of LRP5 and documented the binding of RUNX2 to these sites in vitro. To further explore this relationship in vivo, in this thesis, an osteoblast differentiation protocol using the hFOB cell line was employed. Transcriptional levels of RUNX2 and LRP5, together with OCN, SOST and ALP were evaluated along 21 days of differentiation in 5 stages. RUNX2 and LRP5 proteins were evaluated along 21 days of differentiation. RUNX2 occupancy of the 5 binding sites on the LRP5 promoter, chromatin immunoprecipitation assays were performed at 3 time-points during hFOB differentiation. Osteoblast differentiation model based on the hFOB displayed characteristic osteoblastic markers of mineralization and alkaline phosphatase activity. Expression of LRP5, RUNX2 and also ALP, SOST and OCN was observed in hFOB cell line. RUNX2 showed steady increase reaching a maximum of 4-fold at day 14. All the other genes analyzed showed a peak at day 3, more than 5-fold for SOST and above 4-fold for ALP. OCN and SOST showed a clear decrease after day 3, while ALP showed a slow decrease, and LRP5 maintained relatively similar mRNA levels. Both RUNX2 and LRP5 proteins were detected in hFOB at day 0, 3 and 7. Low levels of LRP5 were also observed at day 14. Overall, the protein levels of both RUNX2 and LRP5 decrease during differentiation of hFOB cell lines. Binding of RUNX2 transcription factor to the promoters of the CDKN1A and SERPINE1 genes was observed in day 7. And RUNX2 binding to CDKN1A and SERPINE1 promoters in hFOB cells was described for the first time. Subsequently, binding of RUNX2 to the five RUNX2 elements in the LRP5 promoter was assessed in chromatin from undifferentiated (day 0) and differentiated (days 7 and 21) hFOB cells. Binding at all five sites was observed at day 7 (above 3-fold enrichment in all cases), while it was negligible at days 0 and 21. No acceptable correlation was observed between RUNX2 binding and LRP5 expression, which leaves the functional effect of RUNX2 binding to the LRP5 promoter as an unsolved question.
L'osteoporosi es caracteritza per una baixa massa òssia i un deteriorament de la microarquitectura del teixit ossi. LRP5 és un membre de la superfamília de receptors de lipoproteïnes de baixa densitat, que actua com a co-receptor de la via de Wnt. LRP5 té una gran influència en la densitat mineral òssia. Runx2 és un membre de la família de factors de transcripció Runt amb un paper essencial en el control de la determinació i la diferenciació dels osteoblasts. La seva expressió és necessària per a la regulació dels gens de l'esquelet. Fins fa poc, Runx2 i LRP5 no havien estat connectats directament. Estudis recents han revelat la presència de cinc llocs d'unió de Runx2 en una regió de 2,9 kb upstream de LRP5 i s’ha documentat la unió de Runx2 a aquests llocs in vitro. Per explorar aquesta relació in vivo, en aquesta tesi es va emprar un protocol de diferenciació d’osteoblasts utilitzant la línia cel·lular hFOB. Es van avaluar els nivells de transcripció de RUNX2 i LRP5, juntament amb els de OCN, SOST i ALP al llarg de 21 dies de diferenciació. També es va avaluar les proteïnes Runx2 i LRP5. Per provar la ocupació de Runx2 als 5 llocs d'unió del promotor de LRP5, es van realitzar assaigs d'immunoprecipitació de cromatina durant la diferenciació de les cèl·lules hFOB, (dies 0, 7 i 21). Només es va observar unió en tots els cinc llocs en el dia 7, i no en els dies 0 i 21. La unió de Runx2 al promotor de LRP5 es descriu per primera vegada en aquesta tesi.

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Em programas de melhoramento de frangos de corte, as aves são selecionadas principalmente para características de desempenho e carcaça, acarretando em mudanças no tamanho, forma e função dos órgãos, o que implica em alterações fisiológicas importantes durante o desenvolvimento das aves. A seleção intensa dos frangos de corte resultou em problemas locomotores, diminuição da resistência óssea dos animais e aumento da mortalidade. Com isso, técnicas de biologia molecular podem ser utilizadas na seleção assistida por marcadores com o intuito de identificar genes candidatos para auxiliar no processo de seleção de características relacionadas às desordens ósseas. Os objetivos do presente trabalho foram estimar os parâmetros genéticos para características relacionadas à integridade óssea do fêmur e peso aos 42 dias de idade e estudar as associações dos genes RUNX2 (Runt-related transcription factor 2) e TNFSF11 (Tumor necrosis fator (ligand) superfamily, member 11) com características de importância econômica em frangos de corte no intuito de fornecer suporte para o programa de melhoramento genético de aves e direcionar o processo de seleção visando a redução dos problemas relacionadas às desordens ósseas. Estimativas de parâmetros genéticos, fenotípicos e ambientais foram obtidas para características associadas à integridade óssea do fêmur e peso aos 42 dias de idade (P42) pelo método de máxima verossimilhança restrita utilizando modelo animal multicaracterística, com efeito fixo de grupo (sexo e incubação) e os efeitos aleatórios genéticos aditivos e residuais. Análises de associações genéticas entre o SNP g.57.397A>G (RUNX2) e SNP g.14.614A>G (TNFSF11) com características de desempenho, composição de carcaça, órgãos e integridade óssea do fêmur foram realizadas com o propósito de avaliar os efeitos desses polimorfismos sobre esse grupo de características, por ...
In the broiler breeding programs, the broilers are selected primarily for performance and carcass traits, resulting in changes in the size, shape and function of organs which implies important physiological changes during the development of the broilers. Intense selection of broilers resulted in locomotors problems, decreased bone strength of animals and increased mortality. Thus, molecular screening techniques began to be used in order to identify candidate genes to assist in the selection of related traits to bone disorders. The objective of this study was estimate genetic parameters for traits related to femoral integrity bone and weight at 42 days and association study of RUNX2 (Runt-related transcription factor 2) and TNFSF11 (Tumor necrosis factor (ligand) superfamily, member 11) genes with economical traits in broilers in order to provide support for the broiler breeding program and direct the selection process to reduce problems related to bone disorders. Estimates of genetic parameters were obtained for traits associated with the femur bone integrity and weight at 42 days of age by the restricted maximum likelihood method using multi-trait animal model with the fixed group effect (sex and incubation), and the random effects and additives residuals. The investigation of polymorphisms in association RUNX2 genes and TNFSF11 and the association study of polymorphisms found and their respective SNPs (Nucleotide single polymorphism) of RUNX2 genes (SNP g.57.397A>G) and TNFSF11 (SNP g.14.614A> g) with the performance, carcass composition, parts and femoral bone integrity in order to evaluate the effects of these polymorphisms about this group of traits to maximum likelihood method. We used the TT reference population database with about 1,500 broilers belonging to Embrapa Swine and Poultry Breeding Program in Concordia/SC, Brazil. Heritability coefficients estimated for weight at 42 days of age (W42) and the related traits to ...

Runx2 est un facteur de transcription indispensable à la différenciation de l'ostéoblaste (Ob) et nécessaire au développement osseux. Chez la souris transgénique (TG), la surexpression de Runx2 spécifiquement dans l'Ob induit un phénotype ostéoporotique précoce et sévère avec apparition de fractures spontanées. Cette ostéoporose est la résultante d'une augmentation de la résorption couplée à un blocage de la différenciation ostéoblastique. Notre étude a eu pour but de déterminer si la voie Wnt/p-Caténine, très importante dans le développement du tissu osseux, était impliquée dans le mécanisme induisant l'ostéoporose chez la souris TG. En particulier nous avons cherché à mettre en évidence l'implication de Runx2 dans la régulation de la voie Wnt et son impact sur le phénotype osseux de la souris Runx2 et son contrôle. Nous avons mis en évidence que l'activité de la p-Caténine est régulée de façon dose-dépendante par l'expression de Runx2 in vitro et in vivo et que son activation peut restaurer l'activité de la P-Caténine et partiellement la différenciation de l'Ob Runx2. Nous avons aussi montré que la stimulation de la voie Wnt induit une restauration du phénotype trabéculaire osseux due à l'inhibition de la résorption osseuse suite à une augmentation de la synthèse d'ostéoprotégérine par l'Ob. En conclusion, alors qu'une restauration de la différenciation ostéoblastique était attendue, nous avons montré que la stimulation de la voie Wnt inhibe la différenciation ostéoclastique et donc la résorption osseuse induite par la surexpression de Runx2. Notre travail montre que la voie Wnt est un régulateur indirect de la résorption osseuse induite par Runx2 dans notre modèle
Runx2 is a transcription factor essentiel for osteoblast differentiation and necessary for bone development. Moreover, transgenic mice overexpressing Runx2 specifically in osteoblast exhibit an early and severe osteoporosis associated with spontaneous fractures. Osteoporosis in mice overexpressing Runx2 is the result of an increased resorption coupled to a blocking of osteoblast differentiation. Our study was designed to determine whether the Wnt/p-Catenin, that is very important in the development of bone tissue, was involved in the mechanism inducing osteoporosis in mice overexpressing Runx2. In particular, we sought to highlight the involvement of Runx2 in the regulation of the Wnt and the impact of this pathway on the bone phenotype in mice overexpressing Runx2 and in their control. We have shown that the activity of p-Catenin is regulated in a dosé-dependent manner by expression of Runx2 in vitro and in vivo, and that stimulation of the Wnt pathway can restore the activity of p-Catenin and partially the differentiation of osteoblasts overexpressing Runx2. We also showed in vivo that stimulation of the Wnt pathway induces a restoration of trabecular bone phenotype. This restoration is due to inhibition of bone resorption with an increase in the synthesis of osteoprotegerin by osteoblasts. In conclusion, while a restoration of osteoblast differentiation was expected, we showed that stimulation of the Wnt signaling inhibits in vivo and in vitro osteoclast differentiation and thus bone resorption induced by overexpression of Runx2. Our work shows that the Wnt pathway is an indirect regulator of bone resorption induced by Runx2 in our model

Orientador: Danísio Prado Munari
Coorientador: Mônica Correa Ledur
Coorientador: Jane de Oliveira Peixoto
Banca: Nedenia Bonvino Stafuzza
Banca: Humberto Tonhati
Banca: Adriana Mercia Guaratini Ibelli
Banca: Claudia Cristina Paro de Paz
Resumo: Em programas de melhoramento de frangos de corte, as aves são selecionadas principalmente para características de desempenho e carcaça, acarretando em mudanças no tamanho, forma e função dos órgãos, o que implica em alterações fisiológicas importantes durante o desenvolvimento das aves. A seleção intensa dos frangos de corte resultou em problemas locomotores, diminuição da resistência óssea dos animais e aumento da mortalidade. Com isso, técnicas de biologia molecular podem ser utilizadas na seleção assistida por marcadores com o intuito de identificar genes candidatos para auxiliar no processo de seleção de características relacionadas às desordens ósseas. Os objetivos do presente trabalho foram estimar os parâmetros genéticos para características relacionadas à integridade óssea do fêmur e peso aos 42 dias de idade e estudar as associações dos genes RUNX2 ("Runt-related transcription factor 2") e TNFSF11 ("Tumor necrosis fator (ligand) superfamily, member 11") com características de importância econômica em frangos de corte no intuito de fornecer suporte para o programa de melhoramento genético de aves e direcionar o processo de seleção visando a redução dos problemas relacionadas às desordens ósseas. Estimativas de parâmetros genéticos, fenotípicos e ambientais foram obtidas para características associadas à integridade óssea do fêmur e peso aos 42 dias de idade (P42) pelo método de máxima verossimilhança restrita utilizando modelo animal multicaracterística, com efeito fixo de grupo (sexo e incubação) e os efeitos aleatórios genéticos aditivos e residuais. Análises de associações genéticas entre o SNP g.57.397A>G (RUNX2) e SNP g.14.614A>G (TNFSF11) com características de desempenho, composição de carcaça, órgãos e integridade óssea do fêmur foram realizadas com o propósito de avaliar os efeitos desses polimorfismos sobre esse grupo de características, por...
Abstract: In the broiler breeding programs, the broilers are selected primarily for performance and carcass traits, resulting in changes in the size, shape and function of organs which implies important physiological changes during the development of the broilers. Intense selection of broilers resulted in locomotors problems, decreased bone strength of animals and increased mortality. Thus, molecular screening techniques began to be used in order to identify candidate genes to assist in the selection of related traits to bone disorders. The objective of this study was estimate genetic parameters for traits related to femoral integrity bone and weight at 42 days and association study of RUNX2 ("Runt-related transcription factor 2") and TNFSF11 ("Tumor necrosis factor (ligand) superfamily, member 11") genes with economical traits in broilers in order to provide support for the broiler breeding program and direct the selection process to reduce problems related to bone disorders. Estimates of genetic parameters were obtained for traits associated with the femur bone integrity and weight at 42 days of age by the restricted maximum likelihood method using multi-trait animal model with the fixed group effect (sex and incubation), and the random effects and additives residuals. The investigation of polymorphisms in association RUNX2 genes and TNFSF11 and the association study of polymorphisms found and their respective SNPs (Nucleotide single polymorphism) of RUNX2 genes (SNP g.57.397A>G) and TNFSF11 (SNP g.14.614A> g) with the performance, carcass composition, parts and femoral bone integrity in order to evaluate the effects of these polymorphisms about this group of traits to maximum likelihood method. We used the TT reference population database with about 1,500 broilers belonging to Embrapa Swine and Poultry Breeding Program in Concordia/SC, Brazil. Heritability coefficients estimated for weight at 42 days of age (W42) and the related traits to ...
Doutor

Runx2 a été identifié en tant que facteur de transcription jouant un rôle majeur au cours de la différenciation ostéoblastique. C'est un médiateur essentiel du phénotype osseux et il est impliqué dans la régulation de gènes qui sont importants au cours de l'ostéogenèse (tels que les protéines de la matrice extracellulaire osseuse). Runx2 appartient à la famille de facteurs de transcription Runt qui contient deux autres membres (Runx1 et Runx3) qui, suivant les conditions, peuvent réguler la prolifération et/ou l’apoptose dans les tissus où ils sont exprimés. Depuis son identification, plusieurs études ont montré l’implication de Runx2 dans le contrôle de la prolifération des ostéoblastes. Plus récemment, il a été suggéré qu’il pourrait agir en tant que facteur pro-apoptotique. Dans le cadre de cette thèse nous nous sommes intéressés à la régulation par Runx2, au sein de cellules osseuses, de la prolifération et de l'apoptose induite par une des cytokines majeures modulant le métabolisme osseux, le TNFa. Nous avons développé deux modèles cellulaires: (1) Des clones d’ostéosarcome humain de la lignée SaOs-2 transfectés de façon stable par une forme dominante négative de Runx2 (deltaRunx2). (2) Des cellules souches mésenchymateuses humaines où l'expression de Runx2 est inhibée par des siRNAs (short interference RNA) dirigés contre Runx2. Sur ces modèles nous avons montré que l'inhibition de l'activité de Runx2 induit une augmentation de la prolifération. Cette augmentation s’accompagne d’une élévation de l’expression des yclines A1, B1 et E1 et d’une diminution des inhibiteurs de cycline p21 et p27. De plus, nous avons bservé sur nos deux modèles que l'inhibition de Runx2 protège des effets antiprolifératifs et poptotiques du TNFa. Ceci s’accompagne d’une diminution du rapport Bax/Bcl2 et d'une nhibition de l'expression et de l'activité de la caspase 3. Ces résultats confirment les points ommuns entre les trois Runx. En ce qui concerne le tissu osseux, cette étude confirme tout 'abord que Runx2 représente un élément de contrôle décisif pour l’orientation de la cellule pré-ostéoblastique entre prolifération et différenciation. Elle nous apporte aussi des modèles cellulaires de choix pour en comprendre les mécanismes. D'autre part nous avons mis en évidence que l'expression de Runx2 peut aussi être indispensable à la réception d'un stimulus apoptotique. L'expression de Runx2 augmente au cours de la différenciation des ostéoblastes, ceci implique qu’il puisse jouer un rôle majeur dans la sensibilité aux hormones et aux cytokines au sein des ostéoblastes différenciés
Runx2 was identified as a transcriptional factor which plays a pivotal role in isteoblast differentiation. It’s an essential mediator for osteoblastic phenotype involved in the regulation of genes that are important in commiting cells to the osteoblast lineage (such as proteins of the bone extracellular matrix). Runx2 belongs to the Runt-related transcriptional factor family which contains two other members (Runx1 and Runx3). Depending on conditions, Runx proteins can control proliferation and/or apoptosis in cell types where they are expressed. Increasing evidences are consistent with a requirement of Runx2 for control of osteoblast proliferation and recent data even suggested that Runx2 might act as a proapoptotic factor. In this thesis, we were interested to study in osteoblastic cells the effects of Runx2 level on the proliferation and apoptosis induced by TNFα which is one of the major cytokines modulating bone tissue metabolism. Thus we had developed two cellular models : (1) Human osteosarcoma cell line SaOs-2 stably transfected with Runx2 dominant negative construct (deltaRunx2). (2) Human mesenchymal stem cells in which Runx2 expression was inhibited by siRNA directed against Runx2. In this models, we had demonstrated that the inhibition of Runx2 activity induced an increase in proliferation. This increase was accompanied by a rise in cyclins A1, B1 and E1 expression and a decrease in the cyclins inhibitor p21 and p27. Moreover we observed that the inhibition of Runx2 protected the cells from the antiproliferative and the apoptotic effects induced by TNFα. This was accompanied by a decrease in the ratio Bax/Bcl2 and an inhibition in the expression and the activity of caspase 3. These results increased the common points between the three members of Runx proteins. This study confirmed that Runx2 represents a critical link between cell fate, proliferation and growth control. Moreover, we had shown that Runx2 expression could be essential to the reception of apoptotic stimulus. It brings us cellular models to understand the mechanisms involved for these regulations. Runx2 expression increases during osteoblastic differentiation, thus our results suggested that Runx2 might be implicated in the regulation of expression elements which they are involved in hormones and cytokines sensitivity during this process

La régulation transcriptionnelle des premières étapes de la différenciation ostéoblastique est clairement orchestrée par runx2, gène indispensable à la mise en place du squelette et primordial dans le maintien des fonctions ostéoblastiques chez l'adulte. Cependant bien que de nombreuses études soient faites sur ce facteur transcriptionnel, son action ostéogénique passe par la régulation de gènes restant à identifier. Ce travail de recherche consistait en la mise au point de modèles cellulaires permettant de mettre en évidence de nouveaux gènescibles de runx2. La lignée d'ostéosarcome humain SaOs-2 a été transfectée par des constructions plasmidiques permettant la surexpression de Runx2 ou d'une forme mutante dominante négative. Des analyses par Differential Display des profils géniques des clones sélectionnés ont été réalisées et la vérification des différences observées, par RT-PCR en temps réel, Western-blot ou gel retard ont permis d'identifier cinq gènes sous la dépendance de Runx2, nous conduisant principalement à trois grands axes de réflexion. 1 ) La modulation de l'expression de Runx2 est reflétée par un changement global de la synthèse des protéines, de la prolifération cellulaire ou du cycle cellulaire avec l'intervention de RPS24, elF-4AI et SelM. 2 ) Le rôle de Runx2 dans les tumeurs osseuses ostéoslérotiques se précise et pourrait être reflété par la régulation de CD99, un marqueur des tumeurs de Ewing. 3 ) La transcription de gnas1, codant pou la Gsα, pourraît être directement sous la dépendance de Runx2. Cette dernière observation est sans doute la plus aboutie et génère de nombreuses réflexions telle qu'une fonction possible de Runx2 dans la désensibilisation à long terme de récepteurs important dans la régulation du métabolisme osseux
Runx2 is a key regulator of osteoblast-specific gene expression and controls the expression of multiple target genes during osteoblast differentiation. Althought some transcriptional targets for Runx2 are known, it is believed that the osteogenic action of Runx2 is mediated by aditional target genes and increasing studies are performed in order to identify such Runx2-responsive genes. To identify genes following the modification of Runx2 activity in osteoblastic cell line, SaOs-2 was stably transfected in order to overexpress Runx2 or a dominant negative mutant of Runx2. Comparison of gene expression patterns was performed by differential display on selected SaOs-2 clones and real time RT-PCR, western-blot or gel shift experiments were performed to verify our observations. This strategy allowed us to identify five new genes, SelM, elF-4AI, RPS24, CD99 and GNAS1, which may be under the control of Runx2 and led us to conclude that : 1 ) Runx2 expression seems to be linked to a global change in the cellular metabolism and cell growth with modulation of SelM, elF-4AI, and RPS24. 2 ) CD99/MIC2, marker of Ewing family tumors, may be associated with osteosarcoma cells in which normal Runx2 expression is perturbed by exogenous factors. 3 ) Runx2 is able to bind to the promoter of GNAS1, suggesting a direct regulation of this gene which encodes for the Gsα protein and numerous evidences showed that Gsα coupled receptors regulate bone mass. This regulation of Gsα protein by Runx2 seems to be of particular interest considering evidences on bone metabolism regulation by G protein

Lo studio di questa tesi è stato rivolto all’analisi di specifiche zone regolative del gene umano per il Recettore dell’Estrogeno α (hERα) in cellule di osso, osteoblasti e osteoclasti. In queste cellule la trascrizione del gene ERα è diretta dal promotore distale F il quale, insieme a 6 diversi altri promotori ad attività tessuto specifica, fa parte della complessa regione 5’ del gene. Sulla base di evidenze che sono state raccolte mediante un’analisi bioinformatica, lo studio ha preso in considerazione putativi siti di legame per i fattori (TF) Runx2, NFATc1, AP-1 e ERα, allo scopo di far luce sui meccanismi che regolano l’espressione del gene ERα stesso, il quale gioca un ruolo centrale nel metabolismo osseo. In particolare la ricerca si è concentrata su quei TF che numerosi studi hanno indicato come regolatori essenziali dell’osteoblasteogenesi e dell’osteclasteogenesi, e che pertanto possono risultare buoni candidati per una terapia mirata basata sulla modulazione dell’espressione di geni specifici. E’ stato infatti dimostrato che molte patologie del tessuto osseo sono caratterizzate da attività anomale di alcuni TF, tra cui quelli considerati in questa tesi. MODELLI SPERIMENTALI: Linee cellulari di osteosarcoma, colture primarie di osteoblasti (OBs). METODOLOGIA: RT-PCR e WESTERN BLOT (per l’analisi dell’espressione), EMSA (saggio di ritardo della corsa elettroforetica) e ChIP (saggio di immunoprecipitazione della cromatina) (per l’analisi in vitro e in vivo delle interazioni DNA- proteina), REPORTER GENE ASSAY (per l’analisi dell’attività del promotore), siRNA (per il silenziamento genico), MUTAGENESI SITO SPECIFICA (per la mutazione dei siti di legame) PRINCIPALI RISULTATI OSTEOBLASTI: RUNX2 si lega in modo specifico a due (a e b) dei tre siti identificati. Al sito a (5’- AACCACA-3’) si lega come repressore, al sito b (AACCTCA) come attivatore. La scoperta di questo duplice ruolo di Runx2 suggerisce che il suo coinvolgimento nella regolazione di ERα si fortemente determinato dai livelli di espressione di Runx2 stesso, come confermato dagli esperimenti di silenziamento e iperespressione. ERα si lega per lo più in modo indiretto alla sequenza “half ERE” e AP-1 associandosi in vivo con alcuni membri della famiglia AP-1. Le combinazioni più probabili che sono state evidenziate sono c-jun/c-fos, ATF-2/ ATF-2, and ERα/ATF-2. Queste interazioni sono dipendenti dall’estrogeno e seguono una cinetica oscillatoria. NFATc1 è un regolatore negativo di ERα. Mediante esperimenti di “decoy” è stato possibile modulare positivamente l’espressione di ERα sottraendo con un oligonucleotide specifico il fattore NFATc1. Poiché questo TF è il principale sostenitore dell’attività erosiva della matrice ossea da parte degli Osteoclasti, l’aver scoperto un suo ruolo negativo nella regolazione di ERα, conferma l’attività osteoprotettiva di ERα e apre la strada per possibili nuove strategie terapeutiche per combattere le malattie osteopeniche come l’osteoporosi.

Memoria para optar al título profesional de Bioquímico
El cáncer ovárico epitelial (COE) es el cáncer ovárico más común, siendo en Chile la segunda causa de muerte por neoplasias ginecológicas. Un aspecto importante para la mantención y progresión tumoral es el establecimiento de nueva vasculatura. En COE, NGF y su receptor TRKA son mediadores directos de angiogénesis in vitro, e indirectos al inducir la expresión y secreción de VEGF. El factor transcripcional RUNX2 induce procesos oncogénicos como proliferación, migración e invasión celular. Adicionalmente, se cree que RUNX2 puede ser un mediador de angiogénesis tumoral, ya que en varios modelos de cáncer promueve la expresión de VEGF y sus niveles proteicos se incrementan en hipoxia. En COE, RUNX2 los niveles se relacionan con mal pronóstico y en modelos in vitro promueve proliferación, migración e invasión en células de cáncer ovárico, sin embargo, no existen estudios que busquen vincularlo con angiogénesis en este cáncer. Hipótesis: En cáncer ovárico epitelial se produce un aumento en los niveles de RUNX2, y en la línea celular A2780 de cáncer ovárico sus niveles son incrementados por los factores proangiogénicos NGF e hipoxia cuando se comparan con la línea celular HOSE de epitelio superficial ovárico normal. Objetivos: Establecer si RUNX2 se expresa en cáncer ovárico epitelial, si sus niveles se incrementan con la progresión de la transformación maligna del epitelio ovárico, y determinar si en el modelo de líneas celulares, RUNX2 es aumentado por las condiciones proangiogénicas de hipoxia y estímulo con NGF. Metodología: Se evaluaron los niveles proteicos de RUNX2 en tejidos ováricos (OVI, ovario normal inactivo; TBE-TBO, tumores ováricos Benignos y Borderline; y COE) mediante Western Blot, y a través de inmunohistoquímica se determinó el % células positivas con RUNX2 nuclear. En las líneas celulares HOSE y A2780 se evaluaron los niveles proteicos de RUNX2 en extractos totales y en fracciones subcelulares mediante Western Blot, y su localización subcelular mediante inmunocitoquímica. Adicionalmente, se realizaron estímulos con NGF, CoCl2 y ensayos de hipoxia en ambas líneas celulares, para posteriormente determinar los niveles proteicos de RUNX2 y HIF-1α por Western Blot. Resultados: Se determinó que los tejidos ováricos correspondientes a TBE-TBO y COE presentaron un mayor porcentaje de células positivas para RUNX2 nuclear respecto a lo observado en OVI. Por otro lado, ambas líneas celulares, HOSE y A2780, presentaron RUNX2 con localización principalmente nuclear, mostrando la línea HOSE niveles más altos respecto a la línea A2780. Los estímulos con NGF y CoCl2 no modificaron los niveles proteicos de RUNX2 en las líneas celulares en las condiciones analizadas. Por otra parte, se determinó que en la línea celular HOSE los niveles de RUNX2 disminuyen durante hipoxia, manteniéndose constantes los de HIF-1α. En el caso de las células A2780, no se observaron cambios en los niveles de las proteínas RUNX2 y HIF-1α en condiciones de hipoxia. Conclusiones: El presente trabajo permite concluir que los tejidos transformados de epitelio ovárico poseen un alto porcentaje de células con RUNX2 nuclear. Consistente con esto, la línea celular A2780 presenta altos niveles de RUNX2 en el núcleo, sugiriendo estos antecedentes un rol de RUNX2 en COE. Por otro lado, en las condiciones evaluadas, NGF e hipoxia no produjeron cambios en los niveles de RUNX2 en las líneas celulares. Proyección: Es necesario realizar nuevos ensayos para determinar con certeza si las condiciones proangiogénicas de NGF e hipoxia pueden inducir angiogénesis a través de RUNX2 en COE. En el caso de NGF es necesario evaluar si este puede influenciar la actividad transcripcional de RUNX2, y en el caso de hipoxia, se requiere modificar las condiciones experimentales para determinar con mayor seguridad si la restricción de oxígeno afecta los niveles de RUNX2
Epithelial ovarian cancer (COE) is the most common ovarian cancer, being Chile the second cause of death due to gynecological neoplasias. An important aspect for tumor maintenance and progression is the establishment of new vasculature. In COE, NGF and its TRKA receptor are direct mediators of in vitro angiogenesis, and indirectly by inducing VEGF expression and secretion. RUNX2 transcriptional factor induces oncogenic processes such as cell proliferation, migration and invasion. In addition, it is believed that RUNX2 may be a mediator of tumor angiogenesis, since in several cancer models it promotes the expression of VEGF and its protein levels increase in hypoxia. In COE, RUNX2 levels are related to poor prognosis and in vitro models promote proliferation, migration and invasion in ovarian cancer cells, however, there are no studies that seek to link it with angiogenesis in this cancer. Hypothesis: In epithelial ovarian cancer there is an increase in RUNX2 levels, and in the A2780 cell line of ovarian cancer their levels are increased by the proangiogenic factors NGF and hypoxia when compared to the HOSE cell line of normal ovarian surface epithelium. Objective: To establish whether RUNX2 is expressed in epithelial ovarian cancer, if its levels increase with the progression of malignant transformation of ovarian epithelium, and to determine if RUNX2 is increased in the cell lines model by the proangiogenic conditions of hypoxia and stimulation with NGF. Methods: RUNX2 protein levels in ovarian tissues (OVI, normal ovary, TBE-TBO, Benign ovarian tumors and Borderline, and COE) were evaluated by Western Blot, and by means of immunohistochemistry the percentage of RUNX2 nuclear positive cells was determined. In the HOSE and A2780 cell lines the protein levels of RUNX2 in total extracts and subcellular fractions were evaluated by Western Blot, and its subcellular localization by immunocytochemistry. Additionally, stimuli were performed with NGF, CoCl2 and hypoxia assays in both cell lines, to later determine the protein levels of RUNX2 and HIF-1α by Western Blot. Results: It was determined that the ovarian tissues corresponding to TBE-TBO and COE presented a higher percentage of RUNX2 nuclear positive cells than observed in OVI. On the other hand, both cell lines, HOSE and A2780, presented RUNX2 with mainly nuclear localization, showing the line HOSE levels higher than the line A2780. The stimuli with NGF and CoCl2 did not modify the protein levels of RUNX2 in the cell lines in the conditions analyzed. On the other hand, it was determined that in the HOSE cell line the levels of RUNX2 decrease during hypoxia, keeping those of HIF-1α constant. In the case of A2780 cells, no changes were observed in the levels of the RUNX2 and HIF-1α proteins under conditions of hypoxia. Conclusions: The present work concludes that the transformed tissues of ovarian epithelium possess a high percentage of cells with nuclear RUNX2. Consistent with this, the A2780 cell line presents high levels of RUNX2 in the nucleus, suggesting this background a RUNX2 role in COE. On the other hand, under the conditions evaluated, NGF and hypoxia did not produce changes in the RUNX2 levels in the cell lines. Projection: Further testing is required to determine with certainty whether the proangiogenic conditions of NGF and hypoxia may induce angiogenesis tHRPough RUNX2 in COE. In the case of NGF it is necessary to evaluate if this can influence the transcriptional activity of RUNX2, and in the case of hypoxia, it is necessary to modify the experimental conditions to determine more safely if the oxygen restriction affects RUNX2 levels
Fondecyt

Transcriptional regulation of CD8+ T cell differentiation during acute and chronic viral infections is an intricate web made up of many of transcription factors. While several transcription factors have been elucidated in this process, there are still many more that remain elusive. In this work, we look into the role of two transcription factors, IRF4 and Runx2, and their role in CD8+ T cell terminal effector cells and memory precursor cells during acute LCMV-Armstrong infection. We found that IRF4 expression was regulated by TCR signal strength during infection, and that IRF4 expression levels directly correlated with the magnitude of the effector cell response. IRF4 was also shown to regulate T-bet and Eomes, two transcription factors critical for CD8+ T cell differentiation into effector and memory cells. From these results, we were interested in the potential role of IRF4 during chronic LCMV-clone 13 infection, where ratios of T-bet and Eomes are critical for viral clearance. We found that haplodeficiency of IRF4 in the T cell compartment lead to an increase in the ratio of Eomes to T-bet in T cells, which in turn affected the proportion of Eomeshi versus T-bethi cells and resulted in a loss in ability to clear viral infection. Irf4+/-Eomes+/- compound heterozygous mice were generated to test if decreasing Eomes expression would rescue the Irf4+/- phenotype. Irf4+/-Eomes+/- mice were phenotypically similar to WT mice in terms of Eomes to T-bet ratios, and were able to clear viral infection, demonstrating a critical role of IRF4 in regulating T-bet and Eomes during chronic viral infection. Next we looked into the role of Runx2 during acute LCMV-Armstrong infection and found that Runx2-deficient pathogen-specific CD8+ T cells had a defect in the total number of memory precursor cells compared to WT controls. We further showed that Runx2 was inversely correlated with TCR signal strength, and that Runx2 expression was repressed by IRF4. From these work, we have introduced two more transcription factors that are critical for CD8+ T cells differentiation during acute and chronic viral infection. Given the sheer number of transcription factors known to regulate these processes, having a full understanding of the transcriptional network will allow us to find the best targets for therapeutic intervention for treatments ranging from vaccine development and autoimmunity to cancer immunotherapy and treatment of chronic viral infections.

Cell Biology
Ph.D.
Osteoactivin (OA) is a glycoprotein required for the differentiation of osteoblasts. In osteoblasts, Bone Morphogenetic Protein-2 (BMP-2) activated Smad1 signaling enhances OA expression. However, the transcriptional regulation of OA gene expression by BMP-2 is still unknown. The aim of this study was to characterize BMP-2-induced transcription factors that regulate OA gene expression during osteoblast differentiation. The stimulatory effects of BMP-2 on OA transcription were established by cloning the proximal 0.96kb of rat OA promoter region in a luciferase reporter vector in various osteogenic cell types. Further, by deletion and mutagenesis analyses of the cloned OA promoter, key binding sites for osteogenic transcription factors namely, Runx2, Smad1, Smad4 and homeodomain proteins (Dlx3, Dlx5 and Msx2) were identified and characterized. Utilizing specific siRNAs to knock down Runx2, Smad1, Smad4, Dlx3, Dlx5 or Msx2 proteins in osteoblasts, we found that Runx2, Smad1, Smad4, Dlx3 and Dlx5 proteins up-regulate OA transcription, whereas, Msx2 down-regulated OA gene expression. These specific effects of transcription factors on OA promoter regulation were confirmed by forced expression of transcription factors. Most notably, BMP-2-stimulated cooperative and synergistic interactions between Runx2-Smad1 proteins and Dlx3-Dlx5 proteins that up-regulate OA promoter activity. Electrophoretic mobility shift and supershift assays demonstrated that BMP-2 stimulates interactions between Runx2, Smad1 and Smad4 and homeodomain transcription factors with the OA promoter regions flanking the -585 Runx2 binding site, the -248 Smad binding site and the region between the -852 and the -843 homeodomain binding sites relative to transcription start site. The OA promoter region was occupied by Runx2 and also Dlx3 transcription factors during proliferation stages of osteoblast differentiation. As the osteoblasts progress from proliferation to matrix maturation stages of differentiation, the OA promoter was predominantly occupied by Runx2 and to a lesser extent Dlx5 in response to BMP-2. Finally, during matrix mineralization stages of osteoblast differentiation, BMP-2-induced a robust recruitment of Dlx5, Smad1, Dlx3 and Msx2 proteins with simultaneous dissociation of Runx2 from the OA promoter region. In conclusion, the BMP-2-induced osteogenic transcription factors Runx2, Smad1, Smad4, Dlx3, Dlx5 and Msx2 provide key molecular switches that regulate OA transcription during osteoblast differentiation.
Temple University--Theses

Bone mineral density (BMD) is a reflection of the action of osteoblasts compared to osteoclasts. An imbalance in the activity of osteoblasts or osteoclasts, results in bone disease such as osteoporosis caused by overactive osteoclasts. BMD is influenced by genetic and environmental factors as demonstrated through twin studies, association studies and linkage analysis (Ralston, 1999). Several polymorphisms involved in the determination of BMD have been identified, with Vitamin D receptor and Collagen Type 1 showing reproducible associations. To identify genes influencing BMD two distinct strategies have been employed: 1) To determine if DNA polymorphism within the runt related transcription factor (RUNX2) gene is a determinant of BMD and fracture in women. 2) The identification of RANKL target genes in osteoclastogenesis. RUNX2 is a runt domain transcription factor (Werner et al., 1999) essential for osteoblast differentiation (Lee et al., 1997). RUNX2 gene knock-out mice have no osteoblasts due to a failure in osteoblast differentiation and consequently unmineralised skeletons, (Komori et al., 1997; Otto et al., 1997). In humans, mutations in RUNX2 cause cleidocranial dysplasia (CCD), a disorder characterised by hypoplasia or aplasia of the clavicles, short stature, supernumerary teeth, patent fontanelles and other changes in skeletal patterning and growth (Mundlos et al., 1997). RUNX2 contains a poly-glutamine poly-alanine (polyQ/polyA) repeat where mutations causing cleidocranial dysplasia have been observed. BMD has not been routinely examined in CCD, two studies have identified CCD patients with lower BMD with one fracture case identified (Quack et al., 1999; Bergwitz et al., 2001). The central role of RUNX2 in determining osteoblast differentiation makes RUNX2 a prime candidate gene for regulating adult bone density. To determine if polymorphism was present in the polyQ/polyA tract the repeat was amplified within the upper and lower deciles of femoral neck (FN) BMD in the Geelong Osteoporosis study (GOS). The upper and lower deciles of FN BMD acted as a surrogate for genotyping the entire cohort. This study identified two common variants within the polyA repeat: an 18 base pair deletion (11Ala) and a synonymous alanine codon polymorphism with alleles, GCA and GCG (noted as A and G alleles, respectively). The 11Ala and SNP polymorphism are found on codon 64 and 66 respectively (RUNX2 MRIPV variant). A allele frequencies were significantly different in a comparison of the upper and lower deciles of FN BMD (p=0.019). In 495 randomly selected women of the Geelong Osteoporosis Study (GOS), the A allele was associated with higher BMD at all sites tested. The association was maximal at the ultra-distal radius (p=0.001). In a separate fracture study, the A allele was significantly protective against Colles' fracture in elderly women but not spine and hip fracture. The 11Ala polymorphism was not related to BMD in GOS. To further decipher the role of the RUNX2 A allele we genotyped 992 women from a Scottish cohort. The alleles of RUNX2 within the glutamine/alanine repeat were determined by MspA1I restriction digest. To examine the possible influence on estrogen related therapies or estrogen status on the potential genetic effect conferred by RUNX2, we divided the cohort by menopausal and hormone replacement therapy status. Within postmenopausal Scottish women the RUNX2 A allele was associated with significantly higher FN BMD (p=0.028, n=312) but not lumbar spine (LS) BMD. The A allele was associated with higher FN BMD (p=0.035) within a postmenopausal subgroup of the population (n=312). To investigate the effect of weight on the RUNX2 alleles the Scottish cohort was segregated into thin/normal (BMI ≥ 25 kg/m2) and overweight /obese (BMI > 25 kg/m2). RUNX2 A allele showed a stronger effect on FN BMD in postmenopausal women above the median BMI. The 11Ala RUNX2 deletion allele was significantly associated with decreased LS BMD (p=0.018) within overweight/obese women (n=546). The 11Ala allele was significantly associated with increased levels of pyridinoline (p=0.014) and deoxypyridinoline (p=0.038) in the HRT treated subgroup of the population (n=492). Glutamine variants and an alanine insertion were identified within the group. These data suggest that the RUNX2 11Ala and A alleles exert differing affects on BMD showing preference for different skeletal sites in a weight dependent manner. We genotyped 78 individuals from an osteoarthritic population to elucidate the role of the RUNX2 alleles on markers of bone turnover and inflammation. The RUNX2 11Ala allele was significantly associated with decreased osteocalcin (OC) serum levels (p = 0.01). The RUNX2 A allele was significantly related to reduced tumor necrosis factor alpha (TNF-alpha) serum levels (p = 0.004). RUNX2 is known to bind to the OC promoter. An OC promoter polymorphism is found 7bp upstream from a putative RUNX2 binding site. We hypothesized that OC polymorphism may effect the RUNX2 transactivation of the OC gene and thus affect OC serum levels. OC promoter polymorphism was not related to OC serum levels (n=78). These data present a novel link between RUNX2 alleles and OC and TNF serum levels, providing putative mechanisms of action for the RUNX2 alleles. Further studies in larger populations are required to confirm these findings. Ten individuals within the GOS and the Scottish cohort were found to carry rare mutations of the polyQ/polyA repeat. All polyQ variants had a normal polyA repeat (17 amino acids) and were heterozygous for a normal 23Q/17A allele. Variants observed were 15, 16, 24 and 30Q. One individual was observed with an extended polyA repeat (24A). Patient records indicated otherwise unremarkable clinical history except for fracture in 4/10 individuals from GOS (hip and spine). BMD data from the LS and the FN were expressed as T-scores, a measure that relates BMD in terms of standard deviations below the young normal value. In addition, BMD data were also expressed as Z-scores around the age-mean. Under the null hypothesis, where RUNX2 Q repeat variation has no effect on BMD, Z scores would be expected to be distributed around a mean of zero. However, when all variants were pooled the BMD was significantly lower than expected. This effect persisted when deletion variants were considered alone. The effect was stronger on FN BMD (p=0.001) rather than LS BMD (p=0.096), reflecting either difference in precision of BMD measurements at these sites or perhaps a differential genetic effect on different skeletal sites. These data suggest that polyQ and polyA variants are associated with significantly lower BMD, and may be an important determinant for fracture. Glutamine variants exist at high frequency (~0.7%): this rate of mutation could be important when considering large populations at risk of age related osteoporosis. Considering that these subjects are heterozygous for a normal allele, it suggests that a more severe phenotype might be expected in rare subjects homozygous for glutamine repeat variants. In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles' fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD. - 2) The identification of RANKL target genes in osteoclastogenesis. Osteoclastogenesis is regulated in vivo by the action of osteoblast/stromal cells that express membrane bound, receptor activator of NF-kB ligand (RANKL). Monocytes treated in vitro with a soluble form of RANKL and macrophage colony stimulating factor (M-CSF) differentiate to osteoclasts, whereas monocytes treated with M-CSF alone differentiate to macrophage-like cells. The gene expression profile of human osteoclasts has not been extensively explored. Genes highly expressed by rabbit osteoclasts were identified through random sequencing of an osteoclast cDNA library (Sakai et al., 1995). Differential gene expression of mouse osteoclastogenesis was elucidated by array analysis (Cappellen et al., 2002). To identify genes important for human osteoclastogenesis, total RNA was isolated from monocytes treated for three weeks with either M-CSF alone or with RANKL and M-CSF. RANKL treatment for 3 weeks and 12 hours was investigated in this study, to complement previous data. Differential display was performed on RNA (12 hour treatment with RANKL) and differential gene expression profiles examined. The differential display products were pooled to generate a probe for screening a gene array system derived from a human osteoclast cDNA library. cDNA (3 week treatment with RANKL) hybridisation experiments against the array revealed additional regulated genes. Gene clones that showed significant regulation in M-CSF and RANKL treated cells compared M-CSF treated cells represent genes that are targets for RANKL-specific regulation. Osteopontin, creatine kinase and various mitochondrial genes were up regulated by the treatment of RANKL. Changes in gene expression observed in the array data were confirmed with real-time PCR using mRNA derived from in vitro induced osteoclasts. Cathepsin K gene expression was more than 300 fold greater in osteoclasts compared to macrophage-like cells after one week treatment with RANKL and M-CSF. Cystatin C expression showed a six-fold induction at two weeks of RANKL and M-CSF treatment and cystatin B showed a steady increase in expression. Some of these regulated genes may provide useful targets for influencing BMD.

In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles’ fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2004.
Joseph M. LeDoux, Committee Member ; Julia E. Babensee, Committee Member ; Robert E. Guldberg, Committee Member ; Andres J. Garcia, Committee Chair ; Grace K. Pavlath, Committee Member ; Barbara D. Boyan, Committee Member. Includes bibliographical references.

Non-healing bone defects have a significant socioeconomic impact in the U.S. with approximately 600,000 bone grafting procedures performed annually. Autografts and allografts are clinically the most common treatments; however, autologous donor bone is in limited supply, and allografts often have poor mechanical properties. Therefore, tissue engineering and regenerative medicine strategies are being developed to address issues with clinical bone grafting. The overall objective of this work was to develop bone tissue engineering strategies that enhance healing of orthotopic defects by targeting specific osteogenic cell signaling pathways. The general approach included the investigation of two different tissue engineering strategies, which both focused on directed osteoblastic differentiation to promote bone formation. In the first cell-based strategy, we hypothesized that constitutive overexpression of the osteoblast-specific transcription factor, Runx2, in bone marrow stromal cells (BMSCs) would promote orthotopic bone formation in vivo. We tested this hypothesis by delivering Runx2-modified BMSCs on synthetic scaffolds to critically-sized defects in rats. We found that Runx2-modified BMSCs significantly increased orthotopic bone formation compared to empty defects, cell-free scaffolds and unmodified BMSCs. This gene therapy approach to bone regeneration provides a mineralizing cell source which has clinical relevance. In the second biomaterial-based strategy, we hypothesized that incorporation of the collagen-mimetic peptide, GFOGER, into synthetic bone scaffolds would promote orthotopic bone formation in vivo without the use of cells or growth factors. We tested this hypothesis by passively adsorbing GFOGER onto poly-caprolactone (PCL) scaffolds and implanting them into critically-sized orthotopic defects in rats. We found that GFOGER-coated scaffolds significantly increased bone formation compared to uncoated scaffolds in a dose dependent manner. Development of this cell-free strategy for bone tissue engineering provides an inexpensive therapeutic alternative to clinical bone defect healing, which could be implemented as a point of care application. Both strategies developed in this work take advantage of specific osteoblastic signaling pathways involved in bone healing. Further development of these tissue engineering strategies for bone regeneration will provide clinically-relevant treatment options for healing large bone defects in humans by employing well-controlled signals to promote bone formation and eliminating the need for donor bone.