Journal articles on the topic 'Osteoblasts'
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1
Kim, Jung Ha, Kabsun Kim, Inyoung Kim, Semun Seong, Jeong-Tae Koh, and Nacksung Kim. "The ATF3–OPG Axis Contributes to Bone Formation by Regulating the Differentiation of Osteoclasts, Osteoblasts, and Adipocytes." International Journal of Molecular Sciences 23, no. 7 (March 23, 2022): 3500. http://dx.doi.org/10.3390/ijms23073500.
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Activating transcription factor 3 (ATF3) has been identified as a negative regulator of osteoblast differentiation in in vitro study. However, it was not associated with osteoblast differentiation in in vivo study. To provide an understanding of the discrepancy between the in vivo and in vitro findings regarding the function of ATF3 in osteoblasts, we investigated the unidentified roles of ATF3 in osteoblast biology. ATF3 enhanced osteoprotegerin (OPG) production, not only in osteoblast precursor cells, but also during osteoblast differentiation and osteoblastic adipocyte differentiation. In addition, ATF3 increased nodule formation in immature osteoblasts and decreased osteoblast-dependent osteoclast formation, as well as the transdifferentiation of osteoblasts to adipocytes. However, all these effects were reversed by the OPG neutralizing antibody. Taken together, these results suggest that ATF3 contributes to bone homeostasis by regulating the differentiation of various cell types in the bone microenvironment, including osteoblasts, osteoclasts, and adipocytes via inducing OPG production.
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Bauer, Omri, Amnon Sharir, Ayako Kimura, Shay Hantisteanu, Shu Takeda, and Yoram Groner. "Loss of Osteoblast Runx3 Produces Severe Congenital Osteopenia." Molecular and Cellular Biology 35, no. 7 (January 20, 2015): 1097–109. http://dx.doi.org/10.1128/mcb.01106-14.
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Congenital osteopenia is a bone demineralization condition that is associated with elevated fracture risk in human infants. Here we show thatRunx3, likeRunx2, is expressed in precommitted embryonic osteoblasts and that Runx3-deficient mice develop severe congenital osteopenia. Runx3-deficient osteoblast-specific (Runx3fl/fl/Col1α1-cre), but not chondrocyte-specific (Runx3fl/fl/Col1α2-cre), mice are osteopenic. This demonstrates that an osteoblastic cell-autonomous function of Runx3 is required for proper osteogenesis. Bone histomorphometry revealed that decreased osteoblast numbers and reduced mineral deposition capacity in Runx3-deficient mice cause this bone formation deficiency. Neonatal bone and cultured primary osteoblast analyses revealed a Runx3-deficiency-associated decrease in the number of active osteoblasts resulting from diminished proliferation and not from enhanced osteoblast apoptosis. These findings are supported by Runx3-null culture transcriptome analyses showing significant decreases in the levels of osteoblastic markers and increases in the levels of Notch signaling components. Thus, while Runx2 is mandatory for the osteoblastic lineage commitment, Runx3 is nonredundantly required for the proliferation of these precommitted cells, to generate adequate numbers of active osteoblasts. HumanRUNX3resides on chromosome 1p36, a region that is associated with osteoporosis. Therefore, RUNX3 might also be involved in human bone mineralization.
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Park, Yu-Seong, Hyun-Woo Kim, Jin-Hyeon Hwang, Jung-Young Eom, Dong-Ha Kim, Jinho Park, Hyun-Jin Tae, et al. "Plum-Derived Exosome-like Nanovesicles Induce Differentiation of Osteoblasts and Reduction of Osteoclast Activation." Nutrients 15, no. 9 (April 27, 2023): 2107. http://dx.doi.org/10.3390/nu15092107.
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Osteoblasts and osteoclasts play crucial roles in bone formation and bone resorption. We found that plum-derived exosome-like nanovesicles (PENVs) suppressed osteoclast activation and modulated osteoblast differentiation. PENVs increased the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells and osteoblasts from mouse bone marrow cultures. Notably, PENVs elevated the expression of osteoblastic transcription factors and osteoblast differentiation marker proteins in MC3T3-E1 cells. Higher levels of phosphorylated BMP-2, p38, JNK, and smad1 proteins were detected in PENV-treated MC3T3-E1 cells. Additionally, the number of TRAP-positive cells was significantly decreased in PENV-treated osteoclasts isolated from osteoblasts from mouse bone marrow cultures. Importantly, osteoclastogenesis of marker proteins such as PPAR-gamma, NFATc1, and c-Fos were suppressed by treatment with PENVs (50 μg/mL). Taken together, these results demonstrate that PENVs can be used as therapeutic targets for treating bone-related diseases by improving osteoblast differentiation and inhibiting osteoclast activation for the first time.
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Kim, Jung Ha, Kabsun Kim, Inyoung Kim, Semun Seong, Hyun Kook, Kyung Keun Kim, Jeong-Tae Koh, and Nacksung Kim. "Bifunctional Role of CrkL during Bone Remodeling." International Journal of Molecular Sciences 22, no. 13 (June 29, 2021): 7007. http://dx.doi.org/10.3390/ijms22137007.
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Coupled signaling between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to the maintenance of bone homeostasis. We previously reported that v-crk avian sarcoma virus CT10 oncogene homolog-like (CrkL), which belongs to the Crk family of adaptors, inhibits bone morphogenetic protein 2 (BMP2)-mediated osteoblast differentiation, while enhancing receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated whether CrkL can also regulate the coupling signals between osteoblasts and osteoclasts, facilitating bone homeostasis. Osteoblastic CrkL strongly decreased RANKL expression through its inhibition of runt-related transcription factor 2 (Runx2) transcription. Reduction in RANKL expression by CrkL in osteoblasts resulted in the inhibition of not only osteoblast-dependent osteoclast differentiation but also osteoclast-dependent osteoblast differentiation, suggesting that CrkL participates in the coupling signals between osteoblasts and osteoclasts via its regulation of RANKL expression. Therefore, CrkL bifunctionally regulates osteoclast differentiation through both a direct and indirect mechanism while it inhibits osteoblast differentiation through its blockade of both BMP2 and RANKL reverse signaling pathways. Collectively, these data suggest that CrkL is involved in bone homeostasis, where it helps to regulate the complex interactions of the osteoblasts, osteoclasts, and their coupling signals.
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Ducy, P., and G. Karsenty. "Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene." Molecular and Cellular Biology 15, no. 4 (April 1995): 1858–69. http://dx.doi.org/10.1128/mcb.15.4.1858.
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Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.
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Moriishi, Takeshi, Yosuke Kawai, Ryo Fukuyama, Yuki Matsuo, You-Wen He, Haruhiko Akiyama, Izumi Asahina, and Toshihisa Komori. "Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis." International Journal of Molecular Sciences 24, no. 24 (December 10, 2023): 17319. http://dx.doi.org/10.3390/ijms242417319.
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Bcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis.
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Sutton, Amelia L. M., Xiaoxue Zhang, Diane R. Dowd, Yogendra P. Kharode, Barry S. Komm, and Paul N. MacDonald. "Semaphorin 3B Is a 1,25-Dihydroxyvitamin D3-Induced Gene in Osteoblasts that Promotes Osteoclastogenesis and Induces Osteopenia in Mice." Molecular Endocrinology 22, no. 6 (June 1, 2008): 1370–81. http://dx.doi.org/10.1210/me.2007-0363.
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Abstract The vitamin D endocrine system is important for skeletal homeostasis. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct actions of 1,25(OH)2D3 in bone, relatively little is known of the mechanisms or target genes that are regulated by 1,25(OH)2D3 in skeletal cells. Here, we identify semaphorin 3B (SEMA3B) as a 1,25(OH)2D3-stimulated gene in osteoblastic cells. Northern analysis revealed strong induction of SEMA3B mRNA by 1,25(OH)2D3 in MG-63, ST-2, MC3T3, and primary osteoblastic cells. Moreover, differentiation of these osteogenic cells enhanced SEMA3B gene expression. Biological effects of SEMA3B in the skeletal system have not been reported. Here, we show that osteoblast-derived SEMA3B alters global skeletal homeostasis in intact animals and osteoblast function in cell culture. Osteoblast-targeted expression of SEMA3B in mice resulted in reduced bone mineral density and aberrant trabecular structure compared with nontransgenic littermates. Histomorphometry studies indicated that this was likely due to increased osteoclast numbers and activity. Indeed, primary osteoblasts obtained from SEMA3B transgenic mice stimulated osteoclastogenesis to a greater extent than nontransgenic osteoblasts. This study establishes that SEMA3B is a 1,25(OH)2D3-induced gene in osteoblasts and that osteoblast-derived SEMA3B impacts skeletal biology in vitro and in vivo. Collectively, these studies support a putative role for SEMA3B as an osteoblast protein that regulates bone mass and skeletal homeostasis.
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Giuliani, Nicola, Francesca Morandi, Sara Tagliaferri, Mirca Lazzaretti, Sabrina Bonomini, Monica Crugnola, Cristina Mancini, et al. "The proteasome inhibitor bortezomib affects osteoblast differentiation in vitro and in vivo in multiple myeloma patients." Blood 110, no. 1 (July 1, 2007): 334–38. http://dx.doi.org/10.1182/blood-2006-11-059188.
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The proteasome inhibitor bortezomib may increase osteoblast-related markers in multiple myeloma (MM) patients; however, its potential osteoblastic stimulatory effect is not known. In this study, we show that bortezomib significantly induced a stimulatory effect on osteoblast markers in human mesenchymal cells without affecting the number of osteoblast progenitors in bone marrow cultures or the viability of mature osteoblasts. Consistently we found that bortezomib significantly increased the transcription factor Runx2/Cbfa1 activity in human osteoblast progenitors and osteoblasts without affecting nuclear and cytoplasmatic active β-catenin levels. Consequently a stimulatory effect of bortezomib on bone nodule formation was also demonstrated in osteoblast progenitors. These in vitro observations were confirmed in vivo by the finding of a significant increase in the number of osteoblastic cells × mm2 of bone tissue and in the number of Runx2/Cbfa1-positive osteoblastic cells that was observed in MM patients who responded to bortezomib. Our in vitro and in vivo observations support the hypothesis that a direct stimulatory effect on bone formation process could occur during bortezomib treatment.
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Ogata, Naoshi, Hiroshi Kawaguchi, Ung-il Chung, Sanford I. Roth, and Gino V. Segre. "Continuous Activation of Gαq in Osteoblasts Results in Osteopenia through Impaired Osteoblast Differentiation." Journal of Biological Chemistry 282, no. 49 (September 5, 2007): 35757–64. http://dx.doi.org/10.1074/jbc.m611902200.
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We explored the role of Gαq-mediated signaling on skeletal homeostasis by selectively expressing a constitutively active Gαq (mutation of Q209L) in osteoblasts. Continuous signaling via Gαq in mouse osteoblastic MC3T3-E1 cells impaired differentiation. Mice that expressed the constitutively active Gαq transgene in cells of the osteoblast lineage exhibited severe osteopenia in cortical and trabecular bones. Osteoblast number, bone volume, and trabecular thickness were reduced in transgenic mice, but the osteoclasts were unaffected. Osteoblasts from transgenic mice showed impaired differentiation and matrix formation. In the presence of a protein kinase C inhibitor GF109203X, this impairment was not seen, indicating mediation by the protein kinase C pathway. We propose that continuous activation of the Gαq signal in osteoblasts plays a crucial, previously unrecognized role in bone formation.
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Taichman, Russell S. "Blood and bone: two tissues whose fates are intertwined to create the hematopoietic stem-cell niche." Blood 105, no. 7 (April 1, 2005): 2631–39. http://dx.doi.org/10.1182/blood-2004-06-2480.
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AbstractThe mechanisms of bone and blood formation have traditionally been viewed as distinct, unrelated processes, but compelling evidence suggests that they are intertwined. Based on observations that hematopoietic precursors reside close to endosteal surfaces, it was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). Preceding these observations are studies demonstrating that the disruption or perturbation of normal osteoblastic function has a profound and central role in defining the operational structure of the HSC niche. These observations provide a glimpse of the dimensions and ramifications of HSC-osteoblast interactions. Although more research is required to secure a broader grasp of the molecular mechanisms that govern blood and bone biology, the central role for osteoblasts in hematopoietic stem cell regulation is reviewed herein from the perspectives of (1) historical context; (2) the role of the osteoblast in supporting stem cell survival, proliferation, and maintenance; (3) the participation, if any, of osteoblasts in the creation of a stem cell niche; (4) the molecules that mediate HSC-osteoblast interactions; (5) the role of osteoblasts in stem cell transplantation; and (6) possible future directions for investigation.
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Hernández-Tapia, Laura G., Zdenka Fohlerová, Jan Žídek, Marco A. Alvarez-Perez, Ladislav Čelko, Jozef Kaiser, and Edgar B. Montufar. "Effects of Cryopreservation on Cell Metabolic Activity and Function of Biofabricated Structures Laden with Osteoblasts." Materials 13, no. 8 (April 22, 2020): 1966. http://dx.doi.org/10.3390/ma13081966.
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Biofabrication and maturation of bone constructs is a long-term task that requires a high degree of specialization. This specialization falls onto the hierarchy complexity of the bone tissue that limits the transfer of this technology to the clinic. This work studied the effects of the short-term cryopreservation on biofabricated osteoblast-containing structures, with the final aim to make them steadily available in biobanks. The biological responses studied include the osteoblast post-thawing metabolic activity and the recovery of the osteoblastic function of 3D-bioprinted osteoblastic structures and beta tricalcium phosphate (β-TCP) scaffolds infiltrated with osteoblasts encapsulated in a hydrogel. The obtained structures were cryopreserved at −80 °C for 7 days using dimethyl sulfoxide (DMSO) as cryoprotectant additive. After thawing the structures were cultured up to 14 days. The results revealed fundamental biological aspects for the successful cryopreservation of osteoblast constructs. In summary, immature osteoblasts take longer to recover than mature osteoblasts. The pre-cryopreservation culture period had an important effect on the metabolic activity and function maintain, faster recovering normal values when cryopreserved after longer-term culture (7 days). The use of β-TCP scaffolds further improved the osteoblast survival after cryopreservation, resulting in similar levels of alkaline phosphatase activity in comparison with the non-preserved structures. These results contribute to the understanding of the biology of cryopreserved osteoblast constructs, approaching biofabrication to the clinical practice.
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Zhu, Jiang, Yi Zhang, Nacksung Kim, Yongwon Choi, Gerard Joe, Russell Taichman, and Stephen G. Emerson. "Osteoblasts Support Early B Lymphoiesis as well as Stem Cell Proliferation and Myelopoiesis: Identification of the Mammalian Cellular Analog of the Bursa of Fabricius." Blood 104, no. 11 (November 16, 2004): 508. http://dx.doi.org/10.1182/blood.v104.11.508.508.
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Abstract The self-renewal, survival and differentiation of hematopoietic stem cells (HSC) are greatly influenced by the activities of neighboring osteoblasts and non-osteogenic bone marrow (BM) stromal cells such as fibroblasts, endothelial cells and adipocytes. Previously, we showed that osteoblasts from human long bones support the in vitro self-renewal as well as myeloid differentiation of human CD34+ cord blood cells. Recently, Li’s and Scadden’s groups provided in vivo evidence indicating a primary role of trabecular osteoblasts as a major component of HSC niche and of stromal osteoblastic cells in facilitating the self-renewal of HSCs. We have now asked whether osteoblasts contribute to early lymphopoiesis as well as myelopoiesis, by measuring the cellular outpout of purified HSCs on isolated osteoblasts alone, or with added non-osteoblast stromal cytokines as well. We prepared mature osteoblasts, as monitored and confirmed by homogeneous OPN and CD61 expression, by pretreating osteoblastic cells isolated from neonatal calvaria of C57BL/6 mice (CD45.2) with 1X10−7 M PTH. Purified OB were then co-cultured for 6 days with Lin− BM cells (CD45.1+) isolated from congenic B6 mice(CD45.1) and labeled with CFSE. Osteoblast coculture stimulated the proliferation of Lin− CD45.1+ BM cells 50-fold during culture, with most cells (87%) remaining tightly adherent to the osteoblast monolayer; no live cells were recovered from Lin− BM cell culture without osteoblasts. In addition to mature granulocytes/monocytes, a substantial amount of CD45.1+B220+ B lymphocytes (about 10% of small size cells gated by forward and side scatter), were detected. In contrast, very few CD45.1+Lin-Sca-1+c-Kit+ (LSK) cells or CD45.1+Lin−Sca-1−c-Kit+ (CMP) cells were detected under these conditions. Most B220+ cells attached to osteoblasts were found to be CD43+CD24+ pre-B cells undergoing division. In contrast to the cells recovered attached to the osteoblasts, the pre-B lymphocytes found in suspension were more mature with phenotype of B220+CD43−CD24+. Prevention of direct contact of Lin− BM cells with osteoblasts by Transwell co-culture abrogated the production of pre-B cells in both adherent and suspension compartments, indicating that physical contact is required for the interaction. Interestingly, when 20ng/ml of SCF, 6ng/ml of IL-3, 10ng/ml of IL-6 and 25ng/ml TPO were added to osteoblast/Lin− cell co-culture, B lymphpoiesis was repressed, while the production of CD45.1+LSK HSCs and CMPs was significantly enhanced. These data demonstrate a direct role of osteoblasts in inducing and supporting the early development of B lymphocytes from HSCs or/and common lymphoid progenitors. Additional cytokines, perhaps provided in specific in vivo niches by non-osteogenic stromal cells, cooperate with the stimulatory signals from osteoblasts to promote the survival and expansion of HSCs. Taken together, these results suggest that osteoblasts may be the mammalian analog of the avian Bursa of Fabricius, and that their local degree of proximity to non-osteogenic stromal cells may define specific microniches for stem cell survival, myelopoiesis and/or B lymphopoiesis.
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Di Benedetto, Adriana, Francesca Posa, Claudia Carbone, Stefania Cantore, Giacomina Brunetti, Matteo Centonze, Maria Grano, Lorenzo Lo Muzio, Elisabetta A. Cavalcanti-Adam, and Giorgio Mori. "NURR1 Downregulation Favors Osteoblastic Differentiation of MSCs." Stem Cells International 2017 (2017): 1–10. http://dx.doi.org/10.1155/2017/7617048.
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Mesenchymal stem cells (MSCs) have been identified in human dental tissues. Dental pulp stem cells (DPSCs) were classified within MSC family, are multipotent, can be isolated from adult teeth, and have been shown to differentiate, under particular conditions, into various cell types including osteoblasts. In this work, we investigated how the differentiation process of DPSCs toward osteoblasts is controlled. Recent literature data attributed to the nuclear receptor related 1 (NURR1), a still unclarified role in osteoblast differentiation, while NURR1 is primarily involved in dopaminergic neuron differentiation and activity. Thus, in order to verify if NURR1 had a role in DPSC osteoblastic differentiation, we silenced it during all the processes and compared the expression of the main osteoblastic markers with control cultures. Our results showed that the inhibition of NURR1 significantly increased the expression of osteoblast markers collagen I and alkaline phosphatase. Further, in long time cultures, the mineral matrix deposition was strongly enhanced in NURR1-silenced cultures. These results suggest that NURR1 plays a key role in switching DPSC differentiation toward osteoblasts rather than neuronal or even other cell lines. In conclusion, DPSCs represent a source of osteoblast-like cells and downregulation of NURR1 strongly prompted their differentiation toward the osteoblastogenesis process.
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Skillington, Jeremy, Lisa Choy, and Rik Derynck. "Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes." Journal of Cell Biology 159, no. 1 (October 14, 2002): 135–46. http://dx.doi.org/10.1083/jcb.200204060.
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Mesenchymal cells can differentiate into osteoblasts, adipocytes, myoblasts, or chondroblasts. Whether mesenchymal cells that have initiated differentiation along one lineage can transdifferentiate into another is largely unknown. Using 3T3-F442A preadipocytes, we explored whether extracellular signals could redirect their differentiation from adipocyte into osteoblast. 3T3-F442A cells expressed receptors and Smads required for bone morphogenetic protein (BMP) signaling. BMP-2 increased proliferation and induced the early osteoblast differentiation marker alkaline phosphatase, yet only mildly affected adipogenic differentiation. Retinoic acid inhibited adipose conversion and cooperated with BMP-2 to enhance proliferation, inhibit adipogenesis, and promote early osteoblastic differentiation. Expression of BMP-RII together with BMP-RIA or BMP-RIB suppressed adipogenesis of 3T3-F442A cells and promoted full osteoblastic differentiation in response to retinoic acid. Osteoblastic differentiation was characterized by induction of cbfa1, osteocalcin, and collagen I expression, and extracellular matrix calcification. These results indicate that 3T3-F442A preadipocytes can be converted into fully differentiated osteoblasts in response to extracellular signaling cues. Furthermore, BMP and retinoic acid signaling cooperate to stimulate cell proliferation, repress adipogenesis, and promote osteoblast differentiation. Finally, BMP-RIA and BMP-RIB induced osteoblast differentiation and repressed adipocytic differentiation to a similar extent.
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Kanazawa, Ippei, Ayumu Takeno, Ken-ichiro Tanaka, Masakazu Notsu, and Toshitsugu Sugimoto. "Osteoblast AMP-Activated Protein Kinase Regulates Postnatal Skeletal Development in Male Mice." Endocrinology 159, no. 2 (November 3, 2017): 597–608. http://dx.doi.org/10.1210/en.2017-00357.
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Abstract Studies have shown that AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, plays important roles in osteoblast differentiation and mineralization. However, little is known about in vivo roles of osteoblastic AMPK in bone development. Thus, to investigate in vivo roles of osteoblast AMPK, we conditionally inactivated Ampk in osterix (Osx)–expressing cells by crossing Osx-Cre mice with floxed AMPKα1 to generate mice lacking AMPKα1 in osteoblasts (Ampk−/− mice). Compared with wild-type and Ampk+/− mice, Ampk−/− mice displayed retardation of postnatal bone development, although bone deformity was not observed at birth. Microcomputed tomography showed significant reductions in trabecular bone volume, cortical bone length, and density, as well as increased cortical porosity in femur as well as development defects of skull in 8-week-old Ampk−/− mice. Surprisingly, histomorphometric analysis demonstrated that the number of osteoclasts was significantly increased, although bone formation rate was not altered. Loss of trabecular network connections and mass, as well as shortened growth plates and reduced thickness of cartilage adjacent to the growth plate, was observed in Ampk−/− mice. In primary cultured osteoblasts from calvaria, the expressions of alkaline phosphatase, type 1 collagen, osteocalcin, bone morphogenetic protein 2, Runx2, and osterix were significantly inhibited in Ampk−/− osteoblasts, whereas the expression of receptor activator of nuclear κB ligand (RANKL) and the RANKL/osteoprotegerin ratio were significantly increased. These findings indicate that osteoblastic AMPK plays important roles in bone development in vivo and that deletion of AMPK in osteoblasts decreases osteoblastic differentiation and enhances bone turnover by increasing RANKL expression.
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Kessler, Catherine B., and Anne M. Delany. "Increased Notch 1 Expression and Attenuated Stimulatory G Protein Coupling to Adenylyl Cyclase in Osteonectin-Null Osteoblasts." Endocrinology 148, no. 4 (April 1, 2007): 1666–74. http://dx.doi.org/10.1210/en.2006-0443.
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Osteonectin, or secreted protein acidic and rich in cysteine, is one of the most abundant noncollagen matrix components in bone. This matricellular protein regulates extracellular matrix assembly and maturation in addition to modulating cell behavior. Mice lacking osteonectin develop severe low-turnover osteopenia, and in vitro studies of osteonectin-null osteoblastic cells showed that osteonectin supports osteoblast formation, maturation, and survival. The present studies demonstrate that osteonectin-null osteoblastic cells have increased expression of Notch 1, a well-documented regulator of cell fate in multiple systems. Furthermore, osteonectin-null cells are more plastic and less committed to osteoblastic differentiation, able to pursue adipogenic differentiation given the appropriate signals. Notch 1 transcripts are down-regulated by inducers of cAMP in both wild-type and osteonectin-null osteoblasts, suggesting that the mutant osteoblasts may have a defect in generation of cAMP in response to stimuli. Indeed, many bone anabolic agents signal through increased cAMP. Wild-type and osteonectin-null osteoblasts generated comparable amounts of cAMP in response to forskolin, a direct stimulator of adenylyl cyclase. However, the ability of osteonectin-null osteoblasts to generate cAMP in response to cholera toxin, a direct stimulator of Gs, was attenuated. These data imply that osteonectin-null osteoblasts have decreased coupling of Gs to adenylyl cyclase. Because osteonectin promotes G protein coupling to an effector, our studies support the concept that low-turnover osteopenia can result from reducing G protein coupled receptor activity.
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Mansukhani, Alka, Paola Bellosta, Malika Sahni, and Claudio Basilico. "Signaling by Fibroblast Growth Factors (Fgf) and Fibroblast Growth Factor Receptor 2 (Fgfr2)–Activating Mutations Blocks Mineralization and Induces Apoptosis in Osteoblasts." Journal of Cell Biology 149, no. 6 (June 12, 2000): 1297–308. http://dx.doi.org/10.1083/jcb.149.6.1297.
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Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts.
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Xu, Xiaoxuan, Wei Zhou, Tingfen Deng, Qinghua Cai, and Shunqing Wang. "Cells of the Osteoblast Lineage Cross-Talk with Myeloid-Derived Suppressor Cells to Promote Multiple Myeloma Growth." Blood 142, Supplement 1 (November 28, 2023): 4691. http://dx.doi.org/10.1182/blood-2023-188593.
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Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM) and is highly influenced by the BM tumor microenvironment. Osteolytic bone disease is a hallmark of MM and is partly due to osteoblast (OB) inhibition. MM cells suppress osteoblast maturation and function via inhibition of the critical Runt-related transcription factor 2 (Runx2) in immature OBs. However, immature cells of the osteoblast lineage (e.g. pre-osteoblasts) remain present and even increased in the MM bone microenvironment and their contribution to the progression of MM has not been well understood. We previously developed a syngeneic mouse model of MM in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2 -/- mice), and demonstrated that OB-Runx2 deficiency creates a highly inflammatory BM microenvironment that is responsible for MM cell bone-homing to new bone sites. We therefore hypothesize that pre-osteoblasts play a role as promoters of MM cell survival and proliferation. To determine the direct effect of pre-osteoblasts on MM cells, we established co-cultures of pre-osteoblasts with MM cells. 5TGM1 murine MM cells were cocultured with MC3T3 murine pre-osteoblasts and newborn mouse calvarial pre-osteoblasts respectively. Within 48hrs of co-culture, each of pre-osteoblast populations slightly stimulated proliferation of 5TGM1 MM cells. These data suggest that pre-osteoblasts do not directly affect myeloma growth in vitro. Therefore, we next focus our studies on the role of pre-osteoblasts in MM progression in vivo. Firstly, we used OB-Runx2 deficient mouse model to assess the impact of pre-osteoblast accumulation on MM progression in vivo. 2x10 5 5TGM1-Luciferase MM cells were injected into the right tibia of OB-Runx2 -/- mice and control mice. Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1 MM cells) ELISA demonstrated that, compared with controls, OB-Runx2 -/- mice had a much larger tumor burden. Following, to more thoroughly evaluate the role of pre-osteoblasts in early stage of MM cell engraftment in bones, we used MC3T3 murine pre-osteoblasts to modulate pre-osteoblast populations at the sites of MM engraftment. 2x10 5 5TGM1-Luciferase MM cells were injected directly into the right tibia of NSG mice with or without equal numbers of MC3T3 pre-osteoblasts. Tumors grew significantly larger when co-injected with 5TGM1 MM cells and MC3T3 pre-osteoblasts than injected with 5TGM1 MM cells alone, which was confirmed by luciferin imaging and serum IgG2bκ ELISA. These data suggest that pre-osteoblasts provide critical support for MM progression in vivo. Given that pre-osteoblasts have no direct effect on the growth of MM cells in vitro, we hypothesized that the pre-osteoblastic niche may regulate MM cells via other intermediary cells in BM. We previously showed that OB-Runx2 deficiency induces an immunosuppressive BM microenvironment that is marked by an increase of myeloid-derived suppressor cells (MDSCs). To investigate whether pre-osteoblasts support MM cell survival and proliferation through the functional crosstalk with MDSCs, 5TGM1 MM cells were co-cultured with MC3T3 pre-osteoblasts and mouse calvarial pre-osteoblasts respectively in the presence or absence of MDSCs. Interestingly, pre-osteoblasts significantly promoted the proliferation of MM cells under the existence of MDSCs. To extend these findings in vivo, we next determined whether MDSC depletion can alleviate the promoting effect of pre-osteoblasts on MM progression. Gemcitabine (GEM) is an FDA-approved anti-cancer agent and MDSC inhibitor used in the treatment of solid tumors but not commonly used in MM therapy. We again injected 5TGM1-Luciferase MM cells into the right tibia of NSG mice with or without co-injection of MC3T3 pre-osteoblasts. We treated tumor-bearing mice with PBS or GEM (i.p. 30 mg/kg/week) for 4 weeks. Bioluminescence imaging and serum IgG2bκ ELISA demonstrated that GEM could overcome the proliferative effect of pre-osteoblasts on MM cells in mouse model, and this effect was independent of NK, B and T cells. Taken together, using in vivo and in vitro models, we show that pre-osteoblasts have a positive regulatory impact on MM cells through communication with MDSCs. Ongoing mechanistic studies aim to delineate how pre-osteoblasts modulate the functions of MDSCs to promote MM progression.
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Giardullo, Liberato, Alberto Altomare, Cinzia Rotondo, Addolorata Corrado, and Francesco Paolo Cantatore. "Osteoblast Dysfunction in Non-Hereditary Sclerosing Bone Diseases." International Journal of Molecular Sciences 22, no. 15 (July 26, 2021): 7980. http://dx.doi.org/10.3390/ijms22157980.
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A review of the available literature was performed in order to summarize the existing evidence between osteoblast dysfunction and clinical features in non-hereditary sclerosing bone diseases. It has been known that proliferation and migration of osteoblasts are concerted by soluble factors such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), bone morphogenetic protein (BMP) but also by signal transduction cascades such as Wnt signaling pathway. Protein kinases play also a leading role in triggering the activation of osteoblasts in this group of diseases. Post-zygotic changes in mitogen-activated protein kinase (MAPK) have been shown to be associated with sporadic cases of Melorheostosis. Serum levels of FGF and PDGF have been shown to be increased in myelofibrosis, although studies focusing on Sphingosine-1-phosphate receptor was shown to be strongly expressed in Paget disease of the bone, which may partially explain the osteoblastic hyperactivity during this condition. Pathophysiological mechanisms of osteoblasts in osteoblastic metastases have been studied much more thoroughly than in rare sclerosing syndromes: striking cellular mechanisms such as osteomimicry or complex intercellular signaling alterations have been described. Further research is needed to describe pathological mechanisms by which rare sclerosing non hereditary diseases lead to osteoblast dysfunction.
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Bassett, J. H. Duncan, Anne van der Spek, John G. Logan, Apostolos Gogakos, Jayashree Bagchi-Chakraborty, Elaine Murphy, Clementine van Zeijl, et al. "Thyrostimulin Regulates Osteoblastic Bone Formation During Early Skeletal Development." Endocrinology 156, no. 9 (September 1, 2015): 3098–113. http://dx.doi.org/10.1210/en.2014-1943.
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The ancestral glycoprotein hormone thyrostimulin is a heterodimer of unique glycoprotein hormone subunit alpha (GPA)2 and glycoprotein hormone subunit beta (GPB)5 subunits with high affinity for the TSH receptor. Transgenic overexpression of GPB5 in mice results in cranial abnormalities, but the role of thyrostimulin in bone remains unknown. We hypothesized that thyrostimulin exerts paracrine actions in bone and determined: 1) GPA2 and GPB5 expression in osteoblasts and osteoclasts, 2) the skeletal consequences of thyrostimulin deficiency in GPB5 knockout (KO) mice, and 3) osteoblast and osteoclast responses to thyrostimulin treatment. Gpa2 and Gpb5 expression was identified in the newborn skeleton but declined rapidly thereafter. GPA2 and GPB5 mRNAs were also expressed in primary osteoblasts and osteoclasts at varying concentrations. Juvenile thyrostimulin-deficient mice had increased bone volume and mineralization as a result of increased osteoblastic bone formation. However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts. Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro. These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development.
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Troka, Ildi, Gabriele Griffanti, Lucie Canaff, Geoffrey Hendy, David Goltzman, and Showan Nazhat. "Effect of Menin Deletion in Early Osteoblast Lineage on the Mineralization of an In Vitro 3D Osteoid-like Dense Collagen Gel Matrix." Biomimetics 7, no. 3 (July 22, 2022): 101. http://dx.doi.org/10.3390/biomimetics7030101.
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Bone has a complex microenvironment formed by an extracellular matrix (ECM) composed mainly of mineralized type I collagen fibres. Bone ECM regulates signaling pathways important in the differentiation of osteoblast-lineage cells, necessary for bone mineralization and in preserving tissue architecture. Compared to conventional 2D cell cultures, 3D in vitro models may better mimic bone ECM and provide an environment to support osteoblastic differentiation. In this study, a biomimetic 3D osteoid-like dense collagen gel model was used to investigate the role of the nuclear protein menin plays in osteoblastic differentiation and matrix mineralization. Previous in vitro and in vivo studies have shown that when expressed at later stages of osteoblastic differentiation, menin modulates osteoblastogenesis and regulates bone mass in adult mice. To investigate the role of menin when expressed at earlier stages of the osteoblastic lineage, conditional knockout mice in which the Men1 gene is specifically deleted early (i.e., at the level of the pluripotent mesenchymal stem cell lineage), where generated and primary calvarial osteoblasts were cultured in plastically compressed dense collagen gels for 21 days. The proliferation, morphology and differentiation of isolated seeded primary calvarial osteoblasts from knockout (Prx1-Cre; Men1f/f) mice were compared to those isolated from wild-type (Men1f/f) mice. Primary calvarial osteoblasts from knockout and wild-type mice did not show differences in terms of proliferation. However, in comparison to wild-type cells, primary osteoblast cells derived from knockout mice demonstrated deficient mineralization capabilities and an altered gene expression profile when cultured in 3D dense collagen gels. In summary, these findings indicate that when expressed at earlier stages of osteoblast differentiation, menin is important in maintaining matrix mineralization in 3D dense collagen gel matrices, in vitro.
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Park, Jin-Ho, Su A. Park, Young-Hoon Kang, So Myeong Hwa, Eun-Byeol Koh, Sun-Chul Hwang, Se Heang Oh, and June-Ho Byun. "Zinc Sulfate Stimulates Osteogenic Phenotypes in Periosteum-Derived Cells and Co-Cultures of Periosteum-Derived Cells and THP-1 Cells." Life 11, no. 5 (April 30, 2021): 410. http://dx.doi.org/10.3390/life11050410.
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Coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption maintains both mechanical integrity and mineral homeostasis. Zinc is required for the formation, mineralization, growth, and maintenance of bones. We examined the effects of zinc sulfate on osteoblastic differentiation of human periosteum-derived cells (hPDCs) and osteoclastic differentiation of THP-1 cells. Zinc sulfate enhanced the osteoblastic differentiation of hPDCs; however, it did not affect the osteoclastic differentiation of THP-1 cells. The levels of extracellular signaling-related kinase (ERK) were strongly increased during osteoblastic differentiation in zinc sulfate-treated hPDCs, compared with other mitogen-activated protein kinases (MAPKs). Zinc sulfate also promoted osteogenesis in hPDCs and THP-1 cells co-cultured with the ratio of one osteoclast to one osteoblast, as indicated by alkaline phosphatase levels, mineralization, and cellular calcium contents. In addition, the receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio was decreased in the zinc sulfate-treated co-cultures. Our results suggest that zinc sulfate enhances osteogenesis directly by promoting osteoblastic differentiation and osteogenic activities in osteoblasts and indirectly by inhibiting osteoclastic bone resorption through a reduced RANKL/OPG ratio in co-cultured osteoblasts and osteoclasts.
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Erlebacher, Adrian, Ellen H. Filvaroff, Jian-Qin Ye, and Rik Derynck. "Osteoblastic Responses to TGF-β during Bone Remodeling." Molecular Biology of the Cell 9, no. 7 (July 1998): 1903–18. http://dx.doi.org/10.1091/mbc.9.7.1903.
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Bone remodeling depends on the spatial and temporal coupling of bone formation by osteoblasts and bone resorption by osteoclasts; however, the molecular basis of these inductive interactions is unknown. We have previously shown that osteoblastic overexpression of TGF-β2 in transgenic mice deregulates bone remodeling and leads to an age-dependent loss of bone mass that resembles high-turnover osteoporosis in humans. This phenotype implicates TGF-β2 as a physiological regulator of bone remodeling and raises the question of how this single secreted factor regulates the functions of osteoblasts and osteoclasts and coordinates their opposing activities in vivo. To gain insight into the physiological role of TGF-β in bone remodeling, we have now characterized the responses of osteoblasts to TGF-β in these transgenic mice. We took advantage of the ability of alendronate to specifically inhibit bone resorption, the lack of osteoclast activity in c-fos −/− mice, and a new transgenic mouse line that expresses a dominant-negative form of the type II TGF-β receptor in osteoblasts. Our results show that TGF-β directly increases the steady-state rate of osteoblastic differentiation from osteoprogenitor cell to terminally differentiated osteocyte and thereby increases the final density of osteocytes embedded within bone matrix. Mice overexpressing TGF-β2 also have increased rates of bone matrix formation; however, this activity does not result from a direct effect of TGF-β on osteoblasts, but is more likely a homeostatic response to the increase in bone resorption caused by TGF-β. Lastly, we find that osteoclastic activity contributes to the TGF-β–induced increase in osteoblast differentiation at sites of bone resorption. These results suggest that TGF-β is a physiological regulator of osteoblast differentiation and acts as a central component of the coupling of bone formation to resorption during bone remodeling.
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Grey, Andrew, Qi Chen, Xin Xu, Karen Callon, and Jill Cornish. "Parallel Phosphatidylinositol-3 Kinase and p42/44 Mitogen-Activated Protein Kinase Signaling Pathways Subserve the Mitogenic and Antiapoptotic Actions of Insulin-Like Growth Factor I in Osteoblastic Cells." Endocrinology 144, no. 11 (November 1, 2003): 4886–93. http://dx.doi.org/10.1210/en.2003-0350.
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Abstract IGF-I is an endocrine and paracrine regulator of skeletal homeostasis, principally by virtue of its anabolic effects on osteoblastic cells. In the current study, we examined the intracellular signaling pathways by which IGF-I promotes proliferation and survival in SaOS-2 human osteoblastic cells. Inhibition of each of the phosphatidylinositol-3 kinase (PI-3 kinase), p42/44 MAPK, and p70s6 kinase pathways partially inhibited the ability of IGF-I to stimulate osteoblast proliferation and survival. Because activation of p70s6 kinase is downstream of both PI-3 kinase and p42/44 MAPK activation in osteoblasts treated with IGF-I, this ribosomal kinase represents a convergence point for IGF-I-induced PI-3 kinase and p42/44 MAPK signaling in osteoblastic cells. In addition, abrogation of PI-3 kinase-dependent Akt signaling, which does not inhibit IGF-I-induced p70s6 kinase phosphorylation, also inhibited the antiapoptotic effects of IGF-I in osteoblasts. Finally, interruption of Gβγ signaling partially abrogated the ability of IGF-I to promote osteoblast survival, without inhibiting signaling through PI-3 kinase/Akt, p42/44 MAPKs, or p70s6 kinase. These data suggest that IGF-I signals osteoblast mitogenesis and survival through parallel, partly overlapping intracellular pathways involving PI-3 kinase, p42/44 MAPKs, and Gβγ subunits.
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Ponzetti, Marco, and Nadia Rucci. "Osteoblast Differentiation and Signaling: Established Concepts and Emerging Topics." International Journal of Molecular Sciences 22, no. 13 (June 22, 2021): 6651. http://dx.doi.org/10.3390/ijms22136651.
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Osteoblasts, the cells that build up our skeleton, are remarkably versatile and important cells that need tight regulation in all the phases of their differentiation to guarantee proper skeletal development and homeostasis. Although we know many of the key pathways involved in osteoblast differentiation and signaling, it is becoming clearer and clearer that this is just the tip of the iceberg, and we are constantly discovering novel concepts in osteoblast physiology. In this review, we discuss well-established pathways of osteoblastic differentiation, i.e., the classical ones committing mesenchymal stromal cells to osteoblast, and then osteocytes as well as recently emerged players. In particular, we discuss micro (mi)RNAs, long non-coding (lnc)RNAs, circular (circ)RNAs, and extracellular vesicles, focusing on the mechanisms through which osteoblasts are regulated by these factors, and conversely, how they use extracellular vesicles to communicate with the surrounding microenvironment.
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DI, SHENGMENG, RUI MENG, AIRONG QIAN, ZONGCHENG TIAN, JINGBAO LI, RONG ZHANG, and PENG SHANG. "IMPACT OF OSTEOCLAST PRECURSORS SUBJECTED TO RANDOM POSITIONING MACHINE ON OSTEOBLASTS." Journal of Mechanics in Medicine and Biology 12, no. 04 (September 2012): 1250074. http://dx.doi.org/10.1142/s0219519412005083.
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Osteoblast-osteoclast interaction plays an important role in the bone remodeling. During long duration space flight, astronauts undergo serious bone loss mainly due to the disruption of equivalence between bone formation and bone resorption. Osteoclast precursors often operate under the control of osteoblasts. However, here we show that the osteoclast precursors could in turn influence osteoblasts. RAW264.7 cells, the murine osteoclast precursors, were treated in the simulated weightlessness produced by a Random Positioning Machine (RPM). After 72 h, conditioned mediums (CM) by the RAW264.7 cells from RPM (RCM) or static control (CCM) were collected and were used to culture osteoblastic-like MC3T3-E1 cells. The results showed that the RCM culture inhibited cell viability and slightly altered cell cycle, but the morphology of the MC3T3-E1 cells was not changed by RCM compared to that of CCM. Furthermore, the intracellular ALP level, NO release and expression of osteoblastic marker genes were all down-regulated by RCM culture. These results suggest that osteoclast precursors subjected to RPM exert negative regulation on osteoblasts.
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Filvaroff, E., A. Erlebacher, J. Ye, S. E. Gitelman, J. Lotz, M. Heillman, and R. Derynck. "Inhibition of TGF-beta receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass." Development 126, no. 19 (October 1, 1999): 4267–79. http://dx.doi.org/10.1242/dev.126.19.4267.
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Transforming growth factor-beta (TGF-beta) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro. To explore the role of endogenous TGF-(beta) in osteoblast function in vivo, we have inhibited osteoblastic responsiveness to TGF-beta in transgenic mice by expressing a cytoplasmically truncated type II TGF-beta receptor from the osteocalcin promoter. These transgenic mice develop an age-dependent increase in trabecular bone mass, which progresses up to the age of 6 months, due to an imbalance between bone formation and resorption during bone remodeling. Since the rate of osteoblastic bone formation was not altered, their increased trabecular bone mass is likely due to decreased bone resorption by osteoclasts. Accordingly, direct evidence of reduced osteoclast activity was found in transgenic mouse skulls, which had less cavitation and fewer mature osteoclasts relative to skulls of wild-type mice. These bone remodeling defects resulted in altered biomechanical properties. The femurs of transgenic mice were tougher, and their vertebral bodies were stiffer and stronger than those of wild-type mice. Lastly, osteocyte density was decreased in transgenic mice, suggesting that TGF-beta signaling in osteoblasts is required for normal osteoblast differentiation in vivo. Our results demonstrate that endogenous TGF-beta acts directly on osteoblasts to regulate bone remodeling, structure and biomechanical properties.
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Enríquez, Juana, Ana Elena Lemus, Jesús Chimal-Monroy, Higinio Arzate, Gustavo A. García, Bertha Herrero, Fernando Larrea, and Gregorio Pérez-Palacios. "The effects of synthetic 19-norprogestins on osteoblastic cell function are mediated by their non-phenolic reduced metabolites." Journal of Endocrinology 193, no. 3 (June 2007): 493–504. http://dx.doi.org/10.1677/joe-06-0038.
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The key role of estrogens on osteoblastic cell function is well documented; however, the role of progesterone (P) and synthetic progestins remains controversial. While several reports indicate that P has no significant effects on bone cells, a number of clinical studies have shown that 19-norprogestins restore postmenopausal bone loss. The mechanisms by which 19-norprogestins induce estrogen-like effects on bone cells are not fully understood. To assess whether the actions of 19-norprogestins on osteoblasts are mediated by their non-phenolic metabolites, we studied the effects of norethisterone (NET), levonorgestrel (LNG), and two of their A-ring reduced derivatives upon cell proliferation and differentiation in neonatal rat osteoblasts. Osteoblast function was assessed by determining cell DNA, cell-associated osteocalcin and calcium content, alkaline phosphatase activity, and mineral deposition. P failed to induce changes on osteoblasts, while NET and LNG exerted a number of actions. The most striking finding was that the 3β,5α- and 3α,5α-tetrahydro derivatives of NET and LNG induced osteoblast proliferation and differentiation with higher potency than those exerted by their parent compounds, mimicking the effects of estradiol. Interestingly, osteoblast differentiation and mineral deposition induced by NET and LNG were abolished by finasteride, a 5α-reductases inhibitor, while the potent effect on osteoblast proliferation induced by progestin derivatives was abolished by a steroidal antiestrogen. Results demonstrate that A-ring reduced derivatives of NET and LNG exhibit intrinsic estrogen-like potency on rat osteoblasts, offering a plausible explanation for the mechanism of action of 19-norprogestins in bone restoration in postmenopausal women and providing new insights for hormone replacement therapy research.
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Mahalingam, Chandrika D., Bharat Reddy Sampathi, Sonali Sharma, Tanuka Datta, Varsha Das, Abdul B. Abou-Samra, and Nabanita S. Datta. "MKP1-dependent PTH modulation of bone matrix mineralization in female mice is osteoblast maturation stage specific and involves P-ERK and P-p38 MAPKs." Journal of Endocrinology 216, no. 3 (November 29, 2012): 315–29. http://dx.doi.org/10.1530/joe-12-0372.
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Limited information is available on the role of MAPK phosphatase 1 (MKP1) signaling in osteoblasts. We have recently reported distinct roles for MKP1 during osteoblast proliferation, differentiation, and skeletal responsiveness to parathyroid hormone (PTH). As MKP1 regulates the phosphorylation status of MAPKs, we investigated the involvement of P-ERK and P-p38 MAPKs in MKP1 knockout (KO) early and mature osteoblasts with respect to mineralization and PTH response. Calvarial osteoblasts from 9–14-week-old WT and MKP1 KO male and female mice were examined. Western blot analysis revealed downregulation and sustained expressions of P-ERK and P-p38 with PTH treatment in differentiated osteoblasts derived from KO males and females respectively. Exposure of early osteoblasts to p38 inhibitor, SB203580 (S), markedly inhibited mineralization in WT and KO osteoblasts from both genders as determined by von Kossa assay. In osteoblasts from males, ERK inhibitor U0126 (U), not p38 inhibitor (S), prevented the inhibitory effects of PTH on mineralization in early or mature osteoblasts. In osteoblasts from KO females, PTH sustained mineralization in early osteoblasts and decreased mineralization in mature cells. This effect of PTH was attenuated by S in early osteoblasts and by U in mature KO cells. Changes in matrix Gla protein expression with PTH in KO osteoblasts did not correlate with mineralization, indicative of MKP1-dependent additional mechanisms essential for PTH action on osteoblast mineralization. We conclude that PTH regulation of osteoblast mineralization in female mice is maturation stage specific and involves MKP1 modulation of P-ERK and P-p38 MAPKs.
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Thomas, GP, SU Baker, JA Eisman, and EM Gardiner. "Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts." Journal of Endocrinology 170, no. 2 (August 1, 2001): 451–60. http://dx.doi.org/10.1677/joe.0.1700451.
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Osteoblast-osteoclast coordination is critical in the maintenance of skeletal integrity. The modulation of osteoclastogenesis by immature cells of the osteoblastic lineage is mediated through receptor activator of NF kappa B (RANK), its ligand RANKL, and osteoprotegerin (OPG), a natural decoy receptor for RANKL. Here, the expression of OPG and RANKL in primary mouse osteoblastic cultures was investigated to determine whether the osteoclastogenic stimulus depended on the stage of osteoblastic differentiation and the presence of the calciotrophic hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). OPG mRNA expression was increased in osteoblastic cultures after the onset of mineralisation relative to less mature cultures, but did not alter in response to 1,25-(OH)(2)D(3) treatment. In contrast, basal RANK L mRNA expression did not change during differentiation but was significantly enhanced by 1,25-(OH)(2)D(3) treatment at all times. The stimulatory effects of 1,25-(OH)(2)D(3) on RANKL were lessened in more mature cultures, however. The RANKL/OPG ratio, an index of osteoclastogenic stimulus, was therefore increased by 1,25-(OH)(2)D(3) treatment at all stages of osteoblastic differentiation, but to a lesser degree in cultures after the onset of mineralisation. Thus the 1,25-(OH)(2)D(3)-driven increase in osteoclastogenic potential of immature osteoblasts appears to be mediated by increased RANKL mRNA expression, with mature osteoblasts having relatively decreased osteoclastogenic activity due to increased OPG mRNA expression. These findings suggest a possible mechanism for the recently proposed negative regulatory role of mature osteoblasts on osteoclastogenesis and indicate that the relative proportions of immature and mature osteoblasts in the local microenvironment may control the degree of resorption at each specific bone site.
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Aasebø, Elise, Annette K. Brenner, Maria Hernandez-Valladares, Even Birkeland, Frode S. Berven, Frode Selheim, and Øystein Bruserud. "Proteomic Comparison of Bone Marrow Derived Osteoblasts and Mesenchymal Stem Cells." International Journal of Molecular Sciences 22, no. 11 (May 26, 2021): 5665. http://dx.doi.org/10.3390/ijms22115665.
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Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, and therapeutic targeting of these cells is considered both for malignant and non-malignant diseases. We analyzed global proteomic profiles for osteoblasts derived from ten and MSCs from six healthy individuals, and we quantified 5465 proteins for the osteoblasts and 5420 proteins for the MSCs. There was a large overlap in the profiles for the two cell types; 156 proteins were quantified only in osteoblasts and 111 proteins only for the MSCs. The osteoblast-specific proteins included several extracellular matrix proteins and a network including 27 proteins that influence intracellular signaling (Wnt/Notch/Bone morphogenic protein pathways) and bone mineralization. The osteoblasts and MSCs showed only minor age- and sex-dependent proteomic differences. Finally, the osteoblast and MSC proteomic profiles were altered by ex vivo culture in serum-free media. We conclude that although the proteomic profiles of osteoblasts and MSCs show many similarities, we identified several osteoblast-specific extracellular matrix proteins and an osteoblast-specific intracellular signaling network. Therapeutic targeting of these proteins will possibly have minor effects on MSCs. Furthermore, the use of ex vivo cultured osteoblasts/MSCs in clinical medicine will require careful standardization of the ex vivo handling of the cells.
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Aubin, Jane E. "Advances in the osteoblast lineage." Biochemistry and Cell Biology 76, no. 6 (December 1, 1998): 899–910. http://dx.doi.org/10.1139/o99-005.
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Osteoblasts are the skeletal cells responsible for synthesis, deposition and mineralization of the extracellular matrix of bone. By mechanisms that are only beginning to be understood, stem and primitive osteoprogenitors and related mesenchymal precursors arise in the embryo and at least some appear to persist in the adult organism, where they contribute to replacement of osteoblasts in bone turnover and in fracture healing. In this review, we describe the morphological, molecular, and biochemical criteria by which osteoblasts are defined and cell culture approaches that have helped to clarify transitional stages in osteoblast differentiation. Current understanding of differential expression of osteoblast-associated genes during osteoprogenitor proliferation and differentiation to mature matrix synthesizing osteoblasts is summarized. Evidence is provided to support the hypothesis that the mature osteoblast phenotype is heterogeneous with subpopulations of osteoblasts expressing only subsets of the known osteoblast markers. Throughout this paper, outstanding uncertainties and areas for future investigation are also identified.Key words: skeletal development, differential gene expression, heterogeneity.
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Thi, Mia M., Marcia Urban-Maldonado, David C. Spray, and Sylvia O. Suadicani. "Characterization of hTERT-immortalized osteoblast cell lines generated from wild-type and connexin43-null mouse calvaria." American Journal of Physiology-Cell Physiology 299, no. 5 (November 2010): C994—C1006. http://dx.doi.org/10.1152/ajpcell.00544.2009.
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The gap junction protein connexin43 (Cx43) has been proposed to play key roles in bone differentiation and mineralization, but underlying cellular mechanisms are not totally understood. To further explore roles of Cx43 in these processes, we immortalized calvarial osteoblasts from wild-type and Cx43-null mice using human telomerase reverse transcriptase (hTERT). Osteoblastic (MOB) cell lines were generated from three individual wild-type and three individual Cx43-null mouse calvaria. Average population doubling times of the cell lines were higher than of the primary osteoblasts but did not greatly differ with regard to genotype. Modest to high level of Cx45 expression was detected in MOBs of both genotypes. Most of the cell lines expressed osteoblastic markers [Type I collagen, osteopontin, osteocalcin, parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP), periostin (OSF-2), osterix (Osx), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP)], and mineralization was comparable to that of primary osteoblasts. Two MOB cell lines from each genotype with most robust maintenance of osteoblast lineage markers were analyzed in greater detail, revealing that the Cx43-null cell lines showed a significant delay in early differentiation (up to 9 days in culture). Matrix mineralization was markedly delayed in one of the Cx43-null lines and slightly delayed in the other. These findings comparing new and very stable wild-type and Cx43-null osteoblastic cell lines define a role for Cx43 in early differentiation and mineralization stages of osteoblasts and further support the concept that Cx43 plays important role in the cellular processes associated with skeleton function.
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Hu, Fen, Yali Zhao, Zhenhai Hui, Fulin Xing, Jianyu Yang, Imshik Lee, Xinzheng Zhang, Leiting Pan, and Jingjun Xu. "Regulation of intracellular Ca2+/CaMKII signaling by TRPV4 membrane translocation during osteoblastic differentiation." Biophysics Reports 5, no. 5-6 (November 22, 2019): 254–63. http://dx.doi.org/10.1007/s41048-019-00100-y.
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AbstractBone constantly remodels between resorption by osteoclasts and formation by osteoblasts; therefore the functions of osteoblasts are pivotal for maintaining homeostasis of bone mass. Transient receptor potential vanilloid 4 (TRPV4), a type of mechanosensitive channel, has been reported to be a key regulator in bone remodeling. However, the relationship between TRPV4 and osteoblast function remains largely elusive. Only little is known about the spatial distribution change of TRPV4 during osteoblastic differentiation and related signal events. Based on three-dimensional super-resolution microscopy, our results clearly showed a different distribution of TRPV4 in undifferentiated and differentiated osteoblasts, which reflected the plasma membrane translocation of TRPV4 along with prolonged differentiation. GSK1016790A (GSK101), the most potent agonist of TRPV4, triggered rapid calcium entry and calmodulin-dependent protein kinase II (CaMKII) phosphorylation via TRPV4 activation in a differentiation-dependent manner, indicating that the abundance of TRPV4 at the cell surface resulting from differentiation may be related to the modulation of Ca2+ response and CaMKII activity. These data provide compelling evidences for the plasma membrane translocation of TRPV4 during osteoblastic differentiation as well as demonstrate the regulation of downstream Ca2+/CaMKII signaling.
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Allen, Kahtonna C., Carlos J. Sanchez, Krista L. Niece, Joseph C. Wenke, and Kevin S. Akers. "Voriconazole Enhances the Osteogenic Activity of Human OsteoblastsIn Vitrothrough a Fluoride-Independent Mechanism." Antimicrobial Agents and Chemotherapy 59, no. 12 (August 31, 2015): 7205–13. http://dx.doi.org/10.1128/aac.00872-15.
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ABSTRACTPeriostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblastsin vitroto voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activityin vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity.
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Jo, Sungsin, Subin Yoon, So Young Lee, So Yeon Kim, Hyosun Park, Jinil Han, Sung Hoon Choi, Joong-Soo Han, Jae-Hyuk Yang, and Tae-Hwan Kim. "DKK1 Induced by 1,25D3 Is Required for the Mineralization of Osteoblasts." Cells 9, no. 1 (January 17, 2020): 236. http://dx.doi.org/10.3390/cells9010236.
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1α,25-dihydroxyvitamin D3 (1,25D3), the most popular drug for osteoporosis treatment, drives osteoblast differentiation and bone mineralization. Wnt/β-catenin signaling is involved in commitment and differentiation of osteoblasts, but the role of the Dickkopf-related protein 1 (DKK1), a Wnt antagonist, in osteoblasts remains unknown. Here, we demonstrate the molecular mechanism of DKK1 induction by 1,25D3 and its physiological role during osteoblast differentiation. 1,25D3 markedly promoted the expression of both CCAAT/enhancer binding protein beta (C/EBPβ) and DKK1 at day 7 during osteoblast differentiation. Interestingly, mRNA and protein levels of C/EBPβ and DKK1 in osteoblasts were elevated by 1,25D3. We also found that C/EBPβ, in response to 1,25D3, directly binds to the human DKK1 promoter. Knockdown of C/EBPβ downregulated the expression of DKK1 in osteoblasts, which was partially reversed by 1,25D3. In contrast, overexpression of C/EBPβ upregulated DKK1 expression in osteoblasts, which was enhanced by 1,25D3. Furthermore, 1,25D3 treatment in osteoblasts stimulated secretion of DKK1 protein within the endoplasmic reticulum to extracellular. Intriguingly, blocking DKK1 attenuated calcified nodule formation in mineralized osteoblasts, but not ALP activity or collagen synthesis. Taken together, these observations suggest that 1,25D3 promotes the mineralization of osteoblasts through activation of DKK1 followed by an increase of C/EBPβ.
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Nafila, Idayu, Putri Aulia Sari, Annisa Firdha, and Sri Suparwitri. "Pengaruh faktor umur dan pemberian serbuk biji gandum terhadap jumlah osteoblas pada pergerakan gigi secara ortodontiThe effect of age and wheat seed powder application on the number of osteoblasts on orthodontic tooth movement." Jurnal Kedokteran Gigi Universitas Padjadjaran 32, no. 3 (December 31, 2020): 193. http://dx.doi.org/10.24198/jkg.v32i3.27923.
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Pendahuluan: Pergerakan gigi pada perawatan ortodonti merupakan kombinasi proses resorpsi oleh osteoklas pada daerah tertekan dan aposisi oleh osteoblas pada sisi tertarik sehingga terjadi remodeling tulang. Remodeling tulang dipengaruhi umur dan hormon estrogen. Biji gandum (Triticum aestivum) merupakan fitoestrogen yang memiliki struktur dan fungsi seperti hormon estrogen. Tujuan penelitian menganalisis pengaruh faktor umur dan pemberian serbuk biji gandum terhadap jumlah osteoblas pada pergerakan gigi secara ortodonti pada tikus Sprague dawley muda dan tua. Metode: Jenis penelitian eksperimental laboratoris dengan menggunakan 36 subjek tikus Sprague dawley jantan yang terbagi menjadi kelompok muda (4-5 minggu) dan tua (18-20 minggu). Setiap grup akan dibagi menjadi 2 subgrup untuk menerima perlakuan yaitu: kelompok kontrol dengan perlakuan insisivus maksila digerakan ke distal menggunakan koil spring dan kelompok perlakuan yang diberi serbuk biji gandum dengan dosis 1,08 g dan perlakukan insisivus maksila digerakan ke distal menggunakan koil spring. Subjek hewan coba dieutanasia menggunakan anestesi dosis mematikan dan dilakukan pemotongan rahang atas pada regio kedua insisivus pada hari ke 1, 4, dan 7. Osteoblas dianalisis secara histologi dengan pengecatan hematoksilin eosin. Perhitungan jumlah osteoblas dilakukan dengan menggunakan mikroskop optilab. Hasil: Jumlah osteoblas kelompok muda lebih banyak dibandingkan kelompok tua. Pemberian serbuk biji gandum dapat meningkatkan jumlah osteoblas pada kelompok tua maupun muda. Pemberian serbuk biji gandum menunjukan pengaruh signifikan p<0,001 (p<0,05) terhadap jumlah osteoblas Simpulan: Terdapat pengaruh faktor umur dan pemberian serbuk biji gandum terhadap peningkatan jumlah osteoblas pada pergerakan gigi secara ortodonti, osteoblas pada tikus berumur muda lebih banyak dibandingkan dengan tikus berumur tua.Kata kunci: Osteoblas, pergerakan gigi secara ortodonti, serbuk gandum, fitoestrogen, tikus Sprague dawley. ABSTRACTIntroduction: Tooth movement in orthodontic treatment is a combination of osteoclasts resorption in the stressed area and osteoblast apposition on the pulled side, resulting in bone remodelling. Bone remodelling is influenced by age and estrogen hormone. Wheat seeds (Triticum aestivum) are phytoestrogens with estrogen-like structure and function. This study was aimed to analyse the effect of age and wheat seed powder on the number of osteoblasts on orthodontic tooth movement in young and old Sprague-Dawley rats. Methods: This study was an experimental laboratory using 36 male Sprague-Dawley rats divided into young (4-5 weeks) and old (18-20 weeks) groups. Each group will be divided into two subgroups to receive treatment, namely: the control group with the treatment of the maxillary incisors moved distally using a coil spring and the treatment group treated with wheat seed powder at a dose of 1.08 g and treated the maxillary incisors were moved distally using a coil spring. Animal subjects were euthanised using lethal doses of anaesthesia, and the maxilla was cut in the second region of the incisors on the 1st, 4th, and 7th days. Osteoblasts were analysed histologically by eosin hematoxylin staining. The number of osteoblasts was calculated using an Optylab microscope. Results: The number of osteoblasts in the young group was found to be higher than the old group. The application of wheat seed powder can increase the number of osteoblasts in the young and old groups. The application of wheat seed powder showed a significant (p<0.001 (p<0.05)) on the number of osteoblasts. Conclusion: There was an effect of age and wheat seed powder on the increase in the number of osteoblasts on orthodontic tooth movement. The young rats have more osteoblasts compared to the old rats.Keywords: Osteoblasts, orthodontic tooth movement, wheat seed powder, phytoestrogen, Sprague-Dawley rats.
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Clover, J., R. A. Dodds, and M. Gowen. "Integrin subunit expression by human osteoblasts and osteoclasts in situ and in culture." Journal of Cell Science 103, no. 1 (September 1, 1992): 267–71. http://dx.doi.org/10.1242/jcs.103.1.267.
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The extracellular matrix may be considered as an insoluble local mediator which plays an important role in regulating cell function. Communication between the cell and its matrix occurs via the integrins, a family of transmembrane proteins composed of non-covalently linked alpha and beta subunits. The aim of this study was to establish which integrins are present on human bone cells in situ and in culture, using cryostat sections of undecalcified human bone, osteoclastoma tissue and cultured human osteoblasts. Integrin subunit expression was identified indirectly using alkaline phosphatase anti-alkaline phosphatase conjugates and FITC-labelled secondary antibodies. Subunits expressed by cultured human osteoblast-like cells were then quantified by FACS analysis. Staining patterns observed in situ show that osteoblasts and osteoclasts possess different integrin subunits. Osteoblasts primarily express alpha 1, alpha 3 and beta 1 and weakly express alpha 2. Osteoclasts express alpha 2, alpha V, beta 1 and beta 3. Subunits alpha 4, alpha 5, alpha 6, alpha L, alpha M and beta 2 were not expressed by either of these cell types. Expression of beta 1 by all cells of the osteoblastic lineage was constitutive, but alpha 1 and alpha 3 subunits were expressed by osteoblasts actively synthesizing bone and some of the osteoblast lining cells. All integrin subunits identified on osteoblasts in situ were maintained on culture but there was an increased expression of alpha 2 and alpha V subunits were weakly positive. Expression of alpha 2, alpha 3, alpha V and beta 1 subunits was independent of cell density but expression of alpha 1 was much greater in confluent cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
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Park, Kyung-Ran, Hanna Lee, MyoungLae Cho, and Hyung-Mun Yun. "A Phytochemical Constituent, (E)-Methyl-Cinnamate Isolated from Alpinia katsumadai Hayata Suppresses Cell Survival, Migration, and Differentiation in Pre-Osteoblasts." International Journal of Molecular Sciences 21, no. 10 (May 24, 2020): 3700. http://dx.doi.org/10.3390/ijms21103700.
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Background: (E)-methyl-cinnamate (EMC), a phytochemical constituent isolated from Alpinia katsumadai Hayata, is a natural flavor compound with anti-inflammatory properties, which is widely used in the food and commodity industry. However, the pharmacological effects of methyl-cinnamate on pre-osteoblasts remain unknown. This study aimed to investigate the pharmacological effects and mechanisms of EMC in pre-osteoblast MC3T3-E1 cells (pre-osteoblasts). Methods: Cell viability and apoptosis were evaluated using the MTT assay and TUNEL staining. Cell migration and osteoblast differentiation were examined using migration assays, as well as alkaline phosphatase activity and staining assays. Western blot analysis was used to examine intracellular signaling pathways and apoptotic proteins. Results: EMC decreased cell viability with morphological changes and increased apoptosis in pre-osteoblasts. EMC also induced the cleavage of Poly (ADP-ribose) polymerase (PARP) and caspase-3 and reduced the expression of anti-apoptotic proteins. In addition, EMC increased TUNEL-positive cells in pre-osteoblasts, decreased the activation of mitogen-activated protein kinases, and suppressed cell migration rate in pre-osteoblasts. Subsequently, EMC inhibited the osteoblast differentiation of pre-osteoblasts, as assessed by alkaline phosphatase staining and activity assays. Conclusion: These findings demonstrate that EMC has a pharmacological and biological role in cell survival, migration, and osteoblast differentiation. It suggests that EMC might be a potential phytomedicine for treating abnormalities of osteoblast function in bone diseases.
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Mansukhani, Alka, Davide Ambrosetti, Greg Holmes, Lizbeth Cornivelli, and Claudio Basilico. "Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation." Journal of Cell Biology 168, no. 7 (March 21, 2005): 1065–76. http://dx.doi.org/10.1083/jcb.200409182.
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Activating mutations in fibroblast growth factor receptor 2 (FGFR2) cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts, which form the calvarial bones. Osteoblasts respond to FGF with increased proliferation and inhibition of differentiation. We analyzed the gene expression profiles of osteoblasts expressing FGFR2 activating mutations (C342Y or S252W) and found a striking down-regulation of the expression of many Wnt target genes and a concomitant induction of the transcription factor Sox2. Most of these changes could be reproduced by treatment of osteoblasts with exogenous FGF. Wnt signals promote osteoblast function and regulate bone mass. Sox2 is expressed in calvarial osteoblasts in vivo and we show that constitutive expression of Sox2 inhibits osteoblast differentiation and causes down-regulation of the expression of numerous Wnt target genes. Sox2 associates with β-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain. Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt–β-catenin pathway.
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Guntur, Anyonya R., Phuong T. Le, Charles R. Farber, and Clifford J. Rosen. "Bioenergetics During Calvarial Osteoblast Differentiation Reflect Strain Differences in Bone Mass." Endocrinology 155, no. 5 (May 1, 2014): 1589–95. http://dx.doi.org/10.1210/en.2013-1974.
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Osteoblastogenesis is the process by which mesenchymal stem cells differentiate into osteoblasts that synthesize collagen and mineralize matrix. The pace and magnitude of this process are determined by multiple genetic and environmental factors. Two inbred strains of mice, C3H/HeJ and C57BL/6J, exhibit differences in peak bone mass and bone formation. Although all the heritable factors that differ between these strains have not been elucidated, a recent F1 hybrid expression panel (C3H × B6) revealed major genotypic differences in osteoblastic genes related to cellular respiration and oxidative phosphorylation. Thus, we hypothesized that the metabolic rate of energy utilization by osteoblasts differed by strain and would ultimately contribute to differences in bone formation. In order to study the bioenergetic profile of osteoblasts, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) first in a preosteoblastic cell line MC3T3-E1C4 and subsequently in primary calvarial osteoblasts from C3H and B6 mice at days 7, 14, and 21 of differentiation. During osteoblast differentiation in media containing ascorbic acid and β-glycerophosphate, all 3 cell types increased their oxygen consumption and extracellular acidification rates compared with the same cells grown in regular media. These increases are sustained throughout differentiation. Importantly, C3H calvarial osteoblasts had greater oxygen consumption rates than B6 consistent with their in vivo phenotype of higher bone formation. Interestingly, osteoblasts utilized both oxidative phosphorylation and glycolysis during the differentiation process although mature osteoblasts were more dependent on glycolysis at the 21-day time point than oxidative phosphorylation. Thus, determinants of oxygen consumption reflect strain differences in bone mass and provide the first evidence that during collagen synthesis osteoblasts use both glycolysis and oxidative phosphorylation to synthesize and mineralize matrix.
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Clabaut, Aline, Céline Grare, Gaëlle Rolland-Valognes, Jean-Guillaume Letarouilly, Chantal Bourrier, Thomas L. Andersen, Tanja Sikjær, et al. "Adipocyte-induced transdifferentiation of osteoblasts and its potential role in age-related bone loss." PLOS ONE 16, no. 1 (January 26, 2021): e0245014. http://dx.doi.org/10.1371/journal.pone.0245014.
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Our preliminary findings have lead us to propose bone marrow adipocyte secretions as new contributors to bone loss. Indeed, using a coculture model based on human bone marrow stromal cells, we previously showed that soluble factors secreted by adipocytes induced the conversion of osteoblasts towards an adipocyte-like phenotype. In this study, microarray gene expression profiling showed profound transcriptomic changes in osteoblasts following coculture and confirmed the enrichment of the adipocyte gene signature. Double immunofluorescence microscopic analyses demonstrated the coexpression of adipogenic and osteoblastic specific markers in individual cells, providing evidence for a transdifferentiation event. At the molecular level, this conversion was associated with upregulated expression levels of reprogramming genes and a decrease in the DNA methylation level. In line with these in vitro results, preliminary immunohistochemical analysis of bone sections revealed adipogenic marker expression in osteoblasts from elderly subjects. Altogether, these data suggest that osteoblast transdifferentiation could contribute to decreased bone mass upon ageing.
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Crisan, Liana, Olga Soritau, Mihaela Baciut, Grigore Baciut, and Bogdan Vasile Crisan. "THE INFLUENCE OF LASER RADIATION ON HUMAN OSTEOBLASTS CULTURED ON NANOSTRUCTURED COMPOSITE SUBSTRATES." Medicine and Pharmacy Reports 88, no. 2 (April 29, 2015): 224–32. http://dx.doi.org/10.15386/cjmed-433.
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Background & Aims. Carbon-based nanomaterials such as carbon nanotubes, graphene oxide and graphene have been explored by researchers as well as the industry. Graphene is a new nanomaterial which has commercial and scientific advantages. Laser therapy has proven highly useful in biomedicine, with the use of different laser types and energies for distinct purposes. The low level laser therapy (LLLT) can have anti-inflammatory, analgesic and biostimulant effects. Recent research has shown that laser radiation has different effects on osteoblasts. The aim of this study was to identify the influence of laser radiation on human osteoblastic cells cultured on nanostructured composite substrates.Materials and methods. Four types of substrates were created using colloidal suspensions of nanostructured composites in PBS at a concentration of 30 µg/ml. We used human osteoblasts isolated from patella bone pieces harvested during arthroplasty. Irradiation of osteoblasts cultured on nanostructured composite substrates was made with a semiconductor laser model BTL-10 having a wavelength of 830 nm. The proliferation activity of osteoblast cells was assessed using the MTT assay. After laser irradiation procedure the viability and proliferation of osteoblast cells were analyzed using fluorescein diacetate (FDA) staining. Results. The osteoblast cells viability and proliferation were evaluated with MTT assay at 30 minutes, 24 hours, 5 days and 10 days after laser irradiation. In the first 30 minutes there were no significant differences between the irradiated and non-irradiated cells. At 24 hours after laser irradiation procedure a significant increase of MTT values in case of irradiated osteoblasts cultivated on nanostructured hydroxyapatite, nanostructured hydroxyapatite with gold nanoparticles and 1.6% and 3.15% graphenes composites substrates was observed. A more marked proliferation rate was observed after 10 days of irradiation for irradiated osteoblasts seeded on nanostructured hydroxyapatite with gold nanoparticles and graphenes containing substrate. Using FDA staining we obtained very similar results with MTT test. Conclusions. The association between the 830 nm laser irradiation of osteoblasts and their long-term cultivation of the nanostructured composite substrates induces the cell proliferation and differentiation and, therefore, it will be a useful alternative for bone regeneration therapy.
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Wan, Jun, Tao Ma, Yun Jin, and Shaodong Qiu. "The effects of morin on bone regeneration to accelerate healing in bone defects in mice." International Journal of Immunopathology and Pharmacology 34 (January 2020): 205873842096290. http://dx.doi.org/10.1177/2058738420962909.
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Restoring bone defects are the major challenge facing clinical trial therapy, particularly skull related problems. Morin, a naturally occurring compound, has pro-osteogenesis. This research focuses on assessing the role of morin for its pro-osteogenesis activities. We utilized in vivo and in vitro models to investigate the molecular-level mechanisms of morin’s osteoblastic biological activity. The effectiveness of morin on pro-osteogenesis (100 mg/kg/day) was assessed by monitoring modifications in the bone histomorphometry score, the development of immature osteoblasts from mesenchymal stems cells and improvements in the expression of pro-osteogenic cytokines in skull defected (SD) mice. Quantitative—PCR, Western blot analysis, and immunofluorescence were studied to investigate the signaling pathways. Morin has a substantial in vivo pro-osteogenesis effect which can facilitate the development of osteoblasts, the production of osteoblast related marker genes, and in vitro protein markers for osteoblasts. From a molecular biology standpoint, morin contributes to the development of osteoblasts and stimulation of the Wnt pathway with the activation and translocation of β-catenin nuclei. Our findings from the study revealed that morin may be a beneficial substitute for helping regenerate bone defects.
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Jansen, Ineke D. C., Wikky Tigchelaar-Gutter, Jolanda M. A. Hogervorst, Teun J. de Vries, Paul Saftig, and Vincent Everts. "LAMP-2 Is Involved in Surface Expression of RANKL of Osteoblasts In Vitro." International Journal of Molecular Sciences 21, no. 17 (August 25, 2020): 6110. http://dx.doi.org/10.3390/ijms21176110.
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Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa β (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.
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Bozec, Aline, Latifa Bakiri, Maria Jimenez, Thorsten Schinke, Michael Amling, and Erwin F. Wagner. "Fra-2/AP-1 controls bone formation by regulating osteoblast differentiation and collagen production." Journal of Cell Biology 190, no. 6 (September 13, 2010): 1093–106. http://dx.doi.org/10.1083/jcb.201002111.
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The activator protein-1 (AP-1) transcription factor complex, in particular the Fos proteins, is an important regulator of bone homeostasis. Fra-2 (Fosl2), a Fos-related protein of the AP-1 family, is expressed in bone cells, and newborn mice lacking Fra-2 exhibit defects in chondrocytes and osteoclasts. Here we show that Fra-2–deficient osteoblasts display a differentiation defect both in vivo and in vitro. Moreover, Fra-2–overexpressing mice are osteosclerotic because of increased differentiation of osteoblasts, which appears to be cell autonomous. Importantly, the osteoblast-specific osteocalcin (Oc) gene and collagen1α2 (col1α2) are transcriptional targets of Fra-2 in both murine and human bone cells. In addition, Fra-2, Oc, and col1 are expressed in stromal cells of human chondroblastic and osteoblastic osteosarcomas (Os’s) as well as during osteoblast differentiation of human Os cell lines. These findings reveal a novel function of Fra-2/AP-1 as a positive regulator of bone and matrix formation in mice and humans.
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Barbuto, Richard, and Jane Mitchell. "Regulation of the osterix (Osx, Sp7) promoter by osterix and its inhibition by parathyroid hormone." Journal of Molecular Endocrinology 51, no. 1 (May 16, 2013): 99–108. http://dx.doi.org/10.1530/jme-12-0251.
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Osterix (Osx, Sp7) is a zinc-finger transcription factor belonging to the specificity protein (Sp) family expressed in cells of the osteoblast lineage in the developing skeleton where it regulates expression of a number of osteoblastic genes. We previously reported inhibition of osterix mRNA and protein by parathyroid hormone (PTH) stimulation of cAMP in osteoblasts. We here show that Osx expression in osteoblasts is regulated by Sp proteins as demonstrated by mithramycin A inhibition of Osx mRNA and OSX protein levels. Mutation of putative transcription factor binding sites within the Osx promoter demonstrated a tandem repeat sequence that selectively binds OSX but not other Sp factors expressed in osteoblasts (Sp1, Sp3, or Tieg (Klf10)). Mutation of either or both the repeat sequences inhibited 90% of the promoter activity and also abrogated some of the PTH-mediated inhibition of the promoter. Previous studies have shown growth factor regulation of Osx expression by MAPK proteins, particularly p38 phosphorylation of OSX that increases its transcriptional activity. PTH stimulation of osteoblasts inhibits MAPK components (ERK, JNK, and p38) but inhibition of Osx mRNA and protein expression by PTH was selectively mimicked by p38 inhibition and expression of constitutively active MKK6, which stimulates p38, blocked PTH inhibition of OSX. Together, our studies suggest that OSX autoregulation is a major mechanism in osteoblasts and that PTH stimulation inhibits osterix by inhibition of p38 MAPK regulation of OSX.
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Fulzele, Keertik, Cristina Panaroni, Kenta Mukaihara, Tomoaki Mori, Shrikanta Chattopadhyay, and Noopur S. Raje. "Mature Osteoblasts Promote Multiple Myeloma Survival through Cell-Cell Contact and Immune Modulation Mechanisms." Blood 134, Supplement_1 (November 13, 2019): 1802. http://dx.doi.org/10.1182/blood-2019-131213.
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The bone marrow (BM) microenvironment (TME) is a complex mix of cellular and non-cellular components that has a profound effect on hematopoiesis and hematological malignancies. Multiple myeloma (MM), a tumor of plasma cells that originates in the BM is highly influenced by TME components including stromal cells, osteoclasts, and immune cells. Among these cells, osteoblasts are the most versatile regulators of hematopoiesis and also play an integral role in malignant transformations of leukemia and lung cancer. MM is characterized by osteolytic bone lesions partly due to decreased numbers of osteoblasts. Therapies such as denosumab and zoledronic acid aim to restore bone health by inhibiting osteoclastogenesis. However, the contribution of osteoblasts to the initiation and progression of MM is not well understood. We previously demonstrated that increased osteoblast numbers using Activin A inhibition in an in vivo humanized myeloma model resulted in inhibition of MM growth (Vallet et al., 2010). Given that osteoblast numbers are on the decline in a typical MM patient, we hypothesized that the loss of osteoblasts contributes to the initiation and progression of MM. To assess the effects of osteoblasts on MM cells, MC3T3 murine pre-osteoblasts were differentiated into mature osteoblasts by supplementing media with ascorbic acid and β-glycerophosphate. Co-culture of 5TGM1 murine MM cells with the osteoblasts showed a significant decrease in the proliferation of MM cells by 40% (N=4). Osteoblasts go through distinct stages of maturation i.e., pre-osteoblasts, committed osteoblasts, and mature osteoblasts. These individual osteoblast populations were FACS sorted from mice to isolate pre-osteoblasts from the long bones of Osterix-GFP+ (Osx+) mice, committed osteoblasts from Collagen 2.3-GFP+ (Col2.3+) mice, and mature osteoblasts from Osteocalcin-YFP+ (OCN+) mice. Each of the osteoblast populations was co-cultured with 5TGM1 MM cells. Although all the osteoblast populationssignificantly suppressed MM proliferation, the OCN+ mature osteoblasts suppressed MM proliferation the most (N=3). Therefore, we focused our studies on the role of mature osteoblasts in MM progression. We generated mice in which mature osteoblasts could be postnatally deleted in an inducible and reversible manner. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre) to generate OC-Cre/iDTR mice. The control mice were littermates lacking the OC-Cre allele. The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treatedwith 50 µg/Kg DT once a week beginning at 8-weeks of age. To study MM engraftment and progression, 3x1065TGM1-Luciferase MM cells were injected into the tibia of OC-Cre/iDTR and control mice followed by weeklyinjection of DT for 8-weeks. Bioluminescence imaging (BLI) showed that 4-weeks onwards the OC-iDTR mice, but not the control mice, continued to express and increase the BLI signal (N=6). This data suggeststhatunder physiological conditions, mature osteoblasts activelysuppress MM engraftment and progression. We hypothesized that osteoblasts provide niche support to MM cells via direct cell-to-cell contact. To begin to identify the molecular mechanisms, we compared gene expression changes in primary murine osteoblasts between the undifferentiated and 30-day osteogenic differentiation time points. We also examined the BM TME by quantitative protein antibody arrays at 2-weeks following 5TGM1MM intratibial injection into OC-Cre/iDTR and control mice. The mature osteoblasts showed a significant increase in the expression of integrins, including integrin α4, and several immunomodulatory markers. The cytokine array analysis showed altered expressions of cell-cell communication proteins MAdCAM1, BAFF-R, TACI, and immunomodulatory factors IL-33, IL-17F, and IL-13. Taken together, using in vivo and in vitro models, we show that mature osteoblasts may have a negative regulatory impact on MM cells through cell-cell communication or immunomodulatory mechanisms. Expanding the osteoblast niche may provide novel therapeutic avenues to reduce disease burden and create an environment for long term tumor control. Disclosures Raje: Merck: Consultancy; Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy.
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Cazzaniga, Alessandra, Sara Castiglioni, and Jeanette A. M. Maier. "Conditioned Media from Microvascular Endothelial Cells Cultured in Simulated Microgravity Inhibit Osteoblast Activity." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/857934.
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Background and Aims. Gravity contributes to the maintenance of bone integrity. Accordingly, weightlessness conditions during space flight accelerate bone loss and experimental models in real and simulated microgravity show decreased osteoblastic and increased osteoclastic activities. It is well known that the endothelium and bone cells cross-talk and this intercellular communication is vital to regulate bone homeostasis. Because microgravity promotes microvascular endothelial dysfunction, we anticipated that the molecular cross-talk between endothelial cells exposed to simulated microgravity and osteoblasts might be altered.Results. We cultured human microvascular endothelial cells in simulated microgravity using the rotating wall vessel device developed by NASA. Endothelial cells in microgravity show growth inhibition and release higher amounts of matrix metalloproteases type 2 and interleukin-6 than controls. Conditioned media collected from microvascular endothelial cells in simulated microgravity were used to culture human osteoblasts and were shown to retard osteoblast proliferation and inhibit their activity.Discussion. Microvascular endothelial cells in microgravity are growth retarded and release high amounts of matrix metalloproteases type 2 and interleukin-6, which might play a role in retarding the growth of osteoblasts and impairing their osteogenic activity.Conclusions. We demonstrate that since simulated microgravity modulates microvascular endothelial cell function, it indirectly impairs osteoblastic function.
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Weivoda, Megan M., and Raymond J. Hohl. "Effects of Farnesyl Pyrophosphate Accumulation on Calvarial Osteoblast Differentiation." Endocrinology 152, no. 8 (May 17, 2011): 3113–22. http://dx.doi.org/10.1210/en.2011-0016.
Full textAbstract:
Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. Statins inhibit 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (HMGCR), the first step of the isoprenoid biosynthetic pathway, leading to the depletion of the isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The effects of statins on bone have previously been attributed to the depletion of GGPP, because the addition of exogenous GGPP prevented statin-stimulated osteoblast differentiation in vitro. However, in a recent report, we demonstrated that the specific depletion of GGPP did not stimulate but, in fact, inhibited osteoblast differentiation. This led us to hypothesize that isoprenoids upstream of GGPP play a role in the regulation of osteoblast differentiation. We demonstrate here that the expression of HMGCR and FPP synthase decreased during primary calvarial osteoblast differentiation, correlating with decreased FPP and GGPP levels during differentiation. Zaragozic acid (ZGA) inhibits the isoprenoid biosynthetic pathway enzyme squalene synthase, leading to an accumulation of the squalene synthase substrate FPP. ZGA treatment of calvarial osteoblasts led to a significant increase in intracellular FPP and resulted in inhibition of osteoblast differentiation as measured by osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization. Simultaneous HMGCR inhibition prevented the accumulation of FPP and restored osteoblast differentiation. In contrast, specifically inhibiting GGPPS to lower the ZGA-induced increase in GGPP did not restore osteoblast differentiation. The specificity of HMGCR inhibition to restore osteoblast differentiation of ZGA-treated cultures through the reduction in isoprenoid accumulation was confirmed with the addition of exogenous mevalonate. Similar to ZGA treatment, exogenous FPP inhibited the mineralization of primary calvarial osteoblasts. Interestingly, the effects of FPP accumulation on osteoblasts were found to be independent of protein farnesylation. Our findings are the first to demonstrate that the accumulation of FPP impairs osteoblast differentiation and suggests that the depletion of this isoprenoid may be necessary for normal and statin-induced bone formation.