Journal articles on the topic 'Osteoblast'
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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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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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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.
4
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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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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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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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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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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Yu, Vionnie W. C., Gourgen Ambartsoumian, Lieve Verlinden, Janet M. Moir, Josée Prud'homme, Claude Gauthier, Peter J. Roughley, and René St-Arnaud. "FIAT represses ATF4-mediated transcription to regulate bone mass in transgenic mice." Journal of Cell Biology 169, no. 4 (May 23, 2005): 591–601. http://dx.doi.org/10.1083/jcb.200412139.
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We report the characterization of factor inhibiting activating transcription factor 4 (ATF4)–mediated transcription (FIAT), a leucine zipper nuclear protein. FIAT interacted with ATF4 to inhibit binding of ATF4 to DNA and block ATF4-mediated transcription of the osteocalcin gene in vitro. Transgenic mice overexpressing FIAT in osteoblasts also had reduced osteocalcin gene expression and decreased bone mineral density, bone volume, mineralized volume, trabecular thickness, trabecular number, and decreased rigidity of long bones. Mineral homeostasis, osteoclast number and activity, and osteoblast proliferation and apoptosis were unchanged in transgenics. Expression of osteoblastic differentiation markers was largely unaffected and type I collagen synthesis was unchanged. Mineral apposition rate was reduced in transgenic mice, suggesting that the lowered bone mass was due to a decline in osteoblast activity. This cell-autonomous decrease in osteoblast activity was confirmed by measuring reduced alkaline phosphatase activity and mineralization in primary osteoblast cultures. These results show that FIAT regulates bone mass accrual and establish FIAT as a novel transcriptional regulator of osteoblastic function.
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Giuliani, Nicola, Francesca Morandi, Sara Tagliaferri, Mirca Lazzaretti, Sabrina Bonomini, Monica Crugnola, Daniela Petrò, Cristina Mancini, Eugenia Martella, and Vittorio Rizzoli. "The Proteasome Inhibitor Bortezomib Affects Osteoblastogenesis and Bone Formation In Vitro and In Vivo in Multiple Myeloma Patients." Blood 108, no. 11 (November 16, 2006): 508. http://dx.doi.org/10.1182/blood.v108.11.508.508.
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Abstract It is well established that osteoblast formation and function are profoundly impaired in multiple myeloma (MM) patients. Osteoblastic cells also regulate myeloma cell growth and increasing bone formation result in a reduction of tumoral burden in mice. Recent data suggest that ubiquitin-proteasome pathway, the major cellular degradative system and therapeutic target in myeloma cells, also regulates osteoblast differentiation. Further it has been demonstrated that different proteasome inhibitors may stimulate bone formation in mice. Finally, preliminary observations obtained in MM patients treated with the proteasome inhibitor Bortezomib show an increase of bone specific alkaline phosphatase in responder patients as compared to non-responder ones. Currently it is not know whether the proteasome inhibitor Bortezomib may have a direct effect on osteoblast and bone formation in vitro human cultures and in vivo in MM patients. To clarify this issue first we checked the effect of Bortezomib either on osteoblast differentiation and formation or on osteoblast proliferation, survival and function. In long-term human BM cultures we found that Bortezomib did not reduce the number of both early bone marrow (BM) osteoblast progenitors Colony Forming Unit-Fibroblast (CFU-F) and late ones Colony Forming Bone nodules (CFU-OB). On the other hand we found that Bortezomib (2–3 nM) significantly induced osteoblast phenotype in human mesenchymal cells incubated in presence of osteogenic factors. A stimulatory effect on osteoblast markers was observed after 24 hours of Bortezomib treatment. Consistently we found that Bortezomib significantly increased the activity of the transcription factor Runx2/Cbfa1 in human osteoblast progenitors without affecting the canonical WNT signaling pathway checked by the evaluation of nuclear and cytoplasmatic active beta-catenin levels. Using the human osteoblast like cells MG-63 and immortalized normal osteoblasts (HOBIT) we found that Bortezomib at concentration ranging between 2nM and 5nM did not inhibit osteoblast proliferation or induce osteoblast apoptosis. Similarly, Bortezomib did not affect the expression of osteoblast markers, Runx2/Cbfa1 activity and WNT signaling in both MG-63 and HOBIT cells. To extent our in vitro observation we have evaluated the potential effect of Bortezomib in vivo in MM patients. Bone histomorphometry as well as immunostainig for Runx2/Cbfa1 and beta-catenin was performed on BM biopsies obtained from 15 MM patients before and after 6–8 cycles of Bortezomib administrated in mono-therapy. A significant increase in the number of osteoblastic cells X mm2 of bone tissue and in the number of Runx2/Cbfa1 positive osteoblastic cells was observed only in responder patients showing an early increase of the serum alkaline phosphatase. In conclusion our data indicate that Bortezomib may increase osteoblast differentiation in human mesenchymal cells without affecting the proliferation, survival and function of mature osteoblasts. In vivo and in vitro observations support the hypothesis that both direct and indirect effects on bone formation process could occur during Bortezomib treatment.
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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.
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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.
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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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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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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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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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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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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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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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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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Zhang, K., X. Liu, Y. Tang, Z. Liu, Q. Yi, L. Wang, B. Geng, and Y. Xia. "Fluid Shear Stress Promotes Osteoblast Proliferation and Suppresses Mitochondrial-Mediated Osteoblast Apoptosis Through the miR-214-3p-ATF4 Signaling Axis." Physiological Research 71, no. 4 (August 31, 2022): 527–38. http://dx.doi.org/10.33549/physiolres.934917.
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MicroRNAs (miRNAs) play vital roles in bone metabolism and participate in the mechanically induced bone alterations. The underlying molecular mechanisms by which fluid shear stress (FSS) regulate the proliferative and apoptotic phenotypic changes of osteoblasts remain elusive. The study aimed to investigate the regulatory effects of FSS on osteoblast proliferative and apoptotic phenotypes and the roles of miR-214-3p-ATF4 (activating transcription factor 4) signaling axis in the mechanomodulation processes. FSS promoted the proliferative activity of osteoblasts and suppressed mitochondrial-mediated osteoblast apoptosis. FSS decreased miR-214-3p expression and increased ATF4 expression in MC3T3-E1 osteoblasts. MiR-214-3p inhibited osteoblast proliferative activity and promoted mitochondrial-mediated osteoblast apoptosis. Overexpression of miR-214-3p attenuated FSS-enhanced osteoblast proliferation and FSS-suppressed mitochondrial-mediated osteoblast apoptosis. We validated that ATF4 acted as a target gene of miR-214-3p. Moreover, miR-214 3p regulated osteoblast proliferation and apoptosis through targeting ATF4. Taken together, our study proved that FSS could suppress mitochondrial-mediated osteoblast apoptosis and promote osteoblast proliferation through the miR-214-3p-ATF4 signaling axis.
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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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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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Mackie, E. J., and R. P. Tucker. "Tenascin in bone morphogenesis: expression by osteoblasts and cell type-specific expression of splice variants." Journal of Cell Science 103, no. 3 (November 1, 1992): 765–71. http://dx.doi.org/10.1242/jcs.103.3.765.
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The extracellular matrix glycoprotein, tenascin, is associated in vivo with mesenchyme undergoing osteogenesis and chondrogenesis, but is absent from mature bone and cartilage matrix. The expression of tenascin by osteoblastic cells in vitro has been investigated by immunoblotting and immunocytochemistry. Tenascin was secreted into the medium and deposited in the matrix by human and rat osteoblast-like cell lines, as well as by primary osteoblast-enriched cultures from chick embryo calvarial bones. In primary osteoblast-enriched cultures, extracellular tenascin was found only in cell aggregates expressing the osteoblast marker alkaline phosphatase. Chicken osteoblast cultures synthesized almost exclusively the largest tenascin subunit, whereas fibroblast cultures from periostea of chicken calvariae synthesized approximately equal amounts of all three subunits. In situ hybridization studies of developing chicken bones, using a cDNA probe that hybridizes to all chicken tenascin splice variants, showed specific labelling of both osteogenic and chondrogenic regions of developing endochondral bones. In contrast, a cDNA probe specific for the large tenascin splice variant showed specific hybridization in osteogenic but not chondrogenic regions. Within osteogenic regions, tenascin mRNA was expressed by osteoblasts. A comparison of in situ hybridization and immunohistochemical studies demonstrated that tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable. The results presented here demonstrate that tenascin is synthesized by osteoblasts. Moreover, within developing bones, there are at least three different cell type-specific patterns of expression of tenascin splice variants.
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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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Kassem, Moustapha, Leif Mosekilde, and Erik F. Eriksen. "Effects of fluoride on human bone cells in vitro: differences in responsiveness between stromal osteoblast precursors and mature osteoblasts." European Journal of Endocrinology 130, no. 4 (April 1994): 381–86. http://dx.doi.org/10.1530/eje.0.1300381.
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Kassem M, Mosekilde L, Eriksen EF. Effects of fluoride on human bone cells in vitro: differences in responsiveness between stromal osteoblast precursors and mature osteoblasts. Eur J Endocrinol 1994;130:381–6. ISSN 0804–4643 The cellular effects of sodium fluoride (NaF) on human bone cells in vitro have been variable and dependent on the culture system used. Variability could be attributed to differences in responsiveness to NaF among different populations of cells at various stages of differentiation in the osteoblastic lineage. In this study we compared the effects of NaF in serum-free medium on cultures of more differentiated human osteoblast-like (hOB) cells derived from trabecular bone explants and on osteoblast committed precursors derived from human bone marrow, i.e. human marrow stromal osteoblast-like (hMS(OB)) cells. Sodium fluoride (10−5 mol/l) increased proliferation of hMS(OB) cells (p<0.05, N = 10) but was not mitogenic to hOB cells (p>0.05, N= 10). Alkaline phosphatase (AP) production increased in both hMS(OB) (p<0.05, N=9) and hOB cells (p<0.05, N=9). No significant effects on procollagen type I propeptide production were obtained in either culture. In the presence of 1,25-dihydroxycholecalciferol (10−9 mol/l), NaF enhanced alkaline phosphatase (p<0.05, N=8), procollagen type I propeptide (p<0.05, N=7) and osteocalcin (p<0.05, N=7) production by hMS(OB) cells but not by hOB cells. Our results suggest that osteoblast precursors are more sensitive to NaF action than mature osteoblasts and that the in vivo effects of NaF on bone formation may be mediated by stimulating proliferation and differentiation of committed osteoblast precursors in bone marrow. M Kassem, Mayo Clinic, Endocrine Research Unit, W-Joseph 5-164, Rochester, MN 55904, USA
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Weinstein, Robert S., Robert L. Jilka, Maria Almeida, Paula K. Roberson, and Stavros C. Manolagas. "Intermittent Parathyroid Hormone Administration Counteracts the Adverse Effects of Glucocorticoids on Osteoblast and Osteocyte Viability, Bone Formation, and Strength in Mice." Endocrinology 151, no. 6 (April 21, 2010): 2641–49. http://dx.doi.org/10.1210/en.2009-1488.
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Glucocorticoids act directly on bone cells to decrease production of osteoblasts and osteoclasts, increase osteoblast and osteocyte apoptosis, and prolong osteoclast life span. Conversely, daily injections of PTH decrease osteoblast and osteocyte apoptosis and increase bone formation and strength. Using a mouse model, we investigated whether the recently demonstrated efficacy of PTH in glucocorticoid-induced bone disease results from the ability of this therapeutic modality to counteract at least some of the direct effects of glucocorticoids on bone cells. Glucocorticoid administration to 5- to 6-month-old Swiss-Webster mice for 28 d increased the prevalence of osteoblast and osteocyte apoptosis and decreased osteoblast number, activation frequency, and bone formation rate, resulting in reduced osteoid, wall and trabecular width, bone mineral density, and bone strength. In contrast, daily injections of PTH caused a decrease in osteoblast and osteocyte apoptosis and an increase in osteoblast number, activation frequency, bone formation rate, bone mineral density, and bone strength. The decreased osteocyte apoptosis was associated with increased bone strength. When the two agents were combined, all the adverse effects of glucocorticoid excess on bone were prevented. Likewise, in cultured osteoblastic cells, PTH attenuated the adverse effects of glucocorticoids on osteoblast survival and Wnt signaling via an Akt phosphorylation-dependent mechanism. We conclude that intermittent PTH administration directly counteracts the key pathogenetic mechanisms of glucocorticoid excess on bone, thus providing a mechanistic explanation of its efficacy against glucocorticoid-induced osteoporosis.
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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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Funato, Noriko, Kiyoshi Ohtani, Kimie Ohyama, Takayuki Kuroda, and Masataka Nakamura. "Common Regulation of Growth Arrest and Differentiation of Osteoblasts by Helix-Loop-Helix Factors." Molecular and Cellular Biology 21, no. 21 (November 1, 2001): 7416–28. http://dx.doi.org/10.1128/mcb.21.21.7416-7428.2001.
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ABSTRACT Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21WAF1/Cip1. Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWISTand FGFR3 genes.
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Guo, Ying, Nicole Pischon, Amitha H. Palamakumbura, and Philip C. Trackman. "Intracellular distribution of the lysyl oxidase propeptide in osteoblastic cells." American Journal of Physiology-Cell Physiology 292, no. 6 (June 2007): C2095—C2102. http://dx.doi.org/10.1152/ajpcell.00613.2006.
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Lysyl oxidase plays a critical role in the formation of the extracellular matrix, and its activity is required for the normal maturation and cross-linking of collagen and elastin. An 18-kDa lysyl oxidase propeptide (LOPP) is generated from 50-kDa prolysyl oxidase by extracellular proteolytic cleavage during the biosynthesis of active 30-kDa lysyl oxidase enzyme. The fate and the functions of the LOPP are largely unknown, although intact LOPP was previously observed in osteoblast cultures. We investigated the spatial localization of molecular forms of lysyl oxidase, including LOPP in proliferating and differentiating osteoblasts, by using confocal immunofluorescence microscopy and Western blots of cytoplasmic and nuclear extracts. In the present study, a stage-dependent intracellular distribution of LOPP in the osteoblastic cell was observed. In proliferating osteoblasts, LOPP epitopes were principally associated with the Golgi and endoplasmic reticulum, and mature lysyl oxidase epitopes were found principally in the nucleus and perinuclear region. In differentiating cells, LOPP and mature lysyl oxidase immunostaining showed clear colocalization with the microtubule network. The subcellular distribution of LOPP and its temporal and physical association with microtubules were confirmed by Western blot and far Western blot studies. We also report that N-glycosylated and nonglycosylated LOPP are present in MC3T3-E1 cell cultures. We conclude that LOPP has a stage-dependent intracellular distribution in osteoblastic cells. Future studies are needed to investigate whether the LOPP associations with microtubules or the osteoblast nucleus have functional effects for osteoblast differentiation and bone formation.
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Lemus, Ana E., Juana Enríquez, Ángeles Hernández, René Santillán, and Gregorio Pérez-Palacios. "Bioconversion of norethisterone, a progesterone receptor agonist into estrogen receptor agonists in osteoblastic cells." Journal of Endocrinology 200, no. 2 (November 10, 2008): 199–206. http://dx.doi.org/10.1677/joe-08-0166.
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A number of clinical studies have demonstrated that norethisterone (NET), a potent synthetic progestin, restores postmenopausal bone loss, although its mode of action on bone cells is not fully understood, while the effect of naturally occurring progesterone in bone has remained controversial. A recent report claims that the potent effects of NET on osteoblastic cell proliferation and differentiation, mimicking the action of estrogens, are mediated by non-phenolic NET derivatives. To determine whether osteoblasts possess the enzymes required to bioconvert a progesterone receptor (PR) agonist into A-ring reduced metabolites with affinity to bind estrogen receptor (ER), we studied the in vitro metabolism of [3H]-labeled NET in cultured neonatal rat osteoblasts and the interaction of its metabolic conversion products with cytosolic –osteoblast ER, employing a competition analysis. Results indicated that NET was extensively bioconverted (36.4%) to 5α-reduced metabolites, including 5α-dihydro NET, 3α,5α-tetrahydro NET (3α,5α-NET) and 3β,5α-tetrahydro NET (3β,5α-NET), demonstrating the activities of 5α-steroid reductase and two enzymes of the aldo-keto reductases family. Expression of Srd5a1 in neonatal osteoblast was well demonstrated, whereas Srd5a2 expression was not detected. The most striking finding was that 3β,5α-NET and 3α,5α-NET were efficient competitors of [3H]-estradiol for osteoblast ER binding sites, exhibiting affinities similar to that of estradiol. The results support the concept that the interplay of 5α-steroid reductase and aldo-keto reductases in osteoblastic cells, acting as an intracrine modulator system is capable to bioconvert a PR agonist into ER agonists, offering an explanation of the molecular mechanisms NET uses to enhance osteoblastic cell activities.
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Yamaguchi, Akira, Toshihisa Komori, and Tatsuo Suda. "Regulation of Osteoblast Differentiation Mediated by Bone Morphogenetic Proteins, Hedgehogs, and Cbfa1." Endocrine Reviews 21, no. 4 (August 1, 2000): 393–411. http://dx.doi.org/10.1210/edrv.21.4.0403.
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Abstract Osteoblasts arise from common progenitors with chondrocytes, muscle and adipocytes, and various hormones and local factors regulate their differentiation. We review here regulation of osteoblast differentiation mediated by the local factors such as bone morphogenetic proteins (BMPs) and hedgehogs and the transcription factor, core-binding factor α-1 (Cbfa1). BMPs are the most potent regulators of osteoblast differentiation among the local factors. Sonic and Indian hedgehogs are involved in osteoblast differentiation by interacting with BMPs. Cbfa1, a member of the runt domain gene family, plays a major role in the processes of a determination of osteoblast cell lineage and maturation of osteoblasts. Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation, because Cbfa1-deficient mice completely lacked bone formation due to maturation arrest of osteoblasts. Although the regulatory mechanism of Cbfa1 expression has not been fully clarified, BMPs are an important local factor that up-regulates Cbfa1 expression. Thus, the intimate interaction between local factors such as BMPs and hedgehogs and the transcription factor, Cbfa1, is important to osteoblast differentiation and bone formation.
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Morandi, Francesca, Sara Tagliaferri, Sabrina Bonomini, Mirca Lazzaretti, Luca Ferrari, Vittorio Rizzoli, and Nicola Giuliani. "Beta-Catenin Depended and Independent Effects Induced by Myeloma Cells in Human and Murine Osteoblasts and Osteoblast Progenitors." Blood 108, no. 11 (November 16, 2006): 3433. http://dx.doi.org/10.1182/blood.v108.11.3433.3433.
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Abstract Osteoblast impairment occurs within myeloma (MM) cell infiltration into the bone marrow (BM). Wnt signaling is involved in the regulation of osteoblast formation. Canonical Wnt signaling pathway is activated by Wnt 1/3a that induce the activation of GSK3/Axin complex leading to the stabilization and nuclear translocation of beta-catenin that in turn activates the transcription system Lef1/TCF. Recently it has been reported that MM cells produce the Wnt inhibitors DKK-1 demonstrating a correlation between its expression and the presence of bone lesions in MM patients. However the effect of MM cells on Wnt signaling cascade in osteoblasts and osteoblast progenitors has not been investigated. To clarify this issue, first we checked DKK-1 production by human myeloma cell lines (HMCLs), purified CD138+ MM cells and BM plasma of MM patients by PCR and ELISA. Following we performed a co-culture system with HMCLs or CD138+ MM cells and either human osteoblast line (HOBIT) and with BM osteoprogenitor cells (PreOB) obtained after differentiation from mesenchymal cells or murine osteoprogenitor cell lines C2C12 and MC3T3. Both DKK-1 positive HMCLs (XG-1 and JJN3) and negative ones (RPMI-8226, OPM-2) have been used in co-culture as well as DKK-1 positive and negative purified CD138+ MM cells. Similarly we tested the effect of BM plasma of MM patients positive and negative for DKK-1 production on both human and murine cells. Wnt signaling in osteoblasts and osteoblast progenitors was evaluated either at mRNA level by specific human and murine Wnt Array kits and by quantitative PCR or at protein one by Western blot analysis for GSK3b/Axin and LEF-1/TCF expression. We evaluated active de-phosphorylated beta-catenin and inactive phosphorilated one by westernblot and by ELISA in cytosolic and nuclear extracts. DKK-1 median levels detected in the conditioned media of XG-1 and JJN3, MM cells and in BM plasma of DKK-1 positve MM patients were 0.60 ng/mL and 0.38 and 8.84 (range: 1.55–91) ng/mL respectively. Any significant inhibitory effect on WNT signaling and active beta-catenin expression and levels was not observed in HOBIT and human PreOB after co-culture with both HMCLs and MM cells or BM plasma independently to DKK-1 expression. On the contrary DKK-1 positive MM cells or BM plasma suppressed active beta-catenin expression in murine osteoprogenitor cell lines in presence of BMP-2. Consistently Wnt3a stimulation as well as anti-DKK-1 abs. did not restore the inhibitory effects on osteoblast formation and differentiation induced by MM cells in human PreOB. Consistently any significant difference was not detected on beta-catenin expression by stromal/osteoblastic cells on bone biopsies by immunohistochemistry between osteolytic (n°=10) and non-osteolytic (N°=10) MM patients. The different behavior between human and murine osteoblastic cells was further investigated. We found that both cells expressed significant levels of active beta-catenin however DKK-1 suppressed active nuclear and cytosol beta-catenin at concentration of 20–30 ng/mL in C2C12 and MC3T3 whereas only DKK-1 concentrations higher to 500 ng/mL are able to inhibited beta-catenin in HOBIT and human PreOB as well as osteoblast formation and differentiation in human BM cultures. In conclusion our data indicate that MM cells block canonical Wnt signaling in murine osteoblastic cells but not in human osteoblasts and osteoblast progenitors. Beta-catenin independent mechanisms could be involved in DKK-1 mediated bone destruction in MM patients.
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Zhu, Haitao, Hua Chen, Degang Ding, Shui Wang, Xiaofeng Dai, and Yulong Zhu. "Overexpression of PIK3R1 Promotes Bone Formation by Regulating Osteoblast Differentiation and Osteoclast Formation." Computational and Mathematical Methods in Medicine 2021 (October 14, 2021): 1–13. http://dx.doi.org/10.1155/2021/2909454.
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In an effort to bolster our understanding of regulation of bone formation in the context of osteoporosis, we screened out differentially expressed genes in osteoporosis patients with high and low bone mineral density by bioinformatics analysis. PIK3R1 is increasingly being nominated as a pivotal mediator in the differentiation of osteoblasts and osteoclasts that is closely related to bone formation. However, the specific mechanisms underlying the way that PIK3R1 affects bone metabolism are not fully elucidated. We intended to examine the potential mechanism by which PIK3R1 regulates osteoblast differentiation. Enrichment analysis was therefore carried out for differentially expressed genes. We noted that the estrogen signaling pathway, TNF signaling pathway, and osteoclast differentiation were markedly associated with ossification, and they displayed enrichment in PIK3R1. Based on western blot, qRT-PCR, and differentiation analysis in vitro, we found that upregulation of PIK3R1 enhanced osteoblastic differentiation, as evidenced by increased levels of investigated osteoblast-related genes as well as activities of ALP and ARS, while it notably decreased levels of investigated osteoclast-related genes. On the contrary, downregulation of PIK3R1 decreased levels of osteoblast-related genes and increased levels of osteoclast-related genes. Besides, in vitro experiments revealed that PIK3R1 facilitated proliferation and repressed apoptosis of osteoblasts but had an opposite impact on osteoclasts. In summary, PIK3R1 exhibits an osteoprotective effect via regulating osteoblast differentiation, which can be represented as a promising therapeutic target for osteoporosis.
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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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Wang, Xueying, Hui-Yi Kua, Yuanyu Hu, Ke Guo, Qi Zeng, Qiang Wu, Huck-Hui Ng, et al. "p53 functions as a negative regulator of osteoblastogenesis, osteoblast-dependent osteoclastogenesis, and bone remodeling." Journal of Cell Biology 172, no. 1 (December 27, 2005): 115–25. http://dx.doi.org/10.1083/jcb.200507106.
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p53 is a well known tumor suppressor. We show that p53 also regulates osteoblast differentiation, bone formation, and osteoblast-dependent osteoclast differentiation. Indeed, p53−/− mice display a high bone mass phenotype, and p53−/− osteoblasts show accelerated differentiation, secondary to an increase in expression of the osteoblast differentiation factor osterix, as a result. Reporter assays indicate that p53 represses osterix transcription by the minimal promoter in a DNA-binding–independent manner. In addition, p53−/− osteoblasts have an enhanced ability to favor osteoclast differentiation, in association with an increase in expression of macrophage-colony stimulating factor, which is under the control of osterix. Furthermore, inactivating p53 is sufficient to rescue the osteoblast differentiation defects observed in mice lacking c-Abl, a p53-interacting protein. Thus, these results identify p53 as a novel regulator of osteoblast differentiation, osteoblast-dependent osteoclastogenesis, and bone remodeling.
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Billiard, Julia, Deana S. Way, Laura M. Seestaller-Wehr, Robert A. Moran, Annamarie Mangine, and Peter V. N. Bodine. "The Orphan Receptor Tyrosine Kinase Ror2 Modulates Canonical Wnt Signaling in Osteoblastic Cells." Molecular Endocrinology 19, no. 1 (January 1, 2005): 90–101. http://dx.doi.org/10.1210/me.2004-0153.
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Abstract Ror2 is an orphan receptor tyrosine kinase that plays crucial roles in developmental morphogenesis, particularly of the skeleton. We have identified human Ror2 as a novel regulator of canonical Wnt signaling in osteoblastic (bone-forming) cells with selective activities, enhancing Wnt1 but antagonizing Wnt3. Immunoprecipitation studies demonstrated physical interactions between human Ror2 and mammalian Wnt1 and Wnt3. Functionally, Ror2 antagonized Wnt1- and Wnt3-mediated stabilization of cytosolic β-catenin in osteoblastic cells. However, Ror2 had opposing effects on a more distal step of canonical Wnt signaling: it potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. Despite binding to Ror2, neither Wnt1 nor Wnt3 altered receptor activity as assessed by levels of Ror2 autophosphorylation. The ability of Ror2 to regulate canonical Wnt signaling in osteoblastic cells should have physiological consequences in bone, because Wnt signaling is known to modulate osteoblast survival and differentiation. Expression of Ror2 mRNA was highly regulated in a biphasic manner during human osteoblast differentiation, being virtually undetectable in pluripotent stem cells, increasing 300-fold in committed preosteoblasts, and disappearing again in osteocytes. Furthermore, Ror2 expression in osteoblasts was suppressed by the Wnt antagonist, secreted frizzled-related protein 1. The regulated expression of Ror2 during osteoblast differentiation, its inverse expression pattern with secreted frizzled-related protein 1, and its ability to modulate Wnt signaling in osteoblastic cells suggest that Ror2 may regulate bone formation.
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Alonso-Pérez, Ana, María Guillán-Fresco, Eloi Franco-Trepat, Alberto Jorge-Mora, Miriam López-Fagúndez, Andrés Pazos-Pérez, Antía Crespo-Golmar, José R. Caeiro-Rey, and Rodolfo Gómez. "Improved Protocol to Study Osteoblast and Adipocyte Differentiation Balance." Biomedicines 11, no. 1 (December 22, 2022): 31. http://dx.doi.org/10.3390/biomedicines11010031.
Full textAbstract:
Adipogenesis-osteoblastogenesis balance-rupture is relevant in multiple diseases. Current human mesenchymal stem cells (hMSCs) in vitro differentiation models are expensive, and are hardly reproducible. Their scarcity and variability make an affordable and reliable method to study adipocyte-osteoblast-equilibrium difficult. Moreover, media composition has been inconstant throughout the literature. Our aims were to compare improved differentiation lab-made media with consensus/commercial media, and to identify a cell-line to simultaneously evaluate both MSCs differentiations. Lab-made media were compared with consensus and commercial media in C3H10T1/2 and hMSC, respectively. Lab-made media were tested on aged women primary pre-osteoblast-like cells. To determine the optimum cell line, C3H10T1/2 and hMSC-TERT cells were differentiated to both cell fates. Differentiation processes were evaluated by adipocytic and osteoblastic gene-markers expression and staining. Lab-made media significantly increased consensus medium induction and overcame commercial media in hMSCs differentiation to adipocytes and osteoblasts. Pre-osteoblast-like cells only properly differentiate to adipocyte. Lab-made media promoted adipocyte gene-markers expression in C3H10T1/2 and hMSC-TERT, and osteoblast gene-markers in C3H10T1/2. Oil Red O and Alizarin Red staining supported these findings. Optimized lab-made media were better at differentiating MSCs compared to consensus/commercial media, and evidenced the adipogenic commitment of pre-osteoblast-like cells from aged-women. C3H10T1/2 is an optimum MSC line by which to study adipocyte-osteoblast differentiation balance.
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Bonnelye, E., L. Merdad, V. Kung, and J. E. Aubin. "The Orphan Nuclear Estrogen Receptor–Related Receptor α (Errα) Is Expressed Throughout Osteoblast Differentiation and Regulates Bone Formation in Vitro." Journal of Cell Biology 153, no. 5 (May 21, 2001): 971–84. http://dx.doi.org/10.1083/jcb.153.5.971.
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The orphan nuclear estrogen receptor–related receptor α (ERRα), is expressed by many cell types, but is very highly expressed by osteoblastic cells in which it transactivates at least one osteoblast-associated gene, osteopontin. To study the putative involvement of ERRα in bone, we first assessed its expression in rat calvaria (RC) in vivo and in RC cells in vitro. ERRα mRNA and protein were expressed at all developmental stages from early osteoprogenitors to bone-forming osteoblasts, but protein was most abundant in mature cuboidal osteoblasts. To assess a functional role for ERRα in osteoblast differentiation and bone formation, we blocked its expression by antisense oligonucleotides in either proliferating or differentiating RC cell cultures and found inhibition of cell growth and a proliferation-independent inhibition of differentiation. On the other hand, ERRα overexpression in RC cells increased differentiation and maturation of progenitors to mature bone-forming cells. Our findings show that ERRα is highly expressed throughout the osteoblast developmental sequence and plays a physiological role in differentiation and bone formation at both proliferation and differentiation stages. In addition, we found that manipulation of receptor levels in the absence of known ligand is a fruitful approach for functional analysis of this orphan receptor and identification of potential target genes.
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Choi, J. Y., A. J. van Wijnen, F. Aslam, J. D. Leszyk, J. L. Stein, G. S. Stein, J. B. Lian, and S. Penman. "Developmental association of the beta-galactoside-binding protein galectin-1 with the nuclear matrix of rat calvarial osteoblasts." Journal of Cell Science 111, no. 20 (October 15, 1998): 3035–43. http://dx.doi.org/10.1242/jcs.111.20.3035.
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The protein composition of the nuclear matrix changes significantly as the osteoblast matures from a proliferating pre-osteoblast to an osteocyte embedded in a mineralized matrix. These matrix protein are the result of developmental stage-specific gene expression during osteoblast differentiation. To isolate nuclear matrix proteins unique to the bone phenotype we analyzed nuclear matrix preparations from cultures of rat calvarial osteoblasts by high resolution two-dimensional gel electrophoresis at two different stages: proliferation (day 3) and differentiation (day 18, mineralized). We characterized one protein (14 kDa; pI 5.0), that was detectable only in the nuclear matrix of differentiated osteoblasts. By mass spectrometry and microsequencing, this protein was identified as the beta -galactoside-binding protein galectin-1. Both immunofluorescence staining of nuclear matrix preparations with the galectin-1 antibody and western blot analysis of subcellular fractions confirmed that galectin-1 is only associated with the nuclear matrix in differentiated osteoblasts as the result of differential retention. Galectin-1 protein and mRNA are present throughout osteoblast differentiation. Galectin-1 is present in the cytoplasmic and nuclear fractions in both proliferating and differentiated osteoblasts. However, its only stable binding is to the nuclear matrix of the differentiated osteoblast; but, in proliferating osteoblasts, galectin-1 is not retained in the nuclear matrix. Taken together, our results suggest that developmental association of galectin-1 with the nuclear matrix reflects differential subnuclear binding of galectin-1 during osteoblast differentiation.
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Yano, Shozo, Romuald Mentaverri, Deepthi Kanuparthi, Sanghamitra Bandyopadhyay, Alicia Rivera, Edward M. Brown, and Naibedya Chattopadhyay. "Functional Expression of β-Chemokine Receptors in Osteoblasts: Role of Regulated upon Activation, Normal T Cell Expressed and Secreted (RANTES) in Osteoblasts and Regulation of Its Secretion by Osteoblasts and Osteoclasts." Endocrinology 146, no. 5 (May 1, 2005): 2324–35. http://dx.doi.org/10.1210/en.2005-0065.
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Abstract The expression and functions of receptors for the β-chemokine, regulated upon activation, normal T cell expressed, and secreted (RANTES)/CCL5, were investigated in osteoblasts. Both primary osteoblasts and the MC3T3-E1 osteoblast cell line express the RANTES receptors, CCR1, 3, 4, and 5 (by RT-PCR), which encode functional receptors in osteoblasts as shown by [125I]-RANTES binding followed by Scatchard analysis. Expression of all four RANTES receptor mRNAs in osteoblast is in contrast to the reports of expression of CCR1 being the only RANTES receptor expressed by osteoclasts. Exogenous RANTES elicits chemotaxis of osteoblasts and promotes cell survival via phosphatidylinositol 3-kinase with attendant phosphorylation of Akt. Osteoclastic RANTES, obtained from the conditioned medium of receptor activator of nuclear factor-κB ligand-differentiated RAW264.7 cells also induces chemotaxis of MC3T3-E1 cells. Incubating the conditioned medium with an anti-RANTES neutralizing antibody attenuated this effect. RANTES secretion from osteoblast is inhibited by differentiation promoting hormones, e.g. 1,25 (OH)2D3 and dexamethasone, whereas macrophage inflammatory protein-1α (but not macrophage inflammatory protein-1β) and elevated calcium induce it. Elevated calcium also stimulated RANTES secretion by osteoclasts. Therefore, RANTES is an osteoblast chemoattractant and a survival-promoting molecule whose regulation in osteoblast is varied. Furthermore, RANTES secreted from osteoclasts induces osteoblast chemotaxis. Therefore, expression of RANTES and its receptors in both osteoblasts and osteoclasts could enable this chemokine to act in autocrine/paracrine modes.
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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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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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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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Komori, Toshihisa. "Regulation of Proliferation, Differentiation and Functions of Osteoblasts by Runx2." International Journal of Molecular Sciences 20, no. 7 (April 4, 2019): 1694. http://dx.doi.org/10.3390/ijms20071694.
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Runx2 is essential for osteoblast differentiation and chondrocyte maturation. During osteoblast differentiation, Runx2 is weakly expressed in uncommitted mesenchymal cells, and its expression is upregulated in preosteoblasts, reaches the maximal level in immature osteoblasts, and is down-regulated in mature osteoblasts. Runx2 enhances the proliferation of osteoblast progenitors by directly regulating Fgfr2 and Fgfr3. Runx2 enhances the proliferation of suture mesenchymal cells and induces their commitment into osteoblast lineage cells through the direct regulation of hedgehog (Ihh, Gli1, and Ptch1), Fgf (Fgfr2 and Fgfr3), Wnt (Tcf7, Wnt10b, and Wnt1), and Pthlh (Pthr1) signaling pathway genes, and Dlx5. Runx2 heterozygous mutation causes open fontanelle and sutures because more than half of the Runx2 gene dosage is required for the induction of these genes in suture mesenchymal cells. Runx2 regulates the proliferation of osteoblast progenitors and their differentiation into osteoblasts via reciprocal regulation with hedgehog, Fgf, Wnt, and Pthlh signaling molecules, and transcription factors, including Dlx5 and Sp7. Runx2 induces the expression of major bone matrix protein genes, including Col1a1, Spp1, Ibsp, Bglap2, and Fn1, in vitro. However, the functions of Runx2 in differentiated osteoblasts in the expression of these genes in vivo require further investigation.
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Zou, Zihao, Ranran Dai, Nan Deng, Wei Su, and Ping Liu. "Exosomal miR-1275 Secreted by Prostate Cancer Cells Modulates Osteoblast Proliferation and Activity by Targeting the SIRT2/RUNX2 Cascade." Cell Transplantation 30 (January 1, 2021): 096368972110529. http://dx.doi.org/10.1177/09636897211052977.
Full textAbstract:
Prostate cancer (PCa) is one of the most frequently diagnosed malignancies and the second leading cause of cancer mortality among men worldwide. Modulation of osteoblast activity is involved in PCa metastasis, and miR-1275 is also reported to regulate PCa metastasis; however, the association between cancer-derived exosomal miR-1275 and osteoblast activity is unclear. Here, we isolated exosomes from PC3-derived conditioned medium by ultracentrifugation. We found that miR-1275 could be transferred from PCa cells to osteoblasts via exosomes. Exosomal miR-1275 significantly accelerated the proliferation of osteoblasts and the expression levels of osteoblast-specific genes, such as osteocalcin (OCN), type I collagen (COL-1), and osteopontin (OPN). Moreover, exosomal miR-1275 increased the expression of RUNX2, a master modulator of osteoblast activity, by down-regulation of SIRT2, which in turn influenced the growth and activity of osteoblasts. Our findings indicate that PCa-derived exosomal miR-1275 promotes the proliferation and activity of osteoblasts via modulation of SIRT2/Runx2 signaling.
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Na, WooJin, and Young-Hee Kang. "Aesculetin Promotes Osteoblastogenic Bone Formation Through Enhancing Osteoblast Differentiation and Mineralization." Current Developments in Nutrition 5, Supplement_2 (June 2021): 350. http://dx.doi.org/10.1093/cdn/nzab037_060.
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Abstract Objectives Osteoporosis is a common chronic disease elicited by imbalance between osteoblastic bone formation and osteoclastic bone resorption. Marked increase in bone resorption leads to the aberrant fall in bone mineral density. With increasing age, there is also a significant reduction in bone formation. Aesculetin, a derivative of coumarin, possesses anti-inflammatory and antioxidant effects. The purpose of this study was to identify that aesculetin accelerated bone formation through increased osteoblastic differentiation and mineralization. Methods MC3T3-E1 cells were cultured with 1–10 μM aesculetin in α-MEM supplemented with 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid and 10 μM dexamethasone for up to 21 days. Alkaline phosphatase (ALP) activity and staining, Alizarin red S staining, and Western blotting for induction of target proteins were conducted for the measurement of osteoblastic differentiation and mineralization. Results Aesculetin further enhanced the ALP activity of differentiated MC3T3-E1 cells, showing that aesculetin stimulated the osteoblast differentiation. Alizarin red S staining revealed that calcium deposits highly increased in 1–10 μM aesculetin-treated osteoblasts. In addition, aesculetin further increased cellular expression of the bone-forming markers of bone morphogenetic protein-2, osteopontin and collagen type I in osteoblasts. Conclusions Aesculetin was effective in enhancing osteoblast differentiation and bone mineralization for bone formation, indicating that this compound may be a potential agent for the treatment of osteoporosis. Funding Sources This work was supported by the BK21 FOUR(Fostering Outstanding Universities for Research, 4220200913807) funded by the National Research Foundation of Korea (NRF).
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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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Mackie, E. J., and S. Ramsey. "Modulation of osteoblast behaviour by tenascin." Journal of Cell Science 109, no. 6 (June 1, 1996): 1597–604. http://dx.doi.org/10.1242/jcs.109.6.1597.
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The extracellular matrix protein tenascin is secreted by osteoblasts but absent from mineralized bone matrix. The current study was undertaken to test the hypothesis that tenascin regulates osteoblast behaviour. Three osteoblast-like cell lines UMR-106, ROS-17/2.8 (rat) and SAOS-2 (human) were used to investigate the role of tenascin in osteoblast morphology, differentiation and proliferation. Two of three cell lines adhered specifically to tenascin, remaining round and failing to spread. Tenascin as a substratum stimulated alkaline phosphatase activity (a marker of osteoblast differentiation) in two of three cell lines. Moreover, anti-tenascin in the medium caused a reduction in alkaline phosphatase levels in all three cell lines. Anti-tenascin also inhibited collagen synthesis, an important osteoblast function. Since it seemed possible that tenascin may exert its effects on cell function through its ability to cause cell rounding, the ability of cell shape change alone to influence alkaline phosphatase levels was investigated. Cells were incubated in the presence of cytochalasin D and alkaline phosphatase levels assayed. Alkaline phosphatase activity was not elevated by cytochalasin D treatment, indicating that cell rounding alone is insufficient to mimic the effect of tenascin. Anti-tenascin caused a slight increase in proliferation of SAOS-2 cells, indicating that tenascin is itself inhibitory. In ROS 17/2.8 and UMR-106 cells, in contrast, proliferation was inhibited by anti-tenascin. The results presented here indicate that tenascin is able to stimulate osteoblastic differentiation and that endogenous tenascin helps to maintain the functional state of cultured osteoblast-like cells.
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Giuliani, Nicola, Simona Colla, Francesca Morandi, Sabrina Bonomini, and Vittorio Rizzoli. "Myeloma Cells Block Runx2/Cbfa1 Activity in Human Bone Marrow Osteoblast Progenitors and Inhibit Osteoblast Formation and Differentiation." Blood 104, no. 11 (November 16, 2004): 632. http://dx.doi.org/10.1182/blood.v104.11.632.632.
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Abstract The decreased bone formation contributes to the development of bone lesions in multiple myeloma (MM) patients. Runx2/Cbfa1 is a transcription factor highly restricted to the osteoblastic lineage that has a critical role in the osteoblast formation and differentiation. In this study we investigated the potential effect of myeloma cells on osteoblastogenesis and the role of Runx2/Cbfa1 in the decreased bone formation in MM. In a co-culture system performed in presence or absence of a transwell insert with human myeloma cell lines (HMCLs) (RPMI-8226, U266, XG-1, XG-6, OPM-2) and human bone marrow (BM) pre-osteoblastic cells, obtained from BM stromal cells (BMSC), we observed an inhibition of osteocalcin, alkaline phosphatase and collagen I expression at both mRNA and protein level. Consistently we found that the formation of both Colony Forming Unit Fibroblasts (CFU-F) and Colony Forming Unit Osteoblasts (CFU-OB) were suppressed in long term BM culture by several HMCLs. Moreover, by a gel mobility shift assay (EMSA) we found that Runx-2/Cbfa1 activity was significantly inhibited in BMSC/pre-osteoblastic cells after 48 hours of co-cultures with OPM-2 whereas the level of Runx2/Cbfa1 protein was not affected. The inhibition of osteoblast formation, differentiation and the block of the Runx2/Cbfa1 activity in BMSC/pre-osteoblastic was more pronounced in the cell-to-cell contact conditions as compared to those without the cellular contact. Further we evaluated Runx2/Cbfa1 expression by immunohistochemistry in BM biopsies of 16 MM patients finding a significant reduction of the number of Runx2/Cbfa1 positive cells in osteolytic patients as compared to those without bone lesions (median %: 19.93% vs. 38%; p=0.001) that supports the in vitro results. To identify molecules that could contribute to the inhibitory effect of myeloma cells on osteoblast formation, differentiation and Runx2/Cbfa1 activity, we screened HMCLs and fresh purified CD138+ MM cells for the expression of DKK1, IL-7, noggin, gremlin and secreted frizzled-related protein (sFRP)-2, -3, -4 also evaluating the potential involvement of these osteoblast inhibitors. DKK1 mRNA was expressed by 2 out of 5 HMCLs tested and by 8 out of 12 MM patients tested as well as by BMSC/pre-osteoblastic cells and hOB, however soluble DKK-1 at a widely range of concentration had not effect on the number of CFU-F and CFU-OB in human BM cultures and it did not inhibited Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells. Moreover, blocking anti-DKK1 Ab did not blunted the effect of HMCLs on osteoblast formation in human BM cultures. All the HMCLs and fresh CD138+ MM cells tested expressed IL-7 mRNA, as we have previously reported. IL-7 reduced Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells showing an inhibitory effect on CFU-F and CFU-OB formation in BM culture, moreover blocking anti-IL-7 Ab partially reduced the effect of HMCLs in co-cultures. Finally, we found that HMCLs and fresh CD138+ MM cells did not expressed noggin, gremlin, sFRP-2 and rarely produced sFRP-3 and sFRP-4 whereas BMSC/pre-osteoblastic cells and hOB were positive for these factors even if HMCLs had not effect on their expression in co-cultures. In conclusion our data indicate that human myeloma cells inhibit osteoblast formation and differentiation in human BM cultures blocking Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells through the cell-to-cell contact. Soluble factors produced by myeloma cells as IL-7 could contribute to the inhibitory effect on osteoblastogenesis.
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Sakasai-Sakai, Akiko, Takanobu Takata, and Masayoshi Takeuchi. "The Association between Accumulation of Toxic Advanced Glycation End-Products and Cytotoxic Effect in MC3T3-E1 Cells." Nutrients 14, no. 5 (February 26, 2022): 990. http://dx.doi.org/10.3390/nu14050990.
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In diabetic patients, the metabolism of excess glucose increases the toxicity of the aldehyde group of sugar. Aldehydes, including glyceraldehyde (GA), react with intracellular proteins to form advanced glycation end-products (AGEs), which deteriorate bone quality and cause osteoporosis. One of the causes of osteoporotic fractures is impaired osteoblast osteogenesis; however, the cytotoxic effects of aldehydes and the subsequent formation of AGEs in osteoblasts have not yet been examined in detail. Therefore, the present study investigated the cytotoxicity of intracellular GA and GA-derived AGEs, named toxic AGEs (TAGE), in the mouse osteoblastic cell line MC3T3-E1. Treatment with GA induced MC3T3-E1 cell death, which was accompanied by TAGE modifications in several intracellular proteins. Furthermore, the downregulated expression of Runx2, a transcription factor essential for osteoblast differentiation, and collagen correlated with the accumulation of TAGE. The GA treatment also reduced the normal protein levels of collagen in cells, suggesting that collagen may be modified by TAGE and form an abnormal structure. Collectively, the present results show for the first time that GA and TAGE exert cytotoxic effects in osteoblasts, inhibit osteoblastic differentiation, and decrease the amount of normal collagen. The suppression of GA production and associated accumulation of TAGE has potential as a novel therapeutic target for osteoporosis under hyperglycemic conditions.