Role In Osteoblastogenesis Research Articles

Pathway-based genome-wide association analysis identified the importance of regulation-of-autophagy pathway for ultradistal radius BMD

Wrist fracture is not only one of the most common osteoporotic fractures but also a predictor of future fractures at other sites. Wrist bone mineral density (BMD) is an important determinant of wrist fracture risk, with high heritability. Specific genes underlying wrist BMD variation are largely unknown. Most published genome-wide association studies (GWASs) have focused only on a few top-ranking single-nucleotide polymorphisms (SNPs)/genes and considered each of the identified SNPs/genes independently. To identify biologic pathways important to wrist BMD variation, we used a novel pathway-based analysis approach in our GWAS of wrist ultradistal radius (UD) BMD, examining approximately 500,000 SNPs genome-wide from 984 unrelated whites. A total of 963 biologic pathways/gene sets were analyzed. We identified the regulation-of-autophagy (ROA) pathway that achieved the most significant result (p = .005, qfdr = 0.043, pfwer = 0.016) for association with UD BMD. The ROA pathway also showed significant association with arm BMD in the Framingham Heart Study sample containing 2187 subjects, which further confirmed our findings in the discovery cohort. Earlier studies indicated that during endochondral ossification, autophagy occurs prior to apoptosis of hypertrophic chondrocytes, and it also has been shown that some genes in the ROA pathway (e.g., INFG) may play important roles in osteoblastogenesis or osteoclastogenesis. Our study supports the potential role of the ROA pathway in human wrist BMD variation and osteoporosis. Further functional evaluation of this pathway to determine the mechanism by which it regulates wrist BMD should be pursued to provide new insights into the pathogenesis of wrist osteoporosis. © 2010 American Society for Bone and Mineral Research.

Autocrine Regulation of Interferon γ in Mesenchymal Stem Cells Plays a Role in Early Osteoblastogenesis

Interferon (IFN)gamma is a strong inhibitor of osteoclast differentiation and activity. However, its role in osteoblastogenesis has not been carefully examined. Using microarray expression analysis, we found that several IFNgamma-inducible genes were upregulated during early phases of osteoblast differentiation of human mesenchymal stem cells (hMSCs). We therefore hypothesized that IFNgamma may play a role in this process. We first observed a strong and transient increase in IFNgamma production following hMSC induction to differentiate into osteoblasts. We next blocked this endogenous production using a knockdown approach with small interfering RNA and observed a strong inhibition of hMSC differentiation into osteoblasts with a concomitant decrease in Runx2, a factor indispensable for osteoblast development. Additionally, exogenous addition of IFNgamma accelerated hMSC differentiation into osteoblasts in a dose-dependent manner and induced higher levels of Runx2 expression during the early phase of differentiation. We next examined IFNgamma signaling in vivo in IFNgamma receptor 1 knockout (IFNgammaR1(-/-)) mice. Compared with their wild-type littermates, IFNgammaR1(-/-) mice exhibited a reduction in bone mineral density. As in the in vitro experiments, MSCs obtained from IFNgammaR1(-/-) mice showed a lower capacity to differentiate into osteoblasts. In summary, we demonstrate that the presence of IFNgamma plays an important role during the commitment of MSCs into the osteoblastic lineage both in vitro and in vivo, and that this process can be accelerated by exogenous addition of IFNgamma. These data therefore support a new role for IFNgamma as an autocrine regulator of hMSC differentiation and as a potential new target of bone-forming cells in vivo.

Variation in the bone morphogenetic protein-2 gene: effects on fat and lean body mass in young and elderly women

Bone morphogenetic protein-2 (BMP2) plays a critical role in osteoblastogenesis and adipogenesis from osteoprogenitor cells. The balance between osteogenic and adipogenic effects is influenced by BMP2 concentration, transcription factors and age. BMP2 single nucleotide polymorphisms (SNPs) may contribute to osteoporosis risk, but the relationship between adiposity and body composition has not been explored. We investigated the relationship between BMP2 polymorphisms and body composition in young and elderly women. Population-based association study. Four BMP2 SNPs studied. Total body fat and lean mass measured by DEXA in two cohorts: 'PEAK-25' women aged 25 (+/-0.00) (n=993) and osteoporosis prospective risk assessment (OPRA) women aged 75 (+/-0.00) years (n=1001). We found no association between BMP2 SNPs and fat or lean mass, however, we observed consistent although non-significant trends. Polymorphisms, rs235767 and Ser37Ala, exerted opposing effects on most parameters of soft tissue and bone mass in both cohorts. This relationship appeared to be age specific with large differences between alleles observed (fat mass; Ser37Ala: 14.3% (PEAK-25), -3.5% (OPRA)). These initial results appear to suggest that alleles exerting a beneficial effect in young women may subsequently contribute to phenotypes associated with osteoporosis risk in elderly women. While further analyses in other comparative populations are necessary, in this study of almost 2000 women we observed interesting, although non-significant trends, regarding the effects of variation in the BMP2 gene on parameters of body mass. Although the exact nature of the relationship remains uncertain, we suggest that the mechanisms are influenced by age and environmental factors.

Fidgetin-Like 1 Gene Inhibited by Basic Fibroblast Growth Factor Regulates the Proliferation and Differentiation of Osteoblasts

The FIGNL1 gene was proven to be a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). In this in vitro study, the AAA proteins inhibited osteoblast proliferation and stimulated osteoblast differentiation. We showed that FIGNL1 may play some regulatory role in osteoblastogenesis. The fidgetin-like 1 (FIGNL1) gene encodes a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). Although the FIGNL1 protein localizes to both the nucleus and cytoplasm, the function of FIGNL1 remains unknown. In a previous study, we identified several genes that mediate the anabolic effects of basic fibroblast growth factor (bFGF) on bone by using microarray data. FIGNL1 was one of the genes that downregulated >2-fold in MC3T3-E1 cells after treatment with bFGF. Therefore, this study was aimed to identify and confirm the function of FIGNL1 on osteoblastogenesis. We examined the effect of the FIGNL1 gene on proliferation, differentiation, and apoptosis in mouse osteoblast cells (MC3T3-E1 and mouse primary calvarial cells) using flow cytometry, RT-PCR, cell proliferation assay, and cell death assay. MC3T3-E1 cells and mouse calvarial cells were transfected with small interfering RNA (siRNA) directed against the FIGNL1 or nontargeting control siRNA and examined by cell proliferation and cell death assays. Also, FIGNL1 was fused to enhance green fluorescent protein (EGFP), and the EGFP-fused protein was transiently expressed in MC3T3-E1 cells. Reduced expression of FIGNL1 by bFGF and TGF-beta1 treatment was verified by RT-PCR analysis. Overexpression of FIGNL1 reduced the proliferation of MC3T3-E1 and calvarial cells, more than the mock transfected control cells did. In contrast, siFIGNL1 transfection significantly increased the proliferation of osteoblasts, whereas overexpression of FIGNL1 did not seem to alter apoptosis in osteoblasts. Meanwhile, overexpression of FIGNL1 enhanced the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in osteoblasts. In contrast, siFIGNL1 decreased the expression of ALP and OCN. A pEGFP-FIGNL1 transfected into MCT3-E1 cells had an initially ubiquitous distribution and rapidly translocated to the nucleus 1 h after bFGF treatment. From these results, we proposed that FIGNL1, a subfamily member of the AAA family of proteins, might play some regulatory role in osteoblast proliferation and differentiation. Further analyses of FIGNL1 will be needed to better delineate the mechanisms contributing to the inhibition of proliferation and stimulation of osteoblast differentiation.

On the Molecular Mechanism of DKK1 Inhibition of Osteoblast Differentiation in Multiple Myeloma.

Lytic bone destruction is a severe complication of multiple myeloma growth in the bone. We have demonstrated that Dkk1, an antagonist of Wnt signaling which is essential for osteoblast growth and differentiation, is expressed by myeloma plasma cells leading to increased levels of this protein in serum. Myeloma serum can block osteoblast differentiation in-vitro and a neutralizing anti-DKK1 antibody can inhibit this. These data suggest that MM cells disrupt Wnt signaling and that this promotes uncoupled bone turnover. However, the molecular mechanism responsible for this processes remains to be elucidated. Here we investigated Wnt signaling in osteoblast differentiation and identified the molecular mechanism regulating this process. Using qRT-PCR and sequence confirmation we showed that multiple Frizzled (Fz) mRNAs and LRP5/6 co-receptors were expressed in C2C12 cells and three human osteoblast cell lines, hFOB1.19, MG63 and Saos-2. Furthermore, these cells had high levels of total beta-catenin an indicator of active Wnt signaling. Wnt3a conditioned medium (CM) and recombinant Wnt3a protein increased uncomplexed beta-catenin level as assessed by GST-E-cadherin binding assay. Blocking autocrine Wnt signaling with Dkk1 blocked Wnt-3a-induced increase in beta-catenin levels in dose-dependent fashion. Co-culturing of OPM-2 MM cells ectopically expressing Dkk1 inhibited the Wnt-3a induced beta-catenin stabilization in C2C12 cells. Transfection of C2C12 cells with a dominant negative beta-catenin (DNBC) completely blocked endogenous and Wnt-3a-induced TCF/LEF transcriptional activity as evidenced by TOPflash luciferase activity. Furthermore, BMP-2 induced increase in alkaline phosphatase (ALP) activity was completed abrogated in C2C12 cells expressing DNCB. Silencing LRP5/6 mRNA with small inference RNAs blocked BMP-2 induced increase in ALP expression, suggesting that canonical Wnt pathway contributes to BPM-2 induced OB differentiation. BMP-2 induced phosphorylation of Smad 1, 5 and 8 whereas Wnt-3a had no effect on phosphorylation of these Smads. Moreover, Dkk1 did not block BMP-2 induced phosphorylation of Smad-1, −5 and −8 and did not alter transcriptional activity of Cbfa1, an essential transcriptional factor for OB differentiation. Taken together, these data suggest that Wnt induction of OB cell differentiation through canonical Wnt-beta-catenin pathway is independent of the activation of the Smad pathway. Our results suggested that canonical Wnt signaling pathway plays an important role in osteoblastogenesis and that Dkk1 inhibits OB cell differentiation in a way that may contribute to MM bone lesions. New therapies targeting DKK1 in myeloma may represent a novel treatment strategy.

The Orphan Receptor Tyrosine Kinase Ror2 Promotes Osteoblast Differentiation and Enhances ex Vivo Bone Formation

Ror2 is a receptor tyrosine kinase, the expression of which increases during differentiation of pluripotent stem cells to osteoblasts and then declines as cells progress to osteocytes. To test whether Ror2 plays a role in osteoblastogenesis, we investigated the effects of Ror2 overexpression and down-regulation on osteoblastic lineage commitment and differentiation. Expression of Ror2 in pluripotent human mesenchymal stem cells (hMSCs) by adenoviral infection caused formation of mineralized extracellular matrix, which is the ultimate phenotype of an osteogenic tissue. Concomitantly, Ror2 over-expression inhibited adipogenic differentiation of hMSCs as monitored by lipid formation. Ror2 shifted hMSC fate toward osteoblastogenesis by inducing osteogenic transcription factor osterix and suppressing adipogenic transcription factors CCAAT/enhancer-binding protein alpha and peroxisome proliferator activated receptor gamma. Infection with Ror2 virus also strongly promoted matrix mineralization in committed osteoblast-like MC3T3-E1 cells. Expression of Ror2 in a human preosteocytic cell line by stable transfection also promoted further differentiation, as judged by inhibited alkaline phosphatase activity, potentiated osteocalcin secretion, and increased cellular apoptosis. In contrast, down-regulation of Ror2 expression by short hairpin RNA essentially abrogated dexamethasone-induced mineralization of hMSCs. Furthermore, down-regulation of Ror2 expression in fully differentiated SaOS-2 osteosarcoma cells inhibited alkaline phosphatase activity. We conclude that Ror2 initiates commitment of MSCs to osteoblastic lineage and promotes differentiation at early and late stages of osteoblastogenesis. Finally, using a mouse calvariae ex vivo organ culture model, we demonstrate that these effects of Ror2 result in increased bone formation, suggesting that it may also activate mature osteoblasts.

Calcyclin, a Ca2+ Ion-binding Protein, Contributes to the Anabolic Effects of Simvastatin on Bone

In vitro treatment with a pharmacological dose of simvastatin, a potent pro-drug of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, stimulates bone formation. In our study, simvastatin stimulated differentiation of osteoblasts remarkably in a dose-dependent manner, with minimal effect on proliferation. To identify the mediators of the anabolic effects of simvastatin on osteoblasts, we tried to identify and characterize simvastatin-induced proteins by using proteomic analysis. Calcyclin was significantly up-regulated by more than 10 times, and annexin I was also up-regulated by simvastatin. However, annexin III, vimentin, and tropomyosin were down-regulated. Up-regulated calcyclin mRNA by simvastatin was validated by reverse transcription in mouse calvarial cells. In confocal microscope analysis, green fluorescence protein-calcyclin fusion protein was ubiquitously observed in the of MC3T3-E1 cells transfected with green fluorescence protein-calcyclin cDNA containing plasmid and was quickly concentrated in the nucleus 20 min after simvastatin treatment. Overexpression of calcyclin cDNA stimulated both the proliferation and expression of alkaline phosphatase mRNA significantly, without exposure to simvastatin in MC3T3-E1 cells. However, both the rate of proliferation of the osteoblasts and the expression of alkaline phosphatase mRNA were suppressed significantly 1 day after treatment with the calcyclin-specific small interference RNA, and furthermore, simvastatin did not overcome this suppression in the small interference RNA-pretreated MC3T3-E1 cells. In conclusion, calcyclin is one of the candidate proteins that plays a role in osteoblastogenesis in response to simvastatin, although the precise functions of calcyclin in osteoblast remain to be verified.