Regulation Of BMP-Smad Signaling And Its Function In Osteogenic Differentiation

Bone morphogenetic proteins (BMPs) are members of transforming growth factor-β (TGF-β) receptor superfamily, which were first isolated from demineralized bones in1960s and are capable of inducing ectopic bone growth. BMPs are secreted proteins present in the extracellular and in the serum can be detected. Engaging of BMPs to BMP receptors on the cell surface leads to activation of the receptor kinase activity, which phosphorylates Smadl/5/8. Smad1,5, or8, with Smad4, forms a complex, which is translocated to the nucleus, where it binds to the consensus DNA sequence to regulate the transcription of BMP target genes. Same fine adjustment of the BMP-Smad signaling pathway have also been a variety of mechanisms, especially BMP-Smad signaling pathway negatively regulate the tightness. Receptor signaling pathway in the cell membrane usually been some negative feedback regulation, such as cells within swallow, dephosphorylation and degradation of the signal molecule.BMP-Smad signaling regulates stem cell renewal, cell proliferation, differentiation, migration, and apoptosis, and controls embryo development and postnatal tissue Homeostasis. BMP-Smad signaling pathway also plays a very important role in tumorigenesis. The human and mouse genetics studies have demonstrated that BMPs play an important role in osteoblast proliferation, differentiation and bone formation. The absence of BMP-Smad signaling pathway can cause bone-related diseases, such as osteoporosis. The Mechanism of BMP-Smad signaling pathway in the regulation of osteogenic differentiation is not well understood. And we accidently found that in the study of the stress response to DNA damage, the two genes p53and Atm play an important role in DNA damage and tumorgenesis, and also play a vital role in the osteogenesis. p53knockout mice showed bone sclerosis and Atm knockout mice showed osteoporosis, osteogenic differentiation of function of these mice have been affected. The mechanism of p53and Atm regulating osteogenic differentiation are not clear. After several years of study on BMP-Smad signaling pathway, we found that BMP-Smad signaling pathway plays a regulatory function in the process of p53-/-mice, Atm-/-mice osteogenic differentiation.This study is supported by Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University and Bio-X Center, Shanghai Jiao Tong University. In this study, a novel regulation mechanisim of BMP-Smad signaling pathway was uncovered and a unique epistatic relationship between ATM and p53in osteogenesis was revealed, which is mediated by the BMP-Smad pathway.First, Negative feedback regulation of serum starvation to SmadlSmad proteins are important mediators of the completion of the BMP signal transduction. During our study of BMP-Smad activation in mouse embryonic fibroblasts, we sometimes needed to starve the cells from serum before adding BMPs. To our surprise, we found that Smadl protein levels were increased in the absence of serum. Smadl up-regulation was also observed in other cell lines, suggesting that this is a rather common cellular response. These results suggest that the fetal bovine serum contains bioactive agents that modulate Smadl expression. After the addition of BMP inhibitor Noggin cause upregulation of Smadl protein levels, supporting our hypothesis that withdrawal of BMPs leads to Smadl up-regulation.Here we report a novel regulation mechanism of Smadl in response to blockade of BMP action. Lowering the serum levels or antagonizing BMPs with Noggin led to up-regulation of Smadl, but not Smad5, Smad8, or Smda2/3, at the protein level in several cell lines. The Smadl up-regulation occurs at the level of protein stabilization. Moreover, up-regulated Smadl was relocalized to the perinuclear region, in contrast to proteosome and nucleus localization of Smadl in response to proteosome inhibitor. These alterations seem to have effects on the dynamics and the amplitude of BMP2-induced Smadl reactivation. These findings indicate that depletion or antagonizing BMPs leads to Smadl stabilization and delocalization, thus revealing an unexpected regulation mechanism for BMP-Smad signaling. But for its function and significance requires further study.Second, The role of p53in regulating Smadl during osteoblasts differentiationBMP-Smad signaling pathway plays a key role in the process of osteogenic differentiation. p53knockout mice accelerate osteogenic differentiation, and we want to know whether it has relationship with BMP-Smad signaling pathway. First, We isolated p53+/+and p53-/-osteoblasts in vitro. The results showed that the Smad1protein expression was significantly increased in p53-1-osteoblasts. This indicates that p53may inhibit the expression of Smadl. Smad1expression at the RNA level was inhibited by p53as detected by real-time quantitative PCR. Using luciferase reporter assay, Smadl transcription were also proved to be indirectly regulated by p53in p53-/-osteoblasts. Second, One well-studied route by which p53represses transcription is via its target gene p21, which inhibits CDKs and subsequently leads to Rb hypophosphorylation and E2F1inactivation. Therefore, we speculated p53in osteoblasts might inhibited Smadl expression through p21-E2F1pathway.Using Western Blot to detect the protein expression of p21in p53-/-osteoblasts, the results showed that the deletion of p53reduce the expression of p21levels significantly. In addition, transfected pMSCV-p21in p53-1-osteoblasts also reduced Smadl protein and mRNA levels. Luciferase reporter assay show that p21also suppress promoter activity of Smadl. These results indicate that p53regulate Smadl expression and transcription through target genes p21.Finally, Using MatInspector software from Genomatix, several potential E2F1binding sites were predicted in Smadl gene with3located in the promoter sequences. Using the reporter assay with promoter of Smadl fused to luciferase, we showed that E2F1was able to activate the Smad1promoter, especially the1kb promoter. Lastly, we found that overexpression of E2F1led to an increase in Smadl in osteoblasts. These results indicate that E2F1positively regulates Smadl transcription. To determine whether E2F1directly binds to Smadl promoter, we carried out chromatin-IP experiments in several lines including MC3T3-E1and IMR90-E1A, with similar results obtained. These results, for the first time, indicate a positive role for E2F1in regulating the transcription of Smadl. The above experiment results show that p53negatively regulate Smadl expression through p21-E2F1pathway.Third, The role of ATM in regulating Smadl during osteoblast differentiationTo investigate the mechanism of ATM regulation of Smadl, we isolated Atm+/+and Atm-/-osteoblasts in vitro, using Western Blot to study the protein expression of the BMP signaling pathway in Atm knockout mice. The results showed that the Smadl protein expression was significantly decreased in Atm-/-osteoblasts. ATM is as upstream of p53in DNA damage response and tumor suppression, which is very important for the p53activation and stability. Hence, we speculated that ATM play a role in the regulation of Smadl by p53. We compared expression of p21and p53in Atm+/+and Atm-/-osteoblasts.The results showed that p53and p21expression in Atm-/-osteoblasts was significantly increased. Early studies reported that in some cells including Atm-/-osteoblasts, the expression level of ROS will increase, and ROS could activate p53through multiple signaling pathways. We infer that the increase of p53result from the increase of ROS. Add NAC to reduce the level of ROS in Atm-/-osteoblasts and Atm+/+osteoblasts. Western Blot results showed that NAC reduced the level of p53in Atm-/-and Atm+/+osteoblasts. This suggested that ROS is at least partially responsible for p53up-regulation in Atm-/-osteoblasts.we crossed Atm+/-and p53+/-to generate Atm-/-p53-/-mice, Primary osteoblasts isolated from the Atm-l-p53-/-mices, like p53-/-osteoblasts, Western Blot results showed little expression of p21, suggesting that p21up-regulation in Atm-/-cells requires p53. Moreover, the decrease in Smadl expression and activation was reversed by p53deficiency, as Atm-l-p53-/-osteoblasts behaved like p53-/-osteoblasts in Smadl expression/activation at the basal levels.This part of the study shown that p21-E2F1-mediated Smadl elevation and hyperactivation makes an important contribution to accelerating differentiation of p53-/-osteoblasts. Fourth, The role of BMP-Smad signaling pathway for regulating osteogenetic differentiation in p53-/-and Atm-/-miceFor the research of BMP-Smad signaling pathways in the role of osteoblast differentiation, we attempted to inhibit BMP-Smad signaling in p53-/-and wild type osteoblasts with BMP antagonists Noggin and Chordin or transfected Smadl siRNA shown to inhibits BMP-Smad signaling in osteoblasts. It was found that Noggin and Chordin treatment resulted in a reduction of Osx mRNA and a slow-down of differentiation of wild type and p53-/-osteoblasts. Moreover, transient knockdown of Smadl with siRNA also led to a decrease in the expression of ALP and Osx. Ectopic expression of p21also led to inhibition of osteoblast differentiation, justified by a decrease in the expression of ALP and Osx. These results suggest that p53regulates Smadl expression, Osx expression, and osteogenic differentiation via its target gene p21and that p21regulates Smadl transcription.To investigate that osteoblast differentiation and bone formation defect in Atm-/-mice is due to the rise of p53, we used6-8week mice for bone studies, Atm-/-p53-/-mice showed an increase in bone formation rates, bone formation surface, and the number of osteoblasts in vivo. Real-time quantitative PCR and Von Kossa staining showed significantly increase in the expression of ALP and Osx mRNA and the bone mineralization in Atm-/-p53-/-osteoblast.We attempted to inhibit BMP-Smad signaling in Atm-/-p53-/-osteoblasts with BMP antagonists for the study of the differentiation of osteoblast. The result of real-time PCR and Von Kossa staining proven that Noggin and Chordin treatment resulted in a reduction of ALP and Osx mRNA and a slow-down of bone mineralization. This indicated that upregulation of Smadl expression and activation lead to the increase of osteoblastic differentiation in Atm-/-p53-/-.Conclusion:In summary, in this study we found that serum depletion or Noggin treatment resulted in up-regulation of Smadl and relocalization of the Smadl protein at the perinuclear region. The cause of Smadl upregulation and the effect of Smadl upregulation was also explored. In addition, this study also establishes a link between the anti-proliferating ATM-p53-p21pathway and the developmentally indispensable BMP-Smad pathway. This link explains the altered osteoblastogenesis and bone formation observed in p53-/-mice and Atm-/-mice, and likely other developmental aspects controlled by BMP-Smad signaling.