The Function Of BMP-Smad Signaling In Osteoblastogenesis And Osteoclasgenesis

In life, bone is a rigid yet dynamic organ that is continuously moulded, shaped and repaired, of1/5body weight, and plays an essential role in the structure, protection, movement, hematopoiesis and endocrine secretion of the body. Once growth and development completed, bone undergoes a game process that invqlves two opposing effects:break down and build up. Bone destruction is constantly carried out by haematopoietically derived osteoclasts and the synthesis is carried by MSC derived osteoblasts. Bone mass and structure in adults is maintained locally by the balance between osteoclastic bone resorption and osteoblastic bone formation. Most adult skeletal diseases such as Osteopetrosis and osteoporosis are due to the imbalance in bone remodeling.Bone morphogenetic proteins (BMPs), exist in bone abundantly, was first defined to describe the components in bone matrix that can induce ectopic bone formation. BMP-Smadl singnaling is important in regulation of the differentiation of osteoblast and osteoclast. Moreover, BMP-Smad plays critical roles in embryonic development, adult tissue homeostasis and tumorigenesis.In the BMP-Smad pathway, the signal was relayed from the cell surface to the nucleus by typeⅡand typeⅠreceptors with serine/threonine kinase activity and intracellular Smad proteins. The BMPRⅠand BMPRⅡ activated by ligand binding phosphorylate the receptor-regulated Smads (R-Smads) including Smad1,5and8, of the C’ter minal SXS motif. Then the phosphorylated R-Smads form complexes with Smad4. Smad heterodimers then migrate into the nucleus to regulate gene transcription.Platelet-derived growth factors (PDGFs) are growth factors that promote cell proliferation and migration, plays critical roles in embryonic development and adult tissue homeostasis. There are four PDGFs (A-D) that are expressed in tissue-specific manners. PDGF molecules bind to specific cell surface receptor PDGFRs, which are members of receptor tyrosine kinases, to execute their functions, PDGF-AA mainly activates PDGFRα. PDGFs-elicited signaling events are tightly regulated. One regulatory mechanism is endocytosis and lysosome-mediated degradation of PDGFRs, which requires ligand-binding and PDGFR autophosphorylation. This acts as a feedback regulation mechanism to attenuate PDGF signaling. PDGFs have an important function in bone development. PDGFRoc ablation led to defects in skeletal patterning and maturation. In adults, PDGFs play important roles in wound healing and bone fracture healing, where it act on fibroblasts, MSCs, and other cell types, and as such PDGFs might present a class of therapeutic regents for wound healing and bone regeneration. At the cell level, PDGFs are shown to promote MSC proliferation. While PDGF-BB was reported to inhibit osteoblast differentiation, PDGF-AA’s function in osteoblast differentiation is inconclusive. It remains unclear how PDGFs decide the fate of differentiation or proliferation in MSC.Vitamin D is produced in the skin from7-dehydrocholesterol by ultraviolet (UV). Irradiation, which is also found in small quantities in the diet, but is a common supplement.1,25-dihydroxyvita min D3is the principal hormonal form of vita min D, responsible for most of its biologic actions. In addition, most of the cells, including MSCs/osteoblasts and mnocytes/osteoclasts, can synthesize active vita min D. Vita min D binds to cytoplasmic vitamin D receptor (VDR) to turn on targeted genes and activate signaling pathways including MAPKs and Akt. Vitamin D has skeletal as well as extra-skeletal functions, e.g., reproduction, immunity, cardiovascular system, hair cycle, and cancer. The main function of vitamin D is to increase intestinal absorption of calcium in people with normal dietary calcium, which is essential for bone mineralization. Nutritional vitamin D deficiency, altered vitamin D responsiveness all have rickets and osteomalacia as their main phenotype. Supplementation with vita min D is generally believed to prevent osteoporosis. However, pharmalogical levels of active vitamin D and its analogs have been shown to increase bone mass and density, due to its positive effects on bone formation and/or negative effects on bone resopriton. While some studies have shown that vitamin D promotes osteoclast differenetiation, other studies reported opposite effects for vitamin D. Moreover, vita min D might affect the seceretion of RNAKL (Receptor Activator for Nuclear Factor-κ B Ligand),OPG (osteoprotegerin) and other molcecules to indirectly affect osteoclastogenesis. It is additionally complicated to understand the impact of vitamin D metabolites on bone is by species differences, differences in responsiveness of bone cells according to their stages of differentiation, and differences in responsiveness in terms of the vitamin D metabolite being examined. Thus, how pharmalogical doses of vitamin D execute its anti-osteoprorsis activities remains controversial and warrants further investigation.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, two novel regulation mechanisims of BMP-Smad signaling.1. PDGF-AA induces MSC osteogenic differentiation and migration via Smadl/5/8Genetic studies have shown that PDGF molecules play important roles in MSC proliferation, skeletal development and bone fracture healing, processes that involve bone morphogenetic proteins (BMPs) as well. We found that PDGF-AA activated Smad1/5/8significantly in primary murine MSC cells. However, PDGF molecules failed to activate Smad2/3,the TGFβ-responsive Smads, pointing to a specific link between PDGF-AA and BMP-Smadl/5/8signaling. On the other hand, We found that PDGF-AA-induced Smadl/5/8activation requires BMPR I A, but did not alter the expression of BMP2, BMPR Ⅰ A and BMPR Ⅱ. These results suggest that PDGF-AA activates BMP-Smad1signaling not by altering the expression of BMPs or BMP receptors. To test the functions of PDGF-AA-induced Smadl/5/8activation, we first looked at MSC osteogenic differentiation, a cellular event promoted by BMP-Smad1/5/8signaling via transcription factors such as Runx2, Osterix, and Atf4. the effect of PDGF-AA on MSC migration can be at least partially mediated by the BMP-Smadl and its target genes including Idl, Twist1,and Atf4, justified by a transwell assay. 2. PDGF-AA induced Smadl/5/8activation requires degradation of PDGFRa to release BMPR I AHaving shown the biological significance of PDGF-AA-induced Samd1/5/8activation in MSCs, we wanted to understand the molecular mechanisms underlying Smadl/5/8activation in response to PDGF-AA. We have excluded the possibility that Smadl/5/8activation in response to PDGF-AA was caused by change in BMPs or BMPRs. Down-regulation of PDGFRa was observed in response to PDGF-AA in MSCs. To test whether down-regulation of PDGFRa plays a role in PDGF-AA-induced Smadl/5/8activation, we proved that BMPR I A and PDGFRa constantly form a complex. The above results suggest that PDGFRa, by complexing with BMPR I A, may compete with BMPR II for BMPR Ⅰ A binding and thus interfere with BMPR Ⅰ-BMPR Ⅱ interaction, which is required for BMPR Ⅰ activation and Smadl/5/8phosphorylation. To test this possibility, we carried out co-immunoprecipitation experiments and found that PDGFRa expression resulted in a decrease in BMPR Ⅰ-Ⅱ complex formation. These results suggest that PDGFRa inhibits BMP-Smadl activation by interacting with BMPR Ⅰ A and interfering with BMPR Ⅰ-BMPR Ⅱ interaction.3.1,25-(OH)2D3increases bone mass by repressing osteoclatogenesis via BMP-Smadl pathwayIt is generally believed that VD increases bone mass by promoting bone formation due to increased Ca uptake and/or enhanced osteoblast differentiation. However, we found that1α,25(OH)2D3increased bone mass due to a decrease in bone resorption via stimulating BMP-Smad pathway.We found that1α,25(OH)2D3-treated mice showed a increase bone mass by50%, which is accompanied by a decrease in osteoclast number and eroded surface but no significant change in bone formation rate, osteoblast number and mineral appositeion rate. The result suggested that osteoclast is the major factor of the increase of bone mass,1α,25(OH)2D3increased bone mass by decreasing bone resorption but not increasing bone formation. Biochemical criterion, including the Urine levels of DPD, which is the marker of bone resorption, conform the conclusion. It is now well established that1α,25(OH)2D3stimulate bone resorption, support osteoclast differentiation from their precursors. Whereas, we found the effect of1a,25(OH)2D3is different in various stages of osteoclast development and activation. In the TRAP-staining assay, we found Pre-treatmtent with1α,25(OH)2D3inhibited ex vivo osteoclastogenesis induced by RANKL/MCSF. Whereas, Simultaneous1α,25(OH)2D3treatment promoted ex vivo osteoclastogenesis induced by RANKL/MCSF. The result is verified by real-time PCR, for osteoclast specific genes, i.g PU.1, MITF, C-FOS, NFATC1, CAT K, intergrimb3, TRAP, DC-STAMP, which is inhibition or stimulation in different stages of osteoclast differentiation. These results suggest that la,25(OH)2D3might have biphasic effects on osteoclast differentiation from its precursor:an inhibitory effect at early steps of differentiation but a positive effect on late steps of differentiation.To understand how1α,25(OH)2D3suppresses osteoclastogenesis, we analyzed many relative signaling and found la,25(OH)2D3led to enhanced Smadl activation and increased Smadl protein levels in monocytes and RAW264.7cells, stimulated the BMP-Smadl pathway. The mRNA level of Smadl was up-regulated by1α,25(OH)2D3in monocytes and bone extracted RNA.1α,25(OH)2D3activates the Smadl promoter in reporter assays and the Chromatin Immunoprecipitation assay proved that the vitamin D receptor promoter has Smad1binding sites.BMP2also have biphasic effects in osteoclast differentiation. The TRAP staining and real-time PCR assay demonstrate that BMP2, as well as1α,25(OH)2D3, suppressed osteoclast differentiation induced by RANKL/M-CSF, whereas simultaneous addition of BMP2or addition of BMP2after RANKL and M-CSF promoted osteocast differentiation. Primary monocytes isolated from lysM-Cre; Bmpr1αf1/fl mice showed decreased differentiation. Analysis of the expression of osteoclast differentiation-related genes revealed that the genes expressed in early stage are increased, in contrast, the genes expressed in the late phase are decreased in lysM-Cre; Bmprl1αf1/fl cells. Thus, BMPR I A deficiency enhances the initial differentiation of osteoclasts but impedes the mature of osteoclast. LDN-193189, a BMPR I inhibitor, together with1α,25(OH)2D3, alleviated the inhibitory effect of la,25(OH)2D3on osteocalst differentiation. LDN-193189significantly inhibited1α,25(OH)2D3-induced increase in bone volume, also blocked the negative effects of1α,25(OH)2D3on bone resorption and osteoclastogenesis in vivo.1α,25(OH)2D3ad ministration failed to further increase bone mass in lysM-Cre; Bmpr1αf1/fl mice, nor alter other markers indicating bone resorption. These results demonstrated that the BMP-Smadl signaling mediates the pro-osteogenic effects of1α,25(OH)2D34. A crosstalk between BMP-Smadl and NF-κB pathwayWe wanted to know how1α,25(OH)2D3and BMP-Smadl signaling regulates RANKL/M-CSF-induced osteoclast differentiation. Because RANKL mainly activates the NF-κB pathway to promote differentiation, so we detected the effect of1α,25(OH)2D3on NF-κB. We found that1α,25(OH)2D3and BMP2pre-treatment could up-regulate IκB-α and down-regulate NF-κB in monocyte/osteoclasts by inhibiting the degradation of IκB-α, which are expected to inhibit osteoclast differentiation. However, addition of la,25(OH)2D3or BMP2two days after RANKL/M-CSF did not show any decrease in IκB-α and down-regulate NF-κB in monocyte/osteoclasts.In summary, in this study, we found PDGF-AA promotes MSC osteogenic differentiation through the BMP-Smadl-Runx2/Osx axis and MSC migration via the BMP-Smadl-Twist1axis. Mechanistic studies show that PDGF-AA activates BMP-Smadl signaling by feedback down-regulating PDGFRa, which frees BMPR Ⅰ and allows for BMPR Ⅰ-BMPR Ⅱ complex formation to activate Smadl/5/8, using BMP molecules in the microenvironment. This study establishes a link between PDGF-AA and BMPs pathways, two essential regulators of embryonic development and tissue homeostasis. Both1α,25(OH)2D3and BMPs appear to have biphasic effects on osteoclast differnetation, which are mediated by NF-κB. Moreover, this study established a crosstalk between BMP-Smadl signaling and the NF-κB pathway at the site of IκB-α,which seems to play an important role in osteocaltogenesis and bone resorption.