The CAMP-CREB1-HDAC8 Pathway Regulates Proliferation And Differentiation Of Bone Marrow Stromal Cells In Craniofacial Fibrous Dysplasia

Fibrous dysplasia(FD),a non-hereditary and benign bone disease,is characterized by osteogenesis disorder and high proliferation of bone marrow stromal cells(BMSCs).Craniofacial bones,the tibia and the femur are the most frequent sites of FD lesions and FD in the craniofacial area can result in serious orofacial deformities,bone pain,dental disorders and pathologic fractures.The cAMP-CREB pathway,which is activated by GNAS mutation,is known to be closely associated with the onset of FD.However,so far there is no available targeted therapeutic strategy for FD,as a critical issue that remains largely unknown is how this pathway is involved in FD.Therefore,new and more effective therapeutic methods are urgently needed.Our previous studies have revealed that histone deacetylase 8(HDAC8)inhibits the osteogenic differentiation of BMSCs via epigenetic regulation and HDAC8 upregulation was found in FD BMSCs compared with BMSCs from healthy donors.In addition,we predicted that CREB1 binding regions are present in the promoter of HDAC8.In summary,our study revealed that HDAC8 associates with abnormal biological properties in FD BMSCs and demonstrated functional relationships with BMSC molecules regulated by the cAMP-CREB1-HDAC8 pathway.These results provide insights into the molecular regulation of FD pathogenesis,and offer novel clues to the clinical treatment of FD.Part Ⅰ Cytological features of BMSCs from craniofacial FD lesions Objective: To isolate and culture FD BMSCs in vitro and to study the in vitro biological properties Methods: 1)FD BMSCs were cultured with explants culture method.Cells were passaged for the first time when the colony attached each other;2)FD lesion was confirmed by radiological features,DNA sequence and H&E staining;3)BrdU assays for cell proliferation ability 4)Flow cytometry for cell apoptosis ability;5)ELISA assays for the level of cAMP in BMSCs;6)Real-time PCR and Western blot for the related gene expression;7)Real-time PCR,Western blot and alizarin red staining were used to detected the osteogenic differentiation potential of FD BMSCs.Results:(1)Orthopanotomography performance of the three FD patients showed swelling and deformed image and ground glass-like changes.The R201 H or R201 C mutation in GNAS of FD BMSCs was detected by allele-specific PCR and sequencing.H&E staining showed the immature bone trabeculae surrounded with fibrous tissue.(2)No obvious morphologic differences between FD BMSCs and normal BMSCs were observed.However,FD BMSCs exhibited a much stronger proliferation ability and a much weaker apoptosis ability relative to BMSCs.Next,we observed that the expression of RUNX2,SP7,SPP1 and BGLAP was downregulated in FD BMSCs during osteogenic induction.Alizarin Red staining indicated that fewer calcium deposits were formed by FD BMSCs.There was significantly higher expression of cAMP,p-CREB1 and HDAC8 in FD BMSCs than that in BMSCs,and the expression of TP53 declined in FD BMSCs.Conclusion: These results indicate that HDAC8 may participate in the regulation of the osteogenic differentiation of FD BMSCs,and its ability to suppress apoptosis is likely related to TP53 downregulation.Part Ⅱ The effect of cAMP-CREB1-HDAC8 pathway on proliferation and osteogenic differentiation of FD BMSCs Objective: To analysis role of cAMP-CREB1-HDAC8 in proliferation and osteogenic differentiation of FD BMSCs and to study the mechanism of onset of FD Methods: 1)Normal BMSCs were treated with exogenous cAMP,and FD BMSCs were treated with inhibitor of cAMP(bupivacaine HCl),inhibitor of HDAC8(PCI-34051)and LeV-shHDAC8.Real-time PCR and Western blot for the related gene expression;2)BrdU assays for cell proliferation ability;3)Flow cytometry for cell apoptosis ability;4)Real-time PCR,Western blot and alizarin red staining were used to detected the osteogenic differentiation potential of FD BMSCs;5)Realtime PCR and Western blot for the expression of CREB1,p-CREB1,HDAC8 and TP53 in FD BMSCs interfered with si-CREB1;6)The assessment for combination of CREB1 in HDAC8 promoter region was conducted with Chromatin Immunoprecipitation(ChIP);7)Dual Luciferase Reporter Assay for putative binding region of CREB1 in HDAC8 promoter.Results:(1)The expression of p-CREB1 and HDAC8 in normal BMSCs treated with cAMP significantly increased and the expression of TP53 decreased compared with the control.Importantly,BMSCs with ectopic cAMP expression exhibited similar properties to FD BMSCs: enhanced cell proliferation,slightly decreased cell apoptosis and impaired osteogenesis potential.(2)Contrary to the former,FD BMSCs with inhibitor of cAMP exhibited similar properties to normal BMSCs.(3)The expression of TP53 significantly increased and FD BMSCs treated with HDAC8 inhibitor exhibited decreased cell proliferation,increased cell apoptosis and improved osteogenesis potential.(4)FD BMSCs transfected with LeV-shHDAC8 showed the similar result as that of FD BMSCs treated with HDAC8 inhibitor.(5)CREB1 knockdown reduced the expression of CREB1,p-CREB1 and HDAC8 and increased the expression of TP53.(6)Significant enrichment of CREB1 was identified in four putative binding sites in HDAC8 promoter region.(7)The luciferase activity was significantly increased in reporter containing the binding site 1 and 3.Conclusion: HDAC8 plays an extremely important and direct role in the abnormal phenotypes of FD BMSCs,and CREB1 promotes HDAC8 transcription by directly binding to its promoter region in FD BMSCs.The existence and interrelation of the cAMP-CREB1-HDAC8 axis has been identified and verified.Part Ⅲ Study the role of HDAC8 inhibition or knockdown in the osteogenesis potential of FD BMSCs in vivo Objective: To examine ectopic bone formation in FD BMSCs treated with LeVGFP,LeV-shHDAC8 and PCI-34051 in nude mice Methods: 1)FD BMSCs were attached to each HA/TCP biomaterial,and then the complexes(BMSCs group,FD BMSCs group,LeV-scramble group,LeVshHDAC8 group and PCI-34051 group)were implanted subcutaneously into the backs of 7-week-old athymic nude mice after 12 h.2)Eight weeks after implantation,samples were harvested,fixed with 4% paraformaldehyde,decalcified in 10% ethylene diaminetetraacetic acid(EDTA),embedded in paraffin wax and further sectioned into 4-μm slices.The slices were stained with hematoxylin and eosin(H&E)and Masson trichrome,and immunohistochemistry was performed.Results:(1)The organized bone matrix was formed in the HDAC8-knockdown group and the PCI-treated group,whereas there was a significant amount of fibrous tissue in the negative control.FD BMSCs treated with HDAC8 downregulation induced more ectopic bone formation compared to the control groups.(2)FD BMSCs treated with HDAC8 knockdown or inhibition exhibited increased RUNX2 and BGLAP expression.Conclusion: HDAC8 downregulation could enhance the osteogenesis of FD BMSCs in vivo and HDAC8 may be a potential therapeutic target for FD.