Regulation Of Adipogenic Differentiation By Cyr61 And The Mechanisms Involved In The Processes

Objective:Cyr61(Cysteine-rich 61)protein is an extracellular signal transduction molecule,a member of the CNN protein family.Cyr61 consists of four different domains,which exhibit different biological functions in different microenvironments.Cyr61 protein plays an important role in the biological processes such as cell differentiation,angiogenesis,cell adhesion,and apoptosis.Mesenchymal stem cells are a kind of adult stem cells derived from mesoderm,with the potential of multi-directional differentiation.There is a biological phenomenon of the shift in the process of differentiation of mesenchymal stem cells into adipocytes and osteoblast differentiation.Studies have shown that Cyr61 plays an important role in the regulation of osteoblast differentiation,and the function and mechanism of Cyr61 in adipocyte differentiation is rarely reported.In this study we investigated the effect of Cyr61 on adipocyte differentiation and clarified its regulatory mechanisms.Methods:1.The Cyr61 overexpression plasmid and Cyr61 siRNA were transfected into bone marrow stromal cell line C3H10T1/2 and adipogenic induction was completed when the cell fusion rate reached 100%,The effects of Cyr61 on adipogenic differentiation of C3H10T1/2 cells were investigated by Oil Red O staining,qRT-PCR and Western Blotting.2.Cyr61 siRNA was transfected into mesenchymal stem cell line C3H10T1/2.After 18 hours later,replaced with fresh DMEM complete medium containing Wnt3 a protein.Transfected 48 hours,the translocation of ?-catenin from the cytoplasm into nucleus was detected by immunofluorescence assay.In the meantime,the nuclear protein was extracted.Western blotting was used to investigate the level of ?-catenin and T cell factor-4(TCF-4).The above of experiments to determine the role of Cyr61 on the classic Wnt signaling pathway.3.In order to determine the role of Cyr61 on the mTORC1 signaling pathway.Cyr61 siRNA was transfected into mesenchymal stem cell line C3H10T1/2.After 48 hours later,the total protein was extracted.Western blotting was used to detect the phosphorylation level of mammalian target of rapamycin protein(mTOR)and ribosomal protein S6 kinase,polypeptide 1(S6K1)protein.4.Cyr61 siRNA was transfected into mesenchymal stem cell line C3H10T1/2 and adipogenic induction(added mTOR protein specific inhibitor rapamycin)was completed when the cell fusion rate reached 100%.To observe the effect of Cyr61 on adipogenic differentiation of C3H10T1/2 cells after inhibiting mTORC1 signaling pathway activity and to explore the molecular mechanism of Cyr61 regulating adipocyte differentiation.Results:1.The Cyr61 overexpression plasmid was transfected into mesenchymal stem cell line C3H10T1/2,the expression of Peroxisome proliferator-activated receptor ?(PPAR?),CCAAT enhancer binding protein ?(C/EBP?),Adipocyte fatty acid-binding Protein 2(aP2)and Adipsin were down-regulated.In contrast,siCyr61 was transfected into mesenchymal stem cell line C3H10T1/2,the expression of PPAR ?,C/EBP ?,aP2 and Adipsin were enhanced.Indicating that Cyr61 inhibited the differentiation of C3H10T1/2 cells into adipocytes.2.Cell immunofluorescence assay showed that Cyr61 siRNA could inhibit the translocation of ?-catenin into the nucleus.The nuclear protein was extracted,Western blotting showed that the level of ?-catenin and TCF-4 were decreased.This can be explained that Cyr61 can activate the classic Wnt signaling pathway.3.Cyr61 siRNA can promote the phosphorylation of mTOR protein and S6K1 protein Indicating that Cyr61 was able to inhibit the activity of the mTORC1 signaling pathway.4.Cyr61 siRNA was transfected into mesenchymal stem cell line C3H10T1/2 and adipogenic induction(added mTOR protein specific inhibitor rapamycin)was completed when the cell fusion rate reached 100%.Compared with the control group,adipogenic differentiation was reduced under rapamycin treatment.The expression of PPAR ?,C/EBP ?,aP2 and Adipsin were down-regulated,the phosphorylation of mTOR protein and S6K1 protein were decreased,the process of Cyr61 siRNA promotes adipogenic differentiation was inhibited.This suggests that Cyr61 can inhibit the differentiation of adipocytes by suppressing the activity of the mTORC1 signaling pathway.Conclusion:1.Cyr61 inhibits the differentiation of C3H10T1/2 cells into adipocytes;2.Cyr61 regulates the differentiation of C3H10T1/2 cells into adipocytes by activating the classic Wnt/?-catenin signaling pathway and inhibiting the activity of the mTORC1 signaling pathway.