The Mechanism Of ColXV On Mice Adipocytes Apoptosis Via Extracelluar Matrix Remodeling

Collagen XV?ColXV?is a member of the MULTIPLEXINs collagen subclass consisting of three ColXV?1 chain trimers in the outermost layer of the basement membrane.ColXV plays an important role in the maintenance of skeletal muscle,heart,skin microvessels,ocular vitreous structure and functions,and regulation of tumor cell adhesion,migration by interacting with a variety of extracellular matrix proteins.The increase in the number and volume of individual cell during obesity are dependent on the dynamic changes in the height of the extracellular matrix of the adipocytes.The extensive deposition of collagen causes necrosis and apoptosis.The study found that lacking Col VI in adipose tissue of obese mice,accompanying increased adipocytes volume can significantly reduce cell death and improve systemic insulin resistance?Khan et al.,2009?.The role of ColXV as an important component to link adipocytes basement membrane and extracellular matrix in regulating the occurrence of adipocytes extracellular matrix remodeling and adipocyte apoptosis remains unclear.The aim of this study was to determine the molecular mechanism of ColXV-mediated apoptosis of adipocytes,extracellular matrix remodeling of adipocytes,and the interaction of them.The following results were obtained:1.Col XV promotes adipocytes apoptosis through the AMPK/mTORC1/S6K1 pathway.Overexpression of ColXV in 3T3-L1 cells significantly promoted the expression of Caspase-3,Caspase-9 and Bax,inhibited the expression of Bcl-2?P<0.05?.The effect is the opposite after interference with ColXV.ColXV treatment significantly increased the number of apoptotic adipocytes at early and late stages and promoted the fragmentation of chromatin DNA by Hoechst staining,Annexin V-FITC/PI staining and TUNEL staining respectively?P<0.05?.In the serum-starved and palmitic-induced apoptosis model,Col XV further promoted the mRNA and protein expression of Caspase-3?Cleaved Caspase-3?,Caspase-9?Cleaved Caspase-9?and Bax,and inhibited mRNA and protein levels of Bcl-2?P<0.05?.Overexpression of ColXV promoted the phosphorylation of AMPK(p-AMPKA?1^Thr183?p-AMPKA?2^Thr172)and inhibited the phosphorylation levels of downstream mTORC1(mTORC1^Ser2448)and S6K1(S6K1^Thr389)?P<0.05?.The effect is the opposite after interference with ColXV.At the same time,after adding the inhibitor Rapamycin?Rapa?of mTORC1,overexpression of ColXV significantly inhibited the phosphorylation of mTORC1(mTORC1^Ser2448)and its downstream target S6K1(S6K1^Thr389)?P<0.05?.It suggested that ColXV induces apoptosis in adipocytes by promoting the AMPK/mTORC1/S6K1 pathway.2.ColXV Regulates Apoptosis Induced by Extracellular Matrix Remodeling via FGF2/FGFR1 Signaling.The effects of ColXV on ECM remodeling in adipose tissue were examined at different treatments?normal levels,MMPs inhibitors Ilomastat and Agonist APMA,respectively?.ColXV significantly increased mRNA and protein expression of ECM components such as Col I,Col VI,fibronectin and matrix metalloproteinase inhibitory enzymes TIMP-1 and TIMP-2.ColXV simultaneously inhibited the expression of matrix metalloproteinases?MMP-2 and MMP-9??P<0.05?.The effect was opposite when adipocytes were interfered with ColXV.At the same time,ColXV exacerbate collagen deposition under Ilomastat treatment and relieve APMA-induced degradation in the extracellular matrix of adipocytes.After the adipose tissue was decellularized,the effect of ColXV on ECM remodeling was verified by confocal microscopy on Z-Stack scanning and three-dimensional reconstruction.ColXV reduced the accumulation and binding of MMP-9 and MMP-2 to extracellular ECM components.In addition,overexpression of ColXV inhibited the enrichment of FGF2 on its receptor FGFR1,significantly inhibited the phosphorylation of FGFR1^{?Tyr653/Tyr654?}and downstream targets ERK1/2 and AKT?P<0.05?.Moreover,ColXV reversed the FGF2-induced collagen deposition accompany with increased Col I,TIMP-1 and decreased MMP-2 and MMP-9.Our study shows that ColXV induces remodeling of adipose tissue extracellular matrix and further promotes adipocytes apoptosis by modulating MMP2/TIMPs balance in extracellular matrix and inhibiting FGF2/FGFR1 signaling.