Background To study the molecular mechanisms of CREG(the cellular repressor of E1A-stimulated gene) on proliferation of VSMCs in vitro.Methods The pRc/CMV-CREG plasmid or the pSM2-siCREG plasmid was transferred into h...Background To study the molecular mechanisms of CREG(the cellular repressor of E1A-stimulated gene) on proliferation of VSMCs in vitro.Methods The pRc/CMV-CREG plasmid or the pSM2-siCREG plasmid was transferred into human vascular smooth muscle cells(hVSMCs) to produce the cell clone that over-expression or down-expression of CREG respectively.BrdU assay and FACS cell cycle analysis were used to detect the proliferation of cells.Western blotting and immunocytochemistry show the expression and localization of IGF2R in hVSMCs.RT-PCR and ELISA assay determined the expression and secretion of IGFII factor. Alex488-labeled rhIGFII was used to investigate the endocysis of cells.And the blockade of IGFII internalization by treatment both the neutralized antibody of anti-IGF2R and rsIGF2R detected the effect of IGFII on VSMCs growth. Furthermore,Western blotting and signal pathway inhibitor were used to analysis the activation of PI3K/AKT and ERK on VSMCs proliferation.Results Western blotting identified that the expression of CREG in hVSMCs-CREG cells increased compared to control cell,and the decreased obviously in hVSMCs-siCREG cells.Meanwhile,the overexpression of CREG in cells was detected to inhibit the proliferation of VSMCs and to enhance the distribution of IGF2R in cellular membrane.Furthermore,overexpression of CREG also accelerated the endocysis of IGFII in hVSMCs-CREG,and attenuate the secretion of IGFII into cell medium by ELISA analysis and Alex488 labeled IGFII analysis.Blockade experiments both neutralized antibody of IGF2R and rhIGF2R fragment determined that enhancement of IGFII secretion promoted the VSMCs proliferation,and PI3K/AKT and ERK signal pathway mediated the effect of IGFII on VSMCs.Conclusions Altogether, these data indicate that CREG inhibits the proliferation of hVSMCs through interfering into the internalization pathway of IGF2R-IGFII.展开更多
文摘Background To study the molecular mechanisms of CREG(the cellular repressor of E1A-stimulated gene) on proliferation of VSMCs in vitro.Methods The pRc/CMV-CREG plasmid or the pSM2-siCREG plasmid was transferred into human vascular smooth muscle cells(hVSMCs) to produce the cell clone that over-expression or down-expression of CREG respectively.BrdU assay and FACS cell cycle analysis were used to detect the proliferation of cells.Western blotting and immunocytochemistry show the expression and localization of IGF2R in hVSMCs.RT-PCR and ELISA assay determined the expression and secretion of IGFII factor. Alex488-labeled rhIGFII was used to investigate the endocysis of cells.And the blockade of IGFII internalization by treatment both the neutralized antibody of anti-IGF2R and rsIGF2R detected the effect of IGFII on VSMCs growth. Furthermore,Western blotting and signal pathway inhibitor were used to analysis the activation of PI3K/AKT and ERK on VSMCs proliferation.Results Western blotting identified that the expression of CREG in hVSMCs-CREG cells increased compared to control cell,and the decreased obviously in hVSMCs-siCREG cells.Meanwhile,the overexpression of CREG in cells was detected to inhibit the proliferation of VSMCs and to enhance the distribution of IGF2R in cellular membrane.Furthermore,overexpression of CREG also accelerated the endocysis of IGFII in hVSMCs-CREG,and attenuate the secretion of IGFII into cell medium by ELISA analysis and Alex488 labeled IGFII analysis.Blockade experiments both neutralized antibody of IGF2R and rhIGF2R fragment determined that enhancement of IGFII secretion promoted the VSMCs proliferation,and PI3K/AKT and ERK signal pathway mediated the effect of IGFII on VSMCs.Conclusions Altogether, these data indicate that CREG inhibits the proliferation of hVSMCs through interfering into the internalization pathway of IGF2R-IGFII.
