1.The Molecular Mechanism Of Increased Proliferation In Murine Macrophage RAW264.7 Cells By Inhibition The Expression Of Glucocorticoid Receptor 2.Increased Expression Of RhoB By Non-genotoxic Stress And Its Biological Significance

I The molecular mechanism of increased proliferation in murine macrophageRAW264.7 cells by inhibition the expression of glucocorticoid receptor AbstractIt is well documented that glucocorticoid (GC) inhibit proliferation through glucocorticoid receptor (GR) in many cell types, but its molecular mechanism remains unclear. We inhibited the expression of GR by RNA interference in the murine macrophage RAW264.7 cells and established the cell line [RAW-(GR-)] which stably transfected with GR-siRNA expression vector. We found RAW-(GR-) cells grew much faster than RAW-vector cells which stably transfected with siRNA blank vector by cytometry and MTT assay. The results indicated that knockdown of GR expression promoted cell proliferation in macrophages. In this study, our purpose is to clarify the molecular mechanisms of enhanced proliferation of RAW-(GR-) cells.RhoB is a member of Rho GTPase family and SIRPal, also known as SHPS-1 (SHP substrate 1), is a member of the Ig-like immunoreceptor super family proteins. The levels of RhoB and SIRPal have been recently shown to dramatically decrease with aggressiveness of many tumors, ectopic expression of RhoB and SIRPal antagonizes proliferation, transformation and metastasis of tumor cells. So we assayed the expressions of mRNA and protein of RhoB and SIRPal by real time PCR and Western blot analysis, and observed that the levels of mRNA and protein of RhoB and SIRPal decreased remarkably in RAW-(GR-) cells as compared with RAW-vector cells.To verify the relationship between the decreased RhoB and increased proliferation, RAW-(GR-) cells were transiently transfected with bland vector, wild type RhoB plasmid (RhoB-wt) or constitutively activated RhoB plasmid (RhoB-V14), and we found that the proliferation of RAW-(GR-) cells decreased after transfection with RhoB-wt or RhoB-V14 plasmid compared to that of bland vector. The anti-proliferation function of RhoB was lost in RAW264.7 cells when blocked the expression of RhoB by RNA interference as well as blocked the activity of RhoB by transfection with the dominant negative mutant plasmid. Similarly, SIRPal overexpression also inhibited the proliferation of RAW-(GR-) cells by transfectionwith wild type SIRPal construct (SIRPal-wt). The data presented here demonstrated that down regulation of RhoB and SIRPal were one of the molecular mechanisms of increased proliferation of RAW-(GR-) cellsOur other studies showed that decreased expression of p27, increased expression of cyclinD1, cyclinBl and cdk2 were observed in RAW-(GR-) cells as compared to RAW-vector cells. In addition, virtually no change in expression of p21, phosphated and total ERK1/2 and p38 protein was detected.Knockdown GR expression by RNA interference promoted proliferation of RAW264.7 cells, but GC had no effect on proliferation of RAW264.7 cells, which showed the possibility for GR to function as negative proliferation modifier independent on GC.In conclusion, these results demonstrated that increased proliferation of RAW-(GR-) cells is at least partly due to decreased expression of RhoB and SIRPal. Decreased expression of p27, increased expression of cyclinDl, cyclinBl and cdk2 were also involved in the increased proliferation of RAW-(GR-) cells.