Knockdown of glucocorticoid receptor expression by RNA interference promotes cell proliferation in murine macrophage RAW264.7 cells

Abstract

It is well documented that glucocorticoids (GC) promote arrest in the G1-S transition of the cell cycle in many cell types, resulting in a decrease in proliferation. However, the relationship between glucocorticoid receptor (GR) and the cell-cycle regulation remains unclear. Suppression of GR is important for exploring GR dependent processes. This study applied RNA interference targeting GR to the murine macrophage RAW264.7 cells. Transient transfection of the GR-siRNA expression vector reduced GR synthesis as measured on mRNA and protein level by RT-PCR and Western blot. GR-siRNA also depressesed GR transcriptional activity. A cell line [RAW-(GR−)] stably transfected with GR-siRNA expression vector was then established, the decreased GR level in this cell line was confirmed by Western blot. MTT assay showed RAW-(GR−) cells grew faster than control cells, which indicated that knockdown of GR promoted cell proliferation in macrophages. Further studies showed decreased p27 expression, increased PKC-α expression and enhanced basal and LPS-induced NF-κB activity in RAW-(GR−) cells as compared to the RAW-control cells. In contrast, virtually no change in p21, ERK1/2 and p38 expression was detected. In conclusion, these results indicate that GR itself is an inhibitor of cell proliferation in RAW264.7 cell line. This effect may be associated with the decreased expression of p27, the increased expression of PKC-α, and the activation of NF-κB. As all the experiments are carried out in GC free or serum-free medium, this study also shows the possibility for GR to have some constitutive functions, which are independent on GC activation.