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Updated: Aug 19, 2026
Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages
Published on: November 3, 2014
Knockdown of glucocorticoid receptor expression by RNA interference promotes cell proliferation in murine macrophage
Xiao-Yan Zhu1, Yu-Jian Liu, Jian Lu
1Department of Pathophysiology, Second Military Medical University, Shanghai 200433, PR China. zhuxiaoyan771101@sohu.com
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-alpha expression and enhanced basal and LPS-induced NF-kappaB 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-alpha, and the activation of NF-kappaB. 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.
Insights
Glucocorticoid receptor (GR) normally inhibits cell proliferation in macrophages. Suppressing GR enhances cell growth by affecting p27, PKC-alpha, and NF-kappaB pathways, revealing constitutive GR functions.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Glucocorticoids (GC) are known to inhibit cell proliferation by arresting the cell cycle.
- The precise role of the glucocorticoid receptor (GR) in cell-cycle regulation is not fully understood.
- Investigating GR-dependent processes requires effective methods to suppress GR expression.
Purpose of the Study:
- To elucidate the relationship between glucocorticoid receptor (GR) and cell-cycle regulation in macrophages.
- To determine the effect of GR suppression on macrophage proliferation and associated molecular pathways.
- To explore potential constitutive functions of GR independent of glucocorticoid activation.
Main Methods:
- Utilized RNA interference (RNAi) targeting GR in murine macrophage RAW264.7 cells.
- Established a stable cell line (RAW-(GR-)) with suppressed GR expression via GR-siRNA.
- Quantified GR levels using RT-PCR and Western blot; assessed cell proliferation via MTT assay; analyzed protein expression (p27, PKC-alpha, p21, ERK1/2, p38) and NF-kappaB activity.
Main Results:
- GR-siRNA effectively reduced GR mRNA, protein levels, and transcriptional activity.
- Knockdown of GR in RAW-(GR-) cells significantly increased cell proliferation compared to control cells.
- GR suppression led to decreased p27, increased PKC-alpha, and enhanced basal and LPS-induced NF-kappaB activity, with no significant changes in p21, ERK1/2, or p38.
Conclusions:
- The glucocorticoid receptor (GR) acts as an endogenous inhibitor of cell proliferation in RAW264.7 macrophages.
- GR's inhibitory effect on proliferation is linked to modulation of p27, PKC-alpha, and NF-kappaB signaling pathways.
- Experiments conducted in serum-free media suggest GR possesses constitutive functions independent of GC activation.
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