ABSTRACT
We previously reported that glucocorticoid receptor (GR) blockade (injected with GR antagonist RU486) primed the host responses to lipopolysaccharide. Since decrease of GR and elevated glucocorticoids (GCs) have been always reported as parallel responses, we hypothesize that both GCs and GR play important roles in GR blockade induced priming. We first confirm that the production of nitric oxide (NO), superoxide (O2-), and PKCalpha expression are all increased in peritoneal macrophages from GR blockade rats, indicating that macrophages are primed by GR blockade. Furthermore, using unilateral adrenalectomy rats, we find that the elevated GCs caused by a feedback mechanism following GR blockade may be involved in the process of priming. In vitro experiments in RAW264.7 cells show the inhibitory effect of GCs on NO production, which can be thoroughly blocked by RU486, indicating the increase of NO production in GR blockade rats is due to the elimination of GCs's anti-inflammatory function. In contrast, 10(-7) M corticosterone induces significant increases in O2- release, PKCalpha expression and phosphorylation, which cannot be reversed by RU486, demonstrating a previously unrecognized pro-inflammatory role of GCs in enhancing PM activation through a GR-independent pathway. The effect of GCs on PKCalpha expression even exists in GR deficient COS-7 cells as well as in GR knock-down RAW264.7 cells. In conclusion, both GR impairment and elevation of GCs are involved in the priming of macrophages caused by GR blockade. The findings of the divergent roles of GCs in modulation of inflammation may change therapeutic strategy for inflammatory diseases with GCs.
Subject(s)
Glucocorticoids/physiology , Macrophage Activation/drug effects , Macrophages/drug effects , Mifepristone/pharmacology , Receptors, Glucocorticoid/antagonists & inhibitors , Receptors, Glucocorticoid/physiology , Adrenalectomy , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Gene Expression Regulation, Enzymologic , Glucocorticoids/pharmacology , Male , Mice , Oxygen/metabolism , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Macrophages/cytology , Macrophages/metabolism , RNA Interference , Receptors, Glucocorticoid/deficiency , Receptors, Glucocorticoid/genetics , Animals , Cell Cycle Proteins/metabolism , Cell Line , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Lipopolysaccharides/pharmacology , MAP Kinase Signaling System , Macrophages/drug effects , Mice , NF-kappa B/metabolism , Protein Kinase C/metabolism , Protein Kinase C-alpha , Receptors, Glucocorticoid/metabolism , Transcription, Genetic/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
On the base of a cDNA fragment cloned from a human osteosarcoma cell line (HOS-8603) which was taken as the model of heat shock by DDRT-PCR, the cDNA library was screened by the probe made from this fragment, and the colony containing full length cDNA of this cDNA fragment (HSSG-1) was identified. Sequencing data indicated that this cDNA consisted of 1 456 bp, coding for 276 amino acids. Computer aided homology analysis did not find the same sequence published. The result of RNA dot hybridization suggested that HSSG-1 was expressing in many kinds of organs and cells, and the down regulating of its expression level caused by heat shock was general. Therefore, this may be a gene that is suppressed by heat shock.
