Mechanisms And Biological Significance Of Small G Protein RhoB Expression Induced By Heat Stress

Cell stress is broadly defined to include cellular responses to heat shock, oxidative stress, heavy metals, and toxic chemicals. Heat stress is one of familiar cell stress, which is the total heat burden to which the body is subjected by both external and internal factors such scalding and fever. Small G protein RhoB, which is constitutively poorly expressed, has been reported as an immediate early inducible gene product formed in response to genotoxic stress caused by UV irradiation or alkylating agents and in response to growth factors. However, the effects of non-genotoxic stress on the expression of RhoB are not reported.In this study we detected the mRNA and protein levels of RhoB in scalding rats or cells in heat stress by RT-PCR and Western blot. The expression of RhoB mRNA and protein in livers and lungs of scalding rats were rapidly up-regulated. RhoB mRNA was about 3.2- fold of that in control rats at 4h and decreased at 8h after scalding. The expression of RhoB protein achieved maximum at 8h after scalding. Heat stress also induced RhoB expression in human prostate cancer cell line PC3 cells and mouse macrophage RAW264.7 cells. Our results indicated that the mRNA and protein expressions of RhoB were induced in time-dependent manner by heat stresses in vivo and in vitro.Previous studies showed that RhoB induction by genotoxic stress was mediated through induction of its promoter activity and through stabilization of its mRNA. After heat stress, we observed that the decay rate of RhoB mRNA under actinomycin D treatment (a transcription inhibitor) was slowed down after heat stress. There was a 1.9-fold increase in the half-life of RhoB, which increased from 3.98h to 7.6h when cells were treated by heat stress. Overall the data showed that RhoB up-regulation by heat stress resulted from a increase in RhoB mRNA expression mediated by post-transcriptional mechanisms.To get further insight into the pathway involved in RhoB expression, we studied signal pathways that might possibly be involved. In fact, it has been demonstrated that heat stress could induce an increase in p38 MAPK phosphorylation in vivo and in vitro. To test whether there was a causative correlation, heat stress-induced RhoB up-regulation was performed in the presence of the p38 MAPK-specific inhibitor SB203580 (10μM). The inhibitor decreased heat stress-induced RhoB protein expression. SB203580 itself, however, did not induce RhoB expression to a detectable level. Our result indicated that p38 MAPK activation correlated well with the increase in RhoB expression.Serum- and glucocorticoid-regulated protein kinase (SGK) was originally identified in a differential screen aimed at finding glucocorticoid-inducible transcripts, which correlated with the survival of cells. SGK is also induced in response to other stimuli, such as follicle-stimulating hormone, increased extracellular osmolarity, injury of the brain. We also found SGK mRNA could be induced by heat stress in RAW264.7 cells. Therefore, the effect of RhoB expression induced by heat stress on SGK mRNA expression was investigated. Our results showed that the expression of SGK mRNA was increased when RAW264.7 cells were transfected with wild type RhoB construct and decreased when RhoB expression was knockdowned by RNA interference, which indicated that increase in RhoB expression by heat stress played an important role in SGK expression. Moreover, we found up-regulation of RhoB expression had no effect on HSP70 expression in heat stress.In conclusion, these results demonstrated that RhoB was induced rapidly and remarkably by heat stress both in vivo and intro. Heat stress could stabilize RhoB mRNA. Moreover, Activation of p38 MAPK participated in increased expression of RhoB induced by heat stress. Up-regulation of RhoB expression could promote SGK mRNA expression but had no effect on HSP70 expression in heat stress.