The Mechanisms And Biological Significance Of Small G Protein RhoB Expression Induced By Non-genotoxic Stress

RhoB is a member of the Rho family of small GTPases that regulates actin organization and vesicle trafficking. Our previous studies also found that glucocorticoid (GC) treatment induced the expressions of RhoB via glucocorticoid receptor (GR) in several human cancer cell lines, including ovarian cancer cell HO-8910 and Osteosarcoma cell HOS-8603. It is known to be up-regulated in response to DNA-damaging agents such as UV irradiation and chemotherapeutic drugs (e.g. cisplatin and 5-FU). The increased expression or activation of RhoB contributes to several important processes including apoptosis response to cellular stresses. Favre et al reported that RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling. Although RhoB is known to be up-regulated by genotoxic stress, little is known of the cellular consequences of the induction of RhoB in response to non-genotoxic stress such as trauma, hypoxia and heat stress. Our group had previously reported that GC induced RhoB expression in a dose- and time-dependent fashion via GR in several cell lines. Therefore, we inferred that non-genotoxic stress also could induce the expression of RhoB. In this paper, we investigated the expression change of RhoB in heat shock and hypoxia and explored the potential role and action mechanism of RhoB in heat stress.In this study we detected the mRNA and protein levels of RhoB in scalding rats or cells in heat stress by RT-PCR, Real-time PCR and Western blot. The expression of RhoB mRNA in livers began to increase at 2 hours after scalding, achieved a 7.5-fold increase at 4 hours, at which the level plateaued and began to decrease 8 hours later. In addition, the RhoB protein expression was also induced in a time-dependent manner and reached a maximum at 8 h after scalding. To determine whether the increased RhoB expression is liver-specific, the level of RhoB were also tested in the lung tissue after scalding. The up-regulation of RhoB mRNA and protein could also be observed in lung tissue, achieving a maximum at 4h (a 5.7-fold increase) and 8h (a 6.2-fold increase) respectively. These data demonstrated that heat stress induced RhoB expression in both mRNA and protein level in a time-dependent and non-tissue-specific manner. It has been documented that GC concentration was obviously increased during heat stress in vivo. Furthermore, GC has been proved to induce RhoB expression in our previous work. In order to test whether heat stress could induce RhoB expression directly, three cultured cell lines including human A549, HepG2 and mouse RAW264.7 cells were used. Heat stress also induced RhoB expression in these cells. Overall these data demonstrated that heat stress induced RhoB expression in both mRNA and protein level in a time-dependent manner in ex vivo systemPrevious 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 1.8-fold increase in the half-life of RhoB, which increased from 3.95h to 7.1h when A549 cells were treated by heat stress. Moreover, we got the similar results in RAW264.7 cells. Overall the data showed that RhoB up-regulation by heat stress resulted from an increase in RhoB mRNA expression mediated by post-transcriptional mechanisms.It has been shown that heat stress treatment leads to activation of p38, a member of the mitogen-activated protein kinases (MAPKs) family, although the mechanism is incompletely understood. To determine the possible involvement of p38 in heat stress induction of RhoB, the p38 specific inhibitor SB203580 was used. A549 cells and RAW264.7 cells were pretreated for 1h with SB203580 (10μM) and exposed to heat stress. The results showed that SB203580, which diminished phosphorylation of p38, block the induction of RhoB expression by scalding in both mRNA and protein levels. These results indicate that p38 contributes to heat stress induced RhoB expressionTo address the potential role of RhoB in heat stress (HS) response, we further investigate the consequence of RhoB up-regulation to the HS-induced apoptosis by specific down-regulation of protein expression using specific RNA interference and overexpression using transient transfection. Apoptotic cell death following heat stress was analyzed by Annexin V assays. The results showed that, upon the suppression of RhoB expression, HS-induced apoptosis was remarkably increased compared to mock cells (64.1% vs 49.5% for annexin-V~+ cells, P<0.01). Concomitantly, RhoB siRNA treatment also significantly enhanced caspase-3 activation. In parallel, ectopic expression of RhoB endowed A549 cells with a lower sensitivity to HS-induced apoptosis as estimated by annexin-V~+ cells % and cleavage of caspase-3. These results demonstrate that RhoB plays a crucial role in protecting cells from HS-induced apoptotic cell death and suggest that one of the consequences of RhoB up-regulation is to increase the survival of scalded cellsIn the next phase of analysis, we attempted to investigate the influence of HS-induced RhoB expression on heat shock protein (HSP) genes, which play an essential role in cell survival and apoptosis in response to stress. We analyzed the effect of RhoB overexpression and silencing on HSP70 expression during heat stress. HSP70 mRNA and protein are significantly induced by heat stress to the same extent in spite of ectopic expression or inhibited expression of RhoB.We also investigated the effect of RhoB on the basal transcriptional activity of the transcription factor NF-κB. Our results showed that the basal transcriptional activity of NF-κB was increased when A549 cells or RAW264.7 cells were transfected with wild type RhoB construct and decreased when RhoB expression was knockdowned by RNA interference.Finally, we detected the mRNA and protein levels of RhoB in hypoxia by RT-PCR, Real-time PCR and Western blot. The expression of RhoB mRNA and protein in spleen and lungs of hypoxic rats were up-regulated. Hypoxia stress also induced RhoB expression in human A549 cells and mouse macrophage RAW264.7 cells. Our results indicated that the mRNA and protein expressions of RhoB were induced by hypoxia in vivo and in vitro.In conclusion, these results demonstrated that RhoB was induced rapidly and remarkably by heat stress and hypoxia 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 inhibit the apoptosis induced by heat stress and eveleate the basal transcriptional activity of the transcription factor NF-κB.