The Mechanisms Of Small G Protein RhoB Expression Induced By Hypoxia And Glucocorticoid

Hypoxia is a component of several disease processes. The ensuing hypoxic response is an early pathophysiological manifestation of the macro-angiopathic complications that occur in, for example, mountain sickness, coronary heart disease, stroke. The cellular hypoxic response also has an important role in tumorigenesis. Glucocorticoids are steroid hormones secreted from the adrenal cortex as part of the hypothalamic pituitary adrenal axis. These hormones are released in response to diverse stimuli and play essential roles in regulating the stress response. The interplay between hypoxia and glucocorticoid-mediated cellular responses in physiology and disease has become increasingly appreciated over recent years. Release of glucocorticoids in response to atmospheric hypoxia associated with high altitude in humans has been extensively documented and prophylactic treatment with glucocorticoids has been used to attenuate the associated mountain sickness.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. Therefore, we inferred that hypoxia stress also could induce the expression of RhoB. In this paper, we investigated the expression change of RhoB in hypoxia stress and explored the potential role and action mechanism of RhoB in hypoxia stress.In this study, we putted adult male Sprague-Dawley rat in normal pressure hypoxia chamber filled with 8% O2 and 92% N2. Rats would stay in the cabin for 8 h,16 h,24 h, results showed that hypoxia 16 h RhoB expression in rat lungs increased 2.8 fold compared with control rats (p<0.05). In hypoxia condition, rats will synthesis more corticosterone. So we would make it clear that whether up-regulate expression of RhoB in rat lung was resulted by hypoxia or corticosterone. Rats underwent adrenalectomy, then treated by hypoxia, dexamethasone, both of them or stayed in normoxia conditions as control group, results showed that hypoxia, dexamethasone could up-regulate RhoB, there was a more obvious change when treated all together.Next, we observed the impact of hypoxia, dexamethasone on the expression of RhoB in A549 cell which can imitate typeⅡepithelial cell. A549 cells were cultured in an O2/CO2 controlled incubator adjusted to 1% O2,5% CO2, and balance N2 at 37℃for different time points, real-time PCR showed that RhoB mRNA was up-regulated slowly with prolonged hypoxia exposure time. We also tested the RhoB protein expression with different hypoxia exposure time, protein expression also up-regulated as the hypoxia time increased. We use CoCl2 treatment to imitate hypoxia and get similar results as hypoxic treatment. A549 cells were cultured in 100nM dex for different time points, results showed that the expression levels of RhoB were increased quickly after dexamethasone treatment, and decreased later. The protein expression of RhoB in A549 cells increased in a time-dependent way. The efficacy of glucocorticoid almost executed by glucocorticoid receptor, we use blocking agent RU486 to detect whether dexamethasone up-regulates RhoB through glucocorticoid receptor. As a result, RU486 could block RhoB expression induced by dexamethasone, thus GR is essential for the regulation of RhoB expression by glucocorticoids.We also treated A549 cells by hypoxia, glucocorticoid alone or together for 8 h, real-time PCR results showed that co-treatment had a better effect than treated by hypoxia, glucocorticoid alone. Protein expression displayed the corresponding results as mRNA.Previous studies showed that RhoB induction by genotoxic stress was mediated through induction of its promoter activity and through stabilization of its mRNA. After hypoxia stress, we observed that the decay rate of RhoB mRNA under actinomycin D treatment (a transcription inhibitor) was slowed down after hypoxia stress. There was a 1.5-fold increase in the half-life of RhoB mRNA, which increased from 3.28 h to 4.75 h when A549 cells were treated by hypoxia stress. Overall the data showed that RhoB up-regulation by hypoxia stress partly resulted from an increase in RhoB mRNA expression mediated by post-transcriptional mechanisms. And we also found that RhoB protein induction by hypoxia was mediated through stabilization of the protein.It has been shown that hypoxia stress treatment leads to activation of JNK, a member of the mitogen-activated protein kinases (MAPKs) family, although the mechanism is incompletely understood. To determine the possible involvement of JNK in hypoxia stress induction of RhoB, the JNK specific inhibitor SP600125 was used. A549 cells were pretreated for 1 h with SP600125 (10μM) and exposed to hypoxia stress. The results showed that SP600125, which diminished phosphorylation of JNK, block the induction of RhoB expression by hypoxia in protein levels. These results indicate that JNK contributes to hypoxia stress induced RhoB expressionIn conclusion, these results demonstrated that RhoB was induced rapidly and remarkably by hypoxia stress and dexamethasone both in vivo and intro. Hypoxia stress could stabilize RhoB mRNA and protein. Moreover, Activation of JNK participated in increased expression of RhoB induced by hypoxia stress.