HIF-1α mRNA and protein upregulation involves Rho GTPase expression during hypoxia in renal cell carcinoma

Abstract

The small G proteins of the Rho family are involved in reorganization of the actin cytoskeleton, cell migration and in the regulation of gene transcription. Hypoxia-induced ATP depletion results in the disruption of actin organization which could affect Rho functions. In solid tumors, regions with low oxygen tension stimulate angiogenesis in order to increase oxygen and nutrient supply. This process is mediated by stabilization of the transcriptional factor hypoxia inducible factor 1 (HIF-1), which increases vascular endothelial growth factor (VEGF) production. In this study, we investigated the activities of Rho proteins, which are key regulators of cytoskeleton organization during hypoxia in renal cell carcinoma. Caki-1 cells were exposed to hypoxia (1% O2) and exhibited increased Cdc42, Rac1 and RhoA protein expression. Immunoprecipitation of metabolically labelled RhoA showed that overexpression was at least due to neo-synthesis. The Rho GTPases overexpressed during hypoxia were mainly located at membranes and pull-down assays demonstrated that they were active since they bound GTP. RT-PCR analysis indicated that the increase in RhoA protein expression was also reflected at the mRNA level. Overexpression and activation of Rho proteins were downstream of, and dependent on, the production of reactive oxygen species (ROS) since, in the presence of an inhibitor, both the rise of ROS and upregulation of Rho proteins were abolished. Importantly,preincubation of cells with the toxin C3, which inhibits RhoA, reduced HIF-1α protein accumulation by 84% during hypoxia. Together, these results support a model where ROS upregulate Rho protein expression and where active RhoA is required for HIF-1α accumulation during hypoxia.

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

“…RhoA plays a critical role in cell motility and has been shown to be induced by hypoxia in mouse embryonic fibroblasts (34) and in renal cancer cells (35) previously. Turcotte et al (35) have speculated that intracellular ATP depletion may be involved in hypoxia-induced RhoA activation. The described hypoxia-induced activation of RhoA is in line with our findings of increased p-Cof1 levels in hypoxia.…”

“…Phosphorylation of Cofilin Depends on PHD2 Hydroxylation Activity-RhoA has been shown to be induced by hypoxia in mouse embryonic fibroblasts (34) and in renal cancer cells (35) previously. Exposing HeLa cells to hypoxia resulted in increased p-Cof1 levels in our study (Fig.…”

von Hippel‐Lindau tumor suppressor protein stimulation by thrombin involves RhoA activation

“…Unlike hypoxia, which increases both HIF-1␣ and VHL mRNA and protein expression in renal Caki-1 cells, 10,31 thrombin and other growth factors tested do not stimulate HIF-1␣ protein expression in these cells. The lack of thrombin effect on HIF-1␣ protein expression differs from results obtained in previous studies in vascular smooth muscle cells (VSMC), which showed that HIF-1␣ may be induced by thrombin or by platelet-associated growth factor.…”

“…We have previously reported that hypoxia increased pVHL expression and that this upregulation was RhoA/ Rho kinase dependent in Caki-1 cells. 31 To determine whether Rho kinase was also involved in thrombin-induced pVHL, pretreatment with the pharmacological Rho kinase inhibitor Y27632 was performed. Once again, thrombin increased pVHL expression but the Rho kinase inhibitor did not significantly prevent pVHL expression in these cells (Fig.…”

Overexpression of Csk-binding protein contributes to renal cell carcinogenesis

“…Previous studies have implicated a role of RhoA in RCC carcinogenesis. For instance, active RhoA was required for the accumulation of HIF1a, one of the most important regulators for RCC (Klatte et al, 2007), in trophoblast (Hayashi et al, 2005) and RCC cells (Turcotte et al, 2003) under hypoxia condition. By promoting RhoA activation, overexpressed plasma membrane associated sialidase could increase RCC cell motility (Ueno et al, 2006).…”