HIF-1alpha mRNA and protein upregulation involves Rho GTPase expression during hypoxia in renal cell carcinoma.
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-1alpha 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-1alpha accumulation during hypoxia.
Turcotte
Sandra
S
Laboratoire de médecine moléculaire, Hôpital Sainte-Justine, Université du Québec à Montréal, CP 8888, Succursale centre-ville, Montréal, Québec, Canada H3C 3P8.
Desrosiers
Richard R
RR
Béliveau
Richard
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D001905:Botulinum Toxins / Q000494:pharmacology; D002292:Carcinoma, Renal Cell; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007680:Kidney Neoplasms; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D020764:cdc42 GTP-Binding Protein / Q000235:genetics; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000378:metabolism; D000075024:rhoC GTP-Binding Protein
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D000255:Adenosine Triphosphate; D001905:Botulinum Toxins; C066690:RHOC protein, human; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein