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First published online 15 April 2003
doi: 10.1242/jcs.00427

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

Laboratoire de médecine moléculaire, Hôpital Sainte-Justine, Université du Québec à Montréal, CP 8888, Succursale centre-ville, Montréal, Québec, Canada H3C 3P8

View larger version (10K): [in a new window]   Fig. 1. ATP depletion and cell survival during hypoxia in renal cell carcinoma. Caki-1 cells were exposed to 1% O2 and ATP content was measured for up to 120 minutes using a luciferase assay (A). The cytotoxicity of hypoxia was measured by cleavage of WST-1 in formazan by spectrophotometry (B). ATP levels and cytotoxicity during hypoxia are represented as the percentage values relative to normoxia. Means ± s.e.m. are given for three separate experiments.

View larger version (26K): [in a new window]   Fig. 2. Upregulation of Rho GTPase protein expression by hypoxia in renal cell carcinoma. Caki-1 cells were exposed to 1% O2 from 0 to 6 hours. Post-nuclear supernatants (20 µg of protein) were separated by SDS-PAGE and submitted to immunodetection using Cdc42, Rac1, RhoA, RhoGDI and RhoB antibodies (A). The expression of these Rho GTPases was quantified by densitometric analysis and expressed as means ± s.e.m. for three separate experiments relative to normoxic values (B). Significant differences (P<0.05) from normoxia are indicated by asterisks (*).

View larger version (13K): [in a new window]   Fig. 3. Subcellular distribution of RhoA and Cdc42 proteins during hypoxia. After hypoxic treatment, renal cell carcinoma were fractionated into soluble and crude membrane fractions by centrifugation at 100,000 g for 1 hour. Each fraction (20 µg of protein) was separated by SDS-PAGE then immunodetected using Cdc42 (A) and RhoA antibodies (B). Densitometric analysis of both GTPases was carried out for soluble () and membrane () fractions. Means ± s.e.m. are shown from three independent experiments relative to samples from normoxic cells. Significant differences (P<0.05) from normoxia are indicated by asterisks (*).

View larger version (24K): [in a new window]   Fig. 4. Metabolic labelling of RhoA during hypoxia is localized to membranes. Caki-1 cells were pre-incubated in cystein and methionine free medium for 1 hour, then incubated with 50 µCi/ml of [35S] Met/Cys. Subsequently, cells under normoxia or hypoxia at 1% O2 for 4 hours were fractionated in soluble fractions (S) and crude membranes (M). RhoA was immunoprecipitated, analyzed by SDS-PAGE, and labelled RhoA detected by autoradiography (A). As a control, Caki-1 cells were exposed to normoxia or hypoxia for 4 hours, fractionated in soluble and membrane fractions, then analyzed by western blot and immunodetected with RhoA antibody (A). Densitometric analysis of immunoprecipitated RhoA from cells under normoxia () and hypoxia () conditions is represented (B). Means ± s.e.m. are shown for two different experiments relative to normoxia. Significant differences (P<0.05) from normoxia are indicated by asterisks (*).

View larger version (33K): [in a new window]   Fig. 5. Cdc42 and RhoA activation in Caki-1 cells exposed to 1% O2. Activation states of Cdc42 and RhoA proteins were measured by affinity precipitation with GST-PBD and GST-RBD, respectively. Cells were exposed to 1% O2 from 0 to 6 hours, lysed and incubated with 20 µg fusion proteins. After pull-down precipitation, the GTP-bound forms of Cdc42 and RhoA proteins were analyzed by SDS-PAGE and western blot. Parallel experiments were performed to assess total amounts of Rho GTPases under these conditions. Cdc42 (A) and RhoA (B) were immunodetected with appropriate antibodies. Means ± s.e.m. from densitometric analysis of active GTPases for three independent experiments are shown. Data are expressed as the percentage of relative activity compared with values found in normoxic cells. Significant differences (P<0.05) from normoxia are indicated by asterisks (*).

View larger version (29K): [in a new window]   Fig. 6. Effect of hypoxia on RhoA and Cdc42 mRNA levels in renal cell carcinoma. Total RNA was isolated from Caki-1 cells in normoxia and hypoxia. RT-PCR amplification was performed using primers for RhoA for 15 to 30 cycles in order to define optimal conditions (A). At 25 cycles, RT-PCR analysis for RhoA (183 bp), Cdc42 (400 bp), and -tubulin (321 bp) were carried out on total RNA isolated from cells incubated under hypoxia for up to three hours (B).

View larger version (15K): [in a new window]   Fig. 7. Hypoxia increases ROS production in renal cell carcinoma. Caki-1 cells were incubated with 10 µM DCFH in hypoxic conditions for 0 to 240 minutes. Production of ROS was measured by fluorescence emitted from oxidized DCF. After hypoxia, cells were lyzed and fluorescence was measured at 530 nm (A). To inhibit ROS production, cells were preincubated with 10 uM DPI then exposed to hypoxic or normoxic conditions for 1 hour (B). Fluorescence data are represented relative to normoxic values. Means ± s.e.m. were calculated from three independent experiments. Significant differences (P<0.1) are indicated by asterisks (*).

View larger version (12K): [in a new window]   Fig. 8. Effect of DPI on overexpression of Rho GTPases in hypoxic conditions. Caki-1 cells were pre-treated with 10 µM DPI in normoxia. Cells were then incubated in hypoxic conditions from 0 to 4 hours. Cell lysates (20 µg of protein) were separated by SDS-PAGE, followed by immunodetection. Densitometric analysis of Cdc42 (A) and RhoA (B) expression are presented for cells in hypoxia () and cells in hypoxia plus DPI (). Means ± s.e.m. for three independent experiments are indicated. Data are expressed relative to normoxic values. Significant differences (P<0.05) from normoxia are indicated by asterisks (*).

View larger version (51K): [in a new window]   Fig. 9. HIF-1 and VEGF mRNA expression during hypoxia. Amplification of HIF-1 (487 bp) and VEGF for 15 to 30 cycles was carried out by RT-PCR to determine the linear portion of the amplification reaction (A). Two isoforms of VEGF mRNA were amplified, VEGF165 (627 bp) and VEGF189 (699 bp). Caki-1 cells were exposed to 1% O2 and total RNA was isolated. RT-PCR analysis of HIF-1 at 25 cycles and VEGF at 30 cycles using total RNA from Caki-1 cells under kinetic of hypoxia is shown (B). mRNA -tubulin (321 bp) was used as a control. HIF1-a protein expression was analyzed by its immunoprecipitation from nuclei and immunodectection (C). Two independent experiments were performed.

View larger version (15K): [in a new window]   Fig. 10. RhoA activates HIF-1 and VEGF mRNA levels in normoxia. Caki-1 cells were transfected or not with vector alone (pcDNA3) or with dominant-active RhoA (pcDNA3-RhoAV14-Myc) for 24 hours using lipofectamine. Total RNA was isolated then HIF-1 (A), VEGF (B) and -tubulin (C) mRNA expressions were evaluated by RT-PCR. Two isoforms of VEGF were amplified, VEGF165 and VEGF189. Densitometric analysis was used to assess relative mRNA expression levels. Means ± s.e.m. from two experiments and values are plotted relative to values from normoxic cells from the sum of both isoforms is presented on the histograms. Significant differences (P<0.05) from normoxia are indicated by asterisks (*).

View larger version (18K): [in a new window]   Fig. 11. Exotoxin C3 blocks HIF-1 mRNA and protein overexpression and VEGF mRNA stimulation. Cells were pre-treated with 50 µg/ml of C3 toxin for 24 hours in normoxia. After this, cells were placed in hypoxia (H) or kept in normoxia (N) for 4 hours. Cells were fractionated into soluble and membrane fractions and proteins were separated by SDS-PAGE. Efficiency of ADP-ribosylation by C3 toxin was verified by immunodetection of RhoA, as the ADP-ribosylation reduces protein mobility (A). Alternatively, following incubation with the toxin, cells were exposed to hypoxia or in normoxia for 4 hours and total RNA isolated. The effects of the toxin were evaluated on mRNA levels (B) of HIF-1, VEGF and -tubulin as the negative control, and on protein expression (C) of HIF-1 and -tubulin by RT-PCR or western blot analysis. These experiments were carried out at least twice. Significant differences (P<0.05) from normoxia are indicated by asterisks (*).

View larger version (13K): [in a new window]   Fig. 12. Model for Rho-dependent HIF-1 and angiogenesis activation during hypoxia. This schema summarizes the main steps that we have identified to play crucial roles in the induction of HIF-1 mediated by Rho GTPases. The involvement of ROS production was abolished by DPI and prevented Cdc42 and RhoA induction. Also, the C3 toxin that abolished RhoA expression prevented HIF-1 induction under hypoxic conditions. The kinetics demonstrated that Cdc42, Rac1 and RhoA are upregulated sequentially by hypoxia but it remains to be established whether there is a hierarchy of activation in Rho GTPase cascades (dotted arrows).