STAT3 is a potential modulator of HIF‐1‐mediated VEGF expression in human renal carcinoma cells

Abstract

SPECIFIC AIMS Aberrantly enhanced vascular endothelial growth factor (VEGF) gene expression is associated with increased tumor growth and metastatic spread of solid malignancies including human renal carcinomas. Persistent activation of STAT3 has been linked to tumor-associated angiogenesis. Underlying mechanisms, however, remain poorly understood. Therefore, we examined whether STAT3 modulates the stability and activity of hypoxia-inducible factor-1α (HIF-1α), and in turn enhances VEGF expression. PRINCIPAL FINDINGS 1. STAT3 is activated in ischemic rat kidney and in hypoxic renal carcinoma cells To investigate the activation of STAT3 by hypoxia, we examined the nuclear localization state of active STAT3 using immunoflorescence assays. We found the active forms of STAT3 in the nucleus under hypoxic stimulations. We found the tyrosine 705 phosphorylation of STAT3 in hypoxia-stimulated Caki I cells and obtained similar findings in rat kidneys that had been subjected to ischemia for 2 and 4 h. Therefore, we speculated that an oncogenic target gene could be induced by the phosphorylation of STAT3 in hypoxic tumor cells. 2. STAT3 increases the cellular levels of HIF-1α either by blocking protein degradation or by enhancing HIF-1α synthesis under hypoxic conditions To determine the relationship between HIF-1α and STAT3, we checked HIF-1α protein levels under STAT3-inhibited conditions. AG490 inhibited the expression of HIF-1α as well as the phosphorylation of STAT3 under hypoxic conditions (Fig. 1 ⤻ A). The HIF-1α expression was also decreased in cells transfected with dominant negative mutant STAT3YF (Fig. 1B⤻ ). These data indicate that STAT3 is involved in the hypoxic expression of HIF-1α protein and up-regulates the activity of HIF-1α. To test whether STAT3 increases the stability of HIF-1α protein, we measured the half-life of HIF-1α protein after blocking de novo protein synthesis with cycloheximide. Figure 1C⤻ shows that the half-life of HIF-1α was prolonged to 50 min in the presence of STAT3, whereas that in the absence of STAT3 was 25 min. This result suggests that STAT3 further enhanced HIF-1α protein stability under hypoxia. Figure 1. STAT3 increases the cellular levels of HIF-1α either by blocking protein degradation or by enhancing HIF-1α synthesis under hypoxic conditions. A) Caki I cells were pretreated for 1 h with 30 μM AG490, then were exposed to hypoxia (1% O₂) for 6 h. Total cell extracts were separated on 8% SDS-PAGE and subjected to Western blot assay using anti-phospho-STAT3 (Y705), anti-STAT3, anti-HIF-1α, and anti-β-actin antibodies. B) Caki I cells were transfected with dominant-negative form of STAT3, RcCMV-STAT3YF and incubated for an additional 24 h. Then these cells were exposed to hypoxia (1% O₂) for 6 h. Total cell extracts were separated on 8% SDS-PAGE and subjected to Western blot assay using anti-phospho-STAT3 (Y705), anti-STAT3, anti-HIF-1α, and anti-β-actin antibodies. C) COS7 cells were transfected with wild-type of STAT3 and the cells were incubated with 130 μM DFO, the cells were then incubated 60 μg/mL cycloheximide for the indicated time. Total cellular protein extracts were subjected to Western blot analysis to determine HIF-1α protein level. D) COS7 cells were transfected with wild-type of STAT3 and the cells were incubated with 20 μM MG132 for indicated time. Total cellular protein extracts were subjected to Western blot analysis to determine HIF-1α protein level. The expression level of wild-type of STAT3 was analyzed using anti-STAT3 antibody. Download figure Download PowerPoint We next checked the de novo protein synthesis of HIF-1α after blocking protein degradation with MG132, a proteasome inhibitor. Figure 1D⤻ shows that synthesis of HIF-1α in transfected cells with STAT3 were processed earlier by 2 h than transfected cells with an empty vector. Taken together, STAT3 seems to be a new regulator to enhance HIF-1α stability under hypoxia and to accelerate protein synthesis. 3. STAT3 interacts with HIF-1α and recruits on the human VEGF promoter in response to hypoxia In our studies, coexpression of STAT3, HIF-1α, and p300 remarkably increased the transcriptional activity of VEGF gene under hypoxia. Thus, we hypothesized that the VEGF expression is cooperatively regulated by HIF-1α and STAT3. To test this hypothesis, we first checked the interaction between HIF-1α and STAT3 using the coimmunoprecipitation method. As shown in Fig. 2 ⤻ A, the HIF-1α was coprecipitated with phospho-STAT3 in hypoxic cells, thus demonstrating the association between HIF-1α and phospho-STAT3 under hypoxia. To confirm whether this association is specific to activated STAT3 or not, we used dominant negative mutant of STAT3, STAT3YF expression plasmids. We found that HIF-1α did not bind unphosphorylated STAT3 in COS7 cells transfected with STAT3YF plasmid under hypoxia (Fig. 2A⤻ ). We found that cytokine stimulation also induced the association between STAT3 and HIF-1α. Phosphorylated STAT3 by IL-6 or LIF was associated with overexpressed HIF-1α (Fig. 2A⤻ ). These results demonstrate that association between STAT3 and HIF-1α is dependent on phosphorylation of STAT3. Figure 2. STAT3 interacts with HIF-1α and recruits on the human VEGF promoter in response to hypoxia. A) COS7 cells were transfected with STAT3WT, STAT3YF, or HIF-1α WT and then cells were exposed to hypoxia (1% O₂) for 6 h or treated 10 nM IL-6, LIF for 15 min. Cell extracts were prepared and immunoprecipitated with a anti-phospho-STAT3 specific...