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

Joo Eun Jung^*, Hyun Gyu Lee, Ik Hyun Cho, Doo Hyun Chung, Sun-Hee Yoon^*, Young Mok Yang, Jung Weon Lee, Seongwon Choi^*, Jong-Wan Park^*, Sang-Kyu Ye^*,1 and Myung-Hee Chung^*

Departments of
^* Pharmacology and
Pathology, Seoul National University College of Medicine, Seoul, Korea;
Department of Physiology, College of Dentistry and Dental Research Institute, Seoul National University, Seoul, Korea;
Department of Premedical Science, College of Medicine, and Bio-Food and Drug Research Center, Konkuk University, Chungju, Korea;
Cancer Research Institute, Department of Tumor Biology, Seoul National University College of Medicine, Seoul, Korea

¹Correspondence: Department of Pharmacology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. E-mail: sangkyu{at}snu.ac.kr

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-1 (HIF-1), 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-1 either by blocking protein degradation or by enhancing HIF-1 synthesis under hypoxic conditions
To determine the relationship between HIF-1 and STAT3, we checked HIF-1 protein levels under STAT3-inhibited conditions. AG490 inhibited the expression of HIF-1 as well as the phosphorylation of STAT3 under hypoxic conditions (Fig. 1 A). The HIF-1 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-1 protein and up-regulates the activity of HIF-1. To test whether STAT3 increases the stability of HIF-1 protein, we measured the half-life of HIF-1 protein after blocking de novo protein synthesis with cycloheximide. Figure 1C shows that the half-life of HIF-1 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-1 protein stability under hypoxia.

View larger version (47K): [in this window] [in a new window]   Figure 1. STAT3 increases the cellular levels of HIF-1 either by blocking protein degradation or by enhancing HIF-1 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-1, and anti-ß-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-1, and anti-ß-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-1 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-1 protein level. The expression level of wild-type of STAT3 was analyzed using anti-STAT3 antibody.

We next checked the de novo protein synthesis of HIF-1 after blocking protein degradation with MG132, a proteasome inhibitor. Figure 1D shows that synthesis of HIF-1 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-1 stability under hypoxia and to accelerate protein synthesis.

3. STAT3 interacts with HIF-1 and recruits on the human VEGF promoter in response to hypoxia
In our studies, coexpression of STAT3, HIF-1, and p300 remarkably increased the transcriptional activity of VEGF gene under hypoxia. Thus, we hypothesized that the VEGF expression is cooperatively regulated by HIF-1 and STAT3. To test this hypothesis, we first checked the interaction between HIF-1 and STAT3 using the coimmunoprecipitation method. As shown in Fig. 2 A, the HIF-1 was coprecipitated with phospho-STAT3 in hypoxic cells, thus demonstrating the association between HIF-1 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-1 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-1. Phosphorylated STAT3 by IL-6 or LIF was associated with overexpressed HIF-1 (Fig. 2A ). These results demonstrate that association between STAT3 and HIF-1 is dependent on phosphorylation of STAT3.

View larger version (36K): [in this window] [in a new window]   Figure 2. STAT3 interacts with HIF-1 and recruits on the human VEGF promoter in response to hypoxia. A) COS7 cells were transfected with STAT3WT, STAT3YF, or HIF-1 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 antibody: immunoprecipitates were developed on Western blots with a HIF-1 specific antibody. The expression level of wild-type of STAT3 was analyzed using anti-STAT3 antibody. B) COS7 cells were transfected with series of deletion constructs of GFP-HIF-1, STAT3WT, then cells were exposed to hypoxia (1% O2) for 6 h. Cell extracts were prepared and immunoprecipitated with an anti-phospho-STAT3 specific antibody: immunoprecipitates were developed on Western blots with an anti-GFP specific antibody. The expression level of wild-type of STAT3 was analyzed using anti-STAT3 antibody. C) DNA binding activity of STAT3 in Caki I cells exposed to hypoxia (1% O2) for 6 h detected by EMSA. Nuclear extracts were prepared and incubated in the absence or presence of anti-STAT3 antibody in combination with a 32P-labeled STAT3 oligonucleotide probe corresponding to the putative binding site in human VEGF promoter. D) ChIP assay, soluble chromatin was prepared from Caki I cells exposed to hypoxia (1% O2) for 6 h and immunoprecipitated anti-STAT3, anti- HIF-1, anti-p300, and anti-H3 acetylation antibodies. The final DNA extractions were amplified using pairs of primers that cover putative STAT3 binding site of the human VEGF promoter.

To clarify the binding site of HIF-1 for the association with STAT3, we performed coimmunoprecipitation assays using a series of deletion constructs of HIF-1. Results shown in Fig. 2B indicate that 577-826 regions of HIF-1 were captured by the interaction with STAT3, whereas the amino acids 1-400 and including ODD domain of HIF-1 were not interacted. The coincidence of the putative STAT binding motif (5'-ATCCCTGGACACTTCCCAAAGGAC-3') in the human VEGF promoter led us to the hypothesis that STAT3 binds to this motif and induces transcription of the VEGF gene. Hypoxia induced STAT3 DNA binding activity on the motif, and these STAT3-DNA complexes were supershifted with anti-STAT3 antibody (Fig. 2C ).

To test whether STAT3 in association with HIF-1 recruit to the human VEGF promoter, we performed ChIP assays on chromatin samples from normoxic and hypoxic cells. Significantly increased binding of STAT3 to the promoter was seen in the hypoxia (Fig. 2D ). The binding of HIF-1 and p300 to the promoter was also significantly enhanced under hypoxic conditions (Fig. 2D ). It is well known that p300 binds both STAT3 and HIF-1. Our data suggest that STAT3 should be considered as a new regulator of HIF-1 activity by participating in the transcriptional unit with HIF-1 and p300, which leads to hypoxia-mediated transcriptional activation of HIF-1 target genes such as VEGF (Fig. 3 ).

View larger version (24K): [in this window] [in a new window]   Figure 3. A model for the mechanism on transcription of human VEGF gene by STAT3 associated with HIF-1 under hypoxia. Hypoxic-activated STAT3 increases HIF-1 protein stability and binds to the human VEGF promoter. STAT3 should be considered as a new regulator of HIF-1 activity by participating in the transcriptional unit with HIF-1 and p300, which leads to hypoxia-mediated transcriptional activation of HIF-1 target genes such as VEGF.

CONCLUSIONS AND SIGNIFICANCE

In the present study, we demonstrate that STAT3 is a novel and essential component of HIF-1 regulatory machinery through associating with HIF-1 under hypoxic conditions. To our best knowledge, it is the first finding that hypoxia-phosphorylated STAT3 directly binds HIF-1 and up-regulates HIF-1 stability through delaying protein degradation and accelerating protein synthesis in human renal carcinoma cells.

We found that hypoxia-induced VEGF expression was significantly blocked by STAT3 inhibition. It is well known that HIF-1 is a transcription factor that has been shown to be essential for VEGF transcriptional activation. However, our results demonstrate that STAT3 is a new regulator of VEGF expression under hypoxic conditions and hypoxia-mediated VEGF expression requires binding of both STAT3 and HIF-1 to the VEGF promoter for maximum induction in renal carcinoma cells.

To clarify a molecular mechanism of the cooperation between STAT3 and HIF-1 on VEGF expression, we examined the recruitment of STAT3, HIF-1, and p300 on the human VEGF promoter under hypoxic conditions. We found that STAT3, HIF-1, and p300 all recruit on the human VEGF promoter under hypoxic conditions in human renal carcinoma cells. It was reported that STAT3 could interact with p300, thus modulating a subset of p300 activities. It was also reported that the recruitment of CBP/p300 is mediated by Asn-803 C-terminal transactivation domain in HIF-1. Our results demonstrate that the amino acids 577–826 regions, C-terminal domain of HIF-1 are interacted with STAT3. The p300 may play a role of linker protein between STAT3 and HIF-1 forming multicomplex for transcriptional regulation on VEGF promoter under hypoxia. In our data, coexpression with STAT3, HIF-1, and p300 remarkably increase the transcriptional activity of VEGF gene under hypoxia. This may represents the full activation of VEGF promoter by transcriptional unit forming complex STAT3/ HIF-1/ p300.

Based on these findings, blocking STAT3 is expected to inhibit the VEGF up-regulation that is induced by many oncoproteins and growth signal in solid tumor cells. Our findings suggest a novel function of STAT3 as a new regulator of hypoxia-induced angiogenesis in human renal carcinoma. Therefore, STAT3 inhibition can be an application for clinical therapy in patients with solid tumors by reducing HIF-1 protein induction and VEGF expression.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3099fje

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