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The transcription factor, hypoxia-inducible factor (HIF), regulates the expression of genes involved in angiogenesis in response to hypoxia. In addition, HIF-1 expression and transcriptional targets of HIF, such as VEGF and PAI-1, are strongly upregulated following stimulation with IGF-1. However, it is unclear whether HIF is the major contributing transcription factor to IGF-1 induced VEGF expression. The aim of this study was to address the contribution of HIF-1α and HIF-2α to IGF-1 stimulated VEGF expression, using siRNA knockdown. In addition, we recently described that the oncoprotein, HDM2, is required for IGF-1 induced HIF-1α protein expression (Bardos et al., Mol Cell Biol 2004 24:2905-14). To further analyse the role of HDM2 in HIF transcriptional activity, we silenced HDM2 with siRNA and analysed its effects on VEGF levels following IGF-1 stimulation. HIF-1α, HIF-2α and HDM2 protein expression was concurrently induced by 6 hours in MCF-7 cells following treatment with IGF-1, and a corresponding 7-fold increase in VEGF levels as compared with unstimulated cells was observed. Gene silencing of HIF-1α resulted in a 50% inhibition of IGF-1 induced VEGF, whereas the contribution of HIF-2α to VEGF levels was minimal following IGF-1 stimulation. Knockdown of HDM2 resulted in a decrease of IGF-1-induced HIF-1α expression in MCF-7 cells as well as inhibition of constitutively expressed HIF-1α and HIF-2α in the VHL negative cell lines, RCC4 and 786-0 respectively. Interestingly however, no significant inhibition of IGF-1 induced VEGF was observed following knockdown of HDM2 in MCF-7 cells. These results indicate that HIF-1α is important for IGF-1 regulation of VEGF expression. In addition, HDM2 positively regulates both HIF-1α and HIF-2α protein expression. While HDM2 is required for IGF-1 induced expression of HIF-1α and HIF-2α, it is not essential for IGF-1 induced VEGF expression. Importantly, our data indicate that HDM2/HIF-independent pathways contribute to VEGF levels following IGF-1 induction.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]