Abstract
Purpose: :
Our previous work has shown that the transcription factor STAT3 is a critical modulator of the production of the permeability and angiogenic factor VEGF in response to hypoxic conditions or to elevated glucose levels. Here we investigated the specific relationship between activation of STAT3 and expression/stabilization of the hypoxic-inducible factor 1 alpha (HIF-1), a crucial regulator of VEGF expression, in endothelial cells exposed to hypoxic conditions.
Methods: :
Bovine retinal (REC) and human umbilical vein endothelial cells (HUVEC) were exposed to hypoxic conditions (pO2=2%±0.5) for various times (10’, 30’, 1h, 2h, 3h). Chromatin immunoprecipitation (ChIP) was conducted to determine STAT3 binding to the VEGF or HIF-1 promoter regions. Western analysis was used to assess expression levels of VEGF, HIF-1a and phospho-tyrosine 705-STAT3 (activated). Selective blockade of STAT3 activity was achieved by transfecting the cells with specific siRNA or by adenovirus-mediated transduction of STAT3 inactive mutants. Rac-1 inhibition was obtained by treatment of the cells with 75uM of Rac-1 inhibitor NSC23766. VEGF and HIF1 mRNA levels were assessed by quantitative RT-PCR.
Results: :
STAT3 phosphorylation and migration to the nuclear compartment occurred within 30 minutes after hypoxia stimulation of REC and HUVEC. ChIP analysis revealed that STAT3 binds the VEGF promoter at the same time. Selective inhibition of STAT3 activity by siRNA blocked hypoxia-induced VEGF mRNA and protein formation and also inhibited increases in HIF-1 protein content. In addition, blockade of the small G protein Rac-1, which we have shown to regulate hypoxia-induced STAT3 activation, also blunted hypoxia-induced HIF-1 protein expression. ChIP analysis confirmed these data by showing that hypoxia-stimulated STAT3 binds to the HIF-1 promoter.
Conclusions: :
The results obtained in this study show that hypoxia-stimulated VEGF production in endothelial cells requires Rac-1-dependent STAT3 activation. Furthermore, STAT3 induces VEGF expression via direct stimulation of the VEGF promoter and by inducing HIF-1 transcription.
Keywords: growth factors/growth factor receptors • hypoxia • vascular endothelial growth factor