Abstract 5615: Dual Role of the Small G Protein Rac-1 in the Regulation of Hypoxia-Induced STAT3 Activation in Endothelial Cells
The Signal Transducer and Activator of Transcription 3 (STAT3) is a critical regulator of Vascular Endothelial Growth factor (VEGF) expression and activity in cardiac muscle and vascular endothelial cells. We have previously shown that hypoxia-induced activation of STAT3 is a redox-dependent event and can be blocked by inhibitors of NAD(P)H oxidase activity. Here we investigated the specific role of the small G protein Rac-1 in the regulation of hypoxia-induced STAT3 activation in endothelial cells. Human umbilical vein endothelial cells (HUVEC) and human coronary endothelial cells in culture were subjected to 2% oxygen levels for different times (10min, 30min). Rac-1 inhibition was achieved by transfection of ECs with specific siRNA or by treatments of the cells with 75 μM of the Rac-1 inhibitor NSC23766 (EMD Biosciences). Chromatin immunoprecipitation (ChIP) was conducted to assess STAT3 transcriptional activity. STAT3 phosphorylation and protein-protein association was measured by Western blotting and immunoprecipitation analysis (respectively). We found that inhibition of Rac-1 activity by siRNA or by treatment with NSC23766 blocks STAT3 binding to VEGF promoter in hypoxic conditions compared to treatment of the cells with scramble siRNA or vehicle only. This effect correlated with blockade of hypoxia-induced STAT3 tyrosine phosphorylation at tyrosine 705 and at serine 727. In addition, hypoxia-stimulated Rac-1 binding to STAT3 at 30 minutes and inhibition of Rac-1 activity stimulated STAT3 binding to Protein Inhibitor of STAT3 (PIAS3) responsible for its sumoylation/inactivation. Taken together our results suggest that Rac-1 activity is associated with hypoxia-stimulated STAT3 activation and STAT3-dependent VEGF by regulating its phosphorylation/activation as well its sumoylation via blockade of STAT3 binding to PIAS3. These data disclose a previously uncovered activity of Rac-1 in the regulation of STAT3 inactivation by sumoylation.