Abstract
Recently, we have shown that transient phosphorylation and inhibition of the pro-apoptotic transcription factor, forkhead, by vascular endothelial growth factor (VEGF) is essential for endothelial cell (EC) survival and proliferation. The goal of the present study was to determine whether forkhead (FKHR) also plays a positive role in agonist-mediated gene induction. Human coronary artery ECs were transduced with adenovirus overexpressing constitutively active phosphorylation-resistant triple mutant FKHR or transfected with small interference RNA (siRNA) against FKHR. The cells were then treated in the absence or presence of VEGF and assayed for gene expression using quantitative real-time PCR and Northern blots analyses. The data revealed a novel set of VEGF-responsive genes that require FKHR activity for optimal expression in ECs, including bone morphogenic protein 2, cbp/p300-interacting transactivator 2, decay accelerating factor (DAF), vascular cell adhesion molecule-1 (VCAM-1), manganese superoxide dismutase, endothelial-specific molecule-1, RING1 and YY1-binding protein, and matrix metalloproteinase-10. Consistent with a positive role for FKHR in mediating VEGF induction of DAF and VCAM-1 mRNA, siRNA against FKHR attenuated the effect of VEGF on complement-mediated EC lysis and monocyte adhesion, respectively. VEGF induction of the forkhead-dependent genes was down-regulated by the NF-kappaB inhibitor, constitutively active Ad-IkappaB, and in some cases by the nuclear factor of activated T-cells (NF-AT) inhibitor, cyclosporin. Together, these findings suggest that the VEGF-forkhead signaling axis plays an important functional role in ECs beyond the regulation of cell survival/apoptosis and cell cycle.
Highlights
The mammalian members of the winged helix, or forkhead, transcription factors include FKHR (Foxo1), FKHRL1 (Foxo3a), and AFX (Foxo4)
Consistent with this paradigm, we have recently demonstrated that vascular endothelial growth factor (VEGF) signaling in endothelial cell (EC) results in PI3K-Aktdependent phosphorylation and nuclear exclusion of forkhead transcription factors, with subsequent down-regulation of the forkhead target gene, p27kip1, increased cell proliferation, and decreased apoptosis [19]
A positive role for forkhead in this pathway was evidenced by the observation that VEGF-mediated induction of manganese superoxide dismutase (Mn-SOD) was enhanced by inhibition of PI3K or Akt or by overexpression of triple mutant (TM)-FKHRL1 [38]
Summary
Cell Culture and Reagents—Human coronary artery ECs (HCAECs) were from Clonetics (San Diego, CA) and grown in endothelial growth medium-2-MV (EGM-2) BulletKit (Clonetics) at 37 °C and 5% CO2. Ad and siRNA are effective in serum-starvation medium containing 1% BSA, 250 l of mono- increasing and decreasing intracellular FKHR levels in ECs, clonal anti-endoglin (CD105) antibody (IgG2a) was added to respectively. The remaining HCAECs in the 24-well plate were repressible, forkhead-responsive transcripts, and 2) novel washed with Hanks’ balanced salt solution plus 1% BSA, and the VEGF-inducible, forkhead-responsive genes. After grown to conflu- demonstrated the usual forkhead response pattern observed ence, HCAECs were serum-starved in EBM-2 containing 0.5% in non-ECs, namely agonist (VEGF)-mediated inhibition of fetal bovine serum for 18 h, and treated with 50 ng/ml VEGF for expression, TM-FKHR-mediated induction of mRNA, and. The by FKHR siRNA, suggesting that VEGF requires FKHR for their adhesion intensities were measured with MetaMorph These genes include bone morphogenic ular Devices Corp., Sunnyvale, CA) and cell image analyzer protein 2 (BMP2), cbp/p300-interacting transactivator 2
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