Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

Background: Hypoxia-induced mitogenic factor (HIMF), a lung-specific growth factor, promotes vascular tubule formation in a matrigel plug model. We initially found that HIMF enhances vascular endothelial growth factor ( VEGF) expression in lung epithelial cells. In present work, we tested whether HIMF modulates expression of fetal liver kinase-1 (Flk-1) in endothelial cells, and dissected the possible signaling pathways that link HIMF to Flk-1 upregulation. Methods: Recombinant HIMF protein was intratracheally instilled into adult mouse lungs, Flk-1 expression was examined by immunohistochemistry and Western blot. The promoter-luciferase reporter assay and real-time RT-PCR were performed to examine the effects of HIMF on Flk-1 expression in mouse endothelial cell line SVEC 4-10. The activation of NF-kappa B (NF-kappa B) and phosphorylation of Akt, IKK, and I kappa B alpha were examined by luciferase assay and Western blot, respectively. Results: Intratracheal instillation of HIMF protein resulted in a significant increase of Flk-1 production in lung tissues. Stimulation of SVEC 4 - 10 cells by HIMF resulted in increased phosphorylation of IKK and I kappa B alpha, leading to activation of NF-kappa B. Blocking NF-kappa B signaling pathway by dominant-negative mutants of IKK and I kappa B alpha suppressed HIMF-induced Flk-1 upregulation. Mutation or deletion of NF-kappa B binding site within Flk-1 promoter also abolished HIMF-induced Flk-1 expression in SVEC 4 - 10 cells. Furthermore, HIMF strongly induced phosphorylation of Akt. A dominant-negative mutant of PI-3K, Delta p85, as well as PI-3K inhibitor LY294002, blocked HIMF-induced NF-kappa B activation and attenuated Flk-1 production. Conclusion: These results suggest that HIMF upregulates Flk-1 expression in endothelial cells in a PI-3K/ Akt-NF-kappa B signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis.