Participation of the PI-3K/Akt-NF-kappa B 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-kappaB) and phosphorylation of Akt, IKK, and IkappaBalpha 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 IkappaBalpha, leading to activation of NF-kappaB. Blocking NF-kappaB signaling pathway by dominant-negative mutants of IKK and IkappaBalpha suppressed HIMF-induced Flk-1 upregulation. Mutation or deletion of NF-kappaB 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, Deltap85, as well as PI-3K inhibitor LY294002, blocked HIMF-induced NF-kappaB activation and attenuated Flk-1 production.


CONCLUSIONS
These results suggest that HIMF upregulates Flk-1 expression in endothelial cells in a PI-3K/Akt-NF-kappaB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis.
Tong
Qiangsong
Q
Department of Internal Medicine, Saint Louis University, Saint Louis, MO 63110, USA. qtong@slu.edu
Zheng
Liduan
L
Lin
Li
L
Li
Bo
B
Wang
Danming
D
Huang
Chuanshu
C
Matuschak
George M
GM
Li
Dechun
D
D042501:Angiogenic Proteins / Q000008:administration & dosage / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002417:Cattle; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D036341:Intercellular Signaling Peptides and Proteins; D007442:Intubation, Intratracheal; D008168:Lung / Q000098:blood supply / Q000166:cytology / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000096:biosynthesis* / Q000235:genetics; D020932:Nerve Growth Factor / Q000008:administration & dosage / Q000235:genetics / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000502:physiology*; D011506:Proteins / Q000008:administration & dosage / Q000235:genetics / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000502:physiology*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis* / Q000235:genetics
D042501:Angiogenic Proteins; D036341:Intercellular Signaling Peptides and Proteins; D016328:NF-kappa B; D011506:Proteins; C413576:Retnla protein, mouse; D012710:Serum Albumin, Bovine; D020932:Nerve Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt