HB-EGF-induced VEGF production and eNOS activation depend on both PI3 kinase and MAP kinase in HaCaT cells.
BACKGROUND
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of growth factors that have been implicated in skin patho-physiology. Although endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) appear to be involved in mitogenesis and chemotaxis in epidermal keratinocytes, the activation of eNOS and VEGF production induced by HB-EGF and its signaling mechanism remains undefined.


OBJECTIVE
We examined possible signal transduction pathways by which HB-EGF leads to eNOS activation and VEGF production in human epidermal keratinocyte cell line (HaCaT cells).


METHODS
The phosphorylation of epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK; p42/p44 MAPK), Akt and eNOS were examined by Western blotting analysis. VEGF production was determined by enzyme-linked immunosorbent assay. Various inhibitors were utilized to investigate the signaling mechanisms of eNOS activation and VEGF production.


RESULTS
HB-EGF-induced phosphorylation of EGFR with maximum phosphorylation at 1h. HB-EGF-induced phosphorylation of p42/p44 MAPK in a few minutes. It activated Akt with maximum phosphorylation at 1h and eNOS with maximum phosphorylation at 3h. The HB-EGF-induced eNOS activation was significantly blocked by the p42/p44 MAPK inhibitor U0126 and the phosphatidylinositol 3-kinase (P13K) inhibitor LY294002. HB-EGF increased VEGF production. The HB-EGF-induced VEGF production was blocked by U0126 and LY294002. Finally, the HB-EGF-induced activation of Akt and eNOS was suppressed by VEGF competitive antagonist, CBO-P11.


CONCLUSIONS
These results demonstrate that HB-EGF-induced eNOS activation depends on p42/p44 MAPK, PI3K/Akt pathways and endogenous VEGF in HaCaT cells.
Nakai
Kozo
K
Department of Dermatology, Kagawa University, Japan. kozo@kms.ac.jp
Yoneda
Kozo
K
Moriue
Tetsuya
T
Igarashi
Junske
J
Kosaka
Hiroaki
H
Kubota
Yasuo
Y
D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism* / Q000494:pharmacology; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009569:Nitric Oxide / Q000819:agonists / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000187:drug effects / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000819:agonists / Q000378:metabolism*
D002070:Butadienes; D002867:Chromones; D004791:Enzyme Inhibitors; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D009025:Morpholines; D009570:Nitriles; C113580:U 0126; D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases