An ezrin/calpain/PI3K/AMPK/eNOSs1179 signaling cascade mediating VEGF-dependent endothelial nitric oxide production.
Calpain was recently reported to mediate vascular endothelial growth factor (VEGF)-induced angiogenesis. In the present study, we investigated detailed molecular mechanisms. VEGF (100 ng/mL) induced a marked increase in endothelial cell production of NO(*), specifically detected by electron spin resonance. This response was abolished by inhibition of calpain with N-acetyl-leucyl-leucyl-norleucinal (ALLN) or Calpeptin. Both also diminished membrane-specific calpain activation by VEGF, which was intriguingly attenuated by silencing ezrin with RNA interference. A rapid membrane colocalization of calpain and ezrin occurred as short as 10 minutes after VEGF stimulation. AKT, AMP-dependent kinase (AMPK), and endothelial nitric oxide synthase (eNOS)(s1179) phosphorylations in VEGF-stimulated endothelial cells were markedly enhanced, which were however significantly attenuated by either ALLN, Calpeptin, or ezrin small interfering RNA, as well as by Wortmannin or compound C (respectively for phosphatidylinositol 3-kinase [PI3K] or AMPK). The latter 3 also abolished VEGF induction of NO(*). These data indicate that AMPK and AKT are both downstream of PI3K and that AKT activation is partially dependent on AMPK. The interrelationship between AMPK and AKT, although known to be individually important in mediating VEGF activation of eNOS, is clearly characterized. Furthermore, AMPK/AKT/eNOS(s1179) was found downstream of a calpain/ezrin membrane interaction. These data no doubt provide new insights into the long mystified signaling gap between VEGF receptors and PI3K/AKT or AMPK-dependent eNOS activation. In view of the well-established significance of VEGF-dependent angiogenesis, these findings might have broad and important implications in cardiovascular pathophysiology.
Youn
Ji-Youn
JY
Division of Molecular Medicine, Cardiovascular Research Laboratories, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Wang
Ting
T
Cai
Hua
H
D055372:AMP-Activated Protein Kinases / Q000502:physiology*; D000818:Animals; D001011:Aorta; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D004151:Dipeptides / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology*; D000099149:Ezrin
D055372:AMP-Activated Protein Kinases; D002154:Calpain; D003598:Cytoskeletal Proteins; D004151:Dipeptides; D006861:Hydrogen Peroxide; D007976:Leupeptins; D008565:Membrane Proteins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D000099149:Ezrin; C063261:acetylleucyl-leucyl-norleucinal; C071482:calpeptin