title: Glomerular endothelial PI3 kinase-α couples to VEGFR2, but is not required for eNOS activation.
abstract: Vascular endothelial growth factor (VEGF)-dependent signals are central to many endothelial cell (EC) functions, including survival and regulation of vascular tone. Akt and endothelial nitric oxide synthase (eNOS) activity are implicated to mediate these effects. Dysregulated signaling is characteristic of endothelial dysfunction that sensitizes the glomerular microvasculature to injury. Signaling intermediates that couple VEGF stimulation to eNOS activity remain unclear; hence, we examined the PI3 kinase isoforms implicated to regulate these enzymes. Using a combination of small molecule inhibitors and RNAi to study responses to VEGF in glomerular EC, we observed that the PI3 kinase p110α catalytic isoform is coupled to VEGFR2 and regulates the bulk of Akt activity. Coimmunoprecipitation experiments support a physical association of p110α with VEGFR2. Downstream, Akt-mediated FOXO1 phosphorylation in EC is regulated by p110α. The p110δ isoform contributes a minor amount of VEGF-stimulated Akt activation. However, we observe no effect of p110α or p110δ to regulate VEGF-stimulated eNOS activation via Akt-mediated phosphorylation on eNOS Ser1177, or NO-mediated vasodilation of the afferent arteriole ex vivo. VEGFR2-stimulated eNOS activation and NO production are inhibited by Compound C, an inhibitor of AMP-stimulated kinase, independent of PI3 kinase signaling. PI3 kinase-α/δ-mediated signaling downstream of VEGFR2 activation regulates Akt-dependent survival signals, but our data suggest it is not required to activate eNOS or to elicit NO production in glomerular EC.
authors.affiliation: Department of Medicine, University of Alberta, Edmonton, AB, Canada.
authors.forename: Qiu-Xia
authors.initials: QX
authors.lastname: Zhang
authors.forename: Maryam
authors.initials: M
authors.lastname: Nakhaei-Nejad
authors.forename: George
authors.initials: G
authors.lastname: Haddad
authors.forename: Xuemei
authors.initials: X
authors.lastname: Wang
authors.forename: Rodger
authors.initials: R
authors.lastname: Loutzenhiser
authors.forename: Allan G
authors.initials: AG
authors.lastname: Murray
mesh_terms: D002478:Cells, Cultured; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes; D007678:Kidney Glomerulus / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D034622:RNA Interference; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014664:Vasodilation / Q000187:drug effects
chemical_list: C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D007527:Isoenzymes; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; C516138:dorsomorphin; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D058543:Class Ia Phosphatidylinositol 3-Kinase; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt