Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression.
Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.
Department of Medicine, University of Texas Health Science Center, 7723 Floyd Curl Drive, San Antonio, TX 78229, USA.
Karen
K
Block
Assaad
A
Eid
Kathy K
KK
Griendling
Duck-Yoon
DY
Lee
Yohann
Y
Wittrant
Yves
Y
Gorin
D064413:3-Phosphoinositide-Dependent Protein Kinases; D019943:Amino Acid Substitution; D000804:Angiotensin II / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D005353:Fibronectins / Q000096:biosynthesis* / Q000235:genetics; D005355:Fibrosis; D005786:Gene Expression Regulation / Q000187:drug effects; D005920:Glomerular Mesangium / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D006984:Hypertrophy / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D007674:Kidney Diseases / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D020125:Mutation, Missense; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000378:metabolism / Q000494:pharmacology*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D005353:Fibronectins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat; D019061:src-Family Kinases; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases