Nox4 NAD(P)H Oxidase Mediates Src-dependent Tyrosine Phosphorylation of PDK-1 in Response to Angiotensin II

Abstract

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 oxidationresistant 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 IIinduced 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.Cellular hypertrophy and extracellular matrix accumulation in glomeruli contributes to the pathogenesis of glomerulosclerosis in fibrotic renal diseases (1-5). The octapeptide hormone angiotensin II (Ang II) 2 is the dominant renin-angiotensin system effector (5-7) and is implicated in the pathogenesis of fibrosis of the glomerular microvascular bed. Up-regulation of the renin-angiotensin system plays a key role in the initiation and the progression of glomerular injury via induction of hypertrophy and extracellular matrix expansion in glomerular mesangial cells (MCs) (6 -13).Ang II-induced oxidative stress has emerged as a critical pathogenic factor in the development of renal and vascular diseases (13-16). NAD(P)H oxidases of the Nox family are major sources of reactive oxygen species (ROS) in many nonphagocytic cells, including renal cells (17-21). The Nox proteins correspond to homologues of gp91 phox (or Nox2), the catalytic moiety found in phagocytes (17,18). Seven members of the Nox family have been identified in the human genome: Nox1 to -5 and the dual oxidases Duox1 and -2 (17, 18, 22). The isoform Nox4 (NAD(P)H oxidase 4) is abundant in the vascular system and kidney cortex (16, 17, 19 -22). We ...

Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

“…This conclusion is consistent to our finding that optimal PI3K activation is dependent on assembly of a Shc⅐Grb2⅐p85 complex on SHPS-1 in response to IGF-I (14). PDK1 phosphorylates AKT at Thr-308 (59,60), and NOX4 modulates PDK1 tyrosine phosphorylation, which increases its activity (61). Because NOX4 and PDK1 interact with SHPS-1, it is possible that this interaction is required for optimal PDK1 activity.…”

NOX Enzymes and Pulmonary Disease

“…Currently limited published studies are available on in vivo roles for NOX4 in lung fibrosis; however, studies in kidney fibrosis (12,89,120,143), vascular-remodeling=fibrosis associated with chronic hypertension (4), cardiac fibrosis (39, 112,139), and pancreatic fibrosis (75) suggest a role for the NOX4 isoform in the fibrogenic process. Other NOX isoforms that are reported to contribute to tissue fibrosis in nonpulmonary organ systems include NOX1 (4,75,112,139) and NOX2 (67,75,89,112,143).…”

“…A p47 phox -requiring NOX isoform is required for the development of fibrosis in a murine lung-injury model that is inflammation dependent, and the observed protection in p47 phoxÀ=À mice is associated with enhanced neutrophilic inflammation and matrix metalloproteinase (MMP)-9 activity (70). Significant crosstalk occurs between the reninangiotensin-aldosterone system and TGF-b1 in organ fibrosis (130,147), and this effect is, at least in part, mediated by induction=activation of NOX1, NOX2, NOX4, or a combination of these (4,12,112,120,139,143).…”

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

“…Nox4-derived ROS has been shown to be required for angiotensin II- (8) and IGF-I-stimulated Src activation (9). Differential subcellular localization of Nox4 has been correlated with alteration in specific cellular functions.…”