Title: 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 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. 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. 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Physiol. 2003; 285: R277-R297Crossref PubMed Scopus (21) Google Scholar). Seven members of the Nox family have been identified in the human genome: Nox1 to -5 and the dual oxidases Duox1 and -2 (17Bedard K. Krause K.H. Physiol. Rev. 2007; 87: 245-313Crossref PubMed Scopus (5040) Google Scholar, 18Lassegue B. Clempus R.E. Am. J. Physiol. 2003; 285: R277-R297Crossref PubMed Scopus (21) Google Scholar, 22Geiszt M. Cardiovasc. Res. 2006; 71: 289-299Crossref PubMed Scopus (180) Google Scholar). The isoform Nox4 (NAD(P)H oxidase 4) is abundant in the vascular system and kidney cortex (16Gill P.S. Wilcox C.S. Antioxid. Redox Signal. 2006; 8: 1597-1607Crossref PubMed Scopus (410) Google Scholar, 17Bedard K. Krause K.H. Physiol. Rev. 2007; 87: 245-313Crossref PubMed Scopus (5040) Google Scholar, 19Lambeth J.D. Free Radic. Biol. Med. 2007; 43: 332-347Crossref PubMed Scopus (525) Google Scholar, 20Orient A. Donko A. Szabo A. Leto T.L. Geiszt M. Nephrol. Dial. 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Antioxid. Redox Signal. 2006; 8: 1497-1508Crossref PubMed Scopus (34) Google Scholar). This study indicates that PDK-1 is tyrosine-phosphorylated and activated by Ang II in a redox-dependent manner. We identify Nox4 NAD(P)H oxidase as a critical mediator of PDK-1 tyrosine phosphorylation and activation through Src oxidation. We also demonstrate that Ang II up-regulates Nox4. Furthermore, we establish for the first time that the tyrosine phosphorylation/activation of PDK-1 contributes to MC hypertrophy and fibronectin expression, indicating that this cascade involving PDK-1 plays a central role in the control of the signaling events implicated in Ang II-induced cell injury. Cell Culture, Transfections, and Adenovirus Infection—Rat glomerular MCs were isolated and characterized as described (23Gorin Y. Block K. Hernandez J. Bhandari B. Wagner B. Barnes J.L. Abboud H.E. J. Biol. Chem. 2005; 280: 39616-39626Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). These cells were used between 15th and 30th passages. Selected experiments were performed in primary and early passaged MCs to confirm the data obtained with late passages. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with antibiotic/antifungal solution and 17% fetal bovine serum. Transient transfection of MCs with plasmid DNA (15 μg of vector alone or Myc epitope-tagged mammalian expression construct with PDK-1 mutated at tyrosine 373 (Myc-PDK-1Y373F) or redox-insensitive mutant of Src (SrcC487A)) was performed by electroporation (Gene Pulser; Bio-Rad), as previously described (41Gorin Y. Kim N.H. Feliers D. Bhandari B. Choudhury G.G. Abboud H.E. FASEB J. 2001; 15: 1909-1920Crossref PubMed Scopus (96) Google Scholar). Construction of mutant expression vector Myc-PDK-1Y373F was described previously (28Taniyama Y. Weber D.S. Rocic P. Hilenski L. Akers M.L. Park J. Hemmings B.A. Alexander R.W. Griendling K.K. Mol. Cell. Biol. 2003; 23: 8019-8029Crossref PubMed Scopus (67) Google Scholar). SrcC487A was a generous gift from Dr. P. Chiarugi (University of Florence, Italy) (43Giannoni E. Buricchi F. Raugei G. Ramponi G. Chiarugi P. Mol. Cell. Biol. 2005; 25: 6391-6403Crossref PubMed Scopus (367) Google Scholar). For the RNA interference experiments, a SMART-pool consisting of four short or small interfering RNA (siRNA) duplexes specific for rat Nox4, Src, PDK-1, Pyk-2 (proline-rich tyrosine kinase-2), or p70S6K was obtained from Dharmacon. The SMARTpool of siRNAs was introduced into the cells by double transfection using Oligofectamine or Lipofectamine as described (24Gorin Y. Ricono J.M. Wagner B. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Biochem. J. 2004; 381: 231-239Crossref PubMed Scopus (109) Google Scholar). The siRNAs for Nox4, Src, PDK-1, Pyk-2, or p70S6K were used at a concentration of 100 nm. Scrambled siRNAs (nontargeting siRNA) (100 nm) served as controls to validate the specificity of the siRNAs (42Block K. Ricono J.M. Lee D.Y. Bhandari B. Choudhury G.G. Abboud H.E. Gorin Y. Antioxid. Redox Signal. 2006; 8: 1497-1508Crossref PubMed Scopus (34) Google Scholar). Adenovirus encoding a kinase-inactive form of PDK-1 (AdPDK-1K11N), adenovirus encoding PDK-1 mutated at tyrosine 9 (AdPDK-1Y9F), and adenovirus encoding for wild type PDK-1 (AdWTPDK-1) were described previously (28Taniyama Y. Weber D.S. Rocic P. Hilenski L. Akers M.L. Park J. Hemmings B.A. Alexander R.W. Griendling K.K. Mol. Cell. Biol. 2003; 23: 8019-8029Crossref PubMed Scopus (67) Google Scholar, 44Weber D.S. Taniyama Y. Rocic P. Seshiah P.N. Dechert M.A. Gerthoffer W.T. Griendling K.K. Circ. Res. 2004; 94: 1219-1226Crossref PubMed Scopus (143) Google Scholar, 45Taniyama Y. Hitomi H. Shah A. Alexander R.W. Griendling K.K. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1142-1147Crossref PubMed Scopus (98) Google Scholar). MCs were infected with adenovirus vectors at the indicated multiplicity of infection as previously described (46Block K. Gorin Y. Hoover P. Williams P. Chelmicki T. Clark R.A. Yoneda T. Abboud H.E. J. Biol. Chem. 2007; 282: 8019-8026Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). As a control for the effects of adenovirus infection alone, an adenovirus encoding green fluorescent protein lacking an insert (AdGFP) was used. RNA Extraction, Reverse Transcription (RT)-PCR—Primers for rat Nox4 were designed as described previously (24Gorin Y. Ricono J.M. Wagner B. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Biochem. J. 2004; 381: 231-239Crossref PubMed Scopus (109) Google Scholar, 46Block K. Gorin Y. Hoover P. Williams P. Chelmicki T. Clark R.A. Yoneda T. Abboud H.E. J. Biol. Chem. 2007; 282: 8019-8026Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar): 5′-GATGTTGGGCCTAGGATTGTGT-3′ (forward primer) and 5′-CAGCCAGGAGGGTGAGTGTCTAA-3′ (reverse primer). Total RNA was isolated from MCs, and the RT-PCR was carried out using the SuperScript One-Step RT-PCR kit (Invitrogen) as described. Amplification was carried out for 35 cycles at 93 °C for 1 min, 85 °C for 1 min, and 72 °C for 1 min. The expected size of the PCR product for Nox4 is 534 bp. End products were resolved on agarose gel, stained with ethidium bromide, and visualized and photographed under UV light. Immunoprecipitation and PDK-1 Activity Assay—MCs grown to near confluence were made quiescent by serum deprivation for 48 h and exposed to serum-free Dulbecco's modified Eagle's medium at 37 °C for the specified duration. The cells were lysed in radioimmune precipitation buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin, and 1% Nonidet P-40) at 4 °C for 30 min. The cell lysates were centrifuged at 10,000 × g for 30 min at 4 °C. Protein was determined in the cleared supernatant using the Bio-Rad protein assay reagent. For immunoprecipitation, equal amounts of protein (100-500 μg) were incubated with sheep anti-PDK-1 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) for 4 h. Protein G-Sepharose beads were added, and the resulting mixture was further incubated at 4 °C for 1 h on a rotating device. The beads were washed three times with radioimmune precipitation buffer and twice with phosphate-buffered saline. The kinase reaction was carried out by incubating the immunobeads in kinase assay buffer (50 mm Tris-HCl, pH 7.4, 10 nm MgCl2, 25 mm β-glycerophosphate, 1 mm dithiothreitol, 10 mm microcystin, and 1 mm Na3VO4) in the presence of 1 μg/ml purified unactivated Akt1/PKBα (Upstate Biotechnology), and 20 μm cold ATP plus 5 μCi of [γ-32P]ATP for 30 min at 30 °C (47Chen H. Nystrom F.H. Dong L.Q. Li Y. Song S. Liu F. Quon M.J. Biochemistry. 2001; 40: 11851-11859Crossref PubMed Scopus (29) Google Scholar). This reaction was stopped by the addition of 2× sample buffer, after which the samples were subjected to 12.5% SDS-polyacrylamide gel electrophoresis, and phosphorylated unactivated Akt/PKB was visualized by autoradiography or using a PhosphorImager. The bands were quantitated by densitometry and/or PhosphorImager analysis. In other experiments, PDK-1 activity was measured with a PDK-1 assay kit (Upstate Biotechnology) according to the manufacturer's recommendations. The immunoprecipitates were prepared as described above, and after washing, the immunobeads were incubated with PDK-1 assay dilution buffer containing inactive SGK1 (serum- and glucocorticoid-regulated protein kinase-1) for 30 min at 30 °C. Then an Akt/SGK substrate peptide and 5 μCi of [γ-32P]ATP was added, and a second kinase reaction was allowed to continue for an additional 10 min at 30 °C. The reactions were spotted on P81 phosphocellulose paper and washed four times with 1% phosphoric acid followed by an acetone rinse. The amount of radioactivity incorporated into the substrate was determined by scintillation counting. Western Blotting Analysis—MC lysates were prepared as described above for the PDK-1 activity assay. For immunoblotting, proteins were separated using SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% low fat milk in Tris-buffered saline and then incubated with a rabbit polyclonal Nox4 antibody (catalog number ab41886; Abcam) (dilution 1:500), a rabbit polyclonal anti-phospho-PDK-1 (Tyr9) (catalog number PP1431; ECM Biosciences) (1:1,000), a rabbit polyclonal anti-phospho-PDK-1 (Tyr373/376) (catalog number 1901-1; Epitomics Inc.) (1:1,000), a rabbit anti-phospho-Src (Tyr416) antibody or a rabbit polyclonal anti-Src (catalog numbers 2101 and 2108; Cell Signaling Technology Inc.) (1:1,000), a rabbit polyclonal anti-p70S6K (catalog number 9202; Cell Signaling Technology Inc.) (1:1,000), a rabbit polyclonal anti-Pyk-2 (catalog number 07-437; Upstate/Millipore) (1:500), a rabbit polyclonal anti-fibronectin antibody (catalog number F3648; Sigma) (1:2,500), or a mouse monoclonal anti-β-actin (1:4,000) (catalog number A2066; Sigma). The appropriate horseradish peroxidase-conjugated secondary antibodies were added, and bands were visualized by enhanced chemiluminescence. Densitometric analysis was performed using NIH Image software. For the detection of PDK-1 total tyrosine phosphorylation, MCs lysates were subjected to immunoprecipitation with anti-PDK1 antibody, and blots were incubated with mouse monoclonal antiphosphotyrosine antibody clone 4G10 (Upstate Biotechnology) or rabbit anti-PDK-1 (Cell Signaling Technology) (1:1,000). Immunofluorescence Confocal Microscopy—MCs grown on 4-well chamber slides were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 5 min. The cells were then blocked with 5% normal goat serum or 5% normal donkey serum in phosphate-buffered saline for 30 min and incubated with appropriate primary antibodies (anti-phospho-Src or anti-fibronectin) for 30 min. Cyanin-3- or fluorescein isothiocyanate-conjugated secondary antibodies were then applied to the appropriate cells for 30 min. The cells were washed 3 times with phosphate-buffered saline, mounted with antifade reagent with 4′,6-diamidino-2-phenylindole, and visualized on an Olympus FV-500 confocal laser scanning microscope. As requested by the reviewer, to estimate the brightness intensity of phospho-Src and fibronectin signals, groups of cells randomly selected from the digital image were outlined (at least five groups for each sample), and the average brightness of the enclosed area was semiquantified using either the Image-Pro Plus 4.5 software (Media Cybernetics) or NIH Image/ImageJ software, as described (23Gorin Y. Block K. Hernandez J. Bhandari B. Wagner B. Barnes J.L. Abboud H.E. J. Biol. Chem. 2005; 280: 39616-39626Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). The data shown represent three separate experiments and are expressed as relative fluorescence intensity. Detection of Intracellular ROS—The peroxide-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescin diacetate (Invitrogen/Molecular Probes) was used to assess the generation of intracellular ROS, as described previously (23Gorin Y. Block K. Hernandez J. Bhandari B. Wagner B. Barnes J.L. Abboud H.E. J. Biol. Chem. 2005; 280: 39616-39626Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 41Gorin Y. Kim N.H. Feliers D. Bhandari B. Choudhury G.G. Abboud H.E. FASEB J. 2001; 15: 1909-1920Crossref PubMed Scopus (96) Google Scholar). This compound is converted by intracellular esterases to 2′,7′-dichlorodihydrofluorescin, which is then oxidized by hydrogen peroxide to the highly fluorescent 2′,7′-dichlorodihydrofluorescein (DCF). Differential interference contrast images were obtained simultaneously using an Olympus inverted microscope with ×40 Aplanfluo objective and an Olympus Fluoview confocal laser-scanning attachment. The DCF fluorescence was measured with an excitation wavelength of 488-nm light, and its emission was detected using a 510-550-nm bandpass filter. Semiquantification of DCF fluorescence was performed as described above. Alternatively, cells were grown in 12- or 24-well plates and serum-deprived for 48 h. Immediately before the experiments, cells were washed with Hank's balanced salt solution and loaded with 50 μm 2′,7′-dichlorodihydrofluorescin diacetate dissolved in Hanks' balanced salt solution for 30 min at 37 °C. They were then incubated with the selected agonist or vehicle for various time periods. Subsequently, DCF fluorescence was detected at excitation and emission wavelengths of 488 and 520 nm, respectively, and measured with a multiwell fluorescence plate reader (Wallac 1420 Victor2; PerkinElmer Life Sciences), as described (42Block K. Ricono J.M. Lee D.Y. Bhandari B. Choudhury G.G. Abboud H.E. Gorin Y. Antioxid. Redox Signal. 2006; 8: 1497-1508Crossref PubMed Scopus (34) Google Scholar, 46Block K. Gorin Y. Hoover P. Williams P. Chelmicki T. Clark R.A. Yoneda T. Abboud H.E. J. Biol. Chem. 2007; 282: 8019-8026Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Protein Synthesis—[3H]Leucine incorporation into trichloroacetic acid-insoluble material was used to assess p