Novel Role of Protein Kinase C Delta Tyr 311 Phosphorylation in Vascular Smooth Muscle Cell Hypertrophy by Angiotensin II

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1 Novel Role of Protein Kinase C Delta Tyr Phosphorylation in Vascular Smooth Muscle Cell Hypertrophy by ngiotensin II Hidekatsu Nakashima, Gerald D. Frank, Heigoro Shirai, kinari Hinoki, Sadaharu Higuchi, Haruhiko Ohtsu, Kunie Eguchi, rchana Sanjay, Mary E. Reyland, Peter J. Dempsey 5, Tadashi Inagami, Satoru Eguchi. Cardiovascular Research Center and Department of Physiology, Temple University School of Medicine, Philadelphia, P.. Department of iochemistry, Vanderbilt University School of Medicine, Nashville, TN.. Department of natomy and Cell iology, Temple University School of Medicine, Philadelphia, P.. Department of Craniofacial iology, School of Dentistry, and Cell and Developmental iology, School of Medicine, University of Colorado Health Science Center, urora, CO. 5. Departments of Pediatrics and Molecular and Integrative Physiology, University of Michigan, nn rbor, MI. Short title: PKC delta in ngiotensin II-induced Hypertrophy These authors contributed equally to this work. Correspondence to Satoru Eguchi, MD, PhD, FH: Cardiovascular Research Center and Department of Physiology, Temple University School of Medicine, N. road Street, Philadelphia, P 9. Tel & FX , seguchi@temple.edu

2 Supplemental Methods and Figures Supplemental Methods Reagents and ntibodies was purchased from Sigma Chemical Co. (St. Louis, MO). PP, PP, G78 and staurosporine were purchased from Calbiochem (La Jolla, C). RNH67 and YM-589 were gifts from Sankyo Pharmaceutical Co Ltd. (Tokyo, Japan) and stellas Pharma Inc. (Tokyo, Japan), respectively. Sources and dilutions of the primary antibodies used are as follows. Phospho-specific antibodies for Tyr -phosphorylated (:,) and Tyr 68 -phosphorylated EGF receptor (:6,) were purchased from iosource (Camarillo, C). Phosphospecific antibody for Ser 7 - phosphorylated kt (:,), total kt antibody (:6,) and cleaved caspase- antibody (:,) were purchased from Cell Signaling (everly, M). Phosphospecific antibody for Tyr -phosphorylated ERK/ (:,) and antibodies against (:,), (:,) and Src (:,) were purchased from Santa Cruz iotechnology (Santa Cruz, C). ntibody for GPDH (:,) was purchased from Chemicon International (Temecula, C). ntibody for GFP (:,) was purchased from D iosciences (Palo lto, C). Cell culture Rat aortic VSMCs were obtained and maintained as described previously. Subcultured cells from passages - were used in the experiments and showed more than 99% positive immunostaining of smooth muscle -actin antibody. For both short-term (- min) and longterm ( days) experiments, cells were cultured to ~8% confluency and then made quiescent by incubating in serum-free media for - days. verage confluency before stimulation is a little more than 8% but still less than 9% since VSMCs do not proliferate significantly after the serum starvation without a mitogen. Statistical nalysis Unless otherwise stated, the data presented in this study are representative of a minimum of three independent experiments yielding similar results. The data were analyzed using one-way NOV followed by a post-hoc modified t-test (Figure, and ) or a two-way NOV (Figure, and 5). Reference. Eguchi S, Matsumoto T, Motley ED, Utsunomiya H, Inagami T. Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells. Possible requirement of Gq-mediated pras activation coupled to a Ca+/calmodulin-sensitive tyrosine kinase. J iol Chem. 996; 7:69-75.

3 Supplemental Figure Legends Figure S:. VSMCs were stimulated with nmol/l for the indicated time periods. and C. VSMCs were pretreated with () T receptor blocker, RNH67 ( µmol/l) or () a selective G q inhibitor, YM-589 ( µmol/l) for min and stimulated with ( nmol/l) for min. D. VSMCs were pretreated with a Src family kinase inhibitor, PP (5 µmol/l) or its negative control, PP (5 µmol/l) for min and stimulated with nmol/l for min. The cell lysates were immunoblotted with phospho-selective antibody which detects Tyr phosphorylation and anti- antibody. The phosphorylation at Tyr was measured by densitometry and shown as mean ± SEM for three independent experiments. P <.5 compared to the control. P <.5 compared to the stimulated control. Figure S:. VSMCs were infected with retrovirus encoding control, wild type tagged with GFP, or YF mutant tagged with GFP. VSMCs were then stimulated with nmol/l for days. Cell volume was analyzed with a Coulter counter.. VSMCs were infected with retrovirus encoding control, wild type, or YF mutant. The cells were stimulated with nmol/l for days. fterwards, cell proliferation was measured by a CellTiter 96 queous cell proliferation/viability assay kit. The data was presented as fold mean ± SEM for three independent experiments. C. VSMCs were infected with adenovirus encoding control or wild type. The cells were stimulated with nmol/l for hours. fterwards, DN synthesis was assessed by a rdu incorporation kit. The data was presented as mean ± SEM (n=5). Figure S:. VSMCs were infected with adenovirus encoding wild type or control empty, and stimulated with nmol/l for hours.. VSMCs were stimulated with nmol/l or mmol/l staurosporine (Staur) for hours. The cell lysates were immunoblotted with cleaved caspase- selective antibody, anti-gpdh antibody and anti- antibody, as indicated. Note that an apoptosis inducer, staurosporine, markedly stimulated cleavage of caspase. Data shown are representative from three independent experiments. Figure S:. VSMCs were infected with adenovirus encoding a kinase-inactive mutant (K76) or control empty, and stimulated with nmol/l for days. Cell volume was analyzed with a Coulter counter.. VSMCs were infected with adenovirus encoding K76 or control. The cells were stimulated with nmol/l for days. fterwards, cell proliferation was measured by a CellTiter 96 queous cell proliferation/viability assay kit. The data was presented as fold mean ± SEM for three independent experiments.

4 Supplement Figure S (min) 5 -p (fold) -ptyr RNH -ptyr C -p (fold) YM D -p (fold) PP PP ptyr -ptyr

5 Supplement Figure S 8 5x x Cell Volume (µm ) Proliferation (%) YF 8 5x x Cell Volume (µm ) C rdu uptake (OD) YF 5x x Cell Volume (µm )

6 Supplement Figure S Cleaved caspase- (9/7 kda) GPDH Staur Cleaved caspase- (9/7 kda) GPDH

7 Supplement Figure S 5 5 Proliferation (%) x x Cell Volume (mm ) K76 5 K76 5x x Cell Volume (mm )