Rear Polarization of the Microtubule-Organizing Center in Neointimal Smooth Muscle Cells Depends on PKCα, ARPC5, and RHAMM
2011; Elsevier BV; Volume: 178; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2010.10.001
ISSN1525-2191
AutoresRosalind Silverman-Gavrila, Lorelei Silverman‐Gavrila, Guangpei Hou, Ming Zhang, Milton P. Charlton, Michelle P. Bendeck,
Tópico(s)Caveolin-1 and cellular processes
ResumoDirected migration of smooth muscle cells (SMCs) from the media to the intima in arteries occurs during atherosclerotic plaque formation and during restenosis after angioplasty or stent application. The polarized orientation of the microtubule-organizing center (MTOC) is a key determinant of this process, and we therefore investigated factors that regulate MTOC polarity in vascular SMCs. SMCs migrating in vivo from the medial to the intimal layer of the rat carotid artery following balloon catheter injury were rear polarized, with the MTOC located posterior of the nucleus. In tissue culture, migrating neointimal cells maintained rear polarization, whereas medial cells were front polarized. Using phosphoproteomic screening and mass spectrometry, we identified ARPC5 and RHAMM as protein kinase C (PKC)-phosphorylated proteins associated with rear polarization of the MTOC in neointimal SMCs. RNA silencing of ARPC5 and RHAMM, PKC inhibition, and transfection with a mutated nonphosphorylatable ARPC5 showed that these proteins regulate rear polarization by organizing the actin and microtubule cytoskeletons in neointimal SMCs. Both ARPC5 and RHAMM, in addition to PKC, were required for migration of neointimal SMCs. Directed migration of smooth muscle cells (SMCs) from the media to the intima in arteries occurs during atherosclerotic plaque formation and during restenosis after angioplasty or stent application. The polarized orientation of the microtubule-organizing center (MTOC) is a key determinant of this process, and we therefore investigated factors that regulate MTOC polarity in vascular SMCs. SMCs migrating in vivo from the medial to the intimal layer of the rat carotid artery following balloon catheter injury were rear polarized, with the MTOC located posterior of the nucleus. In tissue culture, migrating neointimal cells maintained rear polarization, whereas medial cells were front polarized. Using phosphoproteomic screening and mass spectrometry, we identified ARPC5 and RHAMM as protein kinase C (PKC)-phosphorylated proteins associated with rear polarization of the MTOC in neointimal SMCs. RNA silencing of ARPC5 and RHAMM, PKC inhibition, and transfection with a mutated nonphosphorylatable ARPC5 showed that these proteins regulate rear polarization by organizing the actin and microtubule cytoskeletons in neointimal SMCs. Both ARPC5 and RHAMM, in addition to PKC, were required for migration of neointimal SMCs. The directed migration of smooth muscle cells (SMCs) from the tunica media to the tunica intima following endothelial injury is critical for the formation of atherosclerotic plaques and contributes to restenosis after angioplasty or stent application.1De Geest B. The origin of intimal smooth muscle cells: are we on a steady road back to the past?.Cardiovasc Res. 2009; 81: 7-8Crossref PubMed Scopus (4) Google Scholar Directed cell migration involves reorganization of the cytoskeleton, and a key determinant is the polarized localization of the microtubule-organizing center (MTOC) relative to the nucleus. In nonmigrating cells, the MTOC is oriented randomly with respect to the nucleus, whereas in migrating cells the MTOC is polarized and frequently localized anterior to the nucleus. Microtubules are nucleated at centrosomes, with their minus ends anchored at the MTOC; they contribute to polarization of interphase cells during migration and to division spindle assembly during proliferation. Protein phosphorylation by protein kinase C (PKC) is an important regulator of MTOC polarity. PKC isoforms regulating polarity include atypical aPKC,2Tzima E. Kiosses W.B. del Pozo M.A. Schwartz M.A. Localized Cdc42 activation, detected using a novel assay, mediates microtubule organizing center positioning in endothelial cells in response to fluid shear stress.J Biol Chem. 2003; 278: 31020-31023Crossref PubMed Scopus (150) Google Scholar PKCβ,3Volkov Y. Long A. McGrath S. Ni Eidhin D. Kelleher D. Crucial importance of PKC-beta(I) in LFA-1-mediated locomotion of activated T cells.Nat Immunol. 2001; 2: 508-514Crossref PubMed Scopus (128) Google Scholar, 4Chen D. Purohit A. Halilovic E. Doxsey S.J. Newton A.C. Centrosomal anchoring of protein kinase CβII by pericentrin controls microtubule organization, spindle function, and cytokinesis.J Biol Chem. 2003; 279: 4829-4839Crossref PubMed Scopus (77) Google Scholar, 5Volkov Y. Long A. Kelleher D. Inside the crawling T cell: leukocyte function-associated antigen-1 cross-linking is associated with microtubule-directed translocation of protein kinase C isoenzymes beta(I) and delta.J Immunol. 1998; 161: 6487-6495PubMed Google Scholar PKCζ,6Etienne-Manneville S. Manneville J.B. Nicholls S. Ferenczi M.A. Hall A. Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization.J Cell Biol. 2005; 170: 895-901Crossref PubMed Scopus (239) Google Scholar and PKCδ.5Volkov Y. Long A. Kelleher D. Inside the crawling T cell: leukocyte function-associated antigen-1 cross-linking is associated with microtubule-directed translocation of protein kinase C isoenzymes beta(I) and delta.J Immunol. 1998; 161: 6487-6495PubMed Google Scholar We have recently shown that the MTOC is oriented anterior of the nucleus (ie, front polarized) in migrating medial SMCs in vitro,7Sabatini P.J. Zhang M. Silverman-Gavrila R. Bendeck M.P. Langille B.L. Homotypic and endothelial cell adhesions via N-cadherin determine polarity and regulate migration of vascular smooth muscle cells.Circ Res. 2008; 15: 405-412Crossref Scopus (38) Google Scholar which is in accord with other studies of cells migrating in two-dimensional culture.8Kupfer A. Louvard D. Singer S.J. Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound.Proc Natl Acad Sci U S A. 1982; 79: 2603-2607Crossref PubMed Scopus (282) Google Scholar, 9Gregory W.A. Edmondson J.C. Hatten M.E. Mason C.A. Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro.J Neurosci. 1988; 8: 1728-1738PubMed Google Scholar, 10Etienne-Manneville S. Hall A. Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta.Cell. 2001; 106: 489-498Abstract Full Text Full Text PDF PubMed Scopus (843) Google Scholar, 11Nemere I. Kupfer A. Singer S.J. Reorientation of the Golgi apparatus and the microtubule-organizing center inside macrophages subjected to a chemotactic gradient.Cell Motil. 1985; 5: 17-29Crossref PubMed Scopus (48) Google Scholar, 12Yvon A.M. Walker J.W. Danowski B. Fagerstrom C. Khodjakov A. Wadsworth P. Centrosome reorientation in wound-edge cells is cell type specific.Mol Biol Cell. 2002; 13: 1871-1880Crossref PubMed Scopus (83) Google Scholar, 13Gotlieb A.I. May L.M. Subrahmanyan L. Kalnins V.I. Distribution of microtubule organizing centers in migrating sheets of endothelial cells.J Cell Biol. 1981; 91: 589-594Crossref PubMed Scopus (171) Google Scholar, 14Euteneuer U. Schliwa M. Mechanism of centrosome positioning during the wound response in BSC-1 cells.J Cell Biol. 1992; 116: 1157-1166Crossref PubMed Scopus (76) Google Scholar Little is known about polarization of the MTOC in vivo, where cells migrate in three dimensions. We therefore set out to study the polarity of SMCs migrating from media to intima in vivo. Using a novel approach to study MTOC polarity in SMCs, we denuded the endothelium from rat carotid artery to trigger migration to the intimal layer. Confocal microscopy of the intimal surface showed that the MTOCs of invading SMCs were rear polarized. Furthermore, SMCs harvested from an established thickened neointima and grown in culture maintained rear polarization during migration. To study the mechanisms controlling this phenomenon, we used phosphoproteomic analysis and mass spectroscopy to identify proteins with different phosphorylation levels in neointimal SMCs, compared with medial SMCs. The proteins identified had high probability for phosphorylation by PKC, and experiments using selective inhibitors demonstrated that PKCα controlled polarization of the MTOC in neointimal SMCs. We used siRNA to knock down expression and to further investigate the functions of two cytoskeleton-related proteins: actin-related protein 2/3 complex subunit 5 (ARPC5) and receptor for hyaluronan mediated motility (RHAMM). Both proteins were required for rear polarization of the MTOC in neointimal SMCs, both influenced actin organization and microtubule dynamics in migrating neointimal SMCs, and both were required for migration of neointimal SMCs. Mutating the putative PKC phosphorylation sites in ARPC5 also resulted in disruption of rear polarization in neointimal SMCs. Animal experiments were performed in accordance with the guidelines set by the Canadian Council on Animal Care. Male Sprague-Dawley rats were obtained from Charles River (Montreal, PQ, Canada). Six rats were anesthetized by intraperitoneal injection of xylazine at 4.6 mg/kg body weight (Rompum; Bayer, Etobicoke, ON, Canada) and ketamine at 70 mg/kg body weight (Ketaset; Ayerst Veterinarian Laboratories, Guelph, ON, Canada). Rat common carotid arteries were injured by passing a 2F embolectomy balloon catheter along the length of the vessel three times to denude the endothelium. At 4 days after balloon injury, the carotids were perfusion-fixed with 4% paraformaldehyde at 110 mm Hg for 15 minutes. The carotids were excised, cut open longitudinally, and then pinned flat to a block of dental rubber with the intimal surface up. Vessels were permeabilized with 0.2% Triton-X for 5 minutes, and then treated with RNase A at 100 μg/ml for 1 hour. The vessels were stained en face with a mouse monoclonal antibody against γ-tubulin clone GTU-88 diluted 1:200 (Sigma-Aldrich, St. Louis, MO) for 1 hour, then incubated with Alexa Fluor 488 goat anti-mouse secondary antibody diluted 1:50 (Invitrogen, Carlsbad, CA). The carotids were counterstained for 20 minutes with propidium iodide 20 μg/ml (Molecular Probes; Invitrogen) to stain the nuclei. Tissues were transferred to glass slides, and were coverslipped with 9:1 glycerol/PBS. Images of fixed rat carotid arteries were captured with an Olympus FluoView FV1000 confocal microscope (Olympus, Canada) equipped with an Olympus confocal scanning unit, and a 60× oil immersion lens (NA 1.4). We used two laser lines: for the Alexa Fluor 488 labeled anti-mouse antibody, the excitation wavelength was 488 nm and the emission wavelength was 519 nm; for propidium iodide, the excitation wavelength was 543 nm and the emission wavelength was 603 nm. Images were acquired at 15 to 20 Z series of 0.2-μm steps using Olympus FluoView 1.7a software. Images were acquired at room temperature and represent the merge of 15 to 20 Z stacks. Natural autofluorescence of elastin allowed visualization of the internal elastic laminae and its fenestrae, and this marked the boundary between media and intima. Approximately 100 MTOC and nuclei were counted from the intima of each artery, giving a total of 611 cells counted. Three-dimensional images were constructed using Imaris software version 5.5 (Bitplane, Saint Paul, MN) and were saved as AVI files. Medial and neointimal rat carotid artery SMCs were obtained from uninjured and balloon-injured rat carotid arteries as described previously.15Hou G. Mulholland D. Gronska M.A. Bendeck M.P. Type VIII collagen stimulates smooth muscle cell migration and matrix metalloproteinase synthesis after arterial injury.Am J Pathol. 2000; 156: 467-476Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar Uninjured carotid arteries were harvested and stripped of adventitia, and the endothelium was scraped off; medial SMCs were then dispersed by digestion for 1 hour in 0.3 mg/ml elastase type III, 1.8 mg/ml collagenase type I (Worthington Biochemical, Freehold, NJ), 0.44 mg/ml soybean trypsin inhibitor, and 2 mg/ml bovine serum albumin. To obtain neointimal SMCs, left carotid arteries of rats were injured with a balloon catheter; 2 weeks later, the thickened neointima was stripped from the vessel with the aid of a dissecting microscope. Neointimal SMCs were dispersed by digestion with elastase and collagenase as described above. Six carotids were pooled for isolation of medial SMCs, and six neointimas were pooled for isolation of neointimal SMCs. To ensure consistency of the SMC phenotypes across different rats, we obtained and maintained several independent dispersions, which were randomly selected for experiments. Neointimal and medial SMCs were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2% penicillin-streptomycin and were used between passages 5 and 10. Immunostaining for smooth muscle actin confirmed that the harvested cells were SMCs. Dulbecco's modified Eagle's medium, penicillin-streptomycin, trypsin, and fetal calf serum were purchased from Life Technologies (Gaithersburg, MD). SMCs grown to confluence in 60-mm tissue culture dishes either untreated or incubated for 30 minutes with PKC inhibitor bisindolylmaleimide I (BIM I) were wounded along perpendicular axes using a sterilized comb (37 mm wide, with 13- by 1-mm teeth) to create a grid wound.16Ho B. Hou G. Pickering J.G. Hannigán G. Langille B.L. Bendeck M.P. Integrin-linked kinase in the vascular smooth muscle cell response to injury.Am J Pathol. 2008; 173: 278-288Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar After wounding, fresh media with or without PKC inhibitor BIM I was added, and the cells were incubated for 6 hours. Cells were lysed with lysis buffer solution containing 1% SDS, 1 mmol/L phenylmethylsulfonyl fluoride, 0.01 mg/ml leupeptin, 50 mmol/L Tris (pH 7.6), phosphatase inhibitor cocktail III (Calbiochem; EMD Chemicals, Gibbstown, NJ), 20 mmol/L β glycerophosphate, 5 mmol/L EDTA, 5 mmol/L sodium fluoride, 5 mmol/L orthovanadate, 10 mmol/L dithiothreitol, and protease inhibitor cocktail (Roche Diagnostics, Laval, PQ, Canada). Cells were scraped, mixed, and then forced through an insulin syringe to break the DNA. Sample buffer (2×) containing 0.5 mol/L Tris (pH 6.8), 10% SDS, glycerol, and 0.1% bromphenol blue was added 1:1 to protein extracts. Thirty microliters each of samples and molecular markers SM00441 (Fermentas, Burlington, ON, Canada), and Peppermint Stick phosphoprotein molecular weight marker (Molecular Probes; Invitrogen) were loaded onto a 10-well 4% to 15% Tris-HCl gel (Bio-Rad, Hercules, CA) and separated by SDS-polyacrylamide gel electrophoresis at constant voltage of 140 mV on a Bio-Rad Mini-PROTEAN 3 apparatus. Proteins separated by SDS-polyacrylamide gel electrophoresis were visualized by staining for phosphoproteins and subsequently for total proteins as reported previously.17Silverman-Gavrila L.B. Lu T.Z. Prashad R.C. Nejatbakhsh N. Charlton M.P. Feng Z.P. Neural phosphoproteomics of a chronic hypoxia model—Lymnaea stagnalis.Neuroscience. 2009; 161: 621-634Crossref PubMed Scopus (19) Google Scholar, 18Silverman-Gavrila L.B. Charlton M.P. Calcineurin and cytoskeleton in low-frequency depression.J Neurochem. 2009; 109: 716-732Crossref PubMed Scopus (12) Google Scholar The gel was first stained with Pro-Q Diamond (Molecular Probes; Invitrogen), a fluorescent dye specific for phosphoamino acids. The gel was fixed in 50% methanol and 10% acetic acid for 60 minutes, washed in distilled H2O three times for 10 minutes, stained with Pro-Q Diamond for 90 minutes, and destained three times for 30 minutes in destaining solution containing 20% acetonitrile and 50 mmol/L sodium acetate, pH 4. The gel was visualized with an Ettan DIGE Imager system (GE HealthCare, Piscataway, NJ) on Cy3 channel (excitation 540/25 nm, emission 595/25 nm) using Ettan DIGE Imager 1.0 software (GE HealthCare). To ensure that all bands imaged from the gel corresponded to the phosphorylated proteins, we used Peppermint Stick phosphoprotein molecular weight standards (Molecular Probes; Invitrogen) that contain a mixture of phosphorylated and nonphosphorylated proteins as positive and negative control for detection of phosphorylated proteins. Separation by SDS-polyacrylamide gel electrophoresis resolves the mixture in two phosphorylated and two nonphosphorylated protein bands. Images were collected so that, on the marker lane, only the phosphorylated proteins appear as two dark bands; nonphosphorylated proteins are not detected. The same gel was then stained overnight with a luminescent SYPRO Ruby dye (Molecular Probes; Invitrogen) for total proteins. After two 30-minute washes in 10% methanol and 7% acetic acid and one rinse with distilled H2O, the gel was scanned using SYPRO Ruby 1 (480 nm excitation, 595 nm emission) or SYPRO Ruby 2 (390 nm excitation, 595 nm emission) channels. Images were collected such that on the lane containing both phosphorylated and nonphosphorylated proteins the standards showed up as four dark bands corresponding to total proteins. The gel was then analyzed to identify bands that had a change in their phosphorylation level. The protein phosphorylation level was calculated by the ratio of phosphoprotein intensity of the Pro-Q Diamond signal (P) to total protein intensity of the SYPRO signal (T). We measured the intensity of the Pro-Q Diamond signal (P) and the SYPRO Ruby signal (T) from a rectangular area along each band of interest in the gel using ImageJ software version 1.43 (NIH, Bethesda, MD; http://rsb.info.nih.gov/ij/) and the values were imported into Excel 2003 software (Microsoft, Redmond, WA). The calculated value of the band intensity was obtained by subtracting the measured intensity from 255 and then was used to calculate the average phosphorylated/total ratio (P/T) intensity. Because both Pro-Q Diamond and SYPRO Ruby intensities were measured from the same protein band, variation in protein loading could not affect the P/T ratio for individual proteins among the groups. Analysis of band intensity of phosphorylated and total proteins was repeated three times using independent samples. After analysis, the gel was stained with a visible permanent dye, GelCode Blue stain reagent (Pierce; Thermo Fisher Scientific, Rockford, IL). To visualize the protein bands, we imaged the gel with a Kodak Image Station 2000 R system via a thermoelectrically cooled charged-coupled device camera (Eastman Kodak, Rochester, NY). Protein bands of interest were excised from the gel and digested before identification by mass spectrometry. In-gel trypsin digestion of proteins was performed using an In-Gel tryptic digestion kit (Pierce; Thermo Fisher Scientific). Bands of interest were excised, destained twice for 30 minutes each time by incubation at 37°C in destaining solution (containing ammonium bicarbonate and acetonitrile), reduced by incubation in reducing buffer [Tris(2-carboxyethyl)phosphine in digestion buffer that contains ammonium bicarbonate] for 10 minutes at 60°C, alkylated in alkylation buffer containing iodoacetamide in dark for 1 hour at room temperature, washed two times in destaining buffer at 37°C for 15 minutes, shrunk in acetonitrile 15 minutes at room temperature, and digested overnight with constant shaking at 30°C in digestion buffer containing activated trypsin. We further extracted peptides with 1% trifluoroacetic acid, and the tryptic digestions from bands were sent to the Ontario Cancer Biomarker Network Facility Centre for protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (also known as MALDI-TOF MS) as described previously.18Silverman-Gavrila L.B. Charlton M.P. Calcineurin and cytoskeleton in low-frequency depression.J Neurochem. 2009; 109: 716-732Crossref PubMed Scopus (12) Google Scholar Peptide-mass fingerprinting chromatograms of MS/MS data showing spectra peak lists of mass to charge value sets were analyzed against all available proteins from a database using the MASCOT search engine (Matrix Science, London, United Kingdom). The following search parameters were used: enzyme = trypsin; fixed modification = carbamidomethyl; variable modification = oxidation; mass value = MH+; monoisotopic protein mass = unrestricted. Peptide mass tolerance was set to ± 100 to 200 ppm (fraction expressed as parts per million); peptide charge state = 1+ and maximum missed cleavages allowed = 1. Mass fragment spectra were compared with the Swiss-Prot database merged into the UniProt database (Universal Protein Resource, available at http://www.uniprot.org). The NetPhos 2.0 server (available from the Center for Biological Sequence Analysis, Technical University of Denmark at http://www.cbs.dtu.dk/services/NetPhos/) and the PhosphoMotif Finder (available from the Human Protein Reference Database at http://www.hprd.org/PhosphoMotif_finder) were used to determine the predicted serine, threonine, and tyrosine phosphorylation sites in the proteins of interest. Kinase-specific phosphorylation predictions were retrieved from the NetPhosK 1.0 server (http://www.cbs.dtu.dk/services/NetPhosK/). Cells were treated with the following PKC inhibitors: 1 μmol/L PKCα inhibitor BIM I (Calbiochem), 20 nmol/L Ly-333531 (Axon Medchem, Groningen, The Netherlands), 10 μmol/L Rottlerin (Calbiochem), 100 μmol/L PKCε inhibitory peptide (Santa Cruz Biotechnology, Santa Cruz, CA), 100 μmol/L PKCζ inhibitory peptide (Tocris Bioscience, Ellisville, MO), and 100 μmol/L PKC scrambled peptide substrate (Tocris Bioscience), or with GSK-3 LiCl inhibitor at 30 mmol/L concentration, or with 10−4 mol/L or 10−5 mol/L cytochalasin D starting 30 minutes before wounding and with the treatment continued for 6 hours after wounding. SMCs were transfected in suspension with target-specific validated siRNA sequences (Ambion Biosciences, Austin, TX): RHAMM-specific siRNA sequence (Silencer predesigned siRNA no. 48767, 197416, and 197415), ARPC5-specific siRNA sequence (Silencer predesigned siRNA no. 281070, 281071, and 281072), with nontargeting siRNA Silencer negative control siRNAs used as a negative control for non-sequence-specific effects. Medial and neointimal SMCs were trypsinized, spun down, and resuspended in standard growth medium without antibiotics such that 2.5 ml medium contained 200,000 cells. Ninety picomoles of either the nontargeting or RHAMM-specific or ARPC5-specific duplex diluted in 500 μL Opti-MEM I reduced serum medium (Gibco; Invitrogen) and 5 μL of Lipofectamine RNAiMAX (Invitrogen) was added to wells. The mixtures were gently mixed and incubated for 20 minutes at room temperature. To each well containing RNAi duplex-Lipofectamine RNAiMAX complexes, 200,000 cells were added, to give a final RNA concentration of 30 nmol/L for RHAMM- or ARPC5-specific duplex. At 24 hours after transfection, antibiotic-free medium was replaced with standard growth medium; once the cells reached confluence, they were lysed to assess the attenuation of RHAMM and ARPC5 expression by Western blotting. Neointimal and medial SMCs transfected with the most potent siRNA for ARPC5 (Silencer predesigned siRNA no. 281071) or for RHAMM (Silencer predesigned siRNA no. 48767) were plated in Petri dishes with coverslips. After confluence, they were wounded with a needle and fixed at 6 hours after wounding. The attenuation of protein expression level after siRNA for RHAMM and ARPC5 was verified by Western blotting of cell lysates probed with antibodies specific for RHAMM, ARPC5, and actin-related protein 2/3 complex subunit 3 (ARPC3). After siRNA, protein concentration was measured by a Bio-Rad detergent-compatible microplate assay using a Kinetic microplate reader (Molecular Devices, Sunnyvale, CA). Ten micrograms of proteins from cell lysates were resolved by electrophoresis on a SDS-polyacrylamide gel. Proteins were transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, MA) in 25 mmol/L Tris-HCl, 250 mmol/L glycine, 0.1% (w/v) SDS, pH 8.3, for 18 hours at 30 V at 4°C in a Bio-Rad mini trans-blot system. The polyvinylidene difluoride membranes were blocked with 3% bovine serum albumin in PBS overnight and then were probed with primary antibodies: rabbit polyclonal anti-RHAMM antibody H-90 diluted 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit monoclonal anti-ARPC5 antibody anti-p16-Arc diluted 1:4000 (Novus Biologicals, Littleton, CO). Next, the membranes were incubated with corresponding goat anti-rabbit IgG horseradish peroxidase−conjugated secondary antibody (Upstate Biotechnology; Millipore). The membrane incubated with ARPC5 antibody was stripped with stripping buffer for 30 minutes (0.1 mol/L glycine-HCl, pH 2.5–3.0), then was reprobed at room temperature for 1 hour with mouse monoclonal anti-ARPC3 antibody anti-p21-Arc diluted 1:3000 (Santa Cruz Biotechnology), and incubated for 1 hour with goat anti-mouse IgG horseradish peroxidase−conjugated secondary antibody diluted 1:2000 (Upstate Biotechnology; Millipore). The membranes were then stripped with stripping buffer for 30 minutes and were reprobed at room temperature for 1 hour with mouse monoclonal anti-α-tubulin antibody diluted 1:1000 as a control for loading variations. Next, the membranes were incubated for 1 hour with goat anti-mouse horseradish peroxidase−conjugated secondary antibody diluted 1:2000 (Upstate Biotechnology; Millipore). An enzymatic chemiluminescence detection kit (RPN 2106) (Amersham Bioscience, Baie d'Urfe, QC, Canada) was used to detect proteins. Probed membranes were exposed to X-ray scientific imaging film (Eastman Kodak) and bands were visualized with a film processor (SRX101A; Konica, Tokyo, Japan). Vectors were designed containing either a wild-type ARPC5 sequence or a mutated nonphosphorylatable ARPC5 sequence. We designed the ARPC5 mutant to contain point mutations in which three serines (at positions 8, 85, and 97) and two threonines (at positions 146 and 150) at putative phosphorylation sites for PKC were replaced with alanine residues. The GCT codon for alanine is the most frequently found in wild-type ARPC5, so we chose this to replace the codons for serine or threonine. The alignment of human, rat, and mouse ARPC5 using UniProt tools (http://www.uniprot.org/align) showed a very high conservation of the ARPC5 gene among these species. All five putative phosphorylation sites had a >50% probability of being phosphorylated by PKC; they were 100% conserved among the three species, and were located in hydrophobic regions of the protein. The wild-type ARPC5 and the mutated ARPC5 sequences were synthesized and cloned into the XhoI/HindIII sites of the pEGFP-N1 vector (GenBank no. U55762) (Clontech, Mountain View, CA) by GenScript (Piscataway, NJ). A Kozak sequence (ACC) was added next to the start codon to promote protein synthesis, and the TAG stop codon was removed to allow continuous gene transcription from ARPC5 to EGFP. GenScript also performed sequencing alignments, vector sequencing to ensure that the flanking sequences of the cloning sites were correct, restriction digest assessment, and PCR amplification to ensure that the size of the inserted fragment was correct and free of contaminating bands. We amplified the plasmids in DH5α cells (Invitrogen), and plasmid DNA was purified using an EndoFree plasmid maxi kit from Qiagen (Germantown, MD). At the start of each experiment, neointimal SMCs were transfected in suspension with ARPC5-specific siRNA sequence as described above to knock down endogenous ARPC5. Next, neointimal SMCs were transfected with either wild-type or mutated ARPC5 plasmid. The siRNA does not interfere with expression of the transfected ARPC5 plasmid because it targets nucleotides 874 to 893 of the mRNA sequence, which are outside the coding sequence in the transfected plasmid. One day before plasmid transfection, cells treated with siRNA for ARPC5 were trypsinized and 25 × 105 cells were plated on slides in six-well plates in 2 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum without antibiotics. Cells were 50% to 80% confluent at the time of transfection. For each well, 2.5 μg plasmid DNA was dissolved in 500 μL Opti-MEM I reduced serum medium without serum (Invitrogen) and was mixed gently. After 5 minutes, 15 μL/well of diluted transfection agent Dharmacon DharmaFect Duo (Thermo Fisher Scientific, Waltham, MA) was added to the diluted DNA mix and was incubated for 30 minutes at room temperature. DNA-transfection agent complexes (500 μL) were added and mixed with the media in wells containing cells. After transfection, cells were grown to confluence in medium containing 10% fetal bovine serum at 37°C in a CO2 incubator. The cells were incubated for 24 hours before testing for gene expression [appearance of green fluorescent protein (GFP) signal]. Transfected neointimal SMCs were wounded in the presence or absence of the PKC inhibitor BIM I and were fixed 6 hours later. Cultured rat aortic SMCs were fixed for 20 minutes in 4% paraformaldehyde for cells that were stained for F-actin and γ-tubulin, and for 2 minutes in methanol for cells that were stained for α-tubulin and γ-tubulin. Cells were permeabilized with 0.2% Triton X-100 for 20 minutes, rinsed three times for 5 minutes each with PBS, and incubated with primary antibodies for 1 hour. Neointimal SMCs were washed three times for 5 minutes each with PBS and were incubated with appropriate secondary antibodies for 30 minutes, followed by washing with PBS three times for 5 minutes each and then incubation for 1 hour with the other primary antibody or with Alexa Fluor 568-labeled phalloidin (Molecular Probes; Invitrogen) to stain F-actin. Cells were either double immunostained with i) goat anti-rabbit polyclonal antibody for γ-tubulin diluted 1:100 (Sigma-Aldrich) and with goat anti-mouse monoclonal antibody for α-tubulin clone DM1-A diluted 1:700 (Sigma-Aldrich), ii) or were stained with mouse monoc
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