Atrophin Proteins Interact with the Fat1 Cadherin and Regulate Migration and Orientation in Vascular Smooth Muscle Cells

Fat1, an atypical cadherin induced robustly after arterial injury, has significant effects on mammalian vascular smooth muscle cell (VSMC) growth and migration. The related Drosophila protein Fat interacts genetically and physically with Atrophin, a protein essential for development and control of cell polarity. We hypothesized that interactions between Fat1 and mammalian Atrophin (Atr) proteins might contribute to Fat1 effects on VSMCs. Like Fat1, mammalian Atr expression increased after arterial injury and in VSMCs stimulated with growth and chemotactic factors including angiotensin II, basic fibroblast growth factor, and platelet-derived growth factor BB. Two distinct Atr2 transcripts, atr2L and atr2S, were identified by Northern analysis; in VSMCs, atr2S mRNA expression was more responsive to stimuli. By immunocytochemistry, Fat1 and Atrs colocalized at cell-cell junctions, in the perinuclear area, and in the nucleus. In coimmunoprecipitation studies, Fat1 interacted with both Atr1 and Atr2; these interactions required Fat1 amino acids 4300–4400 and an intact Atro-box in the Atrs. Knock-down of Atrs by small interfering RNA did not affect VSMC growth but had complex effects on migration, which was impaired by Atr1 knockdown, enhanced by Atr2L knockdown, and unchanged when both Atr2S and Atr2L were depleted. Enhanced migration caused by Atr2L knockdown required Fat1 expression. Similarly, orientation of cells after monolayer denudation was impaired in cells with Atr1 knockdown but enhanced in cells selectively depleted of Atr2L. Together these findings suggest that Fat1 and Atrs act in concert after vascular injury but show further that distinct Atr isoforms have disparate effects on VSMC directional migration. Fat1, an atypical cadherin induced robustly after arterial injury, has significant effects on mammalian vascular smooth muscle cell (VSMC) growth and migration. The related Drosophila protein Fat interacts genetically and physically with Atrophin, a protein essential for development and control of cell polarity. We hypothesized that interactions between Fat1 and mammalian Atrophin (Atr) proteins might contribute to Fat1 effects on VSMCs. Like Fat1, mammalian Atr expression increased after arterial injury and in VSMCs stimulated with growth and chemotactic factors including angiotensin II, basic fibroblast growth factor, and platelet-derived growth factor BB. Two distinct Atr2 transcripts, atr2L and atr2S, were identified by Northern analysis; in VSMCs, atr2S mRNA expression was more responsive to stimuli. By immunocytochemistry, Fat1 and Atrs colocalized at cell-cell junctions, in the perinuclear area, and in the nucleus. In coimmunoprecipitation studies, Fat1 interacted with both Atr1 and Atr2; these interactions required Fat1 amino acids 4300–4400 and an intact Atro-box in the Atrs. Knock-down of Atrs by small interfering RNA did not affect VSMC growth but had complex effects on migration, which was impaired by Atr1 knockdown, enhanced by Atr2L knockdown, and unchanged when both Atr2S and Atr2L were depleted. Enhanced migration caused by Atr2L knockdown required Fat1 expression. Similarly, orientation of cells after monolayer denudation was impaired in cells with Atr1 knockdown but enhanced in cells selectively depleted of Atr2L. Together these findings suggest that Fat1 and Atrs act in concert after vascular injury but show further that distinct Atr isoforms have disparate effects on VSMC directional migration. Migration and proliferation of VSMCs 2The abbreviations used are: VSMC, vascular smooth muscle cell; Atr, atrophin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTOC, microtubule organizing center; siRNA, small interfering RNA; qRT, quantitative reverse transcription; EGF, epidermal growth factor; DAPI, 4′,6′-diamino-2-phenylindole. in the wall of injured blood vessels are critical activities in the pathogenesis of atherosclerosis and related, clinically important vascular diseases. Arterial injury strongly induces expression of the Fat1 cadherin, which has distinct effects on VSMC migration and proliferation (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar). Fat1 and related, atypical cadherins form a subfamily characterized by large extracellular domains containing 34 cadherin motifs, a variable number of EGF repeats, one or two laminin A/G domains, and a single transmembrane domain (2Tanoue T. Takeichi M. J. Cell Sci. 2005; 118: 2347-2353Crossref PubMed Scopus (125) Google Scholar). In vertebrates, the Fat subfamily consists of four members, Fat1, -2, -3, and -4 (or Fat-J), whereas in Drosophila, two members, Fat and Fat-like, have been identified (2Tanoue T. Takeichi M. J. Cell Sci. 2005; 118: 2347-2353Crossref PubMed Scopus (125) Google Scholar, 3Halbleib J.M. Nelson W.J. Genes Dev. 2006; 20: 3199-3214Crossref PubMed Scopus (802) Google Scholar). Although the Drosophila fat (ft) mutation, which causes enlargement of all larval imaginal discs, including wing, leg, eye-antenna, haltere, and genital imaginal discs, was first described by Mohr 85 years ago (4Bryant P.J. Huettner B. Held Jr., L.I. Ryerse J. Szidonya J. Dev. Biol. 1988; 129: 541-554Crossref PubMed Scopus (162) Google Scholar), understanding of how Fat proteins control developmental processes has only recently started to emerge. Distinct functions have been ascribed to Drosophila Fat and Fat-like. The former acts as a suppressor of hyperplastic growth, as mentioned above, and as a mediator of planar cell polarity signals in development (4Bryant P.J. Huettner B. Held Jr., L.I. Ryerse J. Szidonya J. Dev. Biol. 1988; 129: 541-554Crossref PubMed Scopus (162) Google Scholar, 5Fanto M. Clayton L. Meredith J. Hardiman K. Charroux B. Kerridge S. McNeill H. Development. 2003; 130: 763-774Crossref PubMed Scopus (131) Google Scholar). Fat has been identified in several recent studies as a regulator of the Hippo signaling pathway, which controls organ size during development through effects on both cell proliferation and survival (6Cho E. Feng Y. Rauskolb C. Maitra S. Fehon R. Irvine K.D. Nat. Genet. 2006; 38: 1142-1150Crossref PubMed Scopus (359) Google Scholar, 7Silva E. Tsatskis Y. Gardano L. Tapon N. McNeill H. Curr. Biol. 2006; 16: 2081-2089Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 8Thompson B.J. Cohen S.M. Cell. 2006; 126: 767-774Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 9Willecke M. Hamaratoglu F. Kango-Singh M. Udan R. Chen C.L. Tao C. Zhang X. Halder G. Curr. Biol. 2006; 16: 2090-2100Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 10Tyler D.M. Baker N.E. Dev. Biol. 2007; 305: 187-201Crossref PubMed Scopus (89) Google Scholar). Fat-like, on the other hand, performs a crucial morphogenetic role in the formation of tubular organs such as the trachea, possibly by acting as an epithelial spacer (11Castillejo-Lopez C. Arias W.M. Baumgartner S. J. Biol. Chem. 2004; 279: 24034-24043Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In contrast to the effect of Fat mutations, impairment of Fat-like expression does not affect imaginal disc development or planar cell polarity (11Castillejo-Lopez C. Arias W.M. Baumgartner S. J. Biol. Chem. 2004; 279: 24034-24043Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), but whether or not Fat and Fat-like have redundant or overlapping functions in other settings has not been fully explored. Information about the function of vertebrate Fat proteins is relatively limited. Mice with homozygous inactivation of the fat1 locus die perinatally with loss of the renal glomerular slit junctions, fusion of glomerular epithelial cell processes, and defects in forebrain and eye development; growth perturbations were not detected during embryonic skin development or in neurospheres derived from fat1-/- mice (12Ciani L. Patel A. Allen N.D. ffrench-Constant C. Mol. Cell. Biol. 2003; 23: 3575-3582Crossref PubMed Scopus (198) Google Scholar). In cultured cells, Fat1 interacts with Ena/VASP proteins, localizes to filopodial tips and lamellipodia at cellular leading edges, organizes cytoskeletal actin, and promotes migration (13Tanoue T. Takeichi M. J. Cell Biol. 2004; 165: 517-528Crossref PubMed Scopus (131) Google Scholar, 14Moeller M.J. Soofi A. Braun G.S. Li X. Watzl C. Kriz W. Holzman L.B. EMBO J. 2004; 23: 3769-3779Crossref PubMed Scopus (156) Google Scholar). We identified increased Fat1 expression in VSMCs responding to arterial injury and found that knockdown of Fat1 in this cell type limited migration but enhanced proliferation (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar); taken together with the findings in fat1-/- mice (12Ciani L. Patel A. Allen N.D. ffrench-Constant C. Mol. Cell. Biol. 2003; 23: 3575-3582Crossref PubMed Scopus (198) Google Scholar), these results point to cell type- or developmental stage-dependent differences in the ability of Fat1 to regulate growth. Additional evidence for Fat1 function as a tumor suppressor includes the recent report of very frequent homozygous deletion or gene silencing of the fat1 locus in oral squamous cell carcinomas (15Nakaya K. Yamagata H.D. Arita N. Nakashiro K.I. Nose M. Miki T. Hamakawa H. Oncogene. 2007; 26: 5300-5308Crossref PubMed Scopus (73) Google Scholar). As for the other vertebrate Fat proteins, recent reports suggest that Fat2 supports the migration of squamous carcinoma cells (16Matsui S. Utani A. Takahashi K. Mukoyama Y. Miyachi Y. Matsuyoshi N. J. Dermatol. Sci. 2008; 51: 207-210Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), whereas Fat4 controls the orientation of cell divisions and tubule elongation during kidney development in the mouse (17Saburi S. Hester I. Fischer E. Pontoglio M. Eremina V. Gessler M. Quaggin S.E. Harrison R. Mount R. McNeill H. Nat. Genet. 2008; 40: 1010-1015Crossref PubMed Scopus (406) Google Scholar). Genetic analysis in Drosophila demonstrates that Atrophin functions as a transcriptional corepressor during development, with important roles in diverse processes including segmentation and planar polarity (18Zhang S. Xu L. Lee J. Xu T. Cell. 2002; 108: 45-56Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). A link between Fat cadherins and Atrophin was first identified in a yeast two-hybrid screen in Drosophila using a fragment of the Fat cytoplasmic domain (5Fanto M. Clayton L. Meredith J. Hardiman K. Charroux B. Kerridge S. McNeill H. Development. 2003; 130: 763-774Crossref PubMed Scopus (131) Google Scholar). Comparison of ft and atrophin mutants in Drosophila showed similar defects in planar polarity, and double mutants showed strongly enhanced effects on viability, indicating genetic interaction (5Fanto M. Clayton L. Meredith J. Hardiman K. Charroux B. Kerridge S. McNeill H. Development. 2003; 130: 763-774Crossref PubMed Scopus (131) Google Scholar). Whether or not fat-like (ftl) and atrophin interact has not been reported. Whereas the Drosophila genome encodes a single Atrophin, vertebrate genomes harbor two loci that give rise to Atr1 and two forms of Atr2: a long form (Atr2L, also known as RERE) and a short form (Atr2S); Drosophila Atrophin is most like Atr2L in terms of overall structure, suggesting that this long form of the protein may reflect the ancestral gene (19Shen Y. Lee G. Choe Y. Zoltewicz J.S. Peterson A.S. J. Biol. Chem. 2007; 282: 5037-5044Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Interestingly, the Atr isoforms with extended N-terminal domains (Drosophila Atrophin and vertebrate Atr2L) can interact with histone deacetylases (20Zoltewicz J.S. Stewart N.J. Leung R. Peterson A.S. Development. 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar, 21Wang L. Rajan H. Pitman J.L. McKeown M. Tsai C.C. Genes Dev. 2006; 20: 525-530Crossref PubMed Scopus (79) Google Scholar, 22Plaster N. Sonntag C. Schilling T.F. Hammerschmidt M. Dev. Dyn. 2007; 236: 1891-1904Crossref PubMed Scopus (39) Google Scholar), indicating at least one mechanism by which these proteins can act as transcriptional repressors. The importance of the N-terminal sequences is supported in vivo by the observation that, although Atr1-null mice are viable and fertile (19Shen Y. Lee G. Choe Y. Zoltewicz J.S. Peterson A.S. J. Biol. Chem. 2007; 282: 5037-5044Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), Atr2L mutant mice die around embryonic day 9.5 with defects in heart looping, telencephalon, and somite development, as well as loss of Sonic hedgehog (Shh) and fibroblast growth factor (Fgf) 8 expression from anterior signaling centers (20Zoltewicz J.S. Stewart N.J. Leung R. Peterson A.S. Development. 2004; 131: 3-14Crossref PubMed Scopus (81) Google Scholar). In view of our previous findings that Fat1 regulates VSMC growth and migration, we asked whether mammalian Atrs and their distinct isoforms might contribute to these Fat1-mediated effects. Our studies show that Atrs, like Fat1, are induced after arterial injury, that Atrs and Fat1 interact physically, and that, again like Fat1, they regulate migration and orientation. Interestingly, however, the different Atr isoforms have distinct effects on these cellular activities; the short Atrs, Atr1 and Atr2S, promote migration and orientation, whereas the long Atr isoform, Atr2L, inhibits these activities. Taken together, these results suggest a new framework, potentially extending from the cell surface to the nucleus, for regulation of VSMC chemotaxis in the post-injury setting. Rat Carotid Artery Balloon Injury-The rat carotid artery balloon injury model was implemented as described previously (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar). All of the procedures were approved by and in accordance with guidelines established by the Albert Einstein Institute for Animal Studies. Quantitative Reverse Transcription (qRT)-PCR-Total RNA was extracted from vascular tissues or cells by homogenization in TRIzol (Invitrogen), treated with DNase I (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar units/μl; Promega), and used for first-strand cDNA synthesis. The mRNA levels were quantified in triplicate by qRT-PCR in the Mx3000P real time PCR system with the Brilliant SYBR Green qPCR kit (Stratagene). The rat and mouse atr1-specific primers were 5′-ATCCACTCTCACCTACACCTGCAC-3′ (forward), 5′-ATCCGGGTAAGGTGAGACCCTGAG-3′ (reverse) and 5′-ACATGCTCATCACCCATTGCACAG-3′ (forward), 5′-GCTTGTCACTCTCCTTCTTCAGGT-3′ (reverse), respectively. The mouse atr2-specific primers were 5′-GCCCTTCATGTTCAAGCCTGTCAA-3′ (forward), 5′-TCTTCATTGATGGGTGAGGCACGA-3′ (reverse). The rat and mouse atr2S-specific primers were 5′-TCCTTACCTGTAGCTGCGAGTCTT-3′ (forward), 5′-AGAAGTTCTTGCCGTACTGTCGGA-3′ (reverse) and 5′-AATTAAGACTCGCACAGCGTCCAC-3′ (forward), 5′-CTGTTCGCTGTCCTCACTGTCAAA-3′ (reverse), respectively. The rat and mouse atr2L-specific primers were 5′-AGATCTCCAGCTCCTGCTTTGTGT-3′ (forward), 5′-TGACGTTCATGAGTAGATGGTCCC-3′ (reverse) and 5′-CCAGTCATCAAGAATCGGGAACTC-3′ (forward), 5′-GGGCTTTAAACTCTCGAGCAGCAA (reverse), respectively. PCR cycling conditions included 10 min at 95 °C for 1 cycle followed by 40 cycles at 95 °C for 20 s, 58–62 °C for 30 s, and 72 °C for 20 s. A dissociation curve obtained for each PCR product after each run confirmed that signals corresponded to unique amplicons. The expression levels were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels for each sample in parallel assays and analyzed using the comparative ΔΔCt method (23Bustin S.A. J. Mol. Endocrinol. 2000; 25: 169-193Crossref PubMed Scopus (3076) Google Scholar). Cell Culture and Transfection-Primary culture VSMCs were prepared from rat or mouse aortae and maintained as described previously (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar). Human aortic VSMCs were obtained from Clonetics and cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum. VSMC phenotype was validated by immunocytochemistry using an antibody specific for α smooth muscle actin (1:400, Clone 1A4; NeoMarkers), and cells were used between four and eight passages from harvest. 293T and Chinese hamster ovary cell lines (American Tissue Type Collection) were cultured in the medium according to the supplier's recommendations. Angiotensin II, dexamethasone, and transforming growth factor-β were obtained from Sigma, and basic fibroblast growth factor, platelet-derived growth factor BB, interferon-γ, and interleukin-1β were from U.S. Biological. In stimulation experiments, the cells were rendered quiescent by incubation in medium containing 0.4% horse serum for 72 h prior to the addition of fetal bovine serum or other stimulus. Control cultures received an equivalent amount of vehicle. Total cellular protein or total RNA was extracted at designated time points. In transfection experiments, FuGENE 6 (Roche Applied Science) or TransIT-LT1 reagent (Mirus) were used according to the manufacturers' protocols. Northern Analysis-Total RNA (10 μg) was fractionated on 1.2% formaldehyde-agarose gels and transferred to nitrocellulose filters. A rat Atr2 cDNA probe, corresponding to nucleotides 4495–4922 in GenBank™ entry NM_053885, was generated by PCR, random primed in the presence of 32P-labeled dCTP, and hybridized to the filters in QuikHyb (Stratagene). The filters were washed to high stringency at 55 °C in buffer containing 30 mmol/liter sodium chloride, 3 mmol/liter sodium citrate, and 0.1% SDS. Autoradiography was performed with Kodak XAR film at -80 °C for 48 h. 5′-Rapid Amplification of cDNA Ends-To define the 5′ end of the mouse atr2S transcript, we performed 5′-rapid amplification of cDNA ends using Ambion mouse embryo rapid amplification of cDNA ends-ready cDNA according to the manufacturer's instructions. The atr2 gene-specific primers were: 5′-TGGGAGACGTGCTGCGATTA-3′ (outer primer) and 5′-ATGCAGCCTCTTCCTTCACCTT-3′ (inner primer). Amplified cDNA products were cloned and sequenced by standard methodology. This sequence will be supplied as supplemental data. Western Analysis-The cells were homogenized, and the proteins were extracted in radioimmunoassay buffer containing protease inhibitors and resolved by SDS-PAGE with 3–8% Tris acetate or 8% Tris-glycine gels (Invitrogen). The proteins were transferred electrophoretically onto ImmobilonP membranes (Millipore), which were incubated with antisera or monoclonal antibodies specific for Fat1 (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar), Atr1 (1:100, A-19; Santa Cruz), FLAG M2 (1:5000; Sigma), or c-Myc (1:100, 9E10; Santa Cruz). Equivalent protein loading was evaluated with anti-Actin (1:100, C-11; Santa Cruz) antibody. cDNA Constructs-The DelN-Fat1 construct (13Tanoue T. Takeichi M. J. Cell Biol. 2004; 165: 517-528Crossref PubMed Scopus (131) Google Scholar) encodes an N-terminal truncated form of human Fat1 (amino acid residues 3987–4590) in which all the cadherin domains, one EGF motif, and the laminin A-G domain have been removed, and part of the extracellular and all of the transmembrane and intracellular domains (IC) are retained. We generated a similar construct by RT-PCR. A 3XFLAG sequence was integrated in frame into the N-terminal of the cDNA, and the resulting product was subcloned into the p3XFLAG-CMV-13 expression vector (Sigma), yielding an N-terminally FLAG-tagged product defined at the C terminus by the native Fat1 stop codon. Derivative C-terminal truncation constructs, designated DelN-Fat1 4500, DelN-Fat1 4400, and DelN-Fat1 4300, were generated by introducing an additional stop codon at the corresponding amino acid residue (residue 4500, 4400, or 4300) by QuikChange site-directed mutagenesis (Stratagene). The full-length cDNAs encoding human Atr1 and Atr2L (long form, amino acids 1–1506) were obtained from Open Biosystems and subcloned into pCMV·SPORT6 (Invitrogen) and pcDNA-DEST40 (Invitrogen), respectively. These cDNA inserts were also cloned in frame into the pCS2–6XMyc vector to provide C-terminal Myc epitope tags and into the pEGFP-C2 vector (Clontech) to provide N-terminal EGFP epitope tags. The Atr2S (short form, amino acids 495–1506) cDNA was obtained by RT-PCR and subcloned similarly into the pCS2–6XMyc and pEGFP-C2 vector. All of the constructs were confirmed by sequencing. RNA Interference-The mouse Fat1 siRNA has been described previously (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar). The mouse atr siRNAs (Sigma/Proligo) were designed to target 21-nucleotide sequences derived from atr1 (GenBank™ accession number NM007881, starting at positions 719 (GAATGCTAGTGGAGGTGTT), 905 (GAGCTTACCTTCTGCACCA), and 1297 (CTAGTATGTCTGTCTCTAA)), atr2 (GenBank™ accession number XM204015, starting at positions 689 (CTGATTATGTTGACACCTA); 1116 (CAGTTATGATGCCGGCAAA), and 4622 (CAAGTCAGGAGGATTATTA)). Control siRNA included scrambled derivatives of the atr1 1297 sequence, atr2 4622 sequence, an unrelated siRNA based on the Renilla luciferase sequence, and a negative control siRNA (Qiagen). siRNA was transfected with X-tremeGENE reagent (Roche Applied Science) according to the manufacturer's recommendations. Knockdown efficiency was assessed by Western analysis or qRT-PCR. Coimmunoprecipitation-Specific proteins were immunoprecipitated by incubating 300–500 μg of precleared whole cell lysates in immunoprecipitation buffer with 2–5 μg of the corresponding antibodies or normal IgG control at 4 °C for 2 h, followed by incubation with protein G agarose (Invitrogen) at 4 °C overnight with gentle agitation. After extensive washing, the immune complexes were recovered by boiling in sample buffer, and the proteins were detected by Western analysis. Immunocytochemistry-The cells were plated on chamber slides (Becton-Dickinson), fixed, stained, and photographed by epifluorescent microscopy as previously described (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar). Primary antibodies were used at the dilutions indicated: anti-Fat1, 1:1000; anti-Atr1 (A-19; Santa Cruz), 1:100; anti-RERE (REREF1H8; Abcam) 1:50; and anti-Myc (9E10; Santa Cruz), 1:100. After incubating with AlexaFluor-conjugated secondary antibody (1:2000; Molecular Probes) and counterstaining with DAPI, the samples were mounted (Supermount medium; Biogenex) on glass slides, and the signals were visualized using an inverted fluorescent microscope (model IX70; Olympus) equipped with 20×/NA 0.4 and 40×/NA 0.6 LWD objectives and standard fluorescent filter sets, a CCD camera (SensiCam; Cooke), and IPLab software (Scanalytics). Subsequent image processing was performed using Photoshop CS3 and Illustrator CS3 (Adobe Systems). Routine control experiments included omission of the primary antibodies. Lentivirus Preparation and Transduction-Lentivirus was produced using the Virapower Lentiviral kit (Invitrogen) according to the manufacturer's directions. Briefly, cDNA encoding full-length of human Atr1 was cloned into the pLenti6-V5-DEST vector, and virus was generated in the 293FT viral packaging cell line. Equal titers of test or vector control virus were used in subsequent experiments. Mouse VSMCs were infected with virus-containing supernatant in the presence of polybrene, and stably transduced cells were selected with Blasticidin. A total of 16 clones were isolated, and Atr1 expression levels were detected by Western blot. Cell Proliferation Assay-To evaluate DNA synthesis, 1 × 104 cells plated on four-chamber type I collagen-coated chamber slides (Becton Dickinson) were transfected with siRNA, serum-starved (0.4% horse serum) for 48 h, and then stimulated with 10% fetal bovine serum. Bromodeoxyuridine (10 μm, Sigma-Aldrich) was added to cells for 6 h before harvest after 24 h of stimulation. The cells were washed in phosphate-buffered saline, fixed in 4% paraformaldehyde, treated with HCl, and stained sequentially with anti-bromodeoxyuridine antibody (1:200; Abcam) and AlexaFluor 555-conjugated secondary antibody (1:2000; Molecular Probes). The cells were counterstained with DAPI (Molecular Probes). Cell Migration Assay-Cell migration was assessed by 1) in vitro scratch wounding of monolayers and 2) with Transwell 24-well cell culture inserts with 8-μm pores (Costar), as described previously (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar). For the former, cellular progress was photographed and quantitated by planimetry of the denuded area and converted to distance migrated using Image J software. For Transwell assays, quiescent cells were harvested and added (1 × 105/well) to the insert. Culture medium containing 10% fetal bovine serum as chemotactic agent was added to the lower chamber. After 10 h, nonmigrating cells were removed from upper filter surfaces, and the filter was washed, fixed, and stained. We then photographed fifteen randomly selected 100× fields and counted cells that had migrated to the underside of the filter. Microtubule Organizing Center (MTOC) Orientation Assay- The cells were grown to confluence, and the monolayer was denuded in a linear stripe using a pipette tip. The cells were fixed 16 h later with 4% paraformaldehyde, and the MTOC was localized by immunolabeling using anti-pericentrin antibody (1:1000, Abcam) and a secondary goat anti-rabbit labeled with Alexa 488 dye (Invitrogen). Cell nuclei and cytoskeleton were identified with DAPI and rhodamine-phalloidin stains, respectively (Invitrogen). The signals were visualized by epifluorescent microscopy, and cells in which the MTOC localized within the 120° sector facing a line parallel to the wound margin were scored positive. At least 100 cells were examined for each condition. Statistical Analysis-Experiments were repeated a minimum of three times. Comparisons between two groups were analyzed by Student's t test (p < 0.05), and comparisons between three or more groups were assessed by analysis of variance with a Bonferroni/Dunn post hoc test (p < 0.05). The data are presented as the means ± S.E. Atr1 and Atr2 Are Induced after Vascular Injury and by Growth Factors-Like fat1 (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar), both atr1 and atr2 transcripts increased after rat carotid artery balloon injury. For each isoform, mRNA levels peaked 7 days after injury, with levels ∼5-, 4-, and 1.8-fold over base line for atr1, atr2S, and atr2L, respectively (Fig. 1A). We also found induction of Atr1 protein in response to serum (Fig. 1B), again reminiscent of the pattern we found with Fat1 (1Hou R. Liu L. Anees S. Hiroyasu S. Sibinga N.E.S. J. Cell Biol. 2006; 173: 417-429Crossref PubMed Scopus (74) Google Scholar). Although a similar analysis of Atr2 protein was limited by lack of effective antibodies, Northern analysis showed two distinct atr2 transcripts, which we designate atr2L and atr2S, consistent with previous observations (19Shen Y. Lee G. Choe Y. Zoltewicz J.S. Peterson A.S. J. Biol. Chem. 2007; 282: 5037-5044Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Both atr2L and atr2S transcripts increased in response to serum stimulation, albeit with somewhat different kinetics and a greater induction of atr2S (Fig. 1C). Interestingly, relative atr2 isoform levels showed some cell type dependence, because we found a preponderance of the atr2S isoform in VSMCs and of the atr2L isoform in Chinese hamster ovary cells (supplemental Fig. S1). We also tested the response of Atrs to individual factors with known regulatory roles in vascular remodeling. Atr1 protein levels increased ∼2.5-fold in VSMCs treated with angiotensin II, basic fibroblast growth factor, platelet-derived growth factor BB, and interleukin-1β and decreased in cells treated with interferon-γ (Fig. 1D). These treatments, like serum treatment, induced atr2S more strongly than atr2L. Among these factors, the effect of interferon-γ was unique, in that it decreased Atr1 expression while preferentially inducing atr2S mRNA (Fig. 1E). Atr Proteins Colocalize with Fat1 in Different Subcellular Locations-We then determined the subcellular localization of these proteins. Atr1 staining was prominent in the perinuclear area, with less intense signal present at cell leading edges and within the nucleus. Overall, the pattern of Fat1 staining was quite similar, although the Fat1 signal was more prominent at the leading edges (Fig. 2A). Overlap of these signals was apparent in the perinuclear area and at cell-cell junctions and leading edges (Fig. 2A, Merge). Atr2 staining was apparent in cell nuclei and diffusely throughout the cytoplasm and showed overlap with Fat1 in these areas (Fig. 2B). To assess the distribution of the 2 Atr2 isoforms, we transfected cells with constructs encoding epitope-tagged Atr2L or Atr2S. The former localized exclusively to the nucleus, where some overlap with Fat1 was evident (Fig. 2C, Merge). The epitope-tagged Atr2S isoform, on the other hand, was present in the cytoplasm and at cell borders and coincided with Fat1 signal within the nucleus and at cell-cell borders (Fig. 2D). Although transfection, epitope tagging, and overexpression can all spuriously affect protein localization, our findings with liposome-mediated transfection of these C-terminal tagged constructs were consistent with studies of e