FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2078463367> ?p ?o ?g. }

• W2078463367 endingPage "22205" @default.
• W2078463367 startingPage "22193" @default.
• W2078463367 abstract "The development of therapeutic strategies to inhibit reactive oxygen species (ROS)-mediated damage in blood vessels has been limited by a lack of specific targets for intervention. Targeting ROS-mediated events in the vessel wall is of interest, because ROS play important roles throughout atherogenesis. In early atherosclerosis, ROS stimulate vascular smooth muscle cell (VSMC) growth, whereas in late stages of lesion development, ROS induce VSMC apoptosis, causing atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, mouse aortic VSMC were infected with a retroviral human heart cDNA expression library, and apoptosis was induced in virus-infected cells by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) treatment. A total of 17 different, complete cDNAs were identified from the DMNQ-resistant VSMC clones by PCR amplification and sequencing. The cDNA encoding PP1cγ1 (catalytic subunit of protein phosphatase 1) was present in several independent DMNQ-resistant VSMC clones. DMNQ increased mitochondrial ROS production, caspase-3/7 activity, DNA fragmentation, and decreased mitochondrial transmembrane potential in VSMC while decreasing PP1cγ1 activity and expression. Depletion of PP1cγ1 expression by short hairpin RNA significantly enhanced basal as well as DMNQ-induced VSMC apoptosis. PP1cγ1 overexpression abrogated DMNQ-induced JNK1 activity, p53 Ser15 phosphorylation, and Bax expression and protected VSMC against DMNQ-induced apoptosis. In addition, PP1cγ1 overexpression attenuated DMNQ-induced caspase-3/7 activation and DNA fragmentation. Inhibition of p53 protein expression using small interfering RNA abrogated DMNQ-induced Bax expression and significantly attenuated VSMC apoptosis. Together, these data indicate that PP1cγ1 overexpression promotes VSMC survival by interfering with JNK1 and p53 phosphorylation cascades involved in apoptosis. The development of therapeutic strategies to inhibit reactive oxygen species (ROS)-mediated damage in blood vessels has been limited by a lack of specific targets for intervention. Targeting ROS-mediated events in the vessel wall is of interest, because ROS play important roles throughout atherogenesis. In early atherosclerosis, ROS stimulate vascular smooth muscle cell (VSMC) growth, whereas in late stages of lesion development, ROS induce VSMC apoptosis, causing atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, mouse aortic VSMC were infected with a retroviral human heart cDNA expression library, and apoptosis was induced in virus-infected cells by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) treatment. A total of 17 different, complete cDNAs were identified from the DMNQ-resistant VSMC clones by PCR amplification and sequencing. The cDNA encoding PP1cγ1 (catalytic subunit of protein phosphatase 1) was present in several independent DMNQ-resistant VSMC clones. DMNQ increased mitochondrial ROS production, caspase-3/7 activity, DNA fragmentation, and decreased mitochondrial transmembrane potential in VSMC while decreasing PP1cγ1 activity and expression. Depletion of PP1cγ1 expression by short hairpin RNA significantly enhanced basal as well as DMNQ-induced VSMC apoptosis. PP1cγ1 overexpression abrogated DMNQ-induced JNK1 activity, p53 Ser15 phosphorylation, and Bax expression and protected VSMC against DMNQ-induced apoptosis. In addition, PP1cγ1 overexpression attenuated DMNQ-induced caspase-3/7 activation and DNA fragmentation. Inhibition of p53 protein expression using small interfering RNA abrogated DMNQ-induced Bax expression and significantly attenuated VSMC apoptosis. Together, these data indicate that PP1cγ1 overexpression promotes VSMC survival by interfering with JNK1 and p53 phosphorylation cascades involved in apoptosis. Enhanced reactive oxygen species (ROS) 3The abbreviations used are: ROSreactive oxygen speciesVSMCvascular smooth muscle cellsDMNQ2,3-dimethoxy-1,4-naphthoquinoneJNKc-Jun N-terminal kinaseDPIdiphenyleneiodonium chlorideDMEMDulbecco's modified Eagle's mediumDCF2′,7′-dichlorofluorescinZ-DEVD-R110bis-N-benzyloxycarbonyl-l-aspartyl-l-glutamyl-l-valyl-aspartic acid amideFBSfetal bovine serumAdGFPadenovirus backbone with green fluorescent protein cDNAshRNAshort hairpin RNATUNELterminal dUTP nick-end labeling. generation plays an important role in the proliferation, migration, or apoptosis of vascular smooth muscle cells (VSMC), all of which have been implicated in the pathophysiology of vascular diseases, including atherosclerosis. VSMC proliferation and migration are important in the development of atherosclerotic lesions, and VSMC apoptosis is a histologic hallmark of advanced atherosclerosis (1Delafontaine P. Song Y.H. Li Y. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 435-444Crossref PubMed Scopus (445) Google Scholar). Additionally, human VSMC isolated from coronary plaques are more susceptible to apoptosis than VSMC isolated from normal arteries (2Bennett M.R. Evan G.I. Schwartz S.M. J. Clin. Invest. 1995; 95: 2266-2274Crossref PubMed Scopus (610) Google Scholar). Apoptosis of VSMC in atherosclerotic plaques is accompanied by numerous other events that increase the likelihood of plaque rupture. These include decreases in collagen and extracellular matrix protein production, decreased fibrous cap thickness, accumulation of macrophages along the shoulder of the plaque, and increases in the size of the necrotic core and amount of cellular debris within the plaque (3Clarke M.C. Figg N. Maguire J.J. Davenport A.P. Goddard M. Littlewood T.D. Bennett M.R. Nat Med. 2006; 12: 1075-1080Crossref PubMed Scopus (532) Google Scholar). A better understanding of the mechanisms that regulate VSMC apoptosis could lead to the development of strategies to stabilize atherosclerotic plaques. reactive oxygen species vascular smooth muscle cells 2,3-dimethoxy-1,4-naphthoquinone c-Jun N-terminal kinase diphenyleneiodonium chloride Dulbecco's modified Eagle's medium 2′,7′-dichlorofluorescin bis-N-benzyloxycarbonyl-l-aspartyl-l-glutamyl-l-valyl-aspartic acid amide fetal bovine serum adenovirus backbone with green fluorescent protein cDNA short hairpin RNA terminal dUTP nick-end labeling. Identifying genes that could protect cells against oxidative stress requires selection of an oxidant and a screening strategy. A number of different approaches have been used to induce oxidative stress in cultured cells and in vivo. The quinones are of particular interest. Quinones are ubiquitous in nature and are also formed as metabolites from a variety of drugs, environmental pollutants, and food derivatives by the action of cytochrome P450 system (4Monks T.J. Hanzlik R.P. Cohen G.M. Ross D. Graham D.G. Toxicol. Appl. Pharmacol. 1992; 112: 2-16Crossref PubMed Scopus (708) Google Scholar). The redox-cycling 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) has been proposed as a model quinone compound to study the role of ROS in cell toxicity and apoptosis (5Watanabe N. Forman H.J. Arch. Biochem. Biophys. 2003; 411: 145-157Crossref PubMed Scopus (87) Google Scholar). It does not react with free thiol groups and is nonalkylating and nonadduct-forming. One-electron reduction of DMNQ by flavoenzymes, such as NADPH-cytochrome P450 reductase or NADH-cytochrome b5 reductase, yields a semiquinone radical, which then reacts with oxygen to form superoxide (O2.¯) and, subsequently, the dismutation product H2O2. An obligatory two-electron reducing cytosolic flavoenzyme NADPH oxidoreductase 1, also known as DT-diaphorase, can compete with one-electron reductases for DMNQ to produce a hydroquinone, which can undergo autoxidation to yield H2O2 and the parental quinone. Retroviral cDNA expression library screening has been used successfully to identify novel oncogenes (6Whitehead I. Kirk H. Kay R. Mol. Cell. Biol. 1995; 15: 704-710Crossref PubMed Google Scholar) and modulators of apoptosis (7Hitoshi Y. Lorens J. Kitada S.I. Fisher J. LaBarge M. Ring H.Z. Francke U. Reed J.C. Kinoshita S. Nolan G.P. Immunity. 1998; 8: 461-471Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 8Sandal T. Ahlgren R. Lillehaug J. Døskeland S.O. Cell Death Differ. 2001; 8: 754-766Crossref PubMed Scopus (23) Google Scholar). The packaging cell lines used in conjunction with retroviral vectors generate high concentrations of infectious virus particles necessary for the efficient infection of large cell populations, including that of primary cells, with a single copy of vector per cell (9Lorens J.B. Sousa C. Bennett M.K. Molineaux S.M. Payan D.G. Curr. Opin. Biotechnol. 2001; 12: 613-621Crossref PubMed Scopus (30) Google Scholar). In addition, retroviral delivery systems have the ability to deliver very large libraries to infected cell populations. These properties make retroviral cDNA expression library screening an ideal strategy to identify genes that protect against oxidative stress. Protein phosphorylation and dephosphorylation play an important regulatory role in apoptosis and other cellular processes (10Hunter T. Karin M. Cell. 1992; 70: 375-387Abstract Full Text PDF PubMed Scopus (1120) Google Scholar, 11Elledge S.J. Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1772) Google Scholar, 12Gjertsen B.T. Doskeland S.O. Biochim. Biophys. Acta. 1995; 1269: 187-199Crossref PubMed Scopus (102) Google Scholar). Protein phosphatase 1 (PP1), 2A (PP2A), 2B (PP2B), and 2C (PP2C) are the four major serine/threonine phosphatases present in eukaryotic cells (13Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2161) Google Scholar). Of these four, PP1 and PP2A account for the majority of cellular phosphatase activity (13Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2161) Google Scholar). The catalytic subunit of PP1 exists in four different isoforms (α, γ1, γ2, and δ), which are targeted to various subcellular compartments by association with distinct regulatory proteins (14Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (402) Google Scholar, 15Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). The interaction of targeting proteins with the catalytic subunit also modulates substrate specificity, enabling unique and independent roles for the various isoforms in regulating discrete cellular processes (15Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). Okadaic acid, a potent inhibitor of PP1 and PP2A, induces apoptosis in several cells, and this was attributed to its preferential inhibition of one or the other isoform depending on the cell type (16Mellgren G. Vintermyr O.K. Boe R. Doskeland S.O. Exp. Cell Res. 1993; 205: 293-301Crossref PubMed Scopus (39) Google Scholar, 17Fernandez-Sanchez M.T. Garcia-Rodriguez A. Diaz-Trelles R. Novelli A. FEBS Lett. 1996; 398: 106-112Crossref PubMed Scopus (46) Google Scholar, 18Morana S.J. Wolf C.M. Li J. Reynolds J.E. Brown M.K. Eastman A. J. Biol. Chem. 1996; 271: 18263-18271Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 19Li D.W. Fass U. Huizar I. Spector A. Eur. J. Biochem. 1998; 257: 351-361Crossref PubMed Scopus (55) Google Scholar). Okadaic acid-induced epithelial cell apoptosis and inhibition of PP1 were correlated with increased expression of tumor suppressor gene p53 and proapoptotic gene Bax (19Li D.W. Fass U. Huizar I. Spector A. Eur. J. Biochem. 1998; 257: 351-361Crossref PubMed Scopus (55) Google Scholar). Serine phosphorylation in both the carboxyl-terminal and N-terminal domains modulates DNA binding ability and transcriptional activity of p53 (20Takenaka I. Morin F. Seizinger B.R. Kley N. J. Biol. Chem. 1995; 270: 5405-5411Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 21Turenne G.A. Paul P. Laflair L. Price B.D. Oncogene. 2001; 20: 5100-5110Crossref PubMed Scopus (81) Google Scholar). It has been shown recently that direct dephosphorylation of p53 at Ser15 and Ser37 by PP1 affects its stability and transcriptional and apoptotic activities (22Haneda M. Kojima E. Nishikimi A. Hasegawa T. Nakashima I. Isobe K. FEBS Lett. 2004; 567: 171-174Crossref PubMed Scopus (39) Google Scholar, 23Li D.W. Liu J.P. Schmid P.C. Schlosser R. Feng H. Liu W.B. Yan Q. Gong L. Sun S.M. Deng M. Liu Y. Oncogene. 2006; 25: 3006-3022Crossref PubMed Scopus (84) Google Scholar). Among the kinases that regulate the p53 pathway are the c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein kinases. JNKs are activated in cells exposed to various environmental stresses (24Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar), and reactive oxygen species (ROS) play an integral part in this activation (25Shen H.M. Liu Z.G. Free Radic. Biol. Med. 2006; 40: 928-939Crossref PubMed Scopus (521) Google Scholar). The 10 members of the JNK family are generated by alternative splicing of transcripts from Jnk1, Jnk2, and Jnk3 genes (24Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). Jnk1 and Jnk2 double knock-out cells are resistant to apoptosis induced by UV, anisomycin, or DNA damage (26Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1550) Google Scholar). Proapoptotic proteins Bax and Bak are necessary for JNK-induced apoptosis, and Bax remains inactive in Jnk-deficient fibroblasts exposed to environmental stress (27Lei K. Nimnual A. Zong W.X. Kennedy N.J. Flavell R.A. Thompson C.B. Bar-Sagi D. Davis R.J. Mol. Cell. Biol. 2002; 22: 4929-4942Crossref PubMed Scopus (443) Google Scholar). JNK is activated by dual phosphorylation at Thr183 and Tyr185 residues (24Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar), which are dephosphorylated by dual specificity mitogen-activated protein kinase phosphatases (28Camps M. Nichols A. Arkinstall S. FASEB J. 2000; 14: 6-16Crossref PubMed Scopus (720) Google Scholar). In this study, we used retrovirus-mediated insertion of a human heart cDNA library into mouse aortic VSMC to create DMNQ-resistant cell clones. The clones contained 17 full-length cDNAs. We chose to investigate the role of PP1cγ1 (catalytic subunit of protein phosphatase 1) in oxidative stress resistance because the cDNA coding for this protein was present in several independent DMNQ-resistant VSMC clones and because of our continued interest in protein phosphorylation as a regulatory mechanism in atherosclerotic disease process in general and VSMC biology in particular. DMNQ treatment induced mitochondrial ROS production and decreased PP1cγ1 activity in VSMC. Overexpression of PP1cγ1 decreased DMNQ-induced phosphorylation of JNK1 and p53 (Ser15) and protected against DMNQ-induced apoptosis. Further, p53 shRNA transfection protected VSMC against DMNQ-induced apoptosis. Together, these results suggest that PP1cγ1 may regulate molecular mechanisms that mediate VSMC apoptosis in atherosclerosis and restenosis. Materials—DMNQ and diphenyleneiodonium chloride (DPI) were obtained from Calbiochem. Rotenone, thenoyltrifluoroacetone, carbonyl cyanide m-chlorophenylhydrazone, oxypurinol, proadifen, phenelzine, indomethacin, nordihydroguaiaretic acid, leptin, and antimycin A were purchased from Sigma. MitoTracker Green FM, MitoSOX Red, and 2′,7′-dichlorofluorescein diacetate were purchased from Molecular Probes (Invitrogen). The antibodies used were anti-PP1cγ1, anti-p53, anti-Bax, and anti-JNK1 (Santa Cruz Biotechnology), anti-phospho-p53 (Ser15) (Cell Signaling Technology), and anti-β-actin (Sigma). Glutathione S-transferase-c-Jun-(1-79) recombinant protein was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). [γ-32P]ATP (6,000 Ci/mmol) was obtained from GE Healthcare. Cell Culture—Aortic VSMC were isolated from 4-month-old male C57BL/6 mice as previously described by us (29Moon S.K. Thompson L.J. Madamanchi N. Ballinger S. Papacon-stantinou J. Horaist C. Runge M.S. Patterson C. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H2779-H2788Crossref PubMed Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) as described previously (30Madamanchi N.R. Li S. Patterson C. Runge M.S. J. Biol. Chem. 2001; 276: 18915-18924Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). All experiments were conducted using VSMC between passages 4 and 11 that were growth-arrested by incubation in DMEM containing 0.1% FBS for 72 h. Quiesced VSMC were treated with 60 μm DMNQ for 16 h. Single cell clones that survived the treatment were isolated by localized trypsinization using cloning cylinders (Sigma) and expanded using the Glasgow modification of Eagle's medium supplemented with 2 mm glutamine, 1 mm sodium pyruvate, 1× nonessential amino acids, 10% (v/v) fetal bovine serum, a 1:1000 dilution of β-mercaptoethanol stock solution (70 μl of β-mercaptoethanol in 20 ml of distilled water), and 500 units/ml of leukocyte-inhibitory factor (Chemicon). Virus Production—A human heart cDNA expression library was constructed in a replication-defective Moloney murine leukemia virus-based vector, pCFB (Stratagene). The unidirectionally oriented cDNA library contained 1 × 106 primary clones and was amplified only once to ensure the best representation. Retrovirus was produced using the MBS mammalian transfection kit (Stratagene). In brief, the ecotropic virus packaging cell line Phoenix-Eco (Orbigen) was transfected with pCFB-human heart cDNA expression library or retroviral plasmid by the calcium phosphate-DNA co-precipitation method. Retroviruses secreted by transfected cells were collected from cell supernatant 24, 48, and 72 h after transfection and immediately applied to VSMC or 293T cells without any freezing and thawing steps. The concentration of infectious particles was determined by exposing 293T cells to virus containing the lacZ reporter gene (pFB-Neo-LacZ; Stratagene). In brief, cells were seeded at a density of 2.5 × 105 cells/well in a 6-well plate, 16 h prior to virus infection. Infection/transduction was performed in the presence of 10 μg/ml DEAE-dextran. After 24 h of incubation at 37 °C, cells were stained for β-galactosidase expression using an in situ β-galactosidase staining kit (Stratagene). The number of blue-stained cells was estimated by light microscopy at ×200 magnification. cDNA Library Screening in VSMC—VSMC (1.0 × 106) were seeded in 15-cm tissue culture dishes. Ten dishes (about 1 × 107 cells) were infected with 2 × 106 infectious particles of human heart cDNA expression library in pCFB retrovirus as described above. 20% infection was achieved as determined by staining for β-galactosidase activity in control cells. Infected cells were incubated for 48 h at 37 °C to achieve maximal expression of genes (ViraPort XR Plasmid cDNA Library protocol; Stratagene). Then VSMC were treated with 60 μm DMNQ for 16 h. Single cell clones that survived DMNQ treatment were isolated as described above. Isolation and Sequencing of Transduced cDNAs—Genomic DNA from 132 clones was isolated using the DNeasy tissue kit (Qiagen) and was used for PCR recovery of cDNA inserts. A 5′ Retro primer (5′-GGCTGCCGACCCCGGGGGTGG-3′) and 3′ Retro primer (5′-CGAACCCCAGAGTCCCGCTCA-3′) specific for the virus vector sequence flanking cDNA inserts were used for PCR synthesis of cDNAs. PCR was run for 33 cycles (15 s at 94 °C, 4 min at 68 °C) and a final extension for 10 min at 72 °C. The PCR products were purified using the QIAquick PCR purification kit (Qiagen) and sequenced using the above mentioned primers. The PCR products were analyzed on agarose gels to determine the length of the cDNAs. Construction of Recombinant Viruses—To make retroviral and adenoviral constructs encoding PP1cγ1 cDNA, PP1cγ1 was subcloned from pEGFP-C1-PP1cγ1 (kindly provided by Dr. Laura Trinkle-Mulcahy (University of Dundee, Scotland)) into pQCXIP (BD Bioscienses) and pShuttle-CMV, respectively. The sequence of PP1cγ1 constructs was confirmed by restriction analysis and DNA sequencing. Retrovirus encoding PP1cγ1 cDNA was generated as described above. Adenovirus encoding PP1cγ1 cDNA was generated after homologous recombination of pShuttle-CMV containing PP1cγ1 cDNA with the adenoviral backbone plasmid pADEasy-1 in BJ5183 Escherichia coli (Stratagene). The recombinant (LE1/E3-deficient) adenoviruses were propagated by the transfection of human embryonic kidney 293 cells using Lipofectamine (Invitrogen). The virus was serially amplified and then purified using the ViraBind adenovirus purification kit (Cell Biolabs, Inc.). A control adenovirus contained an identical adenovirus backbone with green fluorescent protein cDNA (AdGFP). Two mouse pSM2 retroviral shRNAs for p53 and one retroviral scrambled shRNA construct were purchased from Open Biosystems. The sense sequences of p53 shRNA were 5′-ACCAGTCTACTTCCCGCCATAA-3′ (RHS1764-9208347) and 5′-CCCACTACAAGTACATGTGTAA-3′ (RMM1766-98468519). Constructs were transfected into Phoenix-Eco cells for retrovirus packaging and amplification using the MBS mammalian transfection kit. Retrovirus containing supernatant was used to infect VSMC, and transduced cells were selected using puromycin (2.5 μg/ml)-containing medium. Successful suppression of p53 expression was confirmed by Western blot analysis. Overexpression of PP1cγ1 Using the Adenoviral Infection System—Adenoviral infection of nearly confluent VSMC was performed at a multiplicity of infection of 100 in DMEM containing 2% FBS. After 16 h of incubation, the cells were quiesced in DMEM containing 0.1% FBS for 72 h. Retroviral p53 and Pp1cγ1 shRNA Constructs and Infection of Mouse Aortic VSMC—Mouse retroviral shRNAmir against p53 and Pp1cγ1 was purchased from Open Biosystems. The scrambled shRNA encoded a 19-bp scrambled sequence (5'-GCGCGCTTTGTAGGATTCG) with no significant homology to any mouse gene and was cloned into pSUPER.retro.puro vector. Retroviral infection of VSMC was performed by spreading 3 ml of virus supplemented with 10 μg/ml DEAE-dextran on VSMC seeded in 100-mm plates. After 3 h of incubation, an additional 7.0 ml of DMEM containing 10% FBS was added, and the plates were incubated at 37 °C for 21 h. The infection medium was then replaced with fresh DMEM containing 10% FBS. For p53 and Pp1cγ1 shRNA expression, puromycin-resistant clones (selected with 2.5 μg/ml puromycin for 10 days) were expanded prior to their use in experiments. Cell Viability Assay—VSMC viability after DMNQ treatment was determined by either crystal violet staining or the trypan blue exclusion method. In the former method, DMNQ-treated VSMC in 96-well plates were washed with phosphate-buffered saline and stained with 100 μl of crystal violet solution (0.5% (w/v) crystal violet, 1.5% (v/v) formaldehyde, and 1% (v/v) ethanol) for 30 min. After the wells were washed with water, stained cells were lysed with 1% (w/v) deoxycholate solution, and the absorbance was read at 560 nm using a microplate reader. Trypan blue exclusion was determined microscopically by scoring five fields of 100 cells each. Intracellular ROS Measurement—VSMC (8 × 105) were seeded in 24-well glass bottom plates, grown for 48 h in DMEM containing 10% FBS, and quiesced for an additional 24 h in culture medium containing 0.1% FBS. Cells were then treated with Hanks' balanced salt solution containing the H2O2-sensitive fluorophore 2′,7′-dichlorofluorescin diacetate (5 μm) for 30 min at 37 °C in darkness. The cells were treated with fresh 2′,7′-dichlorofluorescin diacetate and DMNQ and incubated for another 30 min. For studying the effect of cellular oxidase inhibitors on DMNQ-induced ROS production, VSMC were pretreated with DPI, oxypurinol, proadifen, phenelzine, indomethacin, or nordihydroguaiaretic acid for 30 min before treatment with 2′,7′-dichlorofluorescein diacetate and DMNQ. The fluorescence of DCF was measured using a microplate reader at an excitation wavelength of 485 nm and emission wavelength of 530 nm. Then cells were lysed, and protein content was quantified. Detection of Mitochondrial ROS—VSMC grown in glass bottom dishes were quiesced and treated with 10 μm DMNQ for 30 min. Cells were washed with Hanks' balanced salt solution and incubated with 5 μm MitoSOX Red and 1 μm MitoTracker Green FM (Molecular Probes) at 37 °C for 10 min. Excess stains were removed, and cells were imaged using Olympus FV500 confocal laser-scanning microscopy. MitoTracker Green FM was visualized at an excitation of 490 nm and an emission of 516 nm, whereas MitoSOX Red was visualized at an excitation of 560 nm and an emission of 600 nm. MitoTracker Green FM preferentially translocates to the mitochondria. MitoSOX Red accumulates in mitochondria and exhibits bright red fluorescence upon oxidation and subsequent binding to mitochondrial DNA. Detection of Changes in Mitochondrial Transmembrane Potential—Changes in mitochondrial transmembrane potential were detected using the MitoCapture apoptosis detection kit (BioVision Research Products), which provides a fluorescence-based method for distinguishing between healthy and apoptotic cells. VSMC, either untreated or treated with 10 μm DMNQ for 15 h, were incubated with diluted MitoCapture solution at 37 °C in a 5% CO2 incubator for 20 min. The cells were washed with the incubation buffer three times and mounted with Vectashield mounting medium (Vector Laboratories). Cells were photographed by fluorescent microscopy. Isolation of Mitochondria—Mitochondria were isolated from 80% confluent VSMC using a Pierce mitochondrial isolation kit according to the manufacturer's instructions with the addition of complete EDTA-free protease inhibitor mixture (Roche Applied Science). Western Blot Analysis—Cells were lysed in radioimmune precipitation buffer (20 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.05 mm sodium fluoride, 1 mm EDTA, 1% Igepal, 0.05% sodium deoxycholate, 0.1% SDS, and protease inhibitors), and Western analysis was performed as described previously (30Madamanchi N.R. Li S. Patterson C. Runge M.S. J. Biol. Chem. 2001; 276: 18915-18924Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Cell lysates containing 50 μg of protein were analyzed in Western blotting experiments. Assessment of Apoptosis—For detection of apoptosis, growth-arrested VSMC were harvested after 16 h of treatment with 10 μm DMNQ. Histone-associated DNA fragmentation in cell lysates was determined using the cell death detection ELISAPLUS kit (Roche Applied Science) in accordance with the manufacturer's instructions. A TUNEL apoptosis detection kit (Upstate Biotechnology, Inc.) was used to measure DNA fragmentation according to the manufacturer's protocol. Briefly, following treatment, the cells were fixed in 4% paraformaldehyde and permeabilized by incubating with 0.5% Tween 20 and 0.2% bovine serum albumin for 15 min. Then cells were incubated with a reaction mix containing biotin-dUTP and terminal deoxynucleotidyltransferase for 60 min. Transfer of biotin-dUTP to the free 3′-OH end of cleaved DNA was visualized by reaction with fluorescein-conjugated avidin (fluorescein isothiocyanate-avidin) for 30 min and photographed by fluorescence microscopy. Cells were counterstained with propidium iodide. Apoptosis was also assessed by measuring caspase-3/7 activities using an Apo-ONE™ homogeneous caspase-3/7 assay kit (Promega). Briefly, growth-arrested VSMC were treated with 10 μm DMNQ for 6 h. The cells were lysed using bifunctional cell lysis/activity buffer, which contained a profluorescent caspase-3/7 consensus substrate, rhodamine 110 bis-N-benzyloxycarbonyl-l-aspartyl-l-glutamyl-l-valyl-aspartic acid amide (Z-DEVD-R110). After incubation at room temperature for 1 h, aliquots (150 μl) were transferred to a 96-well clear bottom plate. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a WALLAC 1420 Multilabel Counter. Measurement of Pp1cγ1 mRNA Expression—Total RNA from VSMC that were either treated or untreated with DMNQ was extracted using the RNeasy Micro kit (Qiagen). Reverse transcription was performed with 1 μg of total RNA using the TaqMan reverse transcription reagents kit (Applied Biosystems). The Pp1cγ1 gene expression assay (catalog number Mm00849631-s1) was purchased from Applied Biosystems. Real time PCR was performed using the ABI PRISM 7900 HT Sequence Detection System and TaqMan PCR Master Mix according to the manufacturer's recommendations. Sequence Detection System software (version 2.1) (Applied Biosystems) was used for raw data analysis. Pp1cγ1 relative expression was calculated using the Relative Expression Software Tool (31Pfaffl M.W. Horgan G.W. Dempfle L. Nucleic Acids Res. 2002; 30: e36Crossref PubMed Google Scholar) and by normalization to 18 S ribosomal expression. Measurement of PP1cγ1 Activity—PP1cγ1 activity was measured using the fluorescence-based RediPlate 96 EnzChek serine/threonine phosphatase assay kit from Molecular Probes. VSMC were lysed in lysis buffer containing 20 mm Tris-Cl, pH 7.4, 132 mm NaCl, 10% glycerol, 1% Triton X-100, and a protease inhibitor mixture. Cell lysates containing 500 μg of protein were immunoprecipitated with anti-PP1cγ1 antibody overnight at 4 °C and then incubated with Protein A-Sepharose beads for another 2 h. The immunoprecipitates were washed three times with lysis buffer and resuspended in 50 μl of assay buffer (50 mm Tris-Cl, pH 7.0, 0.1 mm CaCl2, 2 mm dithiothreitol, 200 μm MnCl2, 125 μg/ml bovine serum albumin, and 0.05% Tween 20) containing 50 μm substrate, 6,8-difluoro-4-methyl-umbelliferyl phosphate. The reaction was performed at 37 °C for 30 min in the dark. 6,8-Difluoro-4-methyl-umbelliferyl fluorescence was measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm, u" @default.
• W2078463367 created "2016-06-24" @default.
• W2078463367 creator A5019213719 @default.
• W2078463367 creator A5069430572 @default.
• W2078463367 creator A5072038293 @default.
• W2078463367 creator A5078941242 @default.
• W2078463367 creator A5086729202 @default.
• W2078463367 date "2008-08-01" @default.
• W2078463367 modified "2023-09-30" @default.
• W2078463367 title "Identification of a Protective Role for Protein Phosphatase 1cγ1 against Oxidative Stress-induced Vascular Smooth Muscle Cell Apoptosis" @default.
• W2078463367 cites W1492302205 @default.
• W2078463367 cites W1657827244 @default.
• W2078463367 cites W1811191752 @default.
• W2078463367 cites W1972514324 @default.
• W2078463367 cites W1972805802 @default.
• W2078463367 cites W1976217600 @default.
• W2078463367 cites W1976873804 @default.
• W2078463367 cites W1983225676 @default.
• W2078463367 cites W1983386156 @default.
• W2078463367 cites W1988177344 @default.
• W2078463367 cites W1994788512 @default.
• W2078463367 cites W2006173249 @default.
• W2078463367 cites W2007683534 @default.
• W2078463367 cites W2007837805 @default.
• W2078463367 cites W2009080781 @default.
• W2078463367 cites W2009776832 @default.
• W2078463367 cites W2010205595 @default.
• W2078463367 cites W2010403420 @default.
• W2078463367 cites W2010565332 @default.
• W2078463367 cites W2014465321 @default.
• W2078463367 cites W2014586657 @default.
• W2078463367 cites W2015985739 @default.
• W2078463367 cites W2021565484 @default.
• W2078463367 cites W2023424326 @default.
• W2078463367 cites W2024306318 @default.
• W2078463367 cites W2024468216 @default.
• W2078463367 cites W2028001397 @default.
• W2078463367 cites W2028169530 @default.
• W2078463367 cites W2032154859 @default.
• W2078463367 cites W2033950216 @default.
• W2078463367 cites W2037141618 @default.
• W2078463367 cites W2037879643 @default.
• W2078463367 cites W2040331457 @default.
• W2078463367 cites W2045563337 @default.
• W2078463367 cites W2047263793 @default.
• W2078463367 cites W2053129567 @default.
• W2078463367 cites W2063971300 @default.
• W2078463367 cites W2066549657 @default.
• W2078463367 cites W2074169536 @default.
• W2078463367 cites W2076485183 @default.
• W2078463367 cites W2077548365 @default.
• W2078463367 cites W2079793296 @default.
• W2078463367 cites W2082797919 @default.
• W2078463367 cites W2084504852 @default.
• W2078463367 cites W2084940422 @default.
• W2078463367 cites W2086127940 @default.
• W2078463367 cites W2090741482 @default.
• W2078463367 cites W2098444271 @default.
• W2078463367 cites W2099249565 @default.
• W2078463367 cites W2107951334 @default.
• W2078463367 cites W2111305451 @default.
• W2078463367 cites W2111650534 @default.
• W2078463367 cites W2113999599 @default.
• W2078463367 cites W2115914856 @default.
• W2078463367 cites W2117089540 @default.
• W2078463367 cites W2119807130 @default.
• W2078463367 cites W2120553816 @default.
• W2078463367 cites W2120715370 @default.
• W2078463367 cites W2123868590 @default.
• W2078463367 cites W2123921117 @default.
• W2078463367 cites W2133835604 @default.
• W2078463367 cites W2136046634 @default.
• W2078463367 cites W2143781287 @default.
• W2078463367 cites W2144817030 @default.
• W2078463367 cites W2146965545 @default.
• W2078463367 cites W2153942172 @default.
• W2078463367 cites W2155645904 @default.
• W2078463367 cites W2156299289 @default.
• W2078463367 cites W2158720649 @default.
• W2078463367 cites W2160345685 @default.
• W2078463367 cites W2167340008 @default.
• W2078463367 cites W2178456244 @default.
• W2078463367 cites W2179375563 @default.
• W2078463367 cites W2209317426 @default.
• W2078463367 cites W2332762993 @default.
• W2078463367 doi "https://doi.org/10.1074/jbc.m803452200" @default.
• W2078463367 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2494915" @default.
• W2078463367 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18540044" @default.
• W2078463367 hasPublicationYear "2008" @default.
• W2078463367 type Work @default.
• W2078463367 sameAs 2078463367 @default.
• W2078463367 citedByCount "27" @default.
• W2078463367 countsByYear W20784633672012 @default.
• W2078463367 countsByYear W20784633672013 @default.
• W2078463367 countsByYear W20784633672014 @default.
• W2078463367 countsByYear W20784633672015 @default.
• W2078463367 countsByYear W20784633672016 @default.
• W2078463367 countsByYear W20784633672022 @default.

• next