Nitric oxide prevents p21 degradation with the ubiquitin-proteasome pathway in vascular smooth muscle cells

被引:30
作者
Kibbe, MR
Nie, SH
Seol, DW
Kovesdi, I
Lizonova, A
Makaroun, M
Billiar, TR
Tzeng, E
机构
[1] Univ Pittsburgh, Dept Surg, Pittsburgh, PA 15261 USA
[2] GenVec Corp, Pittsburgh, PA USA
关键词
D O I
10.1016/S0741-5214(00)90166-6
中图分类号
R61 [外科手术学];
学科分类号
摘要
Purpose: We have shown that gene transfer of the inducible nitric oxide synthase (iNOS) gene to injured arteries inhibits the development of intimal hyperplasia. One mechanism by which nitric oxide (NO) may inhibit this process is through the upregulation of the cyclin-dependent kinase inhibitor p21, which induces a G0/G1 cell cycle arrest, leading to an inhibition of vascular smooth muscle cell (VSMC) proliferation. Because NO induced such a dramatic upregulation of p21 and because p21 is a universal inhibitor of the cell cycle, this study aimed to determine how NO upregulates p21 protein expression in VSMCs. Methods: p21 messenger RNA (mRNA) levels in rat aortic smooth muscle cells (RASMCs) were determined by Northern blot analysis after treatment with S-nitroso-N-acetylpenicillamine (SNAP) or after adenoviral iNOS gene transfer, p21 protein levels in RASMCs in similar conditions were determined by Western blot analysis. Levels of ubiquinated p21 in these same treatment groups were assessed by immunoprecipitation of p21 from RASMCs, followed by Western blot analysis for ubiquitin. Protein tyrosine and protein serine/threonine phosphatase activity after treatment with SNAP, plus or minus the phosphatase inhibitors calyculin A or cantharidin, were measured with P-32-labeled myelin basic protein as a substrate. Results: NO exposure by the NO-donor SNAP or iNOS gene transfer induced a dose-and time-dependent increase in p21 protein expression in RASMCs. p21 mRNA levels were significantly increased after SNAP treatment only at the 6-hour point, but were not increased at 24 hours. In contrast, protein levels were increased from 6 to 24 hours, and transcriptional inhibitors did not inhibit this increase in protein synthesis. The increase in p21 protein expression induced by NO was associated with less of the ubiquinated form of p21 at both early and late points. Furthermore, NO induced an increase ill both protein tyrosine and protein serine/threonine phosphatase activity. Inhibition of these phosphatases with calyculin A or cantharidin prevented the upregulation of p21 protein expression by NO. Conclusion: These data indicate that one mechanism by which NO upregulates p21 protein expression is through the prevention of p21 protein degradation by the ubiquitin-proteasome pathway in association with increased protein tyrosine and serine/threonine phosphatase activity.
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页码:364 / 374
页数:11
相关论文
共 37 条
[1]   ADENOVIRUS-MEDIATED OVER-EXPRESSION OF THE CYCLIN CYCLIN-DEPENDENT KINASE INHIBITOR, P21 INHIBITS VASCULAR SMOOTH-MUSCLE CELL-PROLIFERATION AND NEOINTIMA FORMATION IN THE RAT CAROTID-ARTERY MODEL OF BALLOON ANGIOPLASTY [J].
CHANG, MW ;
BARR, E ;
LU, MM ;
BARTON, K ;
LEIDEN, JM .
JOURNAL OF CLINICAL INVESTIGATION, 1995, 96 (05) :2260-2268
[2]   SINGLE-STEP METHOD OF RNA ISOLATION BY ACID GUANIDINIUM THIOCYANATE PHENOL CHLOROFORM EXTRACTION [J].
CHOMCZYNSKI, P ;
SACCHI, N .
ANALYTICAL BIOCHEMISTRY, 1987, 162 (01) :156-159
[3]   Suppression of apoptosis by nitric oxide via inhibition of interleukin-1 beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases [J].
Dimmeler, S ;
Haendeler, J ;
Nehls, M ;
Zeiher, AM .
JOURNAL OF EXPERIMENTAL MEDICINE, 1997, 185 (04) :601-607
[4]   NITRIC OXIDE-GENERATING VASODILATORS AND 8-BROMO-CYCLIC GUANOSINE-MONOPHOSPHATE INHIBIT MITOGENESIS AND PROLIFERATION OF CULTURED RAT VASCULAR SMOOTH-MUSCLE CELLS [J].
GARG, UC ;
HASSID, A .
JOURNAL OF CLINICAL INVESTIGATION, 1989, 83 (05) :1774-1777
[5]   MOLECULAR-CLONING AND EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE FROM HUMAN HEPATOCYTES [J].
GELLER, DA ;
LOWENSTEIN, CJ ;
SHAPIRO, RA ;
NUSSLER, AK ;
DISILVIO, M ;
WANG, SC ;
NAKAYAMA, DK ;
SIMMONS, RL ;
SNYDER, SH ;
BILLIAR, TR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (08) :3491-3495
[6]   CYTOKINES, ENDOTOXIN, AND GLUCOCORTICOIDS REGULATE THE EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN HEPATOCYTES [J].
GELLER, DA ;
NUSSLER, AK ;
DISILVIO, M ;
LOWENSTEIN, CJ ;
SHAPIRO, RA ;
WANG, SC ;
SIMMONS, RL ;
BILLIAR, TR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (02) :522-526
[7]   Nitric oxide-induced downregulation of cdk2 activity and cyclin A gene transcription in vascular smooth muscle cells [J].
Guo, K ;
Andrés, V ;
Walsh, K .
CIRCULATION, 1998, 97 (20) :2066-2072
[8]   Introduction: Evolving roles for ubiquitin in cellular regulation [J].
Haas, AL .
FASEB JOURNAL, 1997, 11 (13) :1053-1054
[9]  
Haas AL, 1997, FASEB J, V11, P1257
[10]  
Ishida A, 1997, J BIOL CHEM, V272, P10050