Tumor Suppressor p53 But Not cGMP Mediates NO-Induced Expression of p21Waf1/Cip1/Sdi1 in Vascular Smooth Muscle Cells
- Akio Ishida1,3,
- Toshiyuki Sasaguri1,
- Yoshikazu Miwa1,4,
- Chiya Kosaka5,
- Yoji Taba1,3 and
- Takeo Abumiya2
- Departments of 1Bioscience (A.I., T.S., Y.M., Y.T.) and 2Epidemiology (T.A.), National Cardiovascular Center Research Institute; 3Third Department of Internal Medicine (A.I., Y.T.), University of the Ryukyus School of Medicine; 4Second Department of Internal Medicine (Y.M.), Faculty of Medicine, Kyushu University; and 5Department of Clinical Sciences and Laboratory Medicine (C.K.), Kansai Medical University, Osaka, Japan
Abstract
Cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 has been suggested to be involved in the antiproliferative effect of nitric oxide (NO) in vascular smooth muscle cells (VSMCs). To elucidate the mechanism underlying NO-induced p21 expression, we investigated the roles of tumor suppressor p53 and the guanylate cyclase-cGMP pathway. The induction of p21 by the NO donorS-nitroso-N-acetylpenicillamine (SNAP) seemed to be due to transactivation because SNAP elevated the activity of p21 promoter but did not stabilize p21 mRNA and protein. Because SNAP did not stimulate the deletion mutant of p21 promoter that lacked p53 binding sites, we tested the involvement of p53. The expression level of p53 was down-regulated after mitogenic stimulation, whereas it was sustained in the presence of SNAP. SNAP markedly stimulated DNA binding activity of p53. Furthermore, SNAP failed to induce p21 in VSMCs obtained from p53-knock out mice and in A431 cells that contained mutated p53. The antiproliferative effect of SNAP also was attenuated in these cells. NO stimulates guanylate cyclase and its product cGMP has been shown to inhibit VSMC proliferation. However, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, did not prevent SNAP-induced p21 expression. 8-Bromo-cGMP, 3-isobutyl-1-methylxanthine, and their combination did not induce p21. Although 8-bromo-cGMP had a small antiproliferative effect, the elevation of cGMP concentration induced by SNAP was little throughout the G1 phase. The antiproliferative effect of SNAP was not attenuated by Rp-8-bromoguanosine-3′,5′-monophosphorothioate, an inhibitor of cGMP-dependent protein kinase. These results suggested that NO induces p21 through a p53-dependent but cGMP-independent pathway.
Footnotes
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Send reprint requests to: Toshiyuki Sasaguri, M.D., Ph.D., Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. E-mail: sasaguri{at}ri.ncvc.go.jp
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This study was supported in part by grants from the Ministry of Health and Welfare [Research Grants for Cardiovascular Diseases (8A-1 and 9A-4), Science and Technology Agency (Special Coordination Funds for Promoting Science and Technology (Encouragement System of COE)]; Japan Cardiovascular Research Foundation; Ichiro Kanehara Foundation; and Research Foundation for Cancer and Cardiovascular Diseases, Osaka, Japan.
- Abbreviations:
- NO
- nitric oxide
- VSMCs
- vascular smooth muscle cells
- eNOS
- endothelial nitric oxide synthase
- SNAP
- S-nitroso-N-acetylpenicillamine
- iNOS
- inducible nitric oxide synthase
- sGC
- soluble guanylate cyclase
- CHX
- cycloheximide
- IBMX
- 3-isobutyl-1-methylxanthine
- Rp-GMPS
- Rp-8-bromoguanosine-3′,5′-monophosphorothioate
- Rp-AMPS
- Rp-8-bromoadenosine-3′,5′-monophosphorothioate
- ODQ
- 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
- DMEM
- Dulbecco’s modified Eagle’s medium
- FBS
- fetal bovine serum
- G0
- quiescent state
- TdR
- thymidine
- PMSF
- phenylmethylsulfonyl fluoride
- PAGE
- polyacrylamide gel electrophoresis
- RT-PCR
- reverse transcription-polymerase chain reaction
- PKG
- cGMP-dependent protein kinase
- PKA
- cAMP-dependent protein kinase
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- Received April 8, 1999.
- Accepted August 2, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
