EXPERIMENTAL CELL RESEARCH 240, 349–358 (1998) ARTICLE NO. EX983999 The Roles of Protein Kinase C bI and bII in Vascular Smooth Muscle Cell Proliferation 1 Mayumi Yamamoto,* ,2 Mildred Acevedo-Duncan,† Charles E. Chalfant,* Niketa A. Patel,* James E. Watson,‡ and Denise R. Cooper* , ‡ , § *Department of Biochemistry and Molecular Biology, §Internal Medicine, and †Chemistry, University of South Florida College of Medicine; and ‡ J. A. Haley Veterans Hospital, Tampa, Florida 33612 recognized that VSMC proliferation is a major etiologic The role of protein kinase C (PKC) on proliferation event in the formation of atherosclerotic lesions. Abnor- of A10 vascular smooth muscle cells (VSMC) was stud- mal proliferation of VSMC with subsequent formation ied by overexpressing specific PKC-bI and -bII iso- of intimal thickening is thought to play a role in the zymes. PKC-bI and -bII are derived from alternative development of atherosclerosis [1]. Elucidating how splicing of the exon encoding the carboxy-terminal (C- VSMC proliferate is important for identifying the terminal) 50 or 52 amino acids, respectively. The differ- mechanisms contributing to the progression of athero- ential functions of the two isozymes with regard to cell sclerosis and for prevention or treatment of atheroscle- proliferation, DNA synthesis, and the cell cycle were rosis. investigated in A10 cells, a clonal cell line of VSMC Several biological substances including growth fac- from rat aorta, and in A10 cells overexpressing PKC- tors, vasoactive hormones, and eicosanoids promote bI and PKC-bII (bI-A10 and bII-A10). PKC levels were VSMC proliferation [2]. Postreceptor signaling path- increased three- to fourfold in heterogeneous cultures ways for most of them include activation of protein of stably transfected cells. Although doubling time of kinase C (PKC), because following stimulation of A10 cells was 36 h, the cell doubling time in bI-A10 their receptors, membrane phospholipids are hy- cells decreased by 12 h, and, in contrast, the doubling drolyzed by phospholipases to produce 1,2-diacylglyc- time of bII-A10 cells increased by 12 h compared to erol (DAG), a physiological PKC activator [3]. Activa- A10 cells. The increase of [ 3 H]thymidine (TdR) incorpo- tion of PKC in VSMC modulates agonist-stimulated ration was accelerated and increased in bI-A10 cells, phospholipid turnover and regulates cell growth and but slowed and diminished in bII-A10 cells compared proliferation [4]. to A10 and control cells transfected with empty vector. Although the molecular basis for PKC-mediated sig- Cell cycle analysis of bI-A10 cells showed an accelera- nal transduction that leads to mitogenesis remains un- tion of S phase entry while bII-A10 cells slowed S phase clear, PKC plays a role in cell proliferation. PKC activa- entry. These results suggest that PKC-bI and PKC-bII regulate cell proliferation bidirectionally and that tors such as phorbol ester and DAG can replace compe- PKC-bI and PKC-bII may have distinct and opposing tent factors in some cells including Swiss 3T3 cells [5, functions as cell cycle check point mediators during 6], and immediate early genes such as c-fos and c-myc late G 1 phase and may regulate S phase entry in A10 in BALB/c-3T3 cells [7] or in human peripheral blood VSMC.  1998 Academic Press mononuclear cells [8]. PKC is involved, at least par- Key Words: atherosclerosis; vascular smooth muscle tially, in the proliferative action of platelet-derived cell; cell cycle; PKC isozymes. growth factor in VSMC [9]. In contrast, PKC activators such as phorbol esters or bryostatins inhibit the prolif- eration of several cell systems including VSMC [10 – INTRODUCTION 18], inhibiting the cell cycle at the G 1 /S transition [12, Vascular smooth muscle cells (VSMC) 3 play a role in the pathogenesis of atherosclerosis and it is widely acylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; DPBS, Dulbecco’s phosphate-buffered saline buffer; 1 A part of this work was reported at the 79th Annual Meeting of EGTA, ethyleneglycol-bis-(b-aminoethyl ether)N,N,N,N,-tetraace- tic acid; EDTA, ethylene diamin tetra acetic acid; PMSF, phenyl Endocrine Society (Minneapolis, 1997). 2 To whom correspondence and reprint requests should be ad- methyl sulfonyl fluoride; NP-40, nonyl phenoxy polyethoxy ethanol; SDS, sodium dodecyl sulfate; SDS – PAGE, sodium dodecyl sulfate – dressed at J. A. Haley Veterans Hospital, VAR 151, 13000 Bruce B. Downs Blvd., Tampa, FL 33612. Fax: (813) 972-7623. E-mail: polyacrylamide resolving gels electrophoresis; TBS, Tris-buffered sa- line; TBS-T, TBS containing Tween 20; ECL, enhanced chemilumi- myamamot@com1.med.usf.edu. 3 Abbreviations used: PKC, protein kinase C; VSMC, vascular nescence; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo- lium bromide; TCA, trichloroacetic acid; PI, propidium iodide; SD, smooth muscle cell; bI-A10, PKC-bI overexpressing A10 cells; bII- A10, PKC-bII overexpressing A10 cells; TdR, thymidine; DAG, di- standard deviation; ANOVA, analysis of variance. 349 0014-4827/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. AID ECR 3999 / 6i31$$$$$1 04-07-98 22:45:56 ecal