Fukumoto S, Nishizawa Y, Hosoi M, Koyama H, Yamakawa K, Ohno S, Morii H
Second Department of Internal Medicine, Osaka City University Medical School, Osaka 545, Japan.
J Biol Chem. 1997 May 23;272(21):13816-22. doi: 10.1074/jbc.272.21.13816.
To elucidate the physiological role of protein kinase C (PKC) delta, a ubiquitously expressed isoform in vascular smooth muscle cells (VSMC), PKC delta was stably overexpressed in A7r5 cells, rat clonal VSMC. The [3H]thymidine incorporation in A7r5 overexpressed with PKC delta (DVs) was suppressed to 37.1 +/- 16.3% (mean +/- S.D.) of the level in control or A7r5 transfected with vector alone (EVs). The reduction of [3H]thymidine incorporation was strongly correlated with overexpressed PKC levels. Moreover, transient transfection of a dominant negative mutant of PKC delta restored the reduced proliferation in DVs. Flow cytometry analysis demonstrated that DVs were arrested in the G0/G1 phase of the cell cycle. Expression of cyclins D1 and E and retinoblastoma protein phosphorylation were reduced, while the protein levels of p27 were elevated in DVs as compared with EVs. There were no significant differences in the expression of c-fos, c-jun, c-myc, cyclin D2, D3, cyclin-dependent kinase 2, cyclin-dependent kinase 4, and p21 among the clones. We conclude that PKC delta inhibits the proliferation of VSMC by arresting cells in G1 via mainly inhibiting the expression of cyclin D1 and cyclin E.
为阐明蛋白激酶C（PKC）δ在血管平滑肌细胞（VSMC）中普遍表达的亚型的生理作用，PKCδ在大鼠克隆VSMC即A7r5细胞中稳定过表达。与单独转染载体的对照或A7r5细胞（EVs）相比，过表达PKCδ的A7r5细胞（DVs）中[3H]胸苷掺入量被抑制至对照水平的37.1±16.3%（平均值±标准差）。[3H]胸苷掺入量的降低与PKC过表达水平密切相关。此外，PKCδ显性负性突变体的瞬时转染恢复了DVs中降低的增殖。流式细胞术分析表明，DVs停滞在细胞周期的G0/G1期。与EVs相比，DVs中细胞周期蛋白D1和E的表达以及视网膜母细胞瘤蛋白磷酸化降低，而p27蛋白水平升高。各克隆间c-fos、c-jun、c-myc、细胞周期蛋白D2、D3、细胞周期蛋白依赖性激酶2、细胞周期蛋白依赖性激酶4和p21的表达无显著差异。我们得出结论，PKCδ主要通过抑制细胞周期蛋白D1和细胞周期蛋白E的表达使细胞停滞在G1期，从而抑制VSMC的增殖。
