Protein kinase C regulation of corneal endothelial cell proliferation and cell cycle.
Protein kinase C regulation of corneal endothelial cell proliferation and cell cycle.
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发表时间：
2000-12
影响因子：
4.4
通讯作者：
Melanie A. Graham;Melanie A. Graham;Ian Rawe;D. Dartt;N. Joyce
中科院分区：
文献类型：
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作者：
Melanie A. Graham;Melanie A. Graham;Ian Rawe;D. Dartt;N. Joyce
目的探讨蛋白激酶C（PKC）在角膜内皮细胞增殖中的作用。方法采用免疫细胞化学和蛋白质印迹法检测原代培养的大鼠角膜内皮细胞中PKC亚型的表达。对于增殖研究，大鼠角膜内皮细胞的原代培养物在血清饥饿状态下培养48小时，然后与PKC抑制剂星形孢菌素（10（-9）至10（-7）M）、白屈菜红碱（10（-9）至5 × 10（-8）M）或calphostin C（10（-9）至10（-7）M）孵育2小时。使用PKCalpha反义寡核苷酸转染或暴露于豆蔻酰化的、假底物衍生的肽抑制剂1小时（10（-8）至10（-6）M）抑制PKCalpha、β-内酰胺酶γ、β-内酰胺酶和β-内酰胺酶δ的单个PKC亚型。然后用2.5%血清刺激细胞24小时。用溴脱氧尿苷（BrDU）和Ki 67免疫细胞化学法检测细胞增殖。Western blotting法检测细胞周期蛋白E（cyclin E）的表达。结果在角膜内皮细胞中检测到PKCalpha、-ssII、-delta、-beta、-iota、-eta、-gamma和-theta。用星形孢菌素、calphostin C和白屈菜红碱对PKC的最大抑制分别将细胞增殖降低至对照的7%、31%和48%。豆蔻酰化肽抑制PKCalpha和PKCalpha使细胞增殖分别降低至对照的57%和59%。PKCalpha反义寡核苷酸将细胞增殖降低至对照的35%。在用钙磷蛋白C、星形孢菌素、白屈菜红碱和PKCalpha反义处理的细胞中，细胞周期蛋白E蛋白水平分别降低至对照的70%、38%、57%和43%。结论：PKC活性，尤其是PKCalpha和β-PKC活性在促进角膜内皮细胞增殖中起重要作用.抑制PKC活性可阻止G1/S期进程并降低细胞周期蛋白E蛋白水平。
PURPOSE The purpose of this study was to determine the role of protein kinase C (PKC) in corneal endothelial cell proliferation. METHODS Immunocytochemistry and Western blotting were used to define the PKC isoforms expressed in primary cultures of rat corneal endothelial cells. For proliferation studies, primary cultures of rat corneal endothelial cells were serum-starved for 48 hours and incubated for 2 hours with the PKC inhibitors staurosporine (10(-9) to 10(-7) M), chelerythrine (10(-9) to 5 x 10(-8) M), or calphostin C (10(-9) to 10(-7) M). Individual PKC isoforms were inhibited using PKCalpha antisense oligonucleotide transfection or exposure for 1 hour to myristoylated, pseudosubstrate-derived peptide inhibitors against PKCalpha, -alphassgamma, -epsilon, and -delta (10(-8) to 10(-6) M). Cells were then stimulated with 2.5% serum for 24 hours. Cell proliferation was measured with bromodeoxyuridine (BrDU) and Ki67 immunocytochemistry. Protein level of cyclin E was determined by Western blotting. RESULTS PKCalpha, -ssII, -delta, -epsilon, -iota, -eta, -gamma, and -theta were detected in corneal endothelial cells. Maximum inhibition of PKC with staurosporine, calphostin C, and chelerythrine reduced cell proliferation to 7%, 31%, and 48% of control, respectively. Myristoylated peptide inhibition of PKCalpha and -epsilon reduced cell proliferation to 57% and 59% of control, respectively. PKCalpha antisense oligonucleotide reduced cell proliferation to 35% of control. Cyclin E protein level was decreased to 70%, 38%, 57%, and 43% of control in cells treated with calphostin C, staurosporine, chelerythrine, and PKCalpha antisense, respectively. CONCLUSIONS PKC activity, in particular PKCalpha and -epsilon activity, is important in promoting corneal endothelial cell proliferation. Inhibition of PKC activity prohibits G1/S-phase progression and reduces cyclin E protein levels.