Glucocorticoid inhibition of fibroblast proliferation and regulation of the cyclin kinase inhibitor p21(Cip1)
Glucocorticoid inhibition of fibroblast proliferation and regulation of the cyclin kinase inhibitor p21(Cip1)
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DOI：
10.1210/mend.11.5.9923
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发表时间：
1997-05-01
影响因子：
--
通讯作者：
Thompson, EA
中科院分区：
文献类型：
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作者：
Ramalingam, A;Hirai, A;Thompson, EA
糖皮质激素在体内和培养中抑制成纤维细胞的增殖；然而，这种作用的分子机制仍然不清楚。我们致力于阐明糖皮质激素降低小鼠L929成纤维细胞增殖率的机制。在对数中期成纤维细胞中加入地塞米松可延长G1期。这种G1间期的增加与RB-1抑癌基因产物pRB的磷酸化抑制有关，而且可能是由于抑制了RB-1抑癌基因产物的磷酸化。细胞周期蛋白D依赖的蛋白激酶在体外可以抑制pRb的磷酸化。然而，细胞周期蛋白D_1、D_2和D_3的表达没有明显变化。在L929细胞中加入糖皮质激素后，细胞周期蛋白依赖性激酶-4(CDK4)和CDK6的表达没有下调，细胞周期蛋白D/CDK4复合体的丰度没有改变。抑制pRb激酶活性与CDK抑制剂之一p21(Cip1)的丰度增加有关。另一种细胞周期蛋白激酶抑制剂p27(Kip1)的丰度保持不变。加入地塞米松后，与p21(Cip1)结合的CDK4的量迅速增加，而CDK4-PRB的活性平行下降。这些结果表明，糖皮质激素抑制成纤维细胞的增殖是由于p21(Cip1)的诱导，p21(Cip1)与细胞周期蛋白D/CDK4复合体结合并失活。加入地塞米松后2小时内，p21mRNA的丰度增加约5倍。这种效应在糖皮质激素受体为空的L929突变体中不存在，并且从四环素可阻抑表达载体中表达糖皮质激素受体的突变体仅在没有四环素的情况下才显示出p21mRNA的诱导。放线菌酮不阻断p21mRNA的诱导，地塞米松对p21mRNA的表观降解率无明显影响。在加入地塞米松后2小时内，Cip1基因的核连续转录增加，这一作用可被四环素介导的糖皮质激素受体抑制所阻断。
Glucocorticoids inhibit the proliferation of fibroblastic cells in vivo and in culture; however, the molecular mechanism that accounts for this effect has remained obscure. We have undertaken to elucidate the mechanism whereby glucocorticoids decrease the rate of proliferation of mouse L929 fibroblastic cells. Addition of dexamethasone to mid-log phase fibroblasts prolongs G1 phase. This increase in the G1 interval is associated with, and probably due to, inhibition of phosphorylation of the product of the Rb-1 tumor suppressor gene, pRb. Inhibition of pRb phosphorylation by cyclin D-dependent kinases can be demonstrated in vitro. Nevertheless, there is no detectable change in the expression of cyclin D1, cyclin D2, or cyclin D3. Cyclin-dependent kinase-4 (Cdk4) and Cdk6 are not down-regulated in L929 cells after addition of glucocorticoids, and the abundance of cyclin D/Cdk4 complexes does not change. Inhibition of pRb kinase activity is associated with an increase in the abundance of one of the Cdk inhibitors, p21(Cip1). The abundance of another cyclin kinase inhibitor, p27(Kip1), remains constant. The amount of Cdk4 that is bound to p21(Cip1) increases rapidly after addition of dexamethasone, and the activity of Cdk4-pRb kinase decreases in parallel. These results indicate that glucocorticoid inhibition of fibroblast proliferation is due to induction of p21(Cip1), which binds to and inactivates cyclinD/Cdk4 complexes. The abundance of p21 mRNA increases about 5-fold within 2 h after addition of dexamethasone. This effect does not obtain in L929 mutants that are null for the glucocorticoid receptor, and a variant that expresses the glucocorticoid receptor from a tetracycline-repressible expression vector demonstrates induction of p21 mRNA only in the absence of tetracycline. Cycloheximide does not block induction of p21 mRNA, and dexamethasone has no detectable effect on the apparent rate of degradation of p21 mRNA. Nuclear run-on transcription of the Cip1 gene increases within 2 h after addition of dexamethasone, This effect can be blocked by tetracycline-mediated repression of the glucocorticoid receptor.