Perillo B, Tedesco I, Laezza C, Santillo M, Romano A, Aloj S M, Bifulco M
Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università Federico II, Napoli, Italy.
J Biol Chem. 1995 Jun 23;270(25):15237-41. doi: 10.1074/jbc.270.25.15237.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and mRNA levels were significantly reduced in FRTL-5 cells transformed with the Kirsten-Moloney sarcoma virus (KiMol); these cells have lost thyrotropin dependence and express high levels of p21ras. FRTL-5 cells, transformed with a temperature-sensitive mutant of the v-K-ras oncogene (Ats cells: 33 degrees C, permissive; 39 degrees C, nonpermissive), showed significant reduction of HMG-CoA reductase expression when exposed to 33 degrees C. In KiMol cells, as well as in Ats cells at 33 degrees C, the transcription driven by cAMP-responsive element was probed by measuring chloramphenicol acetyl transferase (CAT) levels after transfection with a chimeric plasmid containing the reporter gene linked to the rat reductase promoter. Basal CAT activity in KiMol cells transfected with wild-type promoter was lower than in FRTL-5 cells but was increased by forskolin to the levels attained in thyrotropin-stimulated FRTL-5 cells. Forskolin failed to increase CAT activity in KiMol cells transfected with the plasmid harboring a reductase promoter in which the cAMP-responsive element octamer was mutated to a nonpalindromic sequence. The effect of v-K-ras could be mimicked in FRTL-5 cells by tetradecanoyl phorbol acetate and reverted in KiMol and Ats cells, expressing active Ras protein, by increasing intracellular cAMP and/or by protein kinase C inhibition. The data are consistent with the contention that v-K-ras, through protein kinase C and depletion of intracellular cAMP, is inhibitory for the protein kinase A pathway. This is the first demonstration that active v-K-ras down-regulates HMG-CoA reductase expression.
用 Kirsten-Moloney 肉瘤病毒（KiMol）转化的 FRTL-5 细胞中，3-羟基-3-甲基戊二酰辅酶 A（HMG-CoA）还原酶活性和 mRNA 水平显著降低；这些细胞已失去对促甲状腺激素的依赖性，并高水平表达 p21ras。用 v-K-ras 癌基因的温度敏感突变体转化的 FRTL-5 细胞（Ats 细胞：33℃允许生长；39℃不允许生长），在 33℃培养时 HMG-CoA 还原酶表达显著降低。在 KiMol 细胞以及 33℃的 Ats 细胞中，通过用含有与大鼠还原酶启动子相连的报告基因的嵌合质粒转染后测量氯霉素乙酰转移酶（CAT）水平，来检测由 cAMP 反应元件驱动的转录。用野生型启动子转染的 KiMol 细胞中的基础 CAT 活性低于 FRTL-5 细胞，但用福斯高林可将其提高到促甲状腺激素刺激的 FRTL-5 细胞所达到的水平。福斯高林未能增加用含有还原酶启动子的质粒转染的 KiMol 细胞中的 CAT 活性，该启动子中的 cAMP 反应元件八聚体已突变为非回文序列。佛波酯可在 FRTL-5 细胞中模拟 v-K-ras 的作用，而在表达活性 Ras 蛋白的 KiMol 和 Ats 细胞中，通过增加细胞内 cAMP 和/或抑制蛋白激酶 C 可使其作用逆转。这些数据与以下观点一致，即 v-K-ras 通过蛋白激酶 C 和细胞内 cAMP 的消耗，对蛋白激酶 A 途径具有抑制作用。这是首次证明活性 v-K-ras 下调 HMG-CoA 还原酶表达。
