Auer K L, Park J S, Seth P, Coffey R J, Darlington G, Abo A, McMahon M, Depinho R A, Fisher P B, Dent P
Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
Biochem J. 1998 Dec 15;336 ( Pt 3)(Pt 3):551-60. doi: 10.1042/bj3360551.
In primary rat hepatocytes, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a decrease in DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor (CKI) proteins p21Cip-1/WAF1 and p16INK4a. To evaluate the relative importance of these CKIs in mediating this response, we determined the impact of prolonged MAPK activation on DNA synthesis in primary cultures of hepatocytes derived from mice embryonically deleted (null) for either p21Cip-1/WAF1 or p16INK4a. When MAPK was activated in wild-type mouse hepatocytes for 24 h, via infection with a construct to express an inducible oestrogen receptor-Raf-1 fusion protein (DeltaRaf:ER), the expression of p21Cip-1/WAF1 and p16INK4a CKI proteins increased, cyclin-dependent kinase 2 (cdk2) and cdk4 activities decreased, and DNA synthesis decreased. Inhibition of RhoA GTPase function increased the basal expression of p21Cip-1/WAF1 and p27Kip-1 but not p16INK4a, and enhanced the ability of MAPK signalling to decrease DNA synthesis. Ablation of the expression of CCAATT enhancer-binding protein alpha (C/EBPalpha), but not of the expression of C/EBPbeta, decreased the ability of MAPK signalling to induce p21Cip-1/WAF1. When MAPK was activated in p16INK4a-null hepatocytes for 24 h, the expression of p21Cip-1/WAF1 increased, cdk2 and cdk4 activities decreased and DNA synthesis decreased. In contrast with these findings, prolonged activation of the MAPK pathway in hepatocytes from p21Cip-1/WAF1-null mice enhanced cdk2 and cdk4 activities and caused a large increase in DNA synthesis, despite elevated expression of p16INK4a. Inhibition of RhoA GTPase activity in p21Cip-1/WAF1-null cells partly blunted both the basal levels of DNA synthesis and the ability of prolonged MAPK signalling to increase DNA synthesis. Expression of anti-sense p21Cip-1/WAF1 in either wild-type or p16INK4a-null hepatocytes decreased the ability of prolonged MAPK signalling to increase the expression of p21Cip-1/WAF1, and permitted MAPK signalling to increase both cdk2 and cdk4 activities and DNA synthesis. These results argue that the ability of prolonged MAPK signalling to inhibit DNA synthesis in hepatocytes requires the expression of p21Cip-1/WAF1, and that the increased expression of p16INK4a has a smaller role in the ability of this stimulus to mediate growth arrest. Our results also suggest that RhoA function can modulate DNA synthesis in primary hepatocytes via the expression of p21Cip-1/WAF1 and p27Kip-1.
在原代大鼠肝细胞中，p42/44丝裂原活化蛋白激酶（MAPK）途径的长期激活与DNA合成减少以及细胞周期蛋白依赖性激酶抑制剂（CKI）蛋白p21Cip-1/WAF1和p16INK4a的表达增加有关。为了评估这些CKI在介导这种反应中的相对重要性，我们确定了长期MAPK激活对源自胚胎期缺失（无效）p21Cip-1/WAF1或p16INK4a的小鼠的原代肝细胞培养物中DNA合成的影响。当通过感染表达诱导型雌激素受体-Raf-1融合蛋白（DeltaRaf:ER）的构建体在野生型小鼠肝细胞中激活MAPK 24小时时，p21Cip-1/WAF1和p16INK4a CKI蛋白的表达增加，细胞周期蛋白依赖性激酶2（cdk2）和cdk4活性降低，DNA合成减少。RhoA GTPase功能的抑制增加了p21Cip-1/WAF1和p27Kip-1的基础表达，但不增加p16INK4a的基础表达，并增强了MAPK信号传导减少DNA合成的能力。CCAAT增强子结合蛋白α（C/EBPα）表达的缺失，但不是C/EBPβ表达的缺失，降低了MAPK信号传导诱导p21Cip-1/WAF1的能力。当在p16INK4a无效的肝细胞中激活MAPK 24小时时，p21Cip-1/WAF1的表达增加，cdk2和cdk4活性降低，DNA合成减少。与这些发现相反，尽管p16INK4a表达升高，但来自p21Cip-1/WAF1无效小鼠的肝细胞中MAPK途径的长期激活增强了cdk2和cdk4活性，并导致DNA合成大幅增加。在p21Cip-1/WAF-1无效细胞中抑制RhoA GTPase活性部分减弱了DNA合成的基础水平以及长期MAPK信号传导增加DNA合成的能力。在野生型或p16INK4a无效的肝细胞中反义p21Cip-1/WAF1的表达降低了长期MAPK信号传导增加p21Cip-1/WAF1表达以及允许MAPK信号传导增加cdk2和cdk4活性和DNA合成的能力。这些结果表明，长期MAPK信号传导抑制肝细胞中DNA合成的能力需要p21Cip-1/WAF1的表达，并且p16INK4a表达的增加在这种刺激介导生长停滞的能力中作用较小。我们的结果还表明，RhoA功能可通过p21Cip-1/WAF1和p27Kip-1的表达调节原代肝细胞中的DNA合成。
