Liu Y, Gorospe M, Kokkonen G C, Boluyt M O, Younes A, Mock Y D, Wang X, Roth G S, Holbrook N J
Laboratory of Biological Chemistry, National Institute on Aging, Baltimore, Maryland 21224, USA.
Exp Cell Res. 1998 Apr 10;240(1):40-8. doi: 10.1006/excr.1997.3931.
Treatment of primary cultured hepatocytes from adult (6-month-old) rats with epidermal growth factor (EGF) results in a marked elevation in DNA synthesis, a response that is markedly attenuated in cells of aged (24-month-old) animals. Recently we demonstrated that this age-related attenuation is associated with reduced activation of extracellular signal-regulated kinase (ERK) in response to EGF treatment. In order to gain further insight into the mechanisms responsible for the age-related decline in this proliferative response, we investigated the expression and/or activities of several other regulatory molecules important for G1 to S phase progression in EGF-stimulated young and aged hepatocytes. Induction of cyclin D1 and activation of cyclin-dependent kinase 2 (CDK2) by EGF were found to be diminished in the aged cells. In young cells, prior treatment with rapamycin inhibited the induction of DNA synthesis and activation of CDK2 to levels similar to those seen in aged cells without inhibiting ERK activity and cyclin D1 expression. This suggested that a distinct, ERK-independent, rapamycin-sensitive pathway might also contribute to the proliferative response in hepatocytes and be subject to age-related alterations. Further studies demonstrated that activation of p70 S6 kinase (p70S6k), a rapamycin-sensitive event, following EGF treatment was 40% lower in aged hepatocytes relative to young cells, although the kinetics of activation did not differ in the two age groups. Western blot analysis for p70S6k expression revealed similar levels of proteins in young and aged cells. From these findings, we conclude that deficiencies in both the ERK and p70S6k signaling pathways contribute to the age-related decline in the proliferative response of hepatocytes.
用表皮生长因子（EGF）处理成年（6月龄）大鼠的原代培养肝细胞，会导致DNA合成显著增加，而在老年（24月龄）动物的细胞中，这种反应会明显减弱。最近我们证明，这种与年龄相关的减弱与EGF处理后细胞外信号调节激酶（ERK）的激活减少有关。为了进一步深入了解导致这种增殖反应与年龄相关下降的机制，我们研究了在EGF刺激的年轻和老年肝细胞中，对G1期到S期进程重要的其他几种调节分子的表达和/或活性。发现老年细胞中EGF诱导的细胞周期蛋白D1和细胞周期蛋白依赖性激酶2（CDK2）的激活减少。在年轻细胞中，雷帕霉素预处理可抑制DNA合成的诱导和CDK2的激活，使其水平与未抑制ERK活性和细胞周期蛋白D1表达的老年细胞相似。这表明，一条独立于ERK、对雷帕霉素敏感的独特途径也可能有助于肝细胞的增殖反应，并受到与年龄相关的改变的影响。进一步的研究表明，EGF处理后，老年肝细胞中雷帕霉素敏感的p70 S6激酶（p70S6k）的激活相对于年轻细胞降低了40%，尽管两个年龄组的激活动力学没有差异。对p70S6k表达的蛋白质印迹分析显示，年轻和老年细胞中的蛋白质水平相似。从这些发现中，我们得出结论，ERK和p70S6k信号通路的缺陷都导致了肝细胞增殖反应与年龄相关的下降。
