Beno D W, Brady L M, Bissonnette M, Davis B H
Department of Medicine, University of Chicago, Illinois 60637.
J Biol Chem. 1995 Feb 24;270(8):3642-7. doi: 10.1074/jbc.270.8.3642.
Recent studies have demonstrated that 1,25-dihydroxyvitamin D3 (D3) can activate Raf kinase and induce Egr expression in cultured rat hepatic Ito cells (Lissoos, T. W., Beno, D. W. A., and Davis, B. H. (1993) J. Biol. Chem. 268, 25132-25138). Since Raf is an upstream activator of mitogen-activated protein kinase (MAPK), the current study evaluated the ability of D3 to activate MAPK. D3-activated MAPK and induced its cytoplasmic to perinuclear translocation in Ito cells. MAPK activation was found to be protein kinase C-dependent, which was analogous to previous studies of D3 and Raf activation. To further explore the D3 cascade, a series of transient transfections were performed using dominant negative raf and MAPK mutant plasmids which effectively block Ras-induced Raf and MAPK activity, respectively. D3 induced a marked increase in the expression of a chloramphenicol acetyltransferase reporter gene linked to the Egr promoter (egr-CAT). When the dominant negative Raf plasmid was co-transfected, there was no significant reduction in egr-CAT. In contrast, when the dominant negative MAPK plasmid was co-transfected, egr-CAT induction was completely abolished. These results suggest that 1) D3 stimulates MAPK via a protein kinase C-dependent pathway, 2) D3-induced Egr expression can occur via a pathway independent of Ras-induced Raf, and 3) D3 absolutely requires MAPK activity for Egr expression.
最近的研究表明，1,25-二羟基维生素D3（D3）可激活Raf激酶并在培养的大鼠肝Ito细胞中诱导Egr表达（Lissoos, T. W., Beno, D. W. A., and Davis, B. H. (1993) J. Biol. Chem. 268, 25132-25138）。由于Raf是丝裂原活化蛋白激酶（MAPK）的上游激活剂，因此本研究评估了D3激活MAPK的能力。D3激活了MAPK并诱导其在Ito细胞中从细胞质向核周移位。发现MAPK激活依赖于蛋白激酶C，这与先前关于D3和Raf激活的研究类似。为了进一步探索D3信号级联反应，使用显性负性raf和MAPK突变体质粒进行了一系列瞬时转染，它们分别有效阻断Ras诱导的Raf和MAPK活性。D3诱导与Egr启动子相连的氯霉素乙酰转移酶报告基因（egr-CAT）的表达显著增加。当共转染显性负性Raf质粒时，egr-CAT没有显著降低。相反，当共转染显性负性MAPK质粒时，egr-CAT的诱导被完全消除。这些结果表明：1）D3通过蛋白激酶C依赖的途径刺激MAPK；2）D3诱导的Egr表达可通过独立于Ras诱导的Raf的途径发生；3）D3诱导Egr表达绝对需要MAPK活性。
