Abstract
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.
Highlights
University of Chicago and National Institutes of Health Grants DK 02022, DK 40223, DK 42086, and DK 07074-18.The costs of publication of this article were defrayed in part by the payment of page charges
D3 induces proto-oncogene expression in several cell types and in Ito cells, we have found that D3 induces the expression of the nuclear proto-oncogene, Egr [11]
Recent work suggests that mitogen-fldivated protein kinase (MAPK) represents a key link in transmitting the Raf-generated signal to the nucleus in many cell types [14, 18,19,20,21,22]
Summary
Vol 270, No., Issue of February 24, pp. 3642-3647, 1995 Printed in U.S.A. Protein Kinase C and Mitogen-activated Protein Kinase Are Required for 1,25-Dihydroxyvitamin D3-stimulated Egr Induction*. Recent studies have demonstrated that 1,25-dihydroxyvitamin Ds (D3) can activate Raf kinase and induce Egr expression in cultured rat hepatic Ito cells 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 Codependent pathway, 2) D3-induced Egr expression can occur via a pathway independent of Rasinduced Raf, and 3) D3 absolutely requires MAPK activity for Egr expression. To determine whether D3 required Ras-induced Raf activation and/or MAPK for nuclear signaling, we performed a series of transfection experiments utilizing dominant negative plasmids which abolish either Rasinduced Raf or MAPK.
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