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Stimulation of Phosphorylation of Mitogen-Activated Protein Kinase by 1,25-Dihydroxyvitamin D₃ in Promyelocytic NB4 Leukemia Cells: A Structure-Function Study¹

Department of Biochemistry (X.S., J.E.B., A.W.N.), Division of Biomedical Sciences (A.W.N.), and the Department of Chemistry (W.H.O.), University of California, Riverside, California 92521

Address all correspondence and requests for reprints to: Prof. Anthony W. Norman, Department of Biochemistry, University of California, Riverside, California 92521. E-mail: Norman{at}ucrac1.ucr.edu

Recent studies have shown that 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] actions in cell growth and differentiation are mediated by both its nuclear receptor (VDR_nuc) and its rapid membrane-related effects. In the present study, we investigated the effect of 1,25-(OH)₂D₃ on p42^mapk phosphorylation using human acute promyelocytic leukemia cells (NB4). 1,25-(OH)₂D₃ (10^-8 M) significantly increased p42^mapk phosphorylation in a time- and dose-dependent manner, with the earliest response detectable at 30 sec. Because 1,25-(OH)₂D₃ is a conformationally flexible molecule, we have used a series of conformationally locked (6-s-cis vs. 6-s-trans) analogs to evaluate which shape is optimal for activation. Four 6-s-cis-locked analogs (HF, JM, JN, and JP) and two 6-s-trans-locked analog (JB and JD) were studied. HF, JM, JN, and JP all increased p42^mapk phosphorylation at 1 and 5 min (10^-8 M), but JB and JD had little effect. Analog HL [1ß,25-(OH)₂D₃], a specific antagonist for only the rapid effects of 1,25-(OH)₂D₃, attenuated 1,25-(OH)₂D₃-induced p42^mapk phosphorylation 65–90%. To assess the potential involvement of the VDR_nuc in mediating the analog’s action, the relative abilities of the analogs to compete with [³H]1,25-(OH)₂D₃ for binding in vitro to the VDR_nuc of NB4 cells was measured. All 6-s-cis analogs bound poorly to VDR_nuc (relative competitive index, 0.5–2%) compared with 1,25-(OH)₂D₃ (relative competitive index, 100%). The present studies demonstrate for the first time that in NB4 cells 1,25-(OH)₂D₃ rapidly activates the p42^mapk pathway, and that this effect can be selectively mediated by analogs that can assume a 6-s-cis conformation.

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