Stimulation of phosphorylation of mitogen-activated protein kinase by 1alpha,25-dihydroxyvitamin D3 in promyelocytic NB4 leukemia cells: a structure-function study.
Recent studies have shown that 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] actions in cell growth and differentiation are mediated by both its nuclear receptor (VDRnuc) and its rapid membrane-related effects. In the present study, we investigated the effect of 1alpha,25-(OH)2D3 on p42mapk phosphorylation using human acute promyelocytic leukemia cells (NB4). 1Alpha,25-(OH)2D3 (10[-8] M) significantly increased p42mapk phosphorylation in a time- and dose-dependent manner, with the earliest response detectable at 30 sec. Because 1alpha,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 p42mapk phosphorylation at 1 and 5 min (10[-8] M), but JB and JD had little effect. Analog HL [1beta,25-(OH)2D3], a specific antagonist for only the rapid effects of 1alpha,25-(OH)2D3, attenuated 1alpha,25-(OH)2D3-induced p42mapk phosphorylation 65-90%. To assess the potential involvement of the VDRnuc in mediating the analog's action, the relative abilities of the analogs to compete with [3H]1alpha,25-(OH)2D3 for binding in vitro to the VDRnuc of NB4 cells was measured. All 6-s-cis analogs bound poorly to VDRnuc (relative competitive index, 0.5-2%) compared with 1alpha,25-(OH)2D3 (relative competitive index, 100%). The present studies demonstrate for the first time that in NB4 cells 1alpha,25-(OH)2D3 rapidly activates the p42mapk pathway, and that this effect can be selectively mediated by analogs that can assume a 6-s-cis conformation.
Song
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Department of Biochemistry, University of California, Riverside 92521, USA.
Bishop
J E
JE
Okamura
W H
WH
Norman
A W
AW
D002117:Calcitriol / Q000737:chemistry / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects* / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D018167:Receptors, Calcitriol / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014807:Vitamin D / Q000031:analogs & derivatives
D018167:Receptors, Calcitriol; D014807:Vitamin D; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D002117:Calcitriol