STIMULATION OF PHOSPHORYLATION OF MITOGEN-ACTIVATED PROTEIN-KINASE BY1-ALPHA,25-DIHYDROXYVITAMIN D-3 IN PROMYELOCYTIC NB4 LEUKEMIA-CELLS -A STRUCTURE-FUNCTION STUDY
Xd. Song et al., STIMULATION OF PHOSPHORYLATION OF MITOGEN-ACTIVATED PROTEIN-KINASE BY1-ALPHA,25-DIHYDROXYVITAMIN D-3 IN PROMYELOCYTIC NB4 LEUKEMIA-CELLS -A STRUCTURE-FUNCTION STUDY, Endocrinology, 139(2), 1998, pp. 457-465
Recent studies have shown that 1 alpha,25-dihydroxyvitamin D-3 [1 alph
a,25-(OH)(2)D-3] actions in cell growth and differentiation are mediat
ed by both its nuclear receptor (VDRnuc) and its rapid membrane-relate
d effects. In the present study, we investigated the effect of 1 alpha
,25-(OH)(2)D-3 on p42(mapk) phosphorylation using human acute promyelo
cytic leukemia cells (NB4), 1 alpha,25-(OH)(2)D-3 (10(-8) M) significa
ntly increased p42(mapk) phosphorylation in a time-and dose-dependent
manner, with the earliest response detectable at 30 sec. Because 1 alp
ha,25-(OH)(2)D-3 is a conformationally flexible molecule, we have used
a series of conformationally locked (6-s-cis vs. B-s-trans) analogs t
o evaluate which shape is optimal for activation. Four B-s-cis-locked
analogs (HF, JM, JN, and JP) and two B-s-trans-locked analog (JB and J
D) were studied. HF, JM, JN, and JP all increased p42(mapk) phosphoryl
ation at 1 and 5 min (10(-8) M), but JB and JD had little effect. Anal
og HL [1 beta,25-(OH)(2)D-3], a specific antagonist for only the rapid
effects of 1 alpha,25-(OH)(2)D-3, attenuated 1 alpha,25-(OH)(2)D-3-in
duced p42(mapk) phosphorylation 65-90%. To assess the potential involv
ement of the VDRnuc in mediating the analog's action, the relative abi
lities of the analogs to compete with [H-3]1 alpha,25-(OH)(2)D-3 for b
inding in vitro to the VDRnuc of NB4 cells was measured. All 6-s-cis a
nalogs bound poorly to VDRnuc (relative competitive index, 0.5-2%) com
pared with 1 alpha,25-(OH)(2)D-3 (relative competitive index, 100%). T
he present studies demonstrate for the first time that in NB4 cells 1
alpha,25-(OH)(2)D-3 rapidly activates the p42(mapk) pathway, and that
this effect can be selectively mediated by analogs that can assume a 6
-s-cis conformation.