1alpha,25(OH)2-vitamin D3 signaling in chick enterocytes: enhancement of tyrosine phosphorylation and rapid stimulation of mitogen-activated protein (MAP) kinase.
The steroid hormone 1alpha,25(OH)2-vitamin D3 (1alpha,25(OH)2D3) generates biological responses in intestinal and other cells via both genomic and rapid, nongenomic signal transduction pathways. We examined the hypothesis that 1alpha,25(OH)2D3 action in chick enterocytes may be linked to pathways involving tyrosine phosphorylation. Brief exposure of isolated chick enterocytes to 1alpha,25(OH)2D3 demonstrated increased tyrosine phosphorylation of several cellular proteins (antiphosphotyrosine immunoblots of whole cell lysates) with prominent bands at 42-44, 55-60, and 105-120 Kda. The 42-44 Kda bands comigrated with mitogen-activated protein (MAP) kinase (immunoblotting with anti-MAP kinase antibody) The response occurred within 30 s, peaked at 1 min, and was dose-dependent (0.01-10 nM), with maximal stimulation at 1 nM (three- to fivefold). This effect was specific for 1alpha,25(OH)2D3 since its metabolic precursors 25(OH)D3 and vitamin D3 did not increase MAP kinase tyrosine phosphorylation. The tyrosine kinase inhibitor, genistein, blocked 1alpha,25(OH)2D3-induced tyrosine phosphorylation of MAP kinase, while staurosporine, a PKC inhibitor, attenuated the hormone's effects by 30%. We have evaluated the ability of 1alpha,25(OH)2D3 analogs, which have complete flexibility around the 6,7 carbon-carbon bond (6F) or which are locked in either the 6-s-cis (6C) or the 6-s-trans (6T) shape(s), to activate MAP kinase. Thus, two 6F and one 6C analog stimulated while one 6T analog did not stimulate MAP kinase tyrosine phosphorylation. In addition, 1beta,25(OH)2D3, a known antagonist of 1alpha,25(OH)2D3-mediated rapid responses, blocked the hormone effects on MAP kinase. We conclude that 1alpha,25(OH)2D3 and analogs which can achieve the 6-s-cis shape (6F and 6C) can increase tyrosine phosphorylation and activation of MAP kinase in chick enterocytes.
de Boland
A R
AR
Departamento de Biologia, Bioquimica, y Farmacia, Universidad Nacional del Sur, Bahia Blanca, Argentina.
Norman
A W
AW
D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D002645:Chickens; D004386:Duodenum / Q000166:cytology* / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D004791:Enzyme Inhibitors; D014443:Tyrosine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002117:Calcitriol; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate