TY - JOUR
T1 - 1,25 dihydroxyvitamin D3 stimulates phospholipase C-γ in rat colonocytes
T2 - Role of c-Src in PLC-γ activation
AU - Khare, S.
AU - Bolt, M. J.G.
AU - Wali, R. K.
AU - Skarosi, S. F.
AU - Roy, H. K.
AU - Niedziela, S.
AU - Scaglione-Sewell, B.
AU - Aquino, B.
AU - Abraham, C.
AU - Sitrin, M. D.
AU - Brasitus, T. A.
AU - Bissonnette, M.
PY - 1997/4/15
Y1 - 1997/4/15
N2 - Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-α and -β11 in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-γ (PI-PLC-γ), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-γ, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-γ in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-γ. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-γ in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PPI, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-γ in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time that 1,25(OH)2D3 stimulates PLC-γ as well as c-Src in rat colonocytes, and indicate that PLC-γ is a direct substrate of secosteroid-activated c-Src in these cells.
AB - Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-α and -β11 in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-γ (PI-PLC-γ), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-γ, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-γ in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-γ. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-γ in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PPI, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-γ in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time that 1,25(OH)2D3 stimulates PLC-γ as well as c-Src in rat colonocytes, and indicate that PLC-γ is a direct substrate of secosteroid-activated c-Src in these cells.
KW - 1,25-dihydroxycholecalciferol
KW - basolateral membrane
KW - nonreceptor tyrosine kinase
KW - vitamin D deficiency
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U2 - 10.1172/JCI119350
DO - 10.1172/JCI119350
M3 - Article
C2 - 9109427
AN - SCOPUS:0030962467
SN - 0021-9738
VL - 99
SP - 1831
EP - 1841
JO - Journal of Clinical Investigation
JF - Journal of Clinical Investigation
IS - 8
ER -