Inhibition of 1,25-dihydroxyvitamin-D-induced keratinocyte differentiation by blocking the expression of phospholipase C-gamma1.

ABSTRACT

Keratinocytes produce vitamin D3 and convert it to the most active form, 1,25-dihydroxyvitamin D3, which regulates keratinocyte proliferation and differentiation. Phospholipase C-gamma1 is the most abundant member of the phospholipase C family in keratinocytes and is induced by 1,25-dihydroxyvitamin D3. Therefore, phospholipase C-gamma1 might be important in the signaling pathway mediating 1,25-dihydroxyvitamin-D3-induced keratinocyte differentiation. To test this hypothesis, phospholipase C-gamma1 expression in human keratinocytes was reduced by transfecting the cells with an antisense phospholipase C-gamma1 construct and then evaluating the response of the keratinocyte differentiation markers involucrin and transglutaminase to 1,25-dihydroxyvitamin D3. The results showed that involucrin and transglutaminase protein and mRNA levels were markedly reduced in keratinocytes transfected by the antisense phospholipase C-gamma1 construct. Cotransfection of keratinocytes with the involucrin or transglutaminase promoter construct and the antisense phospholipase C-gamma1 construct showed decreased involucrin or transglutaminase promoter activity in response to 1,25-dihydroxyvitamin D3. To further investigate the mechanism by which phospholipase C-gamma1 regulates keratinocyte differentiation, the calcium and inositol triphosphate levels in keratinocytes transfected by the antisense phospholipase C-gamma1 construct were measured following 1,25-dihydroxyvitamin D3 administration. The increase in keratinocyte intracellular free calcium and inositol triphosphate levels following 1,25-dihydroxyvitamin D3 administration were markedly reduced by the transfection of the antisense phospholipase C-gamma1 construct. These studies indicate that phospholipase C-gamma1 plays a critical role in the signal transduction pathway mediating 1,25-dihydroxyvitamin-D3-induced keratinocyte differentiation at least in part by mediating the increase in inositol triphosphate production and intracellular calcium mobilization following 1,25-dihydroxyvitamin D3 administration.