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

Keratinocytes produce vitamin D-3 and convert it to the most active form, 1 ,25-dihydroxyvitamin D-3, which regulates keratinocyte proliferation and di fferentiation. Phospholipase C-gammal is the most abundant member of the ph ospholipase C family in keratinocytes and is induced by 1,25-dihydroxyvitam in D-3. Therefore, phospholipase C-gammal might be important in the signali ng pathway mediating 1,25-dihydroxyvitamin-133-induced keratinocyte differe ntiation. To test this hypothesis, phospholipase C-gammal expression in hum an keratinocytes was reduced by transfecting the cells with an antisense ph ospholipase C-gammal construct and then evaluating the response of the kera tinocyte differentiation markers involucrin and transglutaminase to 1,25-di hydroxyvitamin D3. The results showed that involucrin and transglutaminase protein and mRNA levels were markedly reduced in keratinocytes transfected by the antisense phospholipase C-gammal construct. Cotransfection of kerati nocytes with the involucrin or transglutaminase promoter construct and the antisense phospholipase C-gammal construct showed decreased involucrin or t ransglutaminase promoter activity in response to 1,25-dihydroxyvitamin D3. To further investigate the mechanism by which phospholipase C-gammal regula tes keratinocyte differentiation, the calcium and inositol triphosphate lev els in keratinocytes transfected by the antisense phospholipase C-gammal co nstruct were measured following 1,25-dihydroxyvitamin D3 administration. Th e increase in keratinocyte intracellular free calcium and inositol triphosp hate levels following 1,25-dihydroxyvitamin D3 administration were markedly reduced by the transfection of the antisense phospholipase C-gammal constr uct. These studies indicate that phospholipase C-gammal plays a critical ro le in the signal transduction pathway mediating 1,25-dihydroxyvitamin-D-3-i nduced keratinocyte differentiation at least in part by mediating the incre ase in inositol triphosphate production and intracellular calcium mobilizat ion following 1,25-dihydroxyvitamin D3 administration.