Inhibition of 1,25-dihydroxyvitamin-D-induced keratinocyte differentiation by blocking the expression of phospholipase C-gamma1.
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.
Xie
Z
Z
Endocrine Unit, VA Medical Center, University of California, San Francisco, California 94121, USA. zjxie@itsa.ucsf.edu
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D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D015603:Keratinocytes / Q000166:cytology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D051966:Phospholipase C gamma; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000235:genetics / Q000494:pharmacology*
D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D011498:Protein Precursors; D012333:RNA, Messenger; D014807:Vitamin D; C032253:involucrin; C097949:1,25-dihydroxyvitamin D; D015544:Inositol 1,4,5-Trisphosphate; D011503:Transglutaminases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium