PPAR gamma is an important transcription factor in 1 alpha,25-dihydroxyvitamin D3-induced involucrin expression.
BACKGROUND
1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the active form of vitamin D, suppresses keratinocyte proliferation, promotes keratinocyte differentiation, and induces involucrin expression. Peroxisome proliferation-activated receptors (PPARs) are ligand-activated transcription factors. It has been reported that PPARs stimulate keratinocyte differentiation and regulate the expression of differentiation molecules.


OBJECTIVE
Keratinocytes treated with 1 alpha,25(OH)2D3 induced PPAR gamma, which was followed by increased involucrin expression. In this study, we investigated whether PPAR gamma is involved in the 1 alpha,25(OH)2D3-induced involucrin expression in human keratinocytes.


METHODS
Subconfluent keratinocytes were treated with 10(-7)M 1 alpha,25(OH)2D3 for the indicated times, and PPAR and involucrin mRNA expression were determined by real-time RT-PCR. The levels of PPARs, involucrin, p38, and phospho-p38 proteins were assayed by Western blotting, and the DNA binding activities of PPAR gamma and AP-1 were investigated by electrophoretic mobility shift assays (EMSA). To examine the role of PPAR gamma in 1 alpha,25(OH)2D3 responses, recombinant adenovirus carrying a dominant-negative form of PPAR gamma (Axdn-PPAR gamma) was constructed and transfected into keratinocytes. The p38 inhibitor SB203580 was added to the cultures to evaluate the involvement of p38 in involucrin expression.


RESULTS
1 alpha,25(OH)2D3 induced PPAR gamma expression and stimulated PPAR gamma activity. The introduction of dn-PPAR gamma inhibited the expression of involucrin mRNA and protein induced by 1 alpha,25(OH)2D3, and suppressed AP-1 DNA binding activity. 1 alpha,25(OH)2D3 also triggered the phosphorylation of p38, which contributes to involucrin induction. Moreover, dn-PPAR gamma prevented the 1 alpha,25(OH)2D3-induced phosphorylation of p38.


CONCLUSIONS
These results suggest that PPAR gamma regulates involucrin expression by controlling the AP-1 signal and p38 activation in 1 alpha,25(OH)2D3-induced keratinocyte differentiation.
Dai
Xiuju
X
Department of Dermatology, Ehime University School of Medicine, Ehime, Japan.
Sayama
Koji
K
Shirakata
Yuji
Y
Tokumaru
Sho
S
Yang
Lujun
L
Tohyama
Mikiko
M
Hirakawa
Satoshi
S
Hanakawa
Yasushi
Y
Hashimoto
Koji
K
D002117:Calcitriol / Q000494:pharmacology*; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D047495:PPAR gamma / Q000502:physiology*; D011498:Protein Precursors / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000235:genetics; D015533:Transcriptional Activation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D047495:PPAR gamma; D011498:Protein Precursors; D018808:Transcription Factor AP-1; C032253:involucrin; D048051:p38 Mitogen-Activated Protein Kinases; D002117:Calcitriol