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Updated: Jul 9, 2026

Generation and Culturing of Primary Human Keratinocytes from Adult Skin
Published on: December 22, 2017
PPAR gamma is an important transcription factor in 1 alpha,25-dihydroxyvitamin D3-induced involucrin expression
Xiuju Dai1, Koji Sayama, Yuji Shirakata
1Department of Dermatology, Ehime University School of Medicine, Ehime, Japan.
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.
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