1,25-Dihydroxyvitamin D3 increases human cystatin A expression by inhibiting the Raf-1/MEK1/ERK signaling pathway of keratinocytes

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The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), regulates proliferation and differentiation of keratinocytes. Cystatin A, a cysteine proteinase inhibitor, is a cornified cell envelope constituent and a differentiation marker of keratinocytes. In the present study, we examined the effect of 1,25(OH)2D3 on the expression of cystatin A of cultured normal human keratinocytes (NHK). 1,25(OH)2D3 suppressed NHK proliferation in a dose-dependent manner with the maximal effect at 1×10−7M. It also stimulated cystatin A promoter activity and its expression with similar dose effects. The increased cystatin A was detected by 24 h and the effect was accompanied by the suppression of ERK activity. Cystatin A promoter activity was not affected by cotransfection of vitamin D3 receptor or retinoid X receptor. Further analyses disclosed that the 12-o-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE), T2 (−272 to −278), in cystatin A promoter is critical for the regulation by 1,25(OH)2D3. Transfection of the dominant-negative form of ERK adenovirus (Ad-dnERK) increased cystatin A promoter activity and its expression, which was markedly augmented by 1,25(OH)2D3 treatment. Transfection of the dominant-active form of Raf-1 (Ad-daRaf-1) or MEK1 (Ad-daMEK1) inhibited 1,25(OH)2D3-dependent cystatin A promoter activity and its expression. Consistent with these results, the MEK1 inhibitor, PD98059, further augmented 1,25(OH)2D3-induced cystatin A promoter activity and its expression. The present study demonstrated that the 1,25(OH)2D3-responsive element in the cystatin A gene is identical to the TRE, T2 (−272 to −278), and that the suppression of Raf-1/MEK1/ERK1,2 signaling pathway increases cystatin A expression of NHK.

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