1,25-dihydroxyvitamin D(3) increases human cystatin A expression by inhibiting the Raf-1/MEK1/ERK signaling pathway of keratinocytes.
The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D3), 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)(2)D3 on the expression of cystatin A of cultured normal human keratinocytes (NHK). 1,25(OH)(2)D3 suppressed NHK proliferation in a dose-dependent manner with the maximal effect at 1x10(-7) M. 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 D(3) 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)(2)D3. 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)(2)D3 treatment. Transfection of the dominant-active form of Raf-1 (Ad-daRaf-1) or MEK1 (Ad-daMEK1) inhibited 1,25(OH)(2)D3-dependent cystatin A promoter activity and its expression. Consistent with these results, the MEK1 inhibitor, PD98059, further augmented 1,25(OH)(2)D3-induced cystatin A promoter activity and its expression. The present study demonstrated that the 1,25(OH)(2)D3-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.
Takahashi
Hidetoshi
H
Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaoka higashi, 078-8510, Asahikawa, Hokkaido, Japan. ht@asahikawa-med.ac.jp
Ibe
Masaki
M
Honma
Masaru
M
Ishida-Yamamoto
Akemi
A
Hashimoto
Yoshio
Y
Iizuka
Hajime
H
D002117:Calcitriol / Q000494:pharmacology*; D002478:Cells, Cultured; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D020218:Response Elements; D015398:Signal Transduction / Q000187:drug effects*
D015891:Cystatins; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D002117:Calcitriol