Expression of human cystatin A by keratinocytes is positively regulated via the Ras/MEKK1/MKK7/JNK signal transduction pathway but negatively regulated via the Ras/Raf-1/MEK1/ERK pathway.
Cystatin A, a cysteine proteinase inhibitor, is a cornified cell envelope constituent expressed in the upper epidermis. We previously reported that a potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate, increases human cystatin A expression by the activation of AP-1 proteins. Here, we delineate the signaling cascade responsible for this regulation. Co-transfection of the cystatin A promoter into normal human keratinocytes together with a dominant active form of ras increased the promoter activity by 3-fold. In contrast, a dominant negative form of ras suppressed basal cystatin A promoter activity. Further analyses disclosed that transfection of dominant negative forms of raf-1, MEK1, ERK1, ERK2, or wild-type MEKK1 all increased cystatin A promoter activity in normal human keratinocytes, whereas wild-type raf-1, ERK1, ERK2, or dominant negative forms of MEKK1, MKK7, or JNK1 suppressed the promoter activity. The increased or decreased promoter activity reflected the expression of cystatin A on mRNA and protein levels. These effects were not observed when a cystatin A promoter with a T2 (-272 to -278) deletion was used. In contrast, transfection of dominant negative forms of MKK3, MKK4, or p38 did not affect cystatin A promoter activity. Immunohistochemical analyses revealed that phosphorylated active extracellular signal-regulated kinases and c-Jun N-terminal kinase were expressed in the nuclei of basal cells and cells in the suprabasal-granular cell layer, respectively. These results indicate that the expression of cystatin A is regulated via mitogen-activated protein kinase pathways positively by Ras/MEKK1/MKK7/JNK and negatively by Ras/Raf/MEK1/ERK.
Takahashi
H
H
Department of Dermatology, Asahikawa Medical College, Asahikawa, Hokkaido 078-8510, Japan. ht@asahikawa-med.ac.jp
Honma
M
M
Ishida-Yamamoto
A
A
Namikawa
K
K
Kiyama
H
H
Iizuka
H
H
D000256:Adenoviridae / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D002273:Carcinogens; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D015891:Cystatins / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D048688:MAP Kinase Kinase 7; D048728:MAP Kinase Kinase Kinase 1; D048055:Mitogen-Activated Protein Kinase 8; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism*
D002273:Carcinogens; D015891:Cystatins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005419:Flavonoids; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D048369:MAP Kinase Kinase 1; D048688:MAP Kinase Kinase 7; C482125:MAP2K1 protein, human; C482569:MAP2K7 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one