Transcriptional factor AP-2gamma increases human cystatin A gene transcription of keratinocytes.
The transcriptional activator protein-2 (AP-2) has been suggested to participate in keratinocyte gene regulation. Cystatin A, a cysteine proteinase inhibitor, is one of the cornified cell envelope constituents and is expressed in the upper epidermis. We report AP-2-dependent transcriptional regulation of cystatin A gene expression of keratinocytes. At least three isoforms of AP-2 (AP-2 alpha, beta, gamma) have been described. Transfection of AP-2alpha, beta and gamma expression vectors into cultured normal human keratinocytes (NHK) resulted in increased cystatin A expression in both mRNA and protein levels. Among the three isoforms AP-2gamma was most potent in inducing cystatin A expression. In contrast, transfection of antisense oriented AP-2gamma expression vector decreased basal AP-2 expression, accompanied by decreased cystatin A mRNA. The fragment, +77 to -478 of 5'-flanking region of human cystatin A gene, was subcloned into chloramphenicol acetyltransferase (CAT) reporter vector (p478CAT). Cotransfection of p478CAT vector with AP-2alpha, beta, and gamma expression vectors resulted in three-, three-, and sixfold increase in the CAT activity, respectively. Transfection of the deleted construct (p478DeltaAP-2CAT, devoid of AP-2-like binding site (-75 to -84)) decreased CAT activity by one-third compared to p478CAT promoter activity. Cotransfection of p478DeltaAP-2CAT with AP-2alpha, beta, and gamma expression vectors had no effect on the decreased promoter activity. Immunohistochemical analysis of human skin showed that AP-2alpha is exclusively expressed in the nuclei of basal cell layer. AP-2gamma is expressed in the nuclei of basal, spinous, and granular cell layers. AP-2beta expression was not observed in the epidermis. Gel mobility shift assay revealed that the AP-2gamma protein specifically binds to oligonucleotides containing AP-2-like binding site of cystatin A gene. These results indicate that AP-2gamma regulates the cystatin A gene expression of epidermal keratinocytes at the transcriptional level.
Takahashi
H
H
Department of Dermatology, Asahikawa Medical College, Asahikawa, 078-8510, Japan.
Oyama
N
N
Itoh
Y
Y
Ishida-Yamamoto
A
A
Kaneko
F
F
Iizuka
H
H
D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D015891:Cystatins / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D015891:Cystatins; D004268:DNA-Binding Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; C493673:TFAP2A protein, human; C493677:TFAP2B protein, human; C493679:TFAP2C protein, human; D050656:Transcription Factor AP-2; D014157:Transcription Factors