Transcriptional activation of psoriasis-associated cytokeratin K17 by interferon-gamma. Analysis of gamma-interferon activation sites.
The acid cytokeratin K17 is inducible by interferon-gamma (IFN-gamma), a characteristic unique for cytokeratins analysed so far. In this report, we analysed the molecular basis of K17 expression by IFN-gamma in epithelial cells. The 5'-flanking region of the K17 gene (positions -1762 to -13), cloned in front of a chloramphenicol acetyl transferase (CAT) reporter gene construct, conferred responsiveness to IFN-gamma but not IFN-alpha in transient transfection assays. Sequence analysis revealed three putative gamma-interferon activation sites (GAS). Band-shift assays and transient transfections with CAT reporter gene constructs were used to characterize and to dissect the functional importance of each of the putative GAS elements. In the band shift assay, GAS3 (positions -1528 to -1515) was found to bind GAF/STAT91 and to compete with tryptophanyl-tRNA synthetase (IFP53/WRS)-GAS for binding to GAF; in contrast, GAS1 (positions -183 to -171) and GAS2 (positions -290 to -277) were neither able to bind to nor to compete for GAF/STAT91. However, deletion constructs and mutational analysis of CAT reporter gene constructs harbouring the 5'-flanking region (positions -1762 to -111) in front of the heterologous promoter revealed that the distal GAS3 site was dispensible, but that alteration of the GAS1 element rendered the promoter uninducible by IFN-gamma. Surprisingly, transfection of a CAT-reporter gene construct harbouring a promoter segment (positions -111 to +13) devoid of the GAS elements revealed enhanced CAT-gene expression upon IFN-gamma treatment. The interaction of GAS1 with the interferon-responsive promoter region in the physiological context remains to be clarified.
Vogel
U
U
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Denecke
B
B
Troyanovsky
S M
SM
Leube
R E
RE
Böttger
E C
EC
D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004252:DNA Mutational Analysis; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007633:Keratins / Q000235:genetics*; D008969:Molecular Sequence Data; D011565:Psoriasis / Q000235:genetics*; D017384:Sequence Deletion; D015533:Transcriptional Activation
D007633:Keratins; D007371:Interferon-gamma; D015500:Chloramphenicol O-Acetyltransferase