Interferon-gamma-dependent Stimulation of Human Involucrin Gene Expression: STAT1 (signal Transduction and Activators of Transcription 1) Protein Activates Involucrin Promoter Activity

Involucrin is one of the precursor proteins of the cornified cell envelope of keratinocytes, and is expressed during the later stages of keratinocyte differentiation. Interferon-gamma (IFN-gamma), a pleiotropic cytokine with anti-proliferative and immunomodulatory activities, is also a potent inducer of squamous differentiation. Using cultured normal human keratinocytes (NHK cells) and simian virus 40-transformed human keratinocytes (SVHK cells), we investigated the effects of IFN-gamma on involucrin gene expression. Expression of involucrin was increased by about 3-fold after treating NHK cells with IFN-gamma (100 units/ml). Northern blot analyses revealed that IFN-gamma increased the expression of involucrin mRNA. The fragment +42 to -2463 in the 5'-flanking region of the human involucrin gene was subcloned into a luciferase reporter vector and the construct (p2463Luc) was transfected into SVHK cells. p2463Luc produced a 3-fold increase in luciferase activity after IFN-gamma treatment. Sequence analysis detected two putative IFN-gamma-responsive regions [G1 (positions -883 to -874) and G2 (-784 to -775)]. Deletion analyses of the p2463Luc vector revealed that the G1 region is critical for the IFN-gamma-dependent up-regulation of the involucrin gene. Gel-shift analyses revealed that STAT1 (signal transduction and activators of transcription 1) protein bound to the G1 region and that involucrin promoter activity was augmented by transfection of a STAT1 expression vector in the presence of IFN-gamma. In contrast, transfection of a STAT1 dominant-negative expression vector suppressed the IFN-gamma-dependent up-regulation of involucrin promoter activity. These results indicate that IFN-gamma stimulates expression of the human involucrin gene via the G1 (-883 to -874) region of the involucrin gene promoter.