Interferon-γ-dependent stimulation of human involucrin gene expression: STAT1 (signal transduction and activators of transcription 1) protein activates involucrin promoter activity

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Abstract
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-γ (IFN-γ), 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-γ on involucrin gene expression. Expression of involucrin was increased by about 3-fold after treating NHK cells with IFN-γ (100 units/ml). Northern blot analyses revealed that IFN-γ increased the expression of involucrin mRNA. The fragment +42 to -2463 in the 5ʹ-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-γ treatment. Sequence analysis detected two putative IFN-γ-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-γ-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-γ. In contrast, transfection of a STAT1 dominant-negative expression vector suppressed the IFN-γ-dependent up-regulation of involucrin promoter activity. These results indicate that IFN-γ stimulates expression of the human involucrin gene via the G1 (-883 to -874) region of the involucrin gene promoter.

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