The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells.
IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.
Yamamoto
Koh
K
Department of Hematology and Oncology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8519, Tokyo, Japan.
Shibata
Fumi
F
Miyasaka
Nobuyuki
N
Miura
Osamu
O
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D019556:COS Cells; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D018664:Interleukin-12 / Q000378:metabolism*; D007694:Killer Cells, Natural / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D008894:Milk Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D054353:Perforin; D010641:Phenotype; D052899:Pore Forming Cytotoxic Proteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050797:STAT4 Transcription Factor; D050799:STAT5 Transcription Factor; D050800:STAT6 Transcription Factor; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014466:Ultraviolet Rays; D015854:Up-Regulation
D016207:Cytokines; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D008894:Milk Proteins; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050797:STAT4 Transcription Factor; C494090:STAT4 protein, human; D050799:STAT5 Transcription Factor; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015534:Trans-Activators; D054353:Perforin; D018664:Interleukin-12