The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

doi:10.1016/S0006-291X(02)02378-1

Biochemical and Biophysical Research Communications

Volume 297, Issue 5, 11 October 2002, Pages 1245-1252

https://doi.org/10.1016/S0006-291X(02)02378-1 Get rights and content

Abstract

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.

Section snippets

Materials and methods

Cell culture and reagents. Recombinant human IL-12 and IL-2 were purchased from R&D Systems (Minneapolis, MN). NKL cells [14], a human NK cell line, were grown in RPMI-1640 medium supplemented with 10% FCS and 2 ng/ml IL-2. COS7 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS. Cells were grown at 37 °C in a 5% CO₂ incubator.

Northern blot analysis. A human perforin cDNA clone [26] was provided by Dr. Gina Spruill. A fragment of perforin cDNA (nucleotides

IL-12 upregulates the perforin gene expression in NKL cells

To examine whether IL-12 regulates the perforin gene expression in NKL cells [14], a human NK cell line, cells were treated with IL-12 and total RNA was extracted at different time points for examination by Northern blot analysis. As demonstrated in Fig. 2, the mRNA level of perforin slightly increased as early as 30 min after stimulation with IL-12 and reached a conspicuously increased level at 3 h after stimulation. Reprobing with a G3DPH probe confirmed equal loading of RNA samples (Fig. 2,

Discussion

The present study has demonstrated that IL-12 activates the perforin gene expression in NK cells, because IL-12 significantly enhanced the perforin mRNA level as well as the perforin gene promoter activity in a human NK cell line, NKL cells (Fig. 2, Fig. 6). Furthermore, we have demonstrated that STAT4, activated by IL-12 in NKL cells, binds in a cooperative manner with the tandem STAT-binding elements located at −1085 and −1059 in the perforin gene promoter region. Although STAT1, STAT3,

Acknowledgements

We thank Dr. James N. Ihle (St. Jude Children’s Research Hospital) for STAT1, STAT3, STAT4, STAT5b, STAT6, and JAK2 expression vectors, Dr. Gina Spruill (University of Miami) for cDNA and genomic DNA of human perforin gene, Drs. Jerome Ritz (Dana-Farber Cancer Institute), K. Oshimi, and N. Kawamata (Juntendo University) for NKL cells, and Ms. Kaori Okada for excellent technical assistance. This study was supported by grants from Ministry of Education, Culture, Sports, Science and Technology of

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The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

Abstract

Section snippets

Materials and methods

IL-12 upregulates the perforin gene expression in NKL cells

Discussion

Acknowledgements

Immunity

Biochem. Biophys. Res. Commun.

Cell

J. Biol. Chem.

Gene

Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity

Annu. Rev. Immunol.

Differential utilization of Janus kinase-signal transducer activator of transcription signaling pathways in the stimulation of human natural killer cells by IL-2, IL-12, and IFN-α

J. Immunol.

Characterization of cytokine differential induction of STAT complexes in primary human T and NK cells

J. Leukoc. Biol.

Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes

Proc. Natl. Acad. Sci. USA

Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4

J. Exp. Med.

Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12

J. Exp. Med.

Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells

Nature

Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice

Nature

Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice

Nature

A central role of perforin in cytolysis?

Annu. Rev. Immunol.