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.2007 Mar 15;109(6):2481-7.
doi: 10.1182/blood-2006-10-050096. Epub 2006 Nov 16.

Hlx homeobox transcription factor negatively regulates interferon-gamma production in monokine-activated natural killer cells

Affiliations

Hlx homeobox transcription factor negatively regulates interferon-gamma production in monokine-activated natural killer cells

Brian Becknell et al. Blood..

Abstract

Natural killer (NK) cells contribute to host immunity, including tumor surveillance, through the production of interferon gamma (IFN-gamma). Although there is some knowledge about molecular mechanisms that induce IFN-gamma in NK cells, considerably less is known about the mechanisms that reduce its expression. Here, we investigate the role of the Hlx transcription factor in IFN-gamma production by NK cells. Hlx expression is induced in monokine-activated NK cells, but with delayed kinetics compared to IFN-gamma. Ectopic Hlx expression decreases IFN-gamma synthesis in primary human NK cells and IFN-gamma promoter activity in an NK-like cell line. Hlx protein levels inversely correlate with those of STAT4, a requisite factor for optimal IFN-gamma transcription. Mechanistically, we provide evidence indicating that Hlx overexpression accelerates dephosphorylation and proteasome-dependent degradation of the active Y693-phosphorylated form of STAT4. Thus, Hlx expression in activated NK cells temporally controls and limits the monokine-induced production of IFN-gamma, in part through the targeted depletion of STAT4.

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Figures

Figure 1
Figure 1
Monokine-dependent Hlx expression by human CD56bright NK cells is delayed with respect to IFN-γ transcription. (A) Delayed induction of Hlx protein with respect to IFN-γ production in primary NK cells. (upper) Total NK cells were stimulated with IL-12/IL-18 for indicated time points, and Hlx protein levels were analyzed by immunoblotting. β-Actin levels were analyzed to ensure equal loading (n = 4 experiments). (lower) In parallel, IFN-γ mRNA levels were analyzed by QRT-PCR. Mean ± SEM from 4 separate experiments is shown. (B) Maximal Hlx protein induction requires monokine costimulation. Total NK cells were stimulated for 72 hours, as indicated, and Hlx and β-actin protein levels were analyzed by immunoblotting (n = 3 experiments). (C) Preferential induction of Hlx protein in the CD56bright NK subset. (upper) FACS-purified unstimulated NK subsets were lysed directly (UN) or after stimulation with the indicated monokine combinations (72 hours). Lysates were analyzed for Hlx and β-actin protein by immunoblotting (n = 3 experiments). (lower) FACS-purified NK subsets were lysed immediately for RNA (UN) or stimulated with IL-12/IL-15 for 12 hours before lysis. Hlx mRNA levels were analyzed by QRT-PCR. Mean ± SEM from 5 separate donors is shown.
Figure 2
Figure 2
Hlx inhibits IFN-γ production by primary CD56bright NK. PINCO- or PINCO-Hlx–infected primary human NK cells were stimulated for 24 hours with IL-12/IL-18 and were subjected to staining for CD56 and IFN-γ. (left) Infected cells were gated on CD56brightEGFP+ or CD56dimEGFP+. (right) IFN-γ staining in each population compared with isotype controls. Results shown are representative of 3 experiments (CD56bright PINCO-Hlx compared with CD56bright PINCO;P < .02).
Figure 3
Figure 3
Hlx inhibits IFN-γ mRNA and protein expression in NK-92 cells. (A) Overexpression of Hlx protein in NK-92 cells. FACS-purified NK-92 cells transduced with PINCO or PINCO-Hlx were subjected to nucleocytoplasmic fractionation and immunoblotting for Hlx. Enrichment of nuclear protein was confirmed by Brg1 staining. Equal loading was confirmed by Ku70 staining. (B) Inhibition of IFN-γ production by Hlx. PINCO- or PINCO-Hlx–infected NK-92 cells were stimulated with IL-12/IL-18, and supernatants were harvested after 24 hours for IFN-γ ELISA. Shown are the mean ± SEM from 5 separate experiments. (C) Time course of IFN-γ mRNA in PINCO-compared with PINCO-Hlx–transduced NK-92 in response to IL-12/IL-18. Shown are results of 1 of 4 representative experiments; error bars represent SD of triplicate PCR reactions. (D) Increased IFN-γ production byHlx−/− NK in response to monokine costimulation. Fetal liver–derived NK of indicated genotypes were subjected to 24-hour stimulation with IL-12 and IL-15, followed by intracellular staining for IFN-γ. Mean ± SEM percentages of IFN-γ+ NK1.1+ cells from 9 experiments with NK cells derived from 9 or more livers of each genotype are shown. *P < .001Hlx+/+ compared withHlx−/−. **P < .005Hlx+/− compared with Hlx−/−).
Figure 4
Figure 4
Hlx inhibitsIFNG promoter activity in DERL-7 cells. (A) The humanIFNG luciferase construct used in this study consisted of 3.6 kB of a 5′ flanking sequence upstream of the transcriptional start site, the firefly luciferase open-reading frame, and 0.8 kB of the intron 1 enhancer element. (B) DERL-7 cells with Hlx or empty vector were transfected with theIFNG luciferase construct, and luciferase activity was measured after 6-hour stimulation with IL-12/IL-18. Mean ±SD of 1 of 3 representative experiments is shown.
Figure 5
Figure 5
Hlx overexpression leads to proteasome-dependent degradation of STAT4. (A) Endogenous Hlx levels increased as total STAT4 levels declined. Primary human NK or NK-92 cells were harvested at the indicated time points after IL-12/IL-18 stimulation, and Hlx, STAT4, and β-actin protein levels were determined by immunoblotting. (n = 4 experiments in primary NK, n = 2 experiments in NK-92). (B) Hlx overexpression decreased total and Y693 pSTAT4 levels in NK-92 after IL-12/IL-18 treatment. FACS-purified PINCO- or Hlx-expressing NK-92 cells were harvested at indicated time points after IL-12/IL-18 stimulation. Total STAT4 and β-actin levels were assessed by immunoblotting (n = 4 experiments). (C) Proteasome inhibition rescued loss of STAT4 in NK-92 cells overexpressing Hlx. Cells were preincubated for 1 hour with 20 mM MG-132 (MG), an equal amount of DMSO vehicle, or medium alone (—), followed by IL-12/IL-18 treatment for the indicated times. Total protein was harvested, and total STAT4 and β-actin levels were assessed by immunoblotting (n = 3 experiments). (D) Diminished Y693 pSTAT4 levels in Hlx-overexpressing NK-92 cells despite inhibition of new protein synthesis. PINCO- or Hlx-expressing NK-92 cells were preincubated for 30 minutes with 10 μM CHX, followed by IL-12/IL-18 treatment for the indicated times. Total cellular protein was harvested, and total STAT4 and β-actin levels were assessed by immunoblotting (the arrowhead indicates the position of the 80 kDa marker). (E) Comparison of CHX compared with ethanol (EtOH) carrier effects on STAT4 expression in PINCO- (P) and Hlx-expressing NK-92 after IL-12/IL-18 stimulation for the indicated times (n = 3 experiments).
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