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. 2014 Jan 15;306(2):E131-49.
doi: 10.1152/ajpendo.00347.2013. Epub 2013 Nov 26.

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription

Susan J Burke  1 , Danhong Lu, Tim E Sparer, Thomas Masi, Matthew R Goff, Michael D Karlstad, J Jason Collier
Affiliations

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription

Susan J Burke et al. Am J Physiol Endocrinol Metab. .

Abstract

Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing β-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1β markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and β-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1β to promote transcription. In addition, IL-1β induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1β induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1β-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic β-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.

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Figures

Fig. 1.
Fig. 1.
Cytokine-mediated activation of CXCL1 and CXCL2 genes in the 832/13 rat β-cell line and isolated rat islets. A and B: 832/13 cells were untreated (NT) or treated with 1 ng/ml IL-1β for indicated times. C and D: 832/13 cells were untreated or treated with increasing concentrations of IL-1β for 3 h. E and F: rat islets were untreated or treated with 10 ng/ml IL-1β, 100 U/ml IFNγ or both cytokines for 6 h. A–F: CXCL1 (A, C, E) and CXCL2 (B, D, F) mRNA levels were measured and normalized to those of the housekeeping gene ribosomal S9 (RS9). *P < 0.05 vs. NT (A, C, D, F), **P < 0.01 vs. NT (A, B, E), ***P < 0.001 vs. NT (A, B); n.s., not significant vs. NT (E, F). G and H: 832/13 cells were transfected with 1.5 kb of the CXCL1 (G) or CXCL2 (H) promoter upstream of the transcriptional start site fused to a luciferase reporter. Posttransfection (24 h), cells were untreated or stimulated for 4 h with 1 ng/ml IL-1β or IL-1β + 100 U/ml IFNγ. Relative promoter luciferase activity is shown. **P < 0.01 vs. NT (G, H). Data are presented as means ± SE from 3–4 individual experiments.
Fig. 2.
Fig. 2.
IL-1β promotes release of the CXCL1 and CXCL2 chemokines and activates peripheral blood neutrophils (PBN) for integrin expression and migration via CXCR2. A and B: 832/13 cells were untreated or treated with 1 ng/ml IL-1β, 100 U/ml IFNγ, or the combination of both cytokines for 0, 3, 6, and 12 h. CXCL1 (A) and CXCL2 (B) release into culture medium was measured by ELISA and normalized to protein content via BCA assay. Values are presented as means ± SE from 3 individual experiments. PBN were unstimulated (PBS control, red lines) or exposed to CXCL1 (blue) or CXCL2 (green) at 100 nM and the level of expression of intergrins CD11a, CD11b, or CD11c measured by flow cytometry (C). PBNs were unstimulated (red) or incubated with a combination of CXCL1/CXCL2 at (1 nM/10 nM, green lines) or CXCL1/CXCL2 at 10 nM/1 nM (blue lines), and levels of integrin expression were measured (D). Plots are representative experiments from 3 replicates. PBNs were exposed to human CXCL1 (10 nM) or 1:10 dilutions of supernatants from control 832/13 (Con) or IL-β exposed (IL-1β) in the presence of CXCR2 inhibitor SB225002 (400 nM) in a chemotaxis assay (E). Bars, are average decrease in migration in the presence of the CXCR2 inhibitor relative to control with no inhibitor. Each bar is the average of 4–6 duplicates ± SD from 6 biological replicates.
Fig. 3.
Fig. 3.
Overexpression of a mutant IκBα protein blocks cytokine-mediated activation and secretion of both CXCL1 and CXCL2. A and B: 832/13 cells were transfected with luciferase reporter plasmids containing −1.5 kb of CXCL1 (A) and CXCL2 (B) promoters. Posttransfection (4 h), adenoviruses expressing βGAL or IκBαSR (SR, superrepressor, which denotes S32A/S36A mutations) were transduced and cultured overnight. Cells were then untreated (open bars) or stimulated with 1 ng/ml IL-1β for 4 h (gray and filled bars). Relative promoter luciferase activity normalized to total protein is shown. *P < 0.05; **P < 0.01 vs. βGAL (gray bars). C and D: 832/13 cells were treated with indicated adenoviruses for 12 h and then stimulated with 1 ng/ml IL-1β for 6 h. Relative mRNA abundance of CXCL1 and CXCL2 was normalized to RS9. **P < 0.01 vs. βGAL (C, D). E and F: 832/13 cells were treated with indicated adenoviruses. Postadenoviral transduction (12 h), cells were treated with IL-1β for an additional 12 h. CXCL1 (E) and CXCL2 (F) release into medium was measured by ELISA and normalized to protein content via BCA assay. **P < 0.01 (E), *P < 0.05 (F). Data are shown as means ± SE from 3 individual experiments.
Fig. 4.
Fig. 4.
NF-κB subunits p65 and p50 are required for cytokine-dependent activation of the CXCL1 and CXCL2 genes. A and B: 832/13 cells were transfected with luciferase reporter constructs containing −1.5 kb of the CXCL1 (A) or CXCL2 (B) promoters (WT) or similar-length constructs wherein upstream conserved NF-κB response elements were mutated (NF-κBm; as shown in schematic). Posttransfection (24 h), cells were stimulated with 1 ng/ml IL-1β for 4 h and luciferase activity was quantified. *P < 0.05 vs. WT (A, B). C–F: 832/13 cells were transfected with duplexes against p65 (C, D) or p50 (E, F), using a scrambled siRNA sequence duplex as a control. Posttransfection (48 h), cells were treated for 6 h with 1 ng/ml IL-1β. Relative CXCL1 and CXCL2 mRNA levels were normalized to RS9. **P < 0.01 vs. siScramble (C, D), *P < 0.05 vs. siScramble (E, F). Data are expressed as means ± SE from 3 independent experiments.
Fig. 5.
Fig. 5.
IκKβ inhibition impairs IL-1β-mediated induction of the CXCL1 and CXCL2 genes. A and B: 832/13 cells were transfected with luciferase reporter constructs containing −1.5 kb of CXCL1 (A) or CXCL2 (B) promoters. The next day, cells were pretreated for 1 h with 2 μM TPCA and then stimulated with 1 ng/ml IL-1β for 4 h. Cells were lysed, and CXCL1 and CXCL2 promoter luciferase activity was quantified. #P < 0.1 vs. DMSO (A, B). C and D: 832/13 cells were pretreated for 1 h with 0.5, 1, or 2 μM TPCA, followed by 6 h stimulation with 1 ng/ml IL-1β. Relative CXCL1 and CXCL2 mRNA abundance was normalized to RS9. **P < 0.01 vs. DMSO (C, D). E and F: 832/13 cells were pretreated individually for 1 h with 10 μM of 3 different p38 inhibitors (SB202190, SB203580, and SB239063) followed by 6 h incubation with 1 ng/ml IL-1β. Relative mRNA abundance of CXCL1 and CXCL2 was calculated; n.s. vs. DMSO (E, F). Data shown represent means ± SE from 3 individual experiments.
Fig. 6.
Fig. 6.
IκKβ drives expression of CXCL1 and CXCL2 genes. A: 832/13 cells were transduced with βGAL or 5 increasing concentrations of IκKβ S177E/S181E (CA IκKβ) overnight. Whole cell lysates were harvested, and immunoblot analysis was performed using antibodies directed against IκKβ, IκBα, and β-actin. B: cells were transfected with 5× NF-κB-Luc and subsequently transduced with indicated adenoviruses (the same concentrations as in Fig. 6A); relative promoter luciferase activity was measured. **P < 0.01 vs. βGAL, ***P < 0.001 vs. βGAL. C and D: 832/13 cells were transfected with −1.5kb-luciferase plasmids (C, CXCL1; D, CXCL2), and 4 h posttransfection, cells were stimulated with adenoviruses expressing βGAL or 3 increasing doses of CA IκKβ. Doses shown correspond to the 3 highest doses seen in the immunoblot for a further 12 h. At the end of the 12 h, cells were lysed and CXCL1 and CXCL2 promoter activity was quantified. *P < 0.05 vs. βGAL, **P < 0.01 vs. βGAL. E and F: 832/13 cells were treated with adenoviruses expressing CA IκKβ and either βGAL or 2 increasing concentrations of IκBαSR (IκBα). CXCL1 (E) and CXCL2 (F) mRNA levels were measured and normalized to those of RS9. **P < 0.01 vs. βGAL. Data shown represent means ± SE from 3 independent experiments. The immunoblot in A is representative of 2 independent experiments.
Fig. 7.
Fig. 7.
Cytokines promote recruitment of p65 and p50 to specific genomic regions in the CXCL1 and CXCL2 gene promoters. A–F: 832/13 cells were untreated or stimulated with 1 ng/ml IL-1β for 15, 30, and 60 min. ChIP assays were carried out using antibodies that immunoprecipitated p65 (A–D) and p50 (E, F). Distal (A, B) and proximal (C–F) NF-κB elements (indicated by arrows in the schematics) in CXCL1 (left) and CXCL2 (right) promoters were targeted for amplification by real-time PCR using recovered DNA as a template. **P < 0.01 vs. NT (A, B, F), *P < 0.05 vs NT (A–F); #P < 0.1 vs. NT (B). Data represent means ± SE from 4 individual experiments.
Fig. 8.
Fig. 8.
NF-κB subunits are recruited to κB genomic response elements within the CXCL1 and CXCL2 gene promoters in a signal-dependent and IκBα-sensitive manner. A: 832/13 cells were stimulated with 1 ng/ml IL-1β for 5, 10, and 15 min or 100 U/ml IFNγ for 5 and 10 min. Whole cell lysates (40 μg) were immunoblotted for PO4-IκBα, total IκBα, PO4-STAT1, and β-actin (as loading control). B: 832/13 cells were treated for 15, 30, and 60 min with 1 ng/ml IL-1β. Whole cell lysates (20 μg) were blotted for abundance of total IκBα with β-actin serving as loading control. C–F: 832/13 cells were cultured with recombinant adenoviruses that express either βGAL or IκBαSR for 12 h. Cells were then stimulated for 15 min with 1 ng/ml IL-1β. ChIP assays were performed with antibodies that immunoprecipitated p65 (C, E) and p50 (D, F). Proximal (C–F) NF-κB elements in the CXCL1 and CXCL2 core promoters were targeted for real-time PCR amplification. **P < 0.01 vs. βGAL + IL-1β. Data in C–F represent means ± SE from 3 individual experiments; immunoblots shown in A and B are representative of 2 independent experiments.
Fig. 9.
Fig. 9.
STAT1 is a key accessory factor for IL-1β-mediated gene transcription. A and B: 832/13 cells were transfected with an siRNA duplex targeting STAT1 or a scrambled sequence (siScramble) as a control. mRNA (A) and protein (B) levels of STAT1 were determined 48 h posttransfection. B: 832/13 cells were treated for 15 min with 1 ng/mL IL-1β or 100 U/ml IFNγ 48 h after siRNA transfection. Blots are from whole cell lysates with β-actin as loading control. C and D: 832/13 cells were transfected with an siSTAT1 duplex. Following 48 h incubation, cells were stimulated for 6 h with 1 ng/ml IL-1β (left). 832/13 cells were treated with adenoviruses expressing 2 increasing concentrations of STAT1 DM (double mutant; Y701F/S727A) for 24 h followed by 6 h stimulation with 1 ng/ml IL-1β (middle). Rat islets were transduced with adenoviruses expressing βGAL or STAT1 Y701F/S727A for 24 h and then treated for a further 6 h with 10 ng/ml IL-β and 100 U/ml IFNγ (right). CXCL1 (C) and CXCL2 (D) mRNA levels were measured. *P < 0.05 vs. siScramble (C, D), **P < 0.01 vs. siScramble (A); #P < 0.1 vs. βGAL (D); *P < 0.05 vs. βGAL (C, D), **P < 0.01 vs. βGAL (C). E and F: 832/13 cells were induced with 1 ng/ml IL-1β for 15 min. ChIP assays were performed with antibodies that immunoprecipitate STAT1 PO4-S727 and IgG as negative control. G and H: 832/13 cells were stimulated for 15 min with 100 U/ml IFNγ. ChIP assays were employed to immunoprecipitate STAT1 PO4-Y701 with IgG as the negative control. Primer sets used for real-time PCR are indicated for CXCL1 (left) and CXCL2 (right). n.s. vs. respective control (E, F, G, H); *P < 0.05 vs. control (F), ***P < 0.001 vs. control (E, G). All experiments are expressed as means ± SE from 3–4 individual experiments. Immunoblot in B was performed on 2 separate occasions.
Fig. 10.
Fig. 10.
IL-1β promotes specific changes in histone acetylation and methylation at regions associated with transcriptional regulation of the CXCL1 and CXCL2 genes. A–H: 832/13 cells were untreated or stimulated with 1 ng/ml IL-1β for 15 or 30 min. ChIP assays were performed using antibodies that immunoprecipitate methylated histone H3 (A, B: lysine 4; C, D: lysine 9; G, H: lysine 27) and acetylated histone H3 (E, F: lysine 14). The core promoter region of the CXCL1 and CXCL2 genes was amplified by real-time PCR. *P < 0.05 vs. NT, **P < 0.01 vs. NT. ChIP assays are represented as means ± SE from 4 individual experiments.
Fig. 11.
Fig. 11.
IL-1β induces signal-specific phosphorylation of RNA Pol II on the CXCL1 and CXCL2 promoters. A–D: 832/13 cells were untreated or stimulated with 1 ng/ml IL-1β for 15, 30, or 60 min. ChIP assays using antisera to immunoprecipitate Pol II CTD PO4-serine 5 (A, B) or Pol II CTD PO4-serine 2 (C, D). The core promoter region and a segment of the coding region, downstream of the transcriptional start site, corresponding to CXCL1(left) and CXCL2 (right) genes were targeted for real-time PCR amplification. *P < 0.05 vs. NT, **P < 0.01 vs. NT. ChIP assays are expressed as means ± SE from 3–4 individual experiments.
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