Histone deacetylase 3 regulates the inflammatory gene expression programme of rheumatoid arthritis fibroblast-like synoviocytes

Abstract

Objectives: Non-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS).

Methods: RA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/β receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1β-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays.

Results: HDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1β-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11.

Conclusions: Inhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases.

Keywords: Cytokines; Fibroblasts; Inflammation; Rheumatoid Arthritis.

Figure 1

Histone deacetylase inhibitors (HDACi) differentially affect global protein acetylation and HDAC activity. (A) Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were incubated with increasing concentrations (20, 100 and 500 nM) of either pan-HDAC1/2, pan-HDAC3/6 and pan-HDAC8 inhibitors or selective HDAC1/2, HDAC3/6 and HDAC8 inhibitors for 4 h. Total cell lysates were analysed by western blotting with antibodies recognising acetylated lysine (Ac Lysine), tubulin and histone 3 (H3). Proteins detected at 52, 18 and 14 kDa with antiacetylated lysine Ab correspond to acetylated tubulin, acetylated H3 and histone 4 (H4), respectively. The results are representative of four independent experiments. (B) RA FLS (n=4) were incubated with different HDACi for 4 h. Cell pellets were incubated with fluorogenic class-specific HDAC substrates and class I and class IIb HDAC activities were measured by fluorescent product release. The values represent arbitrary fluorescence values for cells untreated or treated with HDACi. *p<0.05, ****p<000.1, ratio t test.

Figure 2

Histone deacetylase (HDAC)3/6i recapitulates the effects of pan-HDAC inhibitors (HDACi) on inflammatory gene expression. (A) Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) (n=5) were left untreated or preincubated with increasing concentrations (20, 100 and 500 nM) of pan-HDACi or selective HDACi for 30 min and further stimulated with interleukin (IL)-1β for 24 h. Changes in cell viability were analysed by MTT assay and presented as the mean absorbance±SEM at 590 nm. (B) FLS (n=3) were left untreated or preincubated with 250 nM of indicated HDACi for 30 min, prior to stimulation with IL-1β for 4 h. mRNA levels of 83 IL-1β-responsive genes were determined by quantitative PCR (qPCR) using a customised RT2 Profiler PCR Array. Data are presented on heat map as row Z-scores computed from delta Ct values relative to a panel of five housekeeping genes. (C) RA FLS (n=6) were left untreated or incubated with 250 nM HDAC3/6i for 30 min and further stimulated with IL-1β for 4 h. Total RNA was extracted and changes in mRNA accumulation were analysed by qPCR. *p<0.05, **p<0.01, ***p<0.001 ****p<000.1, ratio t test. (D) RA FLS (n=5) were left untreated or preincubated with increasing concentrations (20, 100 and 500 nM) of HDAC3/6i for 30 min and then stimulated with IL-1β for 24 h. IL-6 and IL-8 production was determined by ELISA. *p<0.05, **p<0.01, repeated measures ANOVA followed by Bonferroni correction for multiple comparison analysis. (E) RA FLS (n=4) were seeded into Boyden chambers, left untreated or preincubated with 250 nM HDAC3/6i for 30 min and then stimulated with IL-1β for 24 h. After 24 h of incubation, the number of invasive cells was determined. Graphs indicate the average number of cells per field. *p<0.05, repeated measures ANOVA followed by Bonferroni correction for multiple comparison analysis.

Figure 3

Histone deacetylase (HDAC)3 silencing suppresses the expression of HDAC3/6i target genes, contrarily to HDAC6 silencing or inhibition. (A) Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) (n=3) were left untreated or incubated with either 250 nM HDAC3/6i or increasing concentrations (250, 500 and 1000 nM) of HDAC6i for 4 h. Protein lysates were analysed by western blotting with antibodies recognising acetylated tubulin and tubulin. (B) RA FLS (n=3) were left untreated or preincubated with 250 nM HDAC3/6i or HDAC6i for 30 min, prior to stimulation with interleukin (IL)-1β for 4 h. Changes in mRNA accumulation were analysed by quantitative PCR (qPCR), and data were presented as expression relative to GAPDH. (C) RA FLS (n=4) were left not transfected or were transfected for 48 h with 20 nM control non-targeting siRNA (siScrb) or 20 nM HDAC3-specific siRNA (siHDAC3). HDAC3 knockdown efficiency was verified at the mRNA level by qPCR. (D) FLS were transfected as in (C) to confirm HDAC3 protein silencing by immunoblotting. Total protein lysates were analysed with Abs recognising HDAC3 or control H3. The signal intensity of five independent experiments was subsequently quantified by densitometry analysis. (E) FLS (n=6) were transfected as in (C) and further stimulated with 1 ng/mL IL-1β for 4 h. Changes in mRNA expression were analysed as in (B). *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001, ratio t test. (F) FLS (n=3) were transfected with either non-targeting siRNA (siScrb) or 20 nM HDAC6-specific siRNA (siHDAC6) using the same protocol as in (C) to confirm HDAC6 silencing on mRNA. (G) Confirmation of HDAC6 silencing on protein level was assessed as in (D), protein lysates were immunoblotted for HDAC6 or control actin, and densitometry analysis of three independent experiments is shown. (H) FLS (n=4) were transfected as in (C) and further stimulated with 1 ng/mL IL-1β for 4 h. Changes in mRNA expression were analysed as in (B). *p<0.05, ratio t test.

Figure 4

Histone deacetylase (HDAC)3 regulates STAT1 phosphorylation, but not STAT1 acetylation. (A) Rheumatoid arthritis fibroblast-like synoviocytes (FLS) (n=3) were left untreated or preincubated with 250 nM HDAC3/6i or HDAC6i for 30 min and further stimulated with interleukin (IL)-1β for 4 h. Protein lysates were analysed by western blotting with antibodies recognising STAT1 Tyr701 phosphorylation (p-STAT1 Tyr701), STAT1 or control H3. A representative of four independent experiments is shown. (B) Transcription factor DNA-binding assays were used to analyse DNA-binding activity of STAT1 in FLS nuclear extracts (n=6). The data are presented as the percentage of the absorbance values relative to unstimulated cells. **p<0.01, Friedman test followed by Dunn's multiple comparison analysis. (C) FLS were transfected as in figure 3C and further stimulated with IL-1β for 4 h. Protein lysates were analysed by western blotting with antibodies recognising phospho-STAT1(Tyr701) and H3 and signal intensity of six independent experiments was subsequently quantified by densitometry analysis. *p<0.05, Wilcoxon matched pairs test. (D and E) FLS were preincubated with 250 nM HDAC3/6i for 30 min and stimulated with IL-1β for either 4 h (D) or 1 and 2 h (E). Cell lysates were precleared, immunoprecipitated with STAT1 Ab and immunoblotted with either STAT1 or acetylated lysine (Ac Lys) Abs. The input represents 10% of the whole cell lysate used for the immunoprecipitation.

Figure 5

Histone deacetylase (HDAC)3/6i effects on STAT1 regulation reflect the suppression of interferon (IFN)-β. (A) Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were stimulated with either 1 ng/mL interleukin (IL)-1β or 1000 U/mL IFN-α and IFN-β at different time points, as shown in the figure. Protein lysates were analysed by western blotting with antibodies recognising phospho-STAT1(Tyr701), STAT1 and H3. (B and C) RA FLS (n=3) were left not transfected or were transfected for 48 h with 20 nM control non-targeting siRNA (siScrb) or 20 nM IFN-α/β receptor alpha chain (IFNAR1)-specific siRNA (siIFNAR1). IFNAR1 knockdown efficiency was verified at the mRNA level by quantitative PCR (qPCR) in (B) and at the protein level by western blot in (C). In (C), total protein lysates were analysed with Abs recognising IFNAR1 or control actin (representative of three independent experiments). (D) FLS (n=2) were transfected as in (B), stimulated with either 1 ng/mL IL-1β for 4 h or with 1000 U/mL IFN-β for 1 h and total protein lysates were processed as in (A). (E) FLS (n=2) were left untreated or incubated with 250 nM HDAC3/6i for 30 min, prior to stimulation with either 1 ng/mL IL-1β for 4 h or with 1000 U/mL IFN-β for 1 h. Protein lysates were analysed as in (A). (F) FLS (n=7) were transfected as in (B) and stimulated with 1 ng/mL IL-1β for 4 h. Changes in mRNA accumulation were analysed by q PCR, and data were presented as the mean±SEM mRNA expression relative to GAPDH. *p<0.05, ratio t test.