Increased activity and expression of histone deacetylase 1 in relation to tumor necrosis factor-alpha in synovial tissue of rheumatoid arthritis

Abstract

Introduction: The purpose of this study was to investigate the profile of histone deacetylase (HDAC) expression in the synovial tissue of rheumatoid arthritis (RA) compared with that of normal control and osteoarthritis (OA), and to examine whether there is a link between HDAC activity and synovial inflammation.

Methods: HDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of total synovial tissue surgically obtained from normal, OA and RA joints. The level of cytoplasmic tumor necrosis factor a (TNFα) fraction was measured by ELISA. Total RNA of synovial tissue was used for RT-PCR of HDAC1-8. In synovial fibroblasts from RA (RASFs), the effects of TNFα on nuclear HDAC activity and class I HDACs (1, 2, 3, 8) mRNA expressions were examined by quantitative real-time PCR. The protein expression and distribution of class I HDACs were examined by Western blotting.

Results: Nuclear HDAC activity was significantly higher in RA than in OA and normal controls and correlated with the amount of cytoplasmic TNFα. The mRNA expression of HDAC1 in RA synovial tissue was higher than in OA and normal controls, and showed positive correlation with TNFα mRNA expression. The protein level of nuclear HDAC1 was higher in RA synovial tissue compared with OA synovial tissue. Stimulation with TNFα significantly increased the nuclear HDAC activity and HDAC1 mRNA expression at 24 hours and HDAC1 protein expression at 48 hours in RASFs.

Conclusions: Our results showed nuclear HDAC activity and expression of HDAC1 were significantly higher in RA than in OA synovial tissues, and they were upregulated by TNFα stimulation in RASFs. These data might provide important clues for the development of specific small molecule HDAC inhibitors.

Figure 1

Total nuclear histone deacetylase (HDAC) and histone deacetylase (HAT) activity in synovial tissues. (a) Nuclear HDAC activity in synovial tissues that were obtained from rheumatoid arthritis (RA, n = 14) patients was significantly increased compared to that from normal controls (n = 3) and osteoarthritis (OA, n = 12). In the box (interquartile range, IQR) and whisker (maximum and minimum) plots, the horizontal line inside the box denoted median and the filled circles denote outliers outside IQR ± 1.5 × IQR. * = P < 0.05 versus normal controls, ** = P < 0.05 versus OA. (b) Nuclear HAT activity was measured in synovial tissues that were obtained from normal controls (n = 3), OA (n = 5) and RA patients (n = 5). There was no significant difference between normal controls, OA and RA synovial tissues.

Figure 2

Correlation between the amount of cytoplasmic TNFα and nuclear HDAC activity in synovial tissues. Both cytoplasimc TNFα and nuclear HDAC activity were measured in synovial tissues that were obtained from osteoarthritis (n = 12), and rheumatoid arthritis (n = 12) patients. The cytoplasmic fraction was obtained during the isolation of the nuclear fraction, and analyzed by ELISA.

Figure 3

HDAC1-8 mRNA expression and correlation between TNFα mRNA and HDAC1 mRNA expressions in synovial tissues. Total RNA was extracted from total synovial tissues that were obtained from normal controls (n = 3), osteoarthritis (OA, n = 8) and rheumatoid arthritis (RA, n = 9) patients. (a) HDAC1-8 mRNA expression was analyzed by quantitative real-time PCR analysis as described in Materials and Methods. * = P < 0.05 versus OA, ** = P < 0.05 versus normal controls. (b) Correlation between TNFα mRNA expression and HDAC1 mRNA in RA synovial tissues (n = 10).

Figure 4

Results of Western blot analysis for nuclear class I HDACs protein expressions in synovial tissues. (a) Class I HDACs (HDAC1, 2, 3, 8) protein expressions in synovial tissues that were obtained from osteoarthritis (OA, n = 3) and rheumatoid arthritis (RA, n = 3) synovial tissues. Lamin A, nuclear membrane protein, served as loading control. (b) Densitometric analysis class I HDACs protein in OA and RA synovial tissue was The data were plotted as means ± SE. * = P < 0.05 versus OA.

Figure 5

Nuclear HDAC activity, class I HDACs mRNA and protein expressions in RASFs after TNFα treatment. RASFs (n = 3) were treated with TNFα (10 ng/ml) and total nuclear protein was extracted at the indicated time points. (a) Nuclear HDAC activity was elevated significantly at 6 h. The data were plotted as means ± SE. * = P < 0.001 versus time 0 h, 24 h. (b) The expression of HDAC1 in RASFs was increased after TNFα treatment, while the expressions of other class I HDACs were not elevated. The increase of mRNA in HDAC1 at 24 h was significantly greater than that in other class I HDACs. For real-time PCR analysis, levels of mRNA were normalized against actin expression and compared with 0 h. * = P < 0.05 versus HDAC2, 3, 8. (c) Quantitative results of Western blot analysis of nuclear class I HDACs protein expression in RASFs. The band intensity of class I HDACs was measured by Image J software. For analysis, levels of protein expression were normalized by lamin A and compared with 0 h. * = P < 0.05 versus HDAC3 and HDAC8 at 48 h.