The superfamily of histone deacetylases comprises HDACs and sirtuins (SIRTs) that regulate many cellular processes by deacetylation of histone and non-histone proteins. Expression and activity of histone deacetylases is altered in rheumatoid arthritis (RA). Smoking is an environmental risk factor for RA. The aim of this study was to examine the influence of cigarette smoke on expression of HDACs and SIRTs in synovial fibroblasts and synovial tissues from RA patients and in joints of mice exposed to cigarette smoke.
Synovial tissues were obtained from smoking (n=5) and non smoking (n=5) RA patients undergoing joint replacement surgery. RA synovial fibroblasts (RASF) from non smokers (n=6–10) were stimulated with freshly prepared 5% cigarette smoke extract (CSE) for 24 hours. Mice were exposed to room air (n=8) or cigarette smoke (n=6) in a whole body exposure chamber for 3 weeks, sacrificed and joints were removed. Expression of HDACs and SIRTs was detected at the mRNA level by Real-time TaqMan and SYBR green PCR and at the protein level by immunoblot analysis.
Stimulation of RASF with CSE significantly enhanced the expression of HDAC1 (x-fold: 2.0±0.4; p=0.04), HDAC2 (1.9±0.3; p=0.02) and HDAC3 (2.4±0.4; p=0.01) at the mRNA level while the expression of HDAC 4–11 remained unchanged. However, the protein levels of HDAC1 and HDAC3 were not altered, whereas the expression of HDAC2 protein was decreased. Among all sirtuins examined the transcriptional level of SIRT4 was 4.3-fold ±1.0 (p=0.01) and SIRT6 mRNA was 2.7-fold±0.5 increased (p=0.02) in CSE stimulated RASF. The elevated expression of SIRT4 and SIRT6 was also detected at the protein level, confirming that stimulation with CSE affects the expression of these two sirtuins in vitro.
Also in joints of mice exposed to cigarette smoke the basal mRNA levels of SIRT4 and SIRT6 were 2.3-fold (DCT smoking 14.6±0.6; DCT control 15.5 ±0.7; p=0.04) and 1.8-fold (DCT smoking 13.6±0.9; DCT control 14.7±1.0; p=0.04) higher as compared to control mice. In human synovial tissue samples from RA patients, smokers had 3.1-fold (DCT smokers 2.12±0.37; DCT non smokers 3.75±0.48; p=0.03) higher mRNA levels of SIRT6 and 2.1-fold higher mRNA levels of HDAC2 (DCT smokers 2.00±0.20; DCT non smokers 3.07±0.34; p=0.03) as compared to non smokers.
In the current study we found that the expression of histone deacetylases, in particular SIRT4, SIRT6 and HDAC2 can be altered in RASF, murine joints and human synovial tissue by environmental factors such as smoking. Since histone deacetylases play an important role in the epigenetic regulation of gene transcription, we suggest that smoking influences gene expression by modification of histone deacetylases.
Disclosure: A. Loeffler, None; P. Kunzler, None; F. Niederer, None; A. Jungel, None; C. Kolling, None; G. Camici, None; B. A. Michel, None; R. E. Gay, None; S. Gay, None; C. Ospelt, None.