Background Since rheumatoid synovial cells produce large amounts of various cytokines and proteases, up to 30% of all newly synthesized proteins are unfolded, leading to a cellular stress condition. Histone-deacetylase (HDAC) 6, a mainly cytoplasmatic member of class II HDACs, plays a key role in the major response pathways to the cytotoxic accumulation of misfolded proteins, which are aggresome formation, autophagy activation, and heat shock protein accumulation.
Methods Expression of HDAC6 mRNA was measured in rheumatoid arthritis synovial fibroblasts (RASF, n=8) and osteoarthritis (OA) SF (n=7) by SYBR green Real-time PCR and normalized to expression levels of GAPDH. HDAC6 protein expression in SF (n=6-7), as well as in synovial tissues of RA and OA patients (n=5) was determined by Western blotting. In silico analysis of the 3’untranslated region of HDAC6 was performed using the miRWalk database. Pooled samples of 3 OASF and 3 RASF cultures were screened for differentially expressed micro RNAs (miR) by expression arrays. Expression levels of miR-221 and miR-222 in cultured SF derived from human RA (n=6) and OA (n=12) patients were measured by TaqMan Real-time PCR and normalized to expression levels of RNU6B. SF were transfected with precursor molecules (pre-miR) of miR-221 and miR-222 or control molecules by lipofectamine (n=2).
Results HDAC6 mRNA expression levels were significantly lower in RASF compared to OASF (RASF: dCt: 14.4±0.4, OASF: dCt: 13.8±0.2; p<0.05). Corresponding results were obtained when HDAC6 protein levels were measured, with a 43% downregulation in SF and a 62% downregulation in synovial tissues from RA compared to OA patients. In silico analysis of the 3’untranslated region predicted that HDAC6 is regulated by miR-221 and miR-222. These miRs were also significantly elevated in RASF compared to OASF in a screen of 260 differentially expressed miRs. Accordingly, the expression levels of miR-221 and miR-222 were higher in RASF compared to OASF (miR-221: RASF dCt: 3.0±0.3, OASF dCt: 4.5±0.3; miR-222: RASF dCt: 6.0±0.5, OASF dCt: 7.9±0.3; p<0.01) and HDAC6 negatively correlated with the expression of miR-221 (Pearson r: 0.78; p=0.01) and miR-222 (Pearson r: 0.67; p<0.05). Furthermore, overexpression of miR-221 and miR-222 by transfection of OASF with pre-miRs decreased HDAC6 levels by 35% ±4.5.
Conclusions Here we show impaired expression of HDAC6 in SF and synovial tissues of RA patients and identified miR-221 and miR-222 as possible mediators of this deregulated HDAC6 expression. Since HDAC6 is a critical component of the cellular stress response, which has most recently been linked to inflammation, lower levels of HDAC6 in RASF might contribute to the pathological inflammatory response seen in these cells.
Acknowledgements IAR Epalinges, FP7 Masterswitch, IMI-BT Cure, EMDO-Stiftung
Disclosure of Interest None Declared