Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C82
Inhibition of HDAC8 and acetylation of Hsp20 regulates contractile activity of human myometrial smooth muscle
M. Karolczak-Bayatti1, M. Sweeney1, S. C. Robson1, S. M. Ulrich2, M. J. Taggart1, N. G. Europe-Finner1
1. Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom. 2. Department of Chemistry, Ithaca College, Ithaca, New York, United States.
Recent evidence has implicated a role for Histone deacetylases 8 (HDAC8) as a cytosolic marker of smooth muscle cells and an important regulator of contractility in vascular smooth muscle cells (1). There is now rising evidence to indicate that acetylation of non-nuclear proteins is an important post-translational modification regulating many cellular functions including the cytoskeletal architecture of cells and in smooth muscle physiology, these include proteins involved in the contractile apparatus (2). The aim of this study was to examine if inhibition of HDAC8 will lead to relaxation of myometrial smooth muscle and to find the putative protein targets of its enzymatic activity. The effect of the specific HDAC8 inhibitor, Compound2 (Comp2, 100µM in DMSO) (3) was tested on contractions in isometrically mounted myometrial strips from biopsies obtained from pregnant women following Caesarean section. Integrals from selected 30 min intervals prior to and post treatment/control were calculated and compared between groups using paired t test. Treatment with Comp2 significantly inhibited (p<0.05) spontaneous (98±2.41 vs. 71±6.9) and oxytocin-induced (94±3.1 vs. 71±4.3) contractions after 20 min lasting up to 1.5h. Importantly, this occurred without elevation of nuclear-resident histone acetylation or marked changes in global gene expression as measured by gene chip arrays (significant change in only 3 out of 47.000 transcripts at 2-fold change, p<0.05, Anova). Co-immunoprecipitation (co-IP) and immunocytochemistry demonstrated that HDAC8 interacts with α-smooth muscle actin and myosin heavy chain (western blotting, n=3), indicating that they can be potential sites of HDAC8 enzymatic activity. However, co-IPs with antibodies for acetylated Lysine residues and lysates from non-pregnant myometrial tissues treated with Comp2 and a pan-HDACs inhibitor Trichostatin A (TSA, 3.3µM) for 24h did not detect acetylation of these proteins. The same results were obtained for known targets of acetylation: calmodulin, Hsp27, 14-3-3 and cofilin. Heat shock protein 20 (Hsp20) was the only protein in which acetylation via inhibition of HDAC8 was observed. An increase in Hsp20 acetylation correlated with decrease in phosphorylation levels of cofilin after 1h and with significant effects after 5h of treatment (140±45 vs. 36±11, C vs Comp2, p<0.05, Anova, post-hoc Kruskal-Wallis test). This indicates a potential molecular mechanism by which Hsp20 acetylation can affect myometrial activity by liberating cofilin and destabilizing actin filaments (4). Our studies show for the first time that inhibition of the non-nuclear lysine deacetylase HDAC8 (or KDAC8) regulates the contractile capacity of human myometrial smooth
Where applicable, experiments conform with Society ethical requirements