HDAC6 Regulates the MRTF-A/SRF Axis and Vascular Smooth Muscle Cell Plasticity

Mengxue Zhang,Go Urabe,Christopher Little,Bowen Wang,Alycia M Kent,Yitao Huang,K Craig Kent,Lian-Wang Guo

doi:10.1016/j.jacbts.2018.08.010

Abstract

Cellular plasticity is fundamental in biology and disease. Vascular smooth muscle cell (SMC) dedifferentiation (loss of contractile proteins) initiates and perpetrates vascular pathologies such as restenosis. Contractile gene expression is governed by the master transcription factor, serum response factor (SRF). Unlike other histone deacetylases, histone deacetylase 6 (HDAC6) primarily resides in the cytosol. Whether HDAC6 regulates SRF nuclear activity was previously unknown in any cell type. This study found that selective inhibition of HDAC6 with tubastatin A preserved the contractile protein (alpha-smooth muscle actin) that was otherwise diminished by platelet-derived growth factor-BB. Tubastatin A also enhanced SRF transcriptional (luciferase) activity, and this effect was confirmed by HDAC6 knockdown. Interestingly, HDAC6 inhibition increased acetylation and total protein of myocardin-related transcription factor A (MRTF-A), a transcription co-activator known to translocate from the cytosol to the nucleus, thereby activating SRF. Consistently, HDAC6 co-immunoprecipitated with MRTF-A. Invivo studies showed that tubastatin A treatment of injured rat carotid arteries mitigated neointimal lesion, which is known to be formed largely by dedifferentiated SMCs. This report is the first to show HDAC6 regulation of the MRTF-A/SRF axis and SMC plasticity, thus opening a new perspective for interventions of vascular pathologies.

Highlights

Another novel finding was that histone deacetylase 6 (HDAC6) inhibition increases acetylation and total protein levels of myocardin-related transcription factor A (MRTF-A), a powerful co-factor that resides in the cytosol but is able to translocate to the nucleus to activate the serum response factor (SRF) transcriptional activity in contractile gene expression
HDAC6 inhibition affords a favorable strategy for effective mitigation of neointima, as our results indicate that the HDAC6 inhibitor abates all the aforementioned pathogenic smooth muscle cell (SMC) phenotypes
Inspired by our initial finding that inactivating HDAC6 maintains SMC marker protein levels under pathogenic stimulation, we have further tracked down a mechanism whereby HDAC6 inhibition enhances MRTF-A protein acetylation and abundance and SRF transcriptional activity downstream of MRTF-A

Summary

Methods

CELL AND ARTERY EXPLANT CULTURES AND PRE-TREATMENT WITH HDAC INHIBITORS. The mouse aortic smooth muscle MOVAS cell line was purchased from ATCC (Manassas, Virginia), and the cell culture was maintained at 37C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, Massachusetts). For pretreatment with HDAC inhibitors, MOVAS cells were starved overnight with 0.5% FBS and added with an HDAC inhibitor 2 h before stimulation with platelet-derived growth factor-BB (PDGF-BB). Rat (see later discussion) aortas deprived of endothelium were cut into strings and cultured in DMEM/F12 containing 0.5% FBS. After a 24-h incubation in the presence of vehicle or 10 mmol/l tubastatin A, the artery explants were pooled and homogenized for immunoprecipitation and Western blotting

Results

Discussion

Conclusion