HDAC1, NOS3, and circadian clock gene expression in the endothelium

Histone deacetylases (HDACs) are in a family of enzymes that have important roles in a number of biological processes. Previous work in our lab has shown that upregulation of HDAC1 deacetylates endothelial nitric oxide synthase (NOS3) and results in decreased NO production in bovine aortic endothelial cells. HDACs have also been demonstrated to serve as regulators of circadian clock gene function. Clock, a core component of the molecular circadian clock, is a histone acetyltransferase, thus acetylation pathways are implicated in the circadian control mechanism. More recently, HDAC1 has been implicated in the circadian clock negative feedback mechanism. Circadian clock genes have an important influence in the endothelium as blood vessels from mice with Bmal1 knockout (Bmal1‐KO) exhibit endothelial dysfunction and reduced NO signaling. We hypothesized that HDAC1 expression displays circadian variation similar to NOS3 and that inhibition of HDAC1 would improve circadian clock gene oscillation in primary mouse aortic endothelial cells (MAECs). Further, we also hypothesized that HDAC1 inhibition improves endothelial NOS‐dependent function in aortic rings from Bmal1‐KO mice. To determine whether basal HDAC1 expression displays circadian variation, protein was harvested from confluent MAECs at 4 hr intervals over a 12 hr period between 10 AM and 10 PM. Western blot analysis (n=3 per time point, p=0.0002) revealed significantly increased HDAC1 expression over the course of the day, with 5‐fold greater expression at 10 PM (0.5±0.05, 1.2±0.28, 2.3±0.19, 2.6±0.16 relative densitometric units). Similarly, nighttime NOS3 protein expression was also elevated relative to daytime expression (p=0.0048). siRNA knockdown of HDAC1 resulted in a 50% reduction in HDAC1 protein expression. Protein expression was quantified relative to actin. 24 hrs after transfection with siRNA, cells were serum shocked with 50% horse serum for circadian synchronization and harvested at 6 hr intervals for 30 hrs (n=4 per group at each time point). QPCR analysis revealed more robust oscillation of core clock genes with HDAC1 siRNA relative to the scramble control. The trough in Bmal1 and Clock expression was at ZT20 whereas Per2 expression peaked at ZT20. At ZT14 Bmal1 expression peaked and Per2 expression showed a trough. Additionally, with HDAC1 knockdown NOS3 mRNA expression peaked at ZT8 and troughed at ZT14 in contrast to the scramble control which did not show temporal variation in mRNA expression. Incubation with the HDAC1 inhibitor MS‐275 (8uM for 1 hr) did not improve the drastically impaired endothelium‐dependent relaxation to acetylcholine in aortic rings from Bmal1‐KO mice while endothelium‐independent relaxation was intact and unaffected by MS‐275 in response to the exogenous NO donor sodium nitroprusside. These findings indicate that HDAC1 displays temporal variation and affects circadian clock and NOS3 gene expression in endothelial cells.