Arginase-2 is cooperatively up-regulated by nitric oxide and histone deacetylase inhibition in human umbilical artery endothelial cells

Abstract

Arginase-2 counteracts endothelial nitric oxide synthase (eNOS) activity in human endothelium, and its expression is negatively controlled by histone deacetylase (HDAC2). Conversely NO inhibits HDAC and previous studies suggest that arginase-2 is up-regulated by NO. We studied whether NO regulates arginase-2 expression in umbilical artery endothelial cells (HUAEC) increasing ARG2 promoter accessibility. HUAEC exposed to NOC-18 (NO donor, 1⿿100μM, 0⿿24h) showed an increase in arginase-2 but a decrease in eNOS mRNA levels in a time-dependent manner, with a maximal effect at 100μM (24h). Conversely NOS inhibition with L-NAME (100μM) reduced arginase-2 mRNA and protein levels, an effect reverted by co-incubation with NOC-18. Treatment with TSA paralleled the effects of NO on arginase-2 and eNOS at mRNA and protein levels, with maximal effect at 10μM. Co-incubation of NOC-18 (100μM) with a sub-maximal concentration of TSA (1μM) potentiated the increase in arginase-2 mRNA levels, whilst L-NAME prevented TSA-dependent arginase-2 induction. The effects on arginase-2 mRNA were paralleled by changes in chromatin accessibility, as well as increased levels of H3K9 and H4K12 acetylation, at ARG2 proximal (⿿579 to ⿿367 and ⿿280 to ⿿73bp from TSS) and core (⿿121 to +126bp from TSS) promoter. Finally NO-dependent arginase-2 induction was prevented by pre-incubation for 10min with the cysteine blocker MMTS (10mM). These data showed for the first time that NO up-regulates arginase-2 expression in primary cultured human endothelial cells by an epigenetic-mediated mechanism increasing ARG2 promoter accessibility suggesting a negative regulatory loop for eNOS activity.