Abstract 354: Nitric Oxide Regulates Chromatin Folding in Human Endothelial Cells via PP2A activation and HDAC-4 Nuclear Shuttling.
Introduction and Background: Laminar shear stress (SS) induces histone H3 and H4 modifications in human umbilical vein endothelial cells (HUVEC). This effect occurs within two hours of SS treatment and decreases between 4 to 8 hours coincidently with a significant increase in intracellular histone de-acetylatase (HDAC) activity that parallels the SS-dependent activation of nitric oxide (NO) production. Since SS-dependent histone modifications and changes in eNOS activity follow a similar time course, it was examined the possibility that NO may modulate chromatin remodeling.
Methods and Results: Immunofluorescence and western blot analyses revealed that, in presence of SS or NO donors, the class II histone deacetylase HDAC-4 localized to the nucleus and this process was abrogated by NO inhibitors. In fact, cells treated with the NO inhibitor S-methyl-thiosourea (SMT) showed a rapid and sustained increase in H3 acetylation on lysine 14 even in absence of SS which was paralleled by a decrease in total nuclear HDAC activity. Consistently, nitroglycerin (NTG) abrogated serum-induced histone H3 acetylation and c-fos expression, confirming that NO may have a role in the regulation of HDAC activity and gene expression in endothelial cells. When phosphorylated, HDAC-4 is predominantly cytosolic and bound to 14 −3−3 chaperonins, becoming nuclear in its unphos-phorylated state. Attempting to dissect the molecular mechanism underlying the effect of NO on HDAC-4 localization, HUVEC were stably engineered with the polyoma virus small T antigen (smT-HUVEC) which functionally inactivates PP2A phosphatases. In this condition, NTG failed to stimulate HDAC-4 nuclear localization compared to mock-transfected cells or cells transfected with a Small-T antigen mutant unable to bind PP2A, while increased the overall phosphatase activity in wild type cells.
Conclusions: SS and/or NO activate a PP2A-related activity important for the regulation of HDAC-4 cellular localization and function which has an impact on the global chromatin landscape of endothelial cells and gives new insights into the molecular mechanism of NO-dependent regulation of gene expression.