Novel Roles of Aryl-Hydrocarbon Receptor ((AhR) on Epigenetic and Post-Translational Modification of Cell-Cycle Regulatory Genes and Enos in Vascular Endothelial Cells---The Therapeutic Potential of Resveratrol/Statins

Our previous several publications have shown that the agonist of aryl-hydrocarbone receptor (AhR), 3MC, caused anti-proliferation and anti-migration through an AhR/FAK/RhoA/PTEN-dependent pathway. Herein, we extent our previous findings to examine novel roles of AhR/RhoA involved in epigenetic modification of chromatin, and posttranslational modification of p21, p27 and eNOS in altering the properties of human umbilical cord vascular endothelial cells (HUVECs). Among them, inactivation of PI3K/Akt by 3MC via RhoA/PTEN activation plays an essential role in the proposed interwoven signaling pathways. We aim to elucidate novel roles of AhR in three aspects; 1) genomic regulation of Rb2/HDAC1 and epigenetic modification of chromatin in the E2F1-driven cell-cycle regulatory genes, 2) post-translational modification of p21 and 27 by phosphorylation via PI3K/Akt in their subcellular localization and thereafter the anti-migratory effect, and 3) eNOS inactivation by 3MC involved in a complex regulation of transcription, post-translation and the interaction with its biochemical partners by diverse signal transduction pathways in HUVECs. Our pilot study showed that 3MC increased retinoblastoma protein2 (Rb2) and HDAC1 expression/nuclear translocation, which occurred in parallel with observed histone H3/H4 deacetylation. Inactivation of HDAC1 by siHDAC1 and apicidin (an HDAC inhibitor) reversed the downregulation of cell cycle regulatory proteins by 3MC. Additionally, Rb2 hypo-phosphorylaion is dependent on 3MC-mediated RhoA/AhR activation through a Ras-dependent pathway. Statins are able to reverse the 3MC effects by blocking nuclear translocation of AhR, HDAC1 and Rb2 (Aim 1). Our preliminary results also showed that 3MC, on one hand, reduced phosphorylation of p21/p27 by RhoA/PTEN-mediated Akt1 inactivation. On the other hand, decreasing cytosolic levels of p21/p27 by 3MC sustained RhoA activation by reducing RhoA inactivation (a positive feedback loop). Increasing RhoA activation is ascribable for the anti-migratory effect of 3MC in HUVECs, which was reversed by Akt1 overexpression and statin treatment (Aim2). Moreover, Akt inactivation is also linked to the observed eNOS dephosphorylation (Aim 3). Effects and the underlying mechanisms of reducing levels of eNOS and HSP90, and the ratio of eNOS dimer/monomer, but augmenting caveolin-1 level, as shown in the preliminary data, will be further unfolded. Interestingly, protein interactions among eNOS interacting proteins (i.e., Akt1, HSP90, and caveolin-1) are essential in the complex regulation of eNOS inactivation by 3MC. According, in Year 1, we intend to elucidate the underlying mechanisms of 3MC-mediated Rb2 hypo-phosphorylation and increased Rb2/HDAC1 recruitment to E2F1 complex in epigenetic modifications of histones, as a result of reducing levels of cell-cycle regulatory genes (i.e., Cdk2/4 and Cyclin D3/E). In Year 2, we intend to examine the molecular mechanisms of PI3K/Akt inactivation by 3MC in altering phosphorylation and subcellular localization of p21/p27. Furthermore, the in vitro effect in sustaining RhoA activation and the anti-migration by 3MC will be substantiated by in vivo angiogenesis. In Year 3, the complex regulation of eNOS inactivation by 3MC including transcription, post-translation and involvement of eNOS’s biochemical partners by diverse signal transduction pathways and its impact in vascular tone will be intensively clarified. Thus, the gain- and loss-of-function of AhR/RhoA/Akt activity will be performed to verify their important roles in these alterations by 3MC. Moreover, functional assays for its reduced NO production and altered vascular tone will be performed both in vitro and in vivo. Based on our pilot study, these above-mentioned phenomena are mediated by AhR/RhoA activation followed by PI3K/Akt inactivation. Therefore, therapeutic approaches using an AhR antagonist, resveratrol, and RhoA inactivators, simvastatin and pravastatin, will be evaluated in 3MC-mediated endothelial dysfunction. Taken together, the underlying molecular actions of AhR agonist will not only shed light on understanding the physiological role of AhR, but also provide a novel molecular basis to develop therapeutic targets in eliminating AhR-mediated injury by xenobiotics in cardiovascular diseases associated with endothelial dysfunction.