Abstract 91: Activation of The Antioxidant Response Element (ARE) in Human Renal Glomerular Endothelial Cells (HRGECs) Reduces Asymmetric Dimethylarginine (ADMA) And Increases Nitric Oxide by Transcriptional Activation of Dimethylarginine Dimethylaminohydrolase (DDAH) and Endothelial Nitric Oxide Synthase (eNOS)
The E2-related factor 2 (Nrf2) binds to AREs and transcriptionally activates several genes that protect against oxidative stress. DDAH phosphorylates and activates eNOS and degrades ADMA which is an endogenous inhibitor of eNOS. We detected 2-3 putative AREs in the promoter regions of the human genes for DDAH-1 and -2, peroxisome proliferator-activated receptor-γ (PPAR-γ) and eNOS. PPAR-γ can increase eNOS expression and eNOS phosphorylation. Therefore, we hypothesized that AREs regulate the DDAH/eNOS/PPAR-γ/ADMA/ NO pathway. Tert-butylhydroquinone (tBHQ) activates Nrf2 by translocation to the nucleus. Incubation of HRGECs for 24 hours with tBHQ enhanced cell proliferation dose-dependently (5-20μM). tBHQ (20μM; n=4) increased medium nitrite by 69 ± 3.7% (P<0.01), intracellular NO (from DAF-FM fluorescence ) by 62 ± 17.1% (P<0.05), and NOS and DDAH activities( from conversion of [3H]-arginine or [14C]-ADMA to [3H]- or [14C]-citrulline) by 58 ± 3.6% and 47 ± 8.5% (P<0.05) and decreased ADMA (by capillary zone electrophoresis) from 1.2±0.03 vs. 2.3±0.16μM/L, (P<0.001, n=6). tBHQ increased the mRNAs and proteins for eNOS by 79 ± 9% and 43 ± 17%, for DDAH-1 by 129 ± 10% and 62 ± 21%, and for DDAH-2 by 119 ± 25% and 48 ± 18% (all P<0.05) and increased the protein expression of PPAR-γ and eNOS phosphorylation and increased tBHQ nuclear translocation markedly. Knockdown of the cell Nrf2 by transfection with a specific siRNA abolished all these effects and attenuated cell proliferation. Chromatin immunoprecipitation (CHIP)-based PCR assays demonstrated that tBHQ enhanced the binding of Nrf2 to one ARE region in the promoters for DDAH-1 and -2 but there was no binding to the eNOS promoter. In conclusion, activation of a specific ARE in the promoter region of the DDAH-1 and -2 genes causes their transcriptional activation. The ensuing enhancement of DDAH activity enhances ADMA metabolism. Nrf2 also increases eNOS expression, NOS activity and NO generation without binding to the ARE for eNOS, likely by upregulation of PPAR-γ. Thus, Nrf2 improves endothelial cell function by coordinating a diverse set of pathways that enhance NO generation.
- © 2012 by American Heart Association, Inc.