Activating the Nrf2-mediated antioxidant response element restores barrier function in the alveolar epithelium of HIV-1 transgenic rats

Abstract

The master transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates the expression of antioxidant and phase II-metabolizing enzymes by activating the antioxidant response element (ARE) and thereby protects cells and tissues from oxidative stress. Pulmonary complications remain the leading cause of death in human immunodeficiency virus (HIV)-1-infected individuals, who display systemic oxidative stress and glutathione deficiency that can be modeled in transgenic rats where HIV-1-related viral proteins decrease glutathione levels and cause epithelial barrier dysfunction within the alveolar space by as yet unknown mechanisms. We hypothesized that HIV-1-related proteins inhibit Nrf2-mediated antioxidant defenses and thereby disrupt the normally tight alveolar epithelial barrier. Nrf2 RNA silencing dampened Nrf2/ARE activity, decreased the expression of the tight junction proteins zonula occludens-1, occludin, and claudin-18, increased paracellular permeability of alveolar epithelial monolayers derived from wild-type rats, and therefore reproduced the effects of HIV-1 transgene expression on the epithelial barrier that we had previously described. In contrast, upregulating Nrf2 activity, either by plasmid-mediated overexpression or treatment with the Nrf2 activator sulforaphane, increased the expression of ARE-dependent antioxidants, including NAD(P)H dehydrogenase, quinone 1 and glutathione, improved the expression of tight junction proteins, and restored the ability to form tight barriers in alveolar epithelial cells from HIV-1 transgenic rats. Taken together, these new findings argue that HIV-1-related proteins downregulate Nrf2 expression and/or activity within the alveolar epithelium, which in turn impairs antioxidant defenses and barrier function, thereby rendering the lung susceptible to oxidative stress and injury. Furthermore, this study suggests that activating the Nrf2/ARE pathway with the dietary supplement sulforaphane could augment antioxidant defenses and lung health in HIV-1-infected individuals.

The relative balance of GM-CSF and TGF-β1 regulates lung epithelial barrier function

“…This was confirmed by quantitative analysis, where overnight treatment with 5 ng/ml TGF-␤1 resulted in a significant increase in the fraction of cells with spikes containing claudin-18 [16.9 Ϯ 1.5% spike-containing cells (n ϭ 8) vs. untreated controls, 11.7 Ϯ 1.8% spike containing cells (n ϭ 8), P Ͻ 0.05]. A comparable effect on tight junction morphology associated with paracellular leak has previously been observed in AECs isolated from alcohol-fed rats (14) and AEC cells treated with BMS-345541 (55). Note that GM-CSF did not significantly decrease spike formation by AECs either in the absence or presence of TGF-␤1 (10.6 Ϯ 0.9%, n ϭ 8, vs. 14.8 Ϯ 1.3%, n ϭ 8).…”

“…AECs were seeded at 2 ϫ 10 5 cells/well in 12-well 0.4-m Transwell permeable supports (Corning Life Sciences). On day 1, the cells were transfected as described (14) using HiPerFect (Qiagen, Gaithersburg, MD) with 30 nM siRNA duplexes from Sigma-Aldrich that were either specific for rat clau-din-18 (cldn-18 siRNA 1: 5=-GUGAUGAUGUGCAUUGCUU -3=; siRNA 2: 5=-CCAUUUACAGGUUAAGAGA -3=) or control siRNA (Sigma MISSION Universal siRNA negative control; no. SIC001).…”

“…To measure claudin-18 mRNA, total RNA was extracted using the Qiagen RNeasy Mini kit. Reverse transcription and real-time PCR (Bio-Rad, Hercules, CA) were performed as previously described using 5=-TGTGGAGCACTCAA-GACCTG-3= and 5=-AGATGGACACGAGGATACCG-3= as forward and reverse primers, respectively (14).…”

The Blood–Brain Barrier

“…NR2F2 also down-regulates expression of Ang-2, a factor known to decrease junctional protein expression. In a similar way, activation of the nrf-2 pathway by oxidative stress activates antioxidant response elements (ARE), which are known to stabilize ZO-1, occludin, and claudin-5 expression (Fan et al 2013). In addition, nrf-2 protects ECs during injury by supressing the expression of inflammatory genes (Chen et al 2006).…”

Oxidative Stress, Redox Regulation and Elite Controllers of HIV Infection: Towards a Functional Cure

“…The master transcription factor known as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is known to regulate the expression of antioxidants through activation of antioxidant response element (ARE). Fan et al showed that upregulation of Nrf2 increased the ARE-dependent antioxidants and that HIV-1-related proteins downregulated Nrf2 expression in HIV-1 transgenic rats [29]. Increased oxidative stress induces Nrf2 and stimulates key antioxidant defence mechanism [30] in HIV cells.…”