EGFR-activated signaling and actin remodeling regulate cyclic stretch-induced NRF2-ARE activation

Abstract

Cyclic stretch (CS) associated with mechanical ventilation (MV) can cause excessive alveolar and endothelial distention, resulting in lung injury and inflammation. Antioxidant enzymes (AOEs) play a major role in suppressing these effects. The transcription factor Nrf2, via the antioxidant response element (ARE), alleviates pulmonary toxicant- and oxidant-induced oxidative stress by up-regulating the expression of several AOEs. Although gene expression profiling has revealed the induction of AOEs in the lungs of rodents exposed to MV, the mechanisms by which mechanical forces, such as CS, regulate the activation of Nrf2-dependent ARE-transcriptional responses are poorly understood. To mimic mechanical stress associated with MV, we have cultured pulmonary alveolar epithelial and endothelial cells on collagen I-coated BioFlex plates and subjected them to CS. CS exposure stimulated ARE-driven transcriptional responses and subsequent AOE expression. Ectopic expression of a dominant-negative Nrf2 suppressed the CS-stimulated ARE-driven responses. Our findings suggest that actin remodeling is necessary but not sufficient for high-level CS-induced ARE activation in both epithelial and endothelial cells. We also found that inhibition of EGFR activity by a pharmacologic agent ablated the CS-induced ARE transcriptional response in both cell types. Additional studies revealed that amphiregulin, an EGFR ligand, regulates this process. We further demonstrated that the PI3K-Akt pathway acts as the downstream effector of EGFR and regulates CS-induced ARE-activation in an oxidative stress-dependent manner. Collectively, these novel findings suggest that EGFR-activated signaling and actin remodeling act in concert to regulate the CS-induced Nrf2-ARE transcriptional response and subsequent AOE expression.

Figure 1.

CS stimulates an Nrf2-dependent ARE-mediated transcriptional response in pulmonary epithelial cells. (A) C10 cells were subjected to static conditions (St) or CS for 90 min. Total RNA was isolated, and the expression of the Gclc and Gpx2 genes, the prototypical targets of Nrf2, was analyzed by real-time PCR. The values obtained for the static control group were normalized to one unit. Results are given as means ± SD (n = 3). *P < 0.005. (B) C10 cells were transfected with 400 ng of the ARE-Luc reporter construct, along with 5 ng of the pRL-TK reference plasmid. After 24 h, cell plates were exposed to CS for 6 or 16 h at 18% elongation, and reporter activity was analyzed as described in Materials and Methods. The luciferase activity of samples exposed to static conditions (open bars) was normalized to one unit. Data shown are the means ± SD (n = 3) for cells from a representative experiment (*P < 0.001, ^#P < 0.0005). A similar qualitative result was obtained in two other independent experiments. (C) Cells were co-transfected with the ARE-Luc and pRL-TK plasmids in the presence of a dominant-negative Nrf2 (dn-Nrf2) expression vector or a corresponding empty vector. Values are means ± SD (n = 6). Open and solid bars represent static and CS-stimulated cells, respectively (*P < 0.001). (D) Nuclear extracts (2.5 μg) isolated from C10 cells exposed to static conditions (lane 2) or CS (lane 3) for 60 min were used in EMSAs with the ³²P-labeled consensus Nrf2 binding sequence, ARE, as a probe. In lane 1, the probe incubated without nuclear extract. To examine Nrf2 binding to the ARE, nuclear extracts were incubated with 2 μg of IgG (lanes 4 and 5) or anti-Nrf2 antibody (α-Nrf2, lanes 6 and 7) for 2 h before the addition of the probe (right panel). The open arrow shows the position of the unbound free probe (FP). A representative autoradiogram from two independent experiments is shown.

Figure 2.

Effect of actin-polymerizing agents on CS-stimulated ARE reporter activity in alveolar and endothelial cells. (A) C10 cells were incubated without (−) or with (+) jasplakinolide (Jasp, 2 μM) for 1 h before CS stimulation for 6 h (*P < 0.001). Open and solid bars represent static and CS-stimulated cells, respectively. (B) C10 cells were stimulated without (−) or with (+) latrunculin (LA, 1 μM), and the luciferase activity was analyzed. (C and D) RPMECs were transfected with ARE-Luc along with a reference plasmid and subjected to 18% elongation for 6 h, and luciferase activity was analyzed. (C) RPMECs were incubated without (−) or with (+) Jasplakinolide (Jasp, 2 μM) before 18% CS. *P < 0.001 (D) The effect of latrunculin (LA, 1 μM) on luciferase activity in RPMECs cultured under static conditions. Data shown are means ± SD (n = 6). (E) The effect of latrunculin (LA) on CS-induced luciferase activity in C10 cells cultured under static conditions. The % increase in ARE-Luc activity produced by CS over vehicle-treated respective static control was considered as 100%. Data shown are means ± SD (n = 4). (F) C10 cells were treated with LA for 30 min and stained for F-actin using Texas Red X–phalloidin as detailed in Materials and Methods. The vehicle DMSO was used as a control. Right panels, the nuclei were stained with DAPI.

Figure 3.

Effect of EGFR inhibition and anti-AREG on CS-stimulated ARE activation. (A) Cells transfected with ARE-Luc were treated with DMSO or AG1478 (10 μM) for 40 min before CS stimulation (*P < 0.001, **P < 0.05). (B) Cells transfected with ARE-Luc were incubated with IgG or anti-AREG antibody (2 μg/ml) for 40 min before CS exposure. Values are means ± SD (n = 4) from a representative experiment of three independent experiments (*P < 0.001). Open and closed bars represent static and CS-stimulated cells, respectively. In C and D, experiments similar to above were performed in RPMECs. Results are from a representative experiment, which was repeated twice (n = 3). *P < 0.0002 and **P < 0.004. Open and solid bars represent static conditions and CS-stimulated cells, respectively.

Figure 4.

PI3K-dependent signaling mediates CS-induced Nrf2-ARE-mediated gene transcription. (A) C10 cells transfected with ARE-Luc were treated either with DMSO or LY294002 (10 μM) and then exposed to static conditions or CS for 6 h (*P < 0.001, ^#P < 0.05). (B) C10 cells were treated either with DMSO or PD98089 (20 μM) for 40 min and then exposed to CS or static conditions (*P < 0.001). (C) C10 cells were transfected with ARE-Luc and pRL-TK along with an empty vector or dn-Akt mutant expression vector. Cells were exposed to CS, and the luciferase activity was analyzed (*P < 0.001, **P < 0.05). Open and solid bars represent static and CS-stimulated cells, respectively. Values are means ± SD (n = 4).

Figure 5.

Effect of AG1478 and LY294002 on CS-stimulated expression of kinases and ARE activation in alveolar epithelial cells. (A) C10 cells were pretreated with DMSO or AG1478 (5 μM) for 40 min and subsequently exposed to static conditions (−) or CS at 18% elongation (+) for 15 min. The activation of Akt and ERK signaling was detected by immunoblotting using native and phospho-specific antibodies as indicated. Results are representative of three independent experiments. (B) The effect of LY294002 (5 μM) on Akt and ERK phosphorylation in static (−) or CS (+)-exposed cells. (C) Cells were pretreated with DMSO (lanes 3 and 4), AG1478 (AG, lanes 5 and 6), or LY294002 (LY, lanes 7 and 8) for 40 min. Nuclear extracts were isolated from cells exposed to static conditions (−) or CS (+) for 60 min, and EMSA was performed using the ARE probe. Extracts were also incubated with a 50-fold excess of unlabeled ARE oligo (CC) before the addition of the labeled probe. Solid and open arrows represent the position of Nrf2 and free probe (FP), respectively. A representative autoradiogram from two independent experiments is shown. (D) RNA was isolated from cells that had been treated with either DMSO or LY294002 (LY) for 40 min before stimulation with CS for 90 min. Cells cultured under static conditions and treated with DMSO or LY were used as a control. Gclc mRNA expression was analyzed by real-time PCR. The percent increase in Gclc expression produced by CS over vehicle-treated static control was considered as 100%.

Figure 6.

Effect of oxidative stress on CS-induced ARE-dependent transcription. (A) C10 cells transfected with ARE-Luc were treated with DMSO or NAC (10 mM) before CS exposure, and luciferase activity was analyzed. *P < 0.001. Open and solid bars represent static and CS-stimulated cells, respectively. (B) C10 cells were treated without or with NAC for 40 min and then subjected to CS exposure (+) for 15 min and immunoblotted with native and phosphospecific Akt and ERK1/2 antibodies. Cells cultured under static conditions (−) were used as a control. Results shown are from a representative experiment, which was repeated twice. (C) Cell extracts (as in B) were blotted and probed with phosphospecific EGFR (Tyr-1068) antibodies. To demonstrate equal protein loading, the membrane was probed with actin antibodies. The results shown are from a representative experiment.

Figure 7.

CS-induced oxidative stress and antioxidant gene expression in lung epithelial cells. (A) C10 cells were cultured to confluence and subjected to different levels of cyclic strain (CS), either 5% or 18% elongation for 15 or 360 min. To detect ROS, cells were stained with DCFH-DA, washed, and photographed (see Materials and Methods for details). Representative figures of DCF stained samples are shown. The static control group showed a very little DCF staining (pictures not shown). (B) The total number of DCF-stained cells from five images per sample (n = 2) observed under green fluorescence were compared with the number of total cells observed under phase contrast from the respective slide. (C) RNA was isolated from cells that had been subjected to static conditions or exposed to 5% or 18% CS for 6 h. Gene expression was analyzed by real-time PCR using gene-specific assays. The expression levels of two house keeping genes, actin and Gapdh, were used to normalize candidate gene expression. The values obtained for the static control group were normalized to one unit. Results are given as means ± SD (n = 3). *P < 0.05. (D) GSSG/GSH ratio of static, 5% and 18% CS exposure samples was determined as detailed in Materials and Methods. The values obtained for the static control group were normalized to 100%. *P < 0.05.