Endothelin-1 induces alveolar epithelial-mesenchymal transition through endothelin type A receptor-mediated production of TGF-beta1

Abstract

Endothelin-1 (ET-1) is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), but the cellular mechanisms underlying the role it plays in this disease are not well characterized. Epithelial-mesenchymal transition (EMT), which was recently demonstrated in alveolar epithelial cells (AEC), may play an important role in the pathogenesis of IPF and other forms of pulmonary fibrosis. Whether ET-1 contributes to the induction of EMT in AEC is unknown. The aims of this study were to evaluate AEC production of ET-1 and to determine if ET-1 induces EMT in AEC. We demonstrate that ET-1 is produced at physiologically relevant levels by primary AEC and is secreted preferentially toward the basolateral surface. We also demonstrate that AEC express high levels of endothelin type A receptors (ET-A) and, to a lesser extent, type B receptors (ET-B), suggesting autocrine or paracrine function for alveolar ET-1. In addition, ET-1 induces EMT through ET-A activation. Furthermore, TGF-beta1 synthesis is increased by ET-1, ET-1 induces Smad3 phosphorylation, and ET-1-induced EMT is attenuated by a TGF-beta1-neutralizing antibody. Thus, ET-1 is an important mediator of EMT in AEC, acting through ET-A-mediated TGF-beta1 production. These findings increase our basic understanding of the role of ET-1 in pulmonary fibrosis and suggest potential roles for AEC-derived ET-1 in the pathogenesis of other alveolar epithelial-mediated lung diseases.

Figure 1.

AEC produce ET-1 and express ET-1 receptors. (A) ELISA analysis of supernatant from primary rat AEC monolayers demonstrates ET-1 production per 24 h at Days 2, 4, and 6 of culture. ET-1 production increased over time and was directed preferentially to the basolateral surface. Values are mean ± SEM (n = 3–5; *P < 0.05 versus apical). (B) Primary AEC stained for lamellar protein (p180, green, Alexa 488) and ET-1 (red, Alexa 594) show ET-1 in the cytoplasm of AEC but in a distinct location from p180. Blue staining represents DAPI-stained nuclei. Deconvoluted high-power images were derived from stacks of images taken at 0.1-μm intervals over a total of 2–3 μm thickness of single cells. The Pearson's coefficient of correlation between p180 and ET-1 was 0.228. (C) Immunoblots for ET-A demonstrated high levels of ET-A in AEC relative to RLF. Immunoblots for ET-B revealed much greater expression of ET-B in endothelial cells (EC) than AEC. Findings by immunoblot analysis were confirmed by three to five independent experiments.

Figure 2.

TGF-β1 increases ET-1 production from AEC and attenuates its polar basolateral secretion. (A) Supernatant from AEC treated with and without TGF-β1 showed increased total ET-1 production by ELISA at Days 4 and 6. (B) TGF-β1 abrogated the basolateral directionality of the secretion of ET-1 by AEC. On Days 4 and 6 the apical: basolateral ratio of secretion increased from < 0.25 in control AEC to > 0.5 in TGF-β1–treated AEC. (C) ET-1 mRNA abundance evaluated by RT-PCR increased at Day 2 in the presence of TGF-β1 but was not elevated on Day 6. (D) Immunoblot evaluating ET-A receptor expression showed no difference between control versus TGF-β1–treated AEC. Immunoblot evaluating ET-B receptor expression showed an increase in AEC ET-B with TGF-β1. GAPDH was used as a loading control. Immunoblots shown are representative of four to five independent studies. Values are mean ± SEM. *P < 0.05 versus control. Lightly shaded bars, control; darkly shaded bars, TGF-β1.

Figure 3.

Figure 3. ET-1 induces EMT in AEC via ET-A. (A) Immunofluorescence analysis demonstrated that ET-1 increases α-SMA expression, and that the increase is inhibited by BQ-123, but not BQ-788. AEC treated with ET-1 also changed morphology from a typical rounded shape to an elongated fibroblast-like morphology (phase images, right panels) with prominent intracellular stress fibers (fluorescent images, middle panels). BQ-123 prevented this morphologic change, while BQ-788 did not. For all immunofluorescence images, green staining represents α-SMA and blue staining represents nuclei stained with DAPI. (B) Immunoblot analysis similarly revealed increased α-SMA expression with ET-1 (150 nM) treatment relative to control. BQ-123, an ET-A antagonist, completely abrogated this effect. BQ-788, an ET-B antagonist, had no effect. For immunoblot analysis, one representative blot is shown. Densitometric analysis (right panel) summarizes four separate experiments. *P < 0.05 compared with control and ET-1 + BQ-123. (C) Immunoblot analysis of the epithelial marker pro–SP-B revealed a dramatic decrease in expression with ET-1 treatment relative to control. BQ-123 prevented this decrease, while BQ-788 did not. Densitometric analysis (right panel) represents three separate experiments. *P < 0.05 compared with control and ET-1 + BQ-123.

Figure 3.

Figure 3. ET-1 induces EMT in AEC via ET-A. (A) Immunofluorescence analysis demonstrated that ET-1 increases α-SMA expression, and that the increase is inhibited by BQ-123, but not BQ-788. AEC treated with ET-1 also changed morphology from a typical rounded shape to an elongated fibroblast-like morphology (phase images, right panels) with prominent intracellular stress fibers (fluorescent images, middle panels). BQ-123 prevented this morphologic change, while BQ-788 did not. For all immunofluorescence images, green staining represents α-SMA and blue staining represents nuclei stained with DAPI. (B) Immunoblot analysis similarly revealed increased α-SMA expression with ET-1 (150 nM) treatment relative to control. BQ-123, an ET-A antagonist, completely abrogated this effect. BQ-788, an ET-B antagonist, had no effect. For immunoblot analysis, one representative blot is shown. Densitometric analysis (right panel) summarizes four separate experiments. *P < 0.05 compared with control and ET-1 + BQ-123. (C) Immunoblot analysis of the epithelial marker pro–SP-B revealed a dramatic decrease in expression with ET-1 treatment relative to control. BQ-123 prevented this decrease, while BQ-788 did not. Densitometric analysis (right panel) represents three separate experiments. *P < 0.05 compared with control and ET-1 + BQ-123.

Figure 3.

Figure 3. ET-1 induces EMT in AEC via ET-A. (A) Immunofluorescence analysis demonstrated that ET-1 increases α-SMA expression, and that the increase is inhibited by BQ-123, but not BQ-788. AEC treated with ET-1 also changed morphology from a typical rounded shape to an elongated fibroblast-like morphology (phase images, right panels) with prominent intracellular stress fibers (fluorescent images, middle panels). BQ-123 prevented this morphologic change, while BQ-788 did not. For all immunofluorescence images, green staining represents α-SMA and blue staining represents nuclei stained with DAPI. (B) Immunoblot analysis similarly revealed increased α-SMA expression with ET-1 (150 nM) treatment relative to control. BQ-123, an ET-A antagonist, completely abrogated this effect. BQ-788, an ET-B antagonist, had no effect. For immunoblot analysis, one representative blot is shown. Densitometric analysis (right panel) summarizes four separate experiments. *P < 0.05 compared with control and ET-1 + BQ-123. (C) Immunoblot analysis of the epithelial marker pro–SP-B revealed a dramatic decrease in expression with ET-1 treatment relative to control. BQ-123 prevented this decrease, while BQ-788 did not. Densitometric analysis (right panel) represents three separate experiments. *P < 0.05 compared with control and ET-1 + BQ-123.

Figure 4.

ET-1 increases total TGF-β1 production from primary AEC and induces Smad3 phosphorylation through ET-A. (A) ELISA was used to quantify total TGF-β1 in the supernatant of AEC treated with and without ET-1. ET-1 increased TGF-β1 production by AEC. BQ-123, an ET-A receptor antagonist, prevented the ET-1–induced increase in TGF-β1 production. BQ-788, an ET-B receptor antagonist, had no effect on TGF-β1 secretion. (B) Immunoblot analysis demonstrated an increase in phosphorylated Smad3 (p-Smad3) by ET-1 after 24 h of treatment. BQ-123 abrogated this effect, and BQ-788 had no effect. Bar graphs represent mean ± SEM (n = 4). *P < 0.05 versus control (no treatment) and ET-1 + BQ-123.

Figure 4.

ET-1 increases total TGF-β1 production from primary AEC and induces Smad3 phosphorylation through ET-A. (A) ELISA was used to quantify total TGF-β1 in the supernatant of AEC treated with and without ET-1. ET-1 increased TGF-β1 production by AEC. BQ-123, an ET-A receptor antagonist, prevented the ET-1–induced increase in TGF-β1 production. BQ-788, an ET-B receptor antagonist, had no effect on TGF-β1 secretion. (B) Immunoblot analysis demonstrated an increase in phosphorylated Smad3 (p-Smad3) by ET-1 after 24 h of treatment. BQ-123 abrogated this effect, and BQ-788 had no effect. Bar graphs represent mean ± SEM (n = 4). *P < 0.05 versus control (no treatment) and ET-1 + BQ-123.

Figure 5.

The induction of EMT by ET-1 is mediated by TGF-β1. (A) Immunoblot analysis of AEC for α-SMA expression revealed ET-1 increased α-SMA expression relative to control. However, the addition of an anti–TGF-β1 monoclonal antibody (anti–TGF-β1 mAb) dramatically attenuated the effect of ET-1. GAPDH was used as a loading control. Immunoblots shown are representative of three independent studies. Densitometric values are mean ± SEM. *P < 0.05 compared with control and ET-1 + Anti–TGF-β1 mAb. (B) Immunofluorescence analysis similarly demonstrated that ET-1 increases α-SMA expression, and that the increase is inhibited by anti–TGF-β1 mAb. Green staining represents α-SMA, while blue staining represents nuclei stained with DAPI.

Figure 6.

ET-1 receptors do not mediate alveolar EMT induced by exogenous TGF-β1. (A) Immunoblot analysis of AEC for α-SMA expression revealed that TGF-β1 (1 ng/ml) increased α-SMA expression dramatically relative to control. The addition of an ET-A (BQ-123) or ET-B (BQ-788) receptor antagonist had no effect on the increase in α-SMA expression. GAPDH was used as a loading control. Immunoblots shown are representative of five independent studies. Densitometric values are mean ± SEM. *P < 0.05 compared with control. (B) Immunofluorescence analysis similarly demonstrated that ET-1 receptor antagonism did not affect the TGF-β1–induced increase in α-SMA expression, or the change in cell morphology to a fibroblast-like shape. Green staining represents α-SMA, while blue staining represents nuclei stained with DAPI.