Hydrogen sulfide attenuates epithelial–mesenchymal transition of human alveolar epithelial cells

https://doi.org/10.1016/j.phrs.2009.10.008Get rights and content

Abstract

We previously reported that the endogenous cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway is implicated in the pathogenesis of bleomycin-induced pulmonary fibrosis in rats, but the exact cellular mechanisms are not well characterized. Epithelial–mesenchymal transition (EMT), induced by transforming growth factor β1 (TGF-β1) in alveolar epithelial cells, plays an important role in the pathogenesis of pulmonary fibrosis. We studied whether H2S could attenuate EMT in cultured alveolar epithelial cells and TGF-β1 treatment suppressed CSE expression in A549 cells. Inhibition of endogenous CSE by dl-propargylglycine led to spontaneous EMT, as manifested by decreased E-cadherin level, increased vimentin expression and fibroblast-like morphologic features. Exogenous H2S applied to TGF-β1-treated A549 cells decreased vimentin expression, increased E-cadherin level and retained epithelial morphologic features. In addition, preincubation with H2S decreased Smad2/3 phosphorylation in A549 cells stimulated by TGF-β1, and H2S-inhibited alveolar EMT was mimicked by treatment with SB505124, a Smad2/3 inhibitor, but not pinacidil, an ATP-sensitive K+ channel (KATP) opener. H2S serves a critical role in preserving an epithelial phenotype and in attenuating EMT in alveolar epithelial cells, mediated, at least in part, by decreased Smad2/3 phosphorylation and not dependent on KATP channel opening.

Introduction

Pulmonary fibrosis is the final common pathway of a diverse group of lung disorders known as interstitial lung diseases and is characterized by fibroblast accumulation, excessive collagen deposition, and matrix remodeling, leading to distorted alveolar architecture, progressive decline in lung function, and, ultimately, death. This recent paradigm suggests that pulmonary fibrosis is a sequence of events that start with alveolar epithelial micro-injuries followed by the formation of fibroblastic foci, and results in an exaggerated deposition of extracellular matrix (ECM), which drives the destruction of the lung parenchyma architecture [1]. Fibroblast and/or myofibroblast activation is a key event playing a critical role in the progression of lung fibrotic disease. Of particular recent interest is the possibility that alveolar epithelial cells contribute directly to fibrosis through epithelial–mesenchymal transition (EMT) to a myofibroblast-like phenotype [2]. Recent studies have demonstrated alveolar EMT both in vitro and in vivo and that most myofibroblast-like cells result from alveolar EMT after injury [3], [4]. Mounting evidence suggests that alveolar EMT is primarily mediated by local production and activation of transforming growth factor β1 (TGF-β1) [3], [4]. However, lung endogenous factors that modify the effects of TGF-β1 and the induction of alveolar EMT have not been identified.

Recently, growing evidence has shown that in addition to nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide (H2S) may be the third gasotransmitter [5], [42]. H2S is endogenously generated by pyridoxal-5′-phosphate-dependent enzymes such as cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), with l-cysteine used as a main substrate [6], [41], [42]. The expression of these enzymes has been detected in various tissues [40]. Both the lung and pulmonary artery are rich in active CSE protein [7], which can endogenously produce and release H2S. The pathophysiological role of H2S in some lung diseases has been explored. The endogenous CSE/H2S pathway participates in the pathophysiological process in lung diseases, such as hypoxia-induced pulmonary hypertension [8], high pulmonary blood flow-induced pulmonary hypertension [9], lung ischemia–reperfusion injury [10], and chronic obstructive pulmonary disease [11].

Our previous studies demonstrated that a deficient endogenous CSE/H2S system is responsible for the development of pulmonary fibrosis induced by bleomycin in rats. Exogenously applied NaHS (the H2S donor) or H2S interfered with lung fibrosis pathogenesis by antagonizing oxidative stress [12] and suppressing migration, proliferation and myofibroblast transdifferentiation in human lung fibroblast cells induced by fetal bovine serum (FBS) and growth factors in vitro [37]. The above findings suggest that H2S is an important regulatory factor in the pathophysiological process of pulmonary fibrosis. However, the potential significance and exact mechanism of H2S in the process is unclear.

We aimed to determine whether CSE is expressed in human lung epithelial A549 cells and investigate the role of H2S in modulation of alveolar EMT induced by TGF-β1 in vitro, to further elucidate the cellular mechanism of H2S-antagonized pulmonary fibrosis.

Section snippets

Materials

H2S-saturated solution (0.09 mol/l at room temperature) was made by bubbling with pure H2S gas (offered by Beijing XianHeYu Co.) and stored at −70 °C. Dimethyl sulfoxide (DMSO), dl-propargylglycine (PPG), pinacidil, glibenclamide and SB505124 were from Sigma (St. Louis, MO, USA). Trizol and TGF-β1 were from Invitrogen (Carlsbad, CA, USA). M-MuLV inverse transcriptase, RNase inhibitor and Taq DNA polymerase were from Promega (Madison, WI, USA). Antibodies against E-cadherin, vimentin, Smad2,

TGF-β1 treatment suppresses CSE expression in A549 cells

Western blot analysis to detect whether A549 cells can generate H2S revealed that A549 cells could express CSE, a 44-kD protein. On incubation with 0.1, 1 and 10 ng/ml TGF-β1, CSE expression was lower, by 15.8%, 19.5% and 23.2%, following 24 h treatment than that in controls (all P < 0.01, Fig. 1).

Inhibition of endogenous H2S production induces A549 cells to undergo EMT

To further evaluate the role of endogenous H2S in the regulation of alveolar EMT, the expression of the epithelial phenotype marker E-cadherin and the mesenchymal phenotype marker vimentin was determined

Discussion

Until recently, H2S was believed to be a toxic environmental pollutant with no physiological significance; however, in the past few years, it has been identified as a physiologically/pathophysiologically relevant endogenous gaseous transmitter, third in line to nitric oxide (NO) and carbon monoxide (CO). It has been reported that inhalation of poisoning H2S causes pulmonary edema and pulmonary interstitial fibrosis [38], [39]. Our previous work showed that the endogenous CSE/H2S pathway

Conclusions

From our overall findings, we postulate that under normal conditions, H2S generated by CSE in the alveolar epithelium functions to maintain alveolar epithelial cell phenotype, thereby contributing to optimal alveolar development. In contrast, with repetitive injury or genetic predispositions that lead to chronic elevations in profibrotic cytokines, including TGF-β1, CSE is downregulated, alveolar epithelial cells transitions to a myofibroblast phenotype, and pulmonary fibrosis ensues. We have

Acknowledgement

This work was supported by the National Natural Sciences Foundation of the People's Republic of China (grant 30572072).

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