Hydrogen sulfide attenuates epithelial–mesenchymal transition of human alveolar epithelial cells
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).
References (43)
- et al.
The MAM (meprin/A5-protein/PTPmu) domain is a homophilic binding site promoting the lateral dimerization of receptor-like protein-tyrosine phosphatase mu
J Biol Chem
(2004) - et al.
The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide
Biochem Biophys Res Commun
(1997) - et al.
Hydrogen sulfide protects rat lung from ischemia–reperfusion injury
Life Sci
(2008) - et al.
Endogenous hydrogen sulfide in patients with COPD
Chest
(2005) - et al.
Fibrosis of the lung and other tissues: new concepts in pathogenesis and treatment
Clin Immunol
(2001) - et al.
The integrin αvβ6 binds and activates latent TGF-β1: a mechanism for regulating pulmonary inflammation and fibrosis
Cell
(1999) - et al.
Transforming growth factor-β regulates tubular epithelialmyofibroblast transdifferentiation in vitro
Kidney Int
(1999) Hydrogen sulfide-releasing anti-inflammatory drugs
Trends Pharmacol Sci
(2007)- et al.
Hydrogen sulfide suppresses migration, proliferation and myofibroblast transdifferentiation of human lung fibroblasts
Pulm Pharmacol Ther
(2009) - et al.
Putative biological roles of hydrogen sulfide in health and disease: a breath of not so fresh air?
Trends Pharmacol Sci
(2008)
The emerging roles of hydrogen sulfide in the gastrointestinal tract and liver
Gastroenterology
Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy
Ann Intern Med
Nitric oxide attenuates epithelial–mesenchymal transition in alveolar epithelial cells
Am J Physiol Lung Cell Mol Physiol
TGF-β-induced EMT: mechanisms and implications for fibrotic lung disease
Am J Physiol Lung Cell Mol Physiol
Transtorming growth factor 1 induces epithelial-to-mesenchymal transition of A549 cells
J Korean Med Sci
Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter?
FASEB J
Effects of hydrogen sulfide on hypoxic pulmonary vascular structural remodeling
Life Sci
Impact of l-arginine on hydrogen sulfide/cystathionine-γ-lyase pathway in rats with high blood flow-induced pulmonary hypertension
Biochem Biophys Res Commun
Hydrogen sulfide attenuates the pathogenesis of pulmonary fibrosis induced by bleomycin in rats
Can J Physiol Pharmacol
Dysfunction of myocardial sarcoplasmic reticulum in rats with myocardial calcification
Life Sci
Idiopathic pulmonary fibrosis: relationship between histopathologic features and mortality
Am J Respir Crit Care Med
Cited by (43)
The Triple Crown: NO, CO, and H<inf>2</inf>S in cancer cell biology
2023, Pharmacology and TherapeuticsEmerging roles of cystathionine β-synthase in various forms of cancer
2022, Redox BiologyCitation Excerpt :β-catenin is constitutively bound to a multiprotein “destruction complex”, whose fate is to be degraded by the proteasome. In the presence of Wnt ligands such as Wnt3, the formation of the destruction complex is prevented, followed by the β-catenin translocation in the nucleus, where it stimulates the expression of genes, such as Snail1 and Twist1, involved in cell proliferation, migration and invasion [346–359]. The role of H2S in the regulation of EMT has been investigated in several models, including lung fibrosis (another disease condition where EMT/MET transitions are pathophysiologically relevant) and in various forms of cancer.
Epithelial-mesenchymal transition: Insights into nickel-induced lung diseases
2021, Seminars in Cancer BiologyCitation Excerpt :One of the endogenous negative regulators of the EMT process is hydrogen sulfide (H2S) [80]. Endogenous H2S plays a number of physiological roles and has been recently identified as a gasotransmitter that is at least as important as the other well-studied gasotransmitters, nitric oxide (NO) and carbon monoxide (CO) [80–82]. In mammalian cells, endogenous H2S is produced through the metabolism of cysteine.