Elsevier

Biochemical Pharmacology

Volume 70, Issue 7, 1 October 2005, Pages 1026-1034
Biochemical Pharmacology

N-Acetyl-l-cysteine suppresses TGF-β signaling at distinct molecular steps: The biochemical and biological efficacy of a multifunctional, antifibrotic drug

https://doi.org/10.1016/j.bcp.2005.07.001Get rights and content

Abstract

The interrelated signaling via TGF-β1 and reactive oxygen species has a profound impact on fibrogenesis and is therefore selected as target for antifibrotic therapies. This prompted us to investigate the influence of the antioxidant N-acetyl-l-cysteine on TGF-β signaling in culture-activated hepatic stellate cells, the most relevant pro-fibrogenic cell type in liver. Dissection of the molecular steps involved in TGF-β signaling revealed that N-acetyl-l-cysteine dose-dependently abrogated the induction of the TGF-β1 signaling reporter gene activation, the phosphorylation of Smad2 and Smad3, and the up-regulation of Smad7 mRNA. By means of Western blot analysis and cross-linking experiments, it was demonstrated that these effects are based on disintegration of TGF-β1 and the TGF-β receptor endoglin, as well as a reduced ligand binding capacity of betaglycan. We conclude that N-acetyl-l-cysteine is a specific inhibitor of TGF-β signaling targeting different components of the TGF-β signaling machinery. In conclusion, these findings suggest that this non-toxic aminothiol downregulates TGF-β signal transduction thereby mediating beneficial effects on experimental liver fibrosis characterized by TGF-β hyperactivity.

Introduction

In liver, the activation of hepatic stellate cells (HSC) results in transdifferentiation of this fibrogenic precursor cell type to the extracellular matrix-producing myofibroblastic phenotype (MFB). This process is regarded as a key issue in fibrogenesis and is therefore in the focus of therapeutic strategies [1]. Numerous studies have identified TGF-β as the major profibrogenic master cytokine, which in concert with other growth factors promotes transdifferentiation of HSC into MFB, stimulation of matrix gene expression, downregulation of matrix degradation and induction of hepatocellular apoptosis, thereby promoting the numerical expansion of MFB. Due to the pleiotropic fibrogenic effects of TGF-β, therapeutic strategies were developed to antagonize or block synthesis of the ligand [2], [3], [4], [5], [6], to interfere with the intracellular Smad signaling pathway [7], and to suppress generation and extracellular activation of TGF-β by MFB and other liver cell types [8].

Of particular interest is the interrelation of TGF-β signaling and reactive oxygen species (ROS) formation; in cultured HSC, TGF-β increases the production of H2O2[9], which in turn induces the expression of α1(I) procollagen mRNA [10]. Catalase, an enzymatic scavenger of H2O2, abrogated TGF-β mediated type I collagen gene expression [10], supporting the hypothesis that H2O2 might act as a mediator of TGF-β signaling. Similarly, H2O2 was identified as mediator in acetaldehyde-induced α1(I) collagen gene expression [11]. Direct profibrogenic effects of H2O2 were also observed in co-cultures of HSC with HepG2 cells overexpressing CYP2E1 [12]. Furthermore, the interruption of the TGF-β autocrine loop by expression of soluble transforming growth factor beta type II receptor was recently shown to inhibit oxidative stress in activated HSC [13].

N-Acetyl-l-cysteine (NAC), a non-toxic aminothiol and synthetic precursor of intracellular glutathione (GSH), is clinically used as a harmless, powerful antioxidant capable of increasing the defense mechanisms against ROS [14]. Pharmacological actions include repletion of intracellular GSH stores, scavenging of toxic radicals, suppression of tumour necrosis factor-α (TNF-α) production, and stimulation of cathepsin B-mediated cleavage of platelet-derived growth factor receptor type β[15]. In cultured HSC it was shown that NAC induces cell cycle arrest through its reducing activity and by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor type β[15], [16], [17]. Moreover, in vivo the naturally occurring amino acid l-cysteine and its derivative NAC were recently suggested to diminish dimethylnitrosamine-induced liver fibrosis by inhibiting the activation and proliferation of HSC [18], [19].

So far, the effect of NAC action on TGF-β induced fibrogenic responses in HSC cultures has not been determined at the molecular level. In this study, we addressed experiments to this question. The data point to NAC elicited disaggregation of the TGF-β1 dimer, a reduced TGF-β1 binding activity of the transforming growth factor type III receptor (TβRIII) betaglycan, and a decomposition of a second accessory TβRIII, i.e. endoglin, recently shown to be expressed in HSC [20]. Furthermore, the demonstration that these alterations are not prevented by administration of proteinases and proteasome inhibitors suggest that the disintegration of individual signaling components is not proteolytically induced. Therefore, the presented data indicate for the first time, that (i) the protective role of the antioxidant agent NAC against the development of hepatic fibrosis is based on a direct blockade of TGF-β1 function and signaling, and (ii) that these effects are direct contributable to the chemical characteristics of NAC.

Section snippets

Isolation and culture of hepatic stellate cells

HSC were isolated from male Sprague–Dawley rats and cultured as previously described [21].

Adenoviral infection of HSC and reporter assays

Adenoviral stocks were prepared following standard protocols outlined in detail elsewhere [6]. In brief, adenoviruses were purified by a two-step-procedure. Firstly, the viral particles were concentrated by CsCl density gradient centrifugation. Secondly, the viruses were further purified through the BD Adeno-X™ Purification Filter system (BD Biosciences, Clontech). The infections were performed at an MOI

NAC inhibits TGF-β-dependent reporter gene expression

HSC were adenovirally infected with a highly sensitive TGF-β reporter construct, i.e. Ad(CAGA)9-MLP-Luc, comprising artificial Smad3/Smad4 binding sites (CAGA-box) derived from the plasminogen activator inhibitor-1 (PAI-1) promoter and part of the major late promoter [24]. Infected HSC were treated with 0.05–1 ng/ml TGF-β1 or left untreated in the absence or presence of 0.5 or 5 mM NAC as indicated (Fig. 1a). The luciferase activity was determined 3 h thereafter showing a dose-dependent

Discussion

NAC is available as an over the counter supplement in health stores and in oral solutions that can be ingested or aerosolized and inhaled. As a non-toxic aminothiol it is suitable as a mucolytic agent [29], in the treatment of acetaminophen (paracetamol) poisoning [30], for reducing the incidence of contrast-induced nephropathy [31], to diminish the symptoms and duration of the flu and the common cold, and in the course of immunodeficiency virus (HIV) infection [32]. Furthermore, it is taken by

Acknowledgements

We thank A. Moustakas (Ludwig Institute for Cancer Research, Uppsala, Sweden) for kindly providing Ad-CA-ALK-5 and P. ten Dijke (The Netherlands Cancer Institute, Amsterdam, The Netherlands) for sending antibodies directed against p-Smads, and C. Stoll (Department of Oral and Maxillofacial Surgery, RWTH University Hospital Aachen, Germany) for performing the statistical analysis. This work was supported by grants from the Federal Ministry of Education and Research of Germany (Network of

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