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Transforming growth factor- (TGF- and downstream Smad signaling pathways have been found to be the most important pathways involved in tissue fibrosis via induction of the profibrotic genes connective tissue growth factor (CTGF) and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1). Cyclosporin A (CsA) is one of the most efficient immunosuppressive drugs that has been shown to induce a rapid activation of TGF-Smad signaling pathway. The present work demonstrates that CsA similar to TGF- causes a rapid phosphorylation of Smad-2 in A549 lung epithelial cells which is abrogated by the addition of neutralizing TGF--antibody. By using the TGF-receptor I kinase inhibitor, activin receptor-like kinase-5 (ALK-5) a critical involvement of TGF- receptor in CsA-triggered Smad signaling was depicted. Furthermore, TGF-release and Smad-2 phosphorylation induced by CsA were highly reduced in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), indicating that ROS is required for TGF- release and Smad-2 phosphorylation induced by CsA. Moreover, the present work demonstrates that CsA via ROS generation and activation of TGF-/Smad signaling cascade can cause an increase in the expression of the profibrotic genes CTGF and TIMP-1 in A549 lung epithelial cells. Collectively, these data demonstrate that CsA causes a rapid activation of the fibrogenic Smad signaling cascade in A549 lung epithelial cells via generation of ROS and subsequent activation of latent TGFβ that is sufficient to elicit fibrogenic cell responses as indicated by increasing the expression of the fibrogenic genes CTGF and TIMP-1 that play an important role in tissue fibrosis. INTRODUCTION Fibrotic diseases have been shown to be associated with excessive deposition of extracellular matrix (ECM) (Wells, 2008; Cox & Erler, 2011; Klingberg et al., 2013). The accumulation of ECM is thought to be primarily due to an impaired ECM turnover (Duymelinck et al., 1998). ECM degradation is mainly regulated by the action of two matrixdegrading enzyme systems, the matrix metalloproteinases and the plasminogen activators and their intrinsic inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) and the plasminogen activator inhibitors, respectively (Woessner, 1991; Eddy, 2000). Transforming growth factor- (TGF- and downstream Smad signaling pathways have been found to be the most important pathways involved in tissue fibrosis via induction of profibrotic genes (Roberts & Sporn, 1990; Gore-Hyer et al., 2002; Ruiz et al., 2003). Generally, TGFAz. J. Pharm Sci. Vol. 50, September, 2014. 2 induces ECM deposition by stimulating the expression of ECM proteins such as connective tissue growth factor (CTGF), reducing synthesis of ECM degrading enzymes such as matrix metalloproteinases-9 (MMP-9) and up-regulating synthesis of proteinase inhibitors such as tissue inhibitors of metalloproteinase-1 (TIMP-1) (Roberts & Sporn, 1990; Gore-Hyer et al., 2002; Ruiz et al., 2003; Weng et al., 2007; Wang et al., 2011). TGF-is usually secreted as latent complex (latent TGF-β) consisting of TGF-β covalently bound to latent TGF-β binding proteins (LTBP) (Roberts, 1998; Okada et al., 2005). Activation of TGF-is achieved by either proteolytic or nonproteolytic events (Annes et al., 2003) and in some cases may include a redox-sensitive mechanism (Barcellos-Hoff & Dix, 1996; Jobling et al., 2006). In this respect, oxidation of the latency-associated peptide can cause a conformational change that releases TGF-β (Barcellos-Hoff & Dix, 1996). Mechanistically, activated TGF- acts through a heteromeric receptor complex. The predominant type 1 receptor for TGF- signaling is thought to be activin receptor-like kinase (ALK)-5 which phosphorylates Smad-2 and Smad3 and subsequently forms a complex with the co-Smad, Smad-4, and translocates into the nucleus. Subsequently, these complexes bind with a high affinity to specific promoter elements, the Smad binding elements (SBE), and thereby can activate the transcription of many TGF--induced target genes, including CTGF (Fukasawa et al., 2004) and TIMP-1 (Marti et al., 1994; Chen et al., 2002). Cyclosporin A (CsA) is one of the most efficient immunosuppressive agents that suppress T-cell activation by inhibiting the cellular phosphatase calcineurin (Schreiber & Crabtree, 1992) and therefore is widely used in organ transplantation and many inflammatory diseases including psoriasis, and rheumatoid arthritis. Recently, CsA has been shown to cause a rapid activation of TGF-/Smad signaling pathway in glomerular mesangial cells and thereby increase the expression of the profibrotic genes CTGF and TIMP-1 (Akool et al., 2005 and 2008). The present work was designed to test the possible modulation of TGF-/Smad signaling pathway by CsA in pulmonary epithelial cells (A549 cells) that are considered as a source of TGF- in the lung (Kwong et al., 2004). MATERIALS AND METHODS Materials The TGF-RI kinase inhibitor [3-(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole (Alk-5 inhibitor), U0126, SP600125 and SB203580 were obtained from Calbiochem (Germany). Diphenylene iodonium (DPI), N-acetyl cysteine (NAC) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Human recombinant TGF1 was purchased from Cell Concepts GmbH (Umkirch, Germany). CsA was purchased from Alexis Biochemicals (Germany). A neutralizing monoclonal TGF1–3 antibody and Human TGF-1 Quantikin-e ELISA Kit were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany). Antibodies specifically raised against phospho-Smad-2, total Smad-2 were derived from Cell Signaling (Frankfurt am Main, Germany). Antibodies against CTGF, TIMP-1, -actin and anti-rabbit HRPlinked IgGs, as well as control IgG were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). The ECL system, and Hyperfilm were purchased from Amersham Pharmacia Biotech (Germany). Az. J. Pharm Sci. Vol. 50, September, 2014. 3 Cell Culture Human A549 lung epithelial/carcinoma cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were cultivated in RPMI 1640, supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 10% heatinactivated FCS and 10mM HEPES buffer (GIBCOBRL, Eggenstein, Germany). Western Blot Analysis For detection of CTGF, TIMP-1, phosphorylated Smad-2 and total Smad-2, wholecell lysates were prepared. Total cell extracts containing 50-100 μg of protein were prepared in sodium dodecyl sulfate (SDS) sample buffer and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis was performed as originally described by Laemmli (1970). In general, a total protein (50-100μg) from each sample was mixed with an equal volume of 2x electrophoresis sample buffer and incubated at 95C for 10 minutes for denaturation. Following gel electrophoresis, the proteins were transferred onto a nitrocellulose (PVDF) membrane by semi-dry electroblotting. After blocking (by shaking the membrane in 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.05% Tween for 1 hour, the membrane was incubated with the primary antibody overnight at 4C followed by incubation with secondary antibodies (coupled to horseradish peroxidase). Signals were detected using enhanced chemi luminescence (ECL) reagent according to the manufacturer’s instructions. ELISA Levels of TGF- in cell-free culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (R&D Systems, Wiesbaden-Nordenstadt, Germany). Statistical Analysis Results are expressed as means ± SD. Statistical analysis was performed using Student’s t test and for multiple comparisons the ANOVA test for significance. P-values below 0.05 were considered as indication for statistically significant differences between conditions compared.

MOLECULAR MECHANISMS OF TGF-/SMAD SIGNALING CASCADE INDUCED BY CYCLOSPORIN A IN A549 LUNG EPITHELIAL CELLS