Abstract
Transforming growth factor-b (TGF-b) 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-b/Smad signaling pathway. The present work demonstrates that CsA similar to TGF-b causes a rapid phosphorylation of Smad-2 in A549 lung epithelial cells which is abrogated by the addition of neutralizing TGF-b-antibody. By using the TGF-b receptor I kinase inhibitor, activin receptor-like kinase-5 (ALK-5) a critical involvement of TGF-b receptor in CsA-triggered Smad signaling was depicted. Furthermore, TGF-b 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-b release and Smad-2 phosphorylation induced by CsA. Moreover, the present work demonstrates that CsA via ROS generation and activation of TGF-b/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.
Highlights
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 present work was designed to test the possible modulation of TGF- /Smad signaling pathway by Cyclosporin A (CsA) in pulmonary epithelial cells (A549 cells) that are considered as a source of TGF- in the lung (Kwong et al, 2004)
To delinate the underlying mechanisms involved in Smad signaling activation by CsA, A549 cells were treated with a specific kinase inhibitor of the type I TGF- receptor denominated as activin receptor-like kinase (ALK)-5 inhibitor, and a neutralizing TGF- antibody before stimulation with CsA
Summary
Fibrotic diseases have been shown to be associated with excessive deposition of extracellular matrix (ECM) (Wells, 2008; Cox & Erler, 2011; Klingberg et al, 2013). 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). The predominant type 1 receptor for TGF- signaling is thought to be activin receptor-like kinase (ALK)-5 which phosphorylates Smad-2 and Smad and subsequently forms a complex with the co-Smad, Smad-4, and translocates into the nucleus 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). 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)
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