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Molecular Mechanisms of TGFβ Receptor-Triggered Signaling Cascades Rapidly Induced by the Calcineurin Inhibitors Cyclosporin A and FK506¹

Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany

The calcineurin inhibitor (CNI)-induced renal fibrosis is attributed to an exaggerated deposition of extracellular matrix, which is mainly due to an increased expression of TGFβ. Herein we demonstrate that the CNI cyclosporin A and tacrolimus (FK506), independent of TGFβ synthesis, rapidly activate TGFβ/Smad signaling in cultured mesangial cells and in whole kidney samples from CNI-treated rats. By EMSA, we demonstrate increased DNA binding of Smad-2, -3, and -4 to a cognate Smad-binding promoter element (SBE) accompanied by CNI-triggered activation of Smad-dependent expression of tissue inhibitor of metalloprotease-1 (TIMP-1) and connective tissue growth factor. Using an activin receptor-like kinase-5 (ALK-5) inhibitor and by small interfering RNA we depict a critical involvement of both types of TGFβ receptors in CNI-triggered Smad signaling and fibrogenic gene expression, respectively. Mechanistically, CNI cause a rapid activation of latent TGFβ, which is prevented in the presence of the antioxidant N-acetyl cysteine. A convergent activation of p38 MAPK is indicated by the partial blockade of CNI-induced Smad-2 activation by SB203580; conversely, both TGFβ-RII and TGFβ are critically involved in p38 MAPK activation by CNI. Activation of both signaling pathways is similarly triggered by reactive oxygen species. Finally, we show that neutralization of TGFβ markedly reduced the CNI-dependent Smad activation in vitro and in vivo. Collectively, this study demonstrates that CNI via reactive oxygen species generation activate latent TGFβ and thereby initiate the canonical Smad pathway by simultaneously activating p38 MAPK, which both synergistically induce Smad-driven gene expression.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the German Research Foundation (DFG) Grants EB 257/3-1, PF 361/2-2, SCHA 1082/2-1, the Excellence Cluster Cardiopulmonary System (ECCPS) EXC 147/1, and the Interdisciplinary Center of Clinical Research, Münster (Schae 2/026/06).

² Address correspondence and reprint requests to Dr. Wolfgang Eberhardt, Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail address: w.eberhardt{at}em.uni-frankfurt.de

³ Abbreviations used in this paper: CsA, cyclosporin A; ALK, activin receptor-like kinase; CNI, calcineurin inhibitors; CTGF, connective tissue growth factor; CyPA, cyclophilin A; DPI, diphenylene iodonium; ECM, extracellular matrix; HDAC, histone deacetlyase; MC, mesangial cells; NAC, N-acetyl cysteine; PEG-SOD, polyethylene glycol-superoxide dismutase; ROS, reactive oxygen species; R-Smad, receptor-bound Smad protein; SBE, Smad binding element; siRNA, small interfering RNA; TIMP-1, tissue inhibitor of matrix metalloproteinase.