Targeting Calpains: A Therapeutic Strategy for the Treatment of TGF Mediated Mesenchymal Transition and Associated Pathologies. D. Kim1, R. Gould1,2, J. Butcher2, H. Dietz1 1) Inst Gen Med, Johns Hopkins Sch Med, Baltimore, MD; 2) Department of Biomedical Engineering, Cornell University, Ithaca, NY.

   Expression profiling studies associate enhanced expression/activity of the calpain family of cysteine proteases with multiple genetic or environmentally-induced TGF-related disease processes including fibrosis and tumor metastasis. The underlying mechanistic connection (if any) remains unknown. We reasoned that this association might relate to TGF-induced mesenchymal transition, the process by which cells of epithelial or endothelial origin lose polarity and cell adhesion and adopt an invasive character and fibrotic synthetic repertoire (EMT). This hypothesis was tested in NMuMG epithelial cells which show striking EMT within 2 days of TGF administration, as evidenced by downregulation of E-cadherin, transition from a cortical to a stress fiber distribution of F-actin, and upregulation of -smooth muscle actin, collagen, and matrix metalloproteinases. As expected, concomitant treatment with a broad inhibitor of TGF signaling (SB431542) prevented EMT in association with attenuation of intracellular TGF signal propagation (phosphorylation of Smad2/3). In contrast, inhibition of calpain activity (as evidenced by failed cleavage of the natural calpain substrate FLNA) with the broad-spectrum calpain inhibitors MDL-28170 or calpeptin abrogated EMT despite maintenance of the pSmad2/3 response. Furthermore, robust EMT inhibition was achieved using either 2-ABP (which is reported to prevent the TRPM7-mediated calcium influx needed for calpain activation) or overexpression of calpastatin, a naturally-occurring and highly specific dimeric calpain inhibitor. Among dimeric calpains, the CAPN1 and CAPN2 large subunits and CAPNS4 small subunit show broad expression, however siRNA-mediated silencing had no effect on EMT. In contrast, we show that the relatively obscure CAPN9 and CAPNS2 subunits only show physiologic expression in the GI tract and skin, respectively, but are potently induced by TGF in both epithelial and endothelial cells; siRNA-mediated silencing of either abrogated EMT in culture systems. Identical provocations prevented EMT in all epithelial and endothelial cell lines tested and showed the capacity to reverse an established mesenchymal phenotype (MET). Taken together, these data suggest that calpain inhibition is an attractive therapeutic strategy for multiple TGF pathologies and lend optimism that CAPN9/S2 inhibition will have a greater influence on pathologic vs. physiologic events and will therefore exhibit a favorable tolerance profile.

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