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Calpain 9 as a therapeutic target in TGF?-induced mesenchymal transition and fibrosis.

ABSTRACT: Fibrosis is a common pathologic outcome of chronic disease resulting in the replacement of normal tissue parenchyma with a collagen-rich extracellular matrix produced by myofibroblasts. Although the progenitor cell types and cellular programs giving rise to myofibroblasts through mesenchymal transition can vary between tissues and diseases, their contribution to fibrosis initiation, maintenance, and progression is thought to be pervasive. Here, we showed that the ability of transforming growth factor-? (TGF?) to efficiently induce myofibroblast differentiation of cultured epithelial cells, endothelial cells, or quiescent fibroblasts is dependent on the induced expression and activity of dimeric calpains, a family of non-lysosomal cysteine proteases that regulate a variety of cellular events through posttranslational modification of diverse substrates. siRNA-based gene silencing demonstrated that TGF?-induced mesenchymal transition of a murine breast epithelial cell line was dependent on induction of expression of calpain 9 (CAPN9), an isoform previously thought to be restricted to the gastrointestinal tract. Mice lacking functional CAPN9 owing to biallelic targeting of Capn9 were viable and fertile but showed overt protection from bleomycin-induced lung fibrosis, carbon tetrachloride-induced liver fibrosis, and angiotensin II-induced cardiac fibrosis and dysfunction. A predicted loss-of-function allele of CAPN9 is common in Southeast Asia, with the frequency of homozygosity matching the prediction of Hardy-Weinberg equilibrium. Together with the highly spatially restricted pattern of CAPN9 expression under physiologic circumstances and the heartiness of the murine knockout, these data provide a strong signature for tolerance of therapeutic strategies for fibrosis aimed at CAPN9 antagonism.

SUBMITTER: Kim DH

PROVIDER: S-EPMC7351287 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Calpain 9 as a therapeutic target in TGFβ-induced mesenchymal transition and fibrosis.

Kim David H DH Beckett James D JD Nagpal Varun V Seman-Senderos Manuel A MA Gould Russell A RA Creamer Tyler J TJ MacFarlane Elena Gallo EG Chen Yichun Y Bedja Djahida D Butcher Jonathan T JT Mitzner Wayne W Rouf Rosanne R Hata Shoji S Warren Daniel S DS Dietz Harry C HC

Science translational medicine 20190701 501

Fibrosis is a common pathologic outcome of chronic disease resulting in the replacement of normal tissue parenchyma with a collagen-rich extracellular matrix produced by myofibroblasts. Although the progenitor cell types and cellular programs giving rise to myofibroblasts through mesenchymal transition can vary between tissues and diseases, their contribution to fibrosis initiation, maintenance, and progression is thought to be pervasive. Here, we showed that the ability of transforming growth f ...[more]

PMID: 31316008

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Project description:Phenotype reprogramming during transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) is an extensive and dynamic process, orchestrated by the integration of biological signaling across multiple time scales. As part of the numerous transcriptional changes necessary for EMT, TGFβ-initiated Smad3 signaling results in remodeling of the redox environment and decreased nucleophilic tone. Because Smad3 itself is susceptible to attenuated activity through antioxidants, the possibility of a positive feedback loop exists, albeit the time scales on which these mechanisms operate are quite different. We hypothesized that the decreased nucleophilic tone acquired during EMT promotes Smad3 signaling, enhancing acquisition and stabilization of the mesenchymal phenotype. Previous findings supporting such a mechanism were characterized independent of each other; we sought to investigate these relationships within a singular experimental context. In this study, we characterized multivariate representations of phenotype as they evolved over time, specifically measuring expression of epithelial/mesenchymal differentiation, redox regulators, and Smad transcription factors. In-cell Western (ICW) assays were developed to evaluate multivariate phenotype states as they developed during EMT. Principal component analysis (PCA) extracted anticorrelations between phospho-Smad3 (pSmad3) and Smad2/Smad4, which reflected a compensatory up-regulation of Smad2 and Smad4 following cessation of TGFβ signaling. Measuring transcript expression following EMT, we identified down-regulation of numerous antioxidant genes concomitant with up-regulation of NADPH oxidase 4 (NOX4) and multiple mesenchymal phenotype markers. TGFβ treatment increased CM-H2DCF-DA oxidation, decreased H2O2 degradation rates, and increased glutathione redox potential. Our findings suggest that the decreased nucleophilic tone during EMT coincides with the acquisition of a mesenchymal phenotype over too long a time scale to enable enhanced Smad3 phosphorylation during initiation of EMT. We further challenged the mesenchymal phenotype following EMT through antioxidant and TGFβ inhibitor treatments, which failed to induce a mesenchymal-epithelial transition (MET). Our characterization of multivariate phenotype dynamics during EMT indicates that the decrease in nucleophilic tone occurs alongside EMT; however, maintenance of the mesenchymal phenotype following EMT is independent of both the nascent redox state and the continuous TGFβ signaling.

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Calpain 9 as a therapeutic target in TGF?-induced mesenchymal transition and fibrosis.

Publications

Calpain 9 as a therapeutic target in TGFβ-induced mesenchymal transition and fibrosis.

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