Calpain 9 As a Therapeutic Target in TGF -Induced Mesenchymal Transition and Fibrosis
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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE FIBROSIS Copyright © 2019 The Authors, some b rights reserved; Calpain 9 as a therapeutic target in TGF -induced exclusive licensee American Association mesenchymal transition and fibrosis for the Advancement David H. Kim1,2*, James D. Beckett1*, Varun Nagpal1, Manuel A. Seman-Senderos1,2, Russell A. Gould1,3, of Science. No claim 4 1,4 1 5 3 to original U.S. Tyler J. Creamer , Elena Gallo MacFarlane , Yichun Chen , Djahida Bedja , Jonathan T. Butcher , Government Works Wayne Mitzner6, Rosanne Rouf5, Shoji Hata7, Daniel S. Warren4, Harry C. Dietz1,8† Fibrosis is a common pathologic outcome of chronic disease resulting in the replacement of normal tissue paren- chyma 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–b (TGFb) to efficiently induce myofibroblast dif- ferentiation 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 TGFb-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 fre- quency 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 knock- out, these data provide a strong signature for tolerance of therapeutic strategies for fibrosis aimed at CAPN9 antagonism. INTRODUCTION broblasts in a process known as mesenchymal transition. The spe- The replacement and distortion of tissue parenchyma with fibrillar cific source of myofibroblasts in fibrotic diseases remains contro- collagens and other extracellular matrix (ECM) proteins—thereby versial and is likely varied; however, the prevailing view is that compromising organ function—is a common feature of chronic dis- mesenchymal transition plays a prominent role in most if not all ease and contributes to a substantial number of deaths in the indus- fibrotic contexts (10). Typical alterations in cellular phenotype that trialized world (1–3). Although collagen deposition is an indispensable accompany TGFb-mediated epithelial-to-mesenchymal or endothelial- component of tissue homeostasis, chronic injury or dysregulation to-mesenchymal transition (EpMT or EnMT, respectively; EMT of wound healing can lead to pathologic scarring, a condition termed collectively) include down-regulation of markers of a mature polarized fibrosis (4). In some instances, provocations that induce tissue fi- cell state (E-cadherin) and induction of mesenchymal markers, brosis have been identified, such as curtained genetic conditions (5–7), such as a-smooth muscle actin (aSMA), vimentin, fibrillar colla- chemical exposures (8), and chronic inflammation secondary to auto- gens, and matrix metalloproteases (MMPs) 2 and 9 (12). Efforts to immune disorders (1). In other cases, such as most idiopathic fate-map cells in fibrotic models of lung, liver, and heart fibrosis pulmonary fibrosis presentations, the specific driver of fibrosis is provide ample evidence for and against a role of EMT in the accumu- unknown. lation of myofibroblasts in vivo (13). Nevertheless, TGFb-SMAD sig- Regardless of the initiating events, all fibrotic disorders show ac- naling induces canonical EMT transcription factors (12), and genetic cumulation of activated fibroblasts that are invasive, synthetic, con- deletion of EMT transcription factors in lung alveolar cells or in tractile, proliferative, and long-lived (9). The profibrotic cytokine hepatocytes blunts experimentally induced organ fibrosis (14, 15). Given transforming growth factor–b (TGFb) can induce differentiation of the clear role of TGFb in fibrosis, we reasoned that a distal molecular a variety of progenitor lineages, including epithelial or endothelial event that is critical for TGFb-induced mesenchymal transition would cells, resident fibroblasts, or pericytes (10, 11), to so-called myofi- be an attractive therapeutic target for multiple etiologies of fibrosis. We were intrigued by the association in the literature between multiple EMT-related disease processes and the increased expres- 1McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 2Cellular and Molecular Medicine Program, sion or activity of calpains—a family of calcium-dependent non- School of Medicine, Baltimore, MD 21205, USA. 3Meinig School of Biomedical lysosomal cysteine proteases that cleave diverse substrates to regulate 4 Engineering, Cornell University, Ithaca, NY 14853, USA. Department of Surgery, cell activities including differentiation, adhesion, invasion, migra- Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 5Depart- ment of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD tion, synthetic repertoire, and survival (16). For example, calpain 21205, USA. 6Department of Environmental Health Sciences, Johns Hopkins University activity has been mechanistically linked to the invasive behavior of Bloomberg School of Public Health, Baltimore, MD 21205, USA. 7Department of epithelial tumors (17–20), normal wound healing (21), cardiac fi- Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, 8 brosis after tissue injury (22–25), and lung fibrosis in response to Tokyo 156-8506, Japan. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. *These authors contributed equally to this work. bleomycin (26). These observations led to our hypothesis that specific †Corresponding author. Email: [email protected] calpain cleavage products are required for mesenchymal transition Kim et al., Sci. Transl. Med. 11, eaau2814 (2019) 17 July 2019 1 of 15 SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE and that inhibition of calpain activity may have therapeutic value in A second broad-spectrum calpain inhibitor, calpeptin (37), also fibrotic disorders. inhibited TGFb-induced EMT in NMuMG cells in association with Of the 15 calpain isoforms expressed by humans, calpain 1 (CAPN1) decreased FLNA cleavage (Fig. 1D). Similar results were obtained with and CAPN2 are the best characterized and are termed the conven- 2-APB (38), a potent inhibitor of calcium channels and the calcium tional classical calpains (27). Active CAPN1 and CAPN2 enzymes influx required for calpains to adopt a catalytically active conforma- consist of a heterodimer formed with small regulatory subunit cal- tion (Fig. 1E) (39, 40). Calpeptin, MDL-28170, and other available pain small subunit 1 (CAPNS1). An alternative subunit, CAPNS2, broad-spectrum calpain inhibitors also antagonize cathepsins, an- is of unknown physiologic function (28). The activity of these con- other class of calcium-dependent proteases. Evidence for the specific ventional dimeric calpains is tightly regulated by the endogenous relevance of calpains included failure of CA-074-OMe, a cathepsin calpain inhibitor calpastatin (CAST). CAST is thought to specifically B and L inhibitor that does not cross-react with calpains, to suppress inhibit all dimeric calpains. CAST binds near the active site cleft of TGFb-induced EMT in NMuMG cells (fig. S1C) (41, 42). dimeric calpains in the presence of calcium and prevents engage- As reported previously, mesenchymal differentiation of NMuMG ment of substrates but is protected from hydrolysis by not binding cells is reversed when TGFb is removed (34). Here, we found that the active site itself (29–31). CAPN1, CAPN2, CAPNS1, and CAST myofibroblasts derived from NMuMG cells showed a decline in aSMA are ubiquitously expressed; however, other calpain isoforms are ex- expression, reorganization of F-actin, and reemergence of epithelial pressed primarily in specific tissues or organs. For example, large marker expression (E-cadherin) upon addition of MDL-28170 de- subunit CAPN9 is reportedly chiefly expressed in the gastrointestinal spite the continued presence of TGFb (Fig. 2). tract (32), whereas CAPNS2 reportedly shows predominant expres- sion in the skin and esophagus (33). CAST-mediated inhibition of mesenchymal transition In this study, we used complementary methods to implicate Capn9 implicates dimeric calpain isoforms and Capns2 in TGFb-induced myofibroblast differentiation in vitro We overexpressed CAST in NMuMG cells by transfecting with a and in multiple experimentally induced models of fibrosis in vivo. CAST-IRES-GFP construct. The expression of CAST, as monitored by Capn9 showed highly restricted physiologic expression but could be po- green fluorescent protein (GFP)