Research ArticleFibrosis

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

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Science Translational Medicine  17 Jul 2019:
Vol. 11, Issue 501, eaau2814
DOI: 10.1126/scitranslmed.aau2814

Counteracting calpain

During fibrosis, myofibroblasts produce extracellular matrix that accumulates and impairs tissue function. Kim et al. found that transforming growth factor–β induced translation of calpain 9, a cysteine protease, which mediated myofibroblast differentiation. Mice lacking calpain 9 were protected from experimentally induced fibrosis in the heart, lung, and liver. The authors identified a common calpain 9 loss-of-function mutation in people that was not associated with markers of intolerance. This study suggests that targeting calpain 9 could have therapeutic potential for inhibiting 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–β (TGFβ) 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 TGFβ-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.

INTRODUCTION

The replacement and distortion of tissue parenchyma with fibrillar collagens and other extracellular matrix (ECM) proteins—thereby compromising organ function—is a common feature of chronic disease and contributes to a substantial number of deaths in the industrialized world (13). Although collagen deposition is an indispensable component of tissue homeostasis, chronic injury or dysregulation of wound healing can lead to pathologic scarring, a condition termed fibrosis (4). In some instances, provocations that induce tissue fibrosis have been identified, such as curtained genetic conditions (57), chemical exposures (8), and chronic inflammation secondary to autoimmune disorders (1). In other cases, such as most idiopathic pulmonary fibrosis presentations, the specific driver of fibrosis is unknown.

Regardless of the initiating events, all fibrotic disorders show accumulation of activated fibroblasts that are invasive, synthetic, contractile, proliferative, and long-lived (9). The profibrotic cytokine transforming growth factor–β (TGFβ) can induce differentiation of a variety of progenitor lineages, including epithelial or endothelial cells, resident fibroblasts, or pericytes (10, 11), to so-called myofibroblasts in a process known as mesenchymal transition. The specific source of myofibroblasts in fibrotic diseases remains controversial and is likely varied; however, the prevailing view is that mesenchymal transition plays a prominent role in most if not all fibrotic contexts (10). Typical alterations in cellular phenotype that accompany TGFβ-mediated epithelial-to-mesenchymal or endothelial-to-mesenchymal transition (EpMT or EnMT, respectively; EMT collectively) include down-regulation of markers of a mature polarized cell state (E-cadherin) and induction of mesenchymal markers, such as α-smooth muscle actin (αSMA), vimentin, fibrillar collagens, and matrix metalloproteases (MMPs) 2 and 9 (12). Efforts to fate-map cells in fibrotic models of lung, liver, and heart fibrosis provide ample evidence for and against a role of EMT in the accumulation of myofibroblasts in vivo (13). Nevertheless, TGFβ-SMAD signaling induces canonical EMT transcription factors (12), and genetic deletion of EMT transcription factors in lung alveolar cells or in hepatocytes blunts experimentally induced organ fibrosis (14, 15). Given the clear role of TGFβ in fibrosis, we reasoned that a distal molecular event that is critical for TGFβ-induced mesenchymal transition would 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 expression or activity of calpains—a family of calcium-dependent non-lysosomal cysteine proteases that cleave diverse substrates to regulate cell activities including differentiation, adhesion, invasion, migration, synthetic repertoire, and survival (16). For example, calpain activity has been mechanistically linked to the invasive behavior of epithelial tumors (1720), normal wound healing (21), cardiac fibrosis after tissue injury (2225), and lung fibrosis in response to bleomycin (26). These observations led to our hypothesis that specific calpain cleavage products are required for mesenchymal transition and that inhibition of calpain activity may have therapeutic value in fibrotic disorders.

Of the 15 calpain isoforms expressed by humans, calpain 1 (CAPN1) and CAPN2 are the best characterized and are termed the conventional classical calpains (27). Active CAPN1 and CAPN2 enzymes consist of a heterodimer formed with small regulatory subunit calpain small subunit 1 (CAPNS1). An alternative subunit, CAPNS2, is of unknown physiologic function (28). The activity of these conventional dimeric calpains is tightly regulated by the endogenous calpain inhibitor calpastatin (CAST). CAST is thought to specifically inhibit all dimeric calpains. CAST binds near the active site cleft of dimeric calpains in the presence of calcium and prevents engagement of substrates but is protected from hydrolysis by not binding the active site itself (2931). CAPN1, CAPN2, CAPNS1, and CAST are ubiquitously expressed; however, other calpain isoforms are expressed primarily in specific tissues or organs. For example, large subunit CAPN9 is reportedly chiefly expressed in the gastrointestinal tract (32), whereas CAPNS2 reportedly shows predominant expression in the skin and esophagus (33).

In this study, we used complementary methods to implicate Capn9 and Capns2 in TGFβ-induced myofibroblast differentiation in vitro and in multiple experimentally induced models of fibrosis in vivo. Capn9 showed highly restricted physiologic expression but could be potently induced in naïve cell types by TGFβ, suggesting the potential of high tolerance for therapeutic strategies for fibrosis aimed at antagonism.

RESULTS

Calpain inhibition prevents TGFβ-induced mesenchymal transition without affecting proximal signaling events

To explore the role of calpain proteases in mesenchymal transition, we initially turned to a robust cell culture–based TGFβ-induced EMT assay using the Namru Mouse Mammary Gland (NMuMG) epithelial cell line (34, 35). TGFβ-stimulated NMuMG cells demonstrated robust phosphorylation of SMAD2 (pSMAD2) accumulation at all assayed time points and expressed αSMA beginning at 48 hours, indicating differentiation into myofibroblasts. Treatment with MDL-28170, a broad-spectrum calpain and cathepsin inhibitor, resulted in a dose-dependent decrease in αSMA expression at 48 and 72 hours after TGFβ stimulation (Fig. 1A). MDL-28170 did not affect TGFβ-dependent phosphorylation (Fig. 1A) or nuclear accumulation (fig. S1A) of SMAD2, suggesting the relevance of events that occur distal to TGFβ receptor–mediated signal transduction.

Fig. 1 Broad-spectrum calpain inhibition attenuates TGFβ-induced EMT in NMuMG cells.

(A) Representative immunoblots (left, bracket indicates identical gel) and quantification (right, normalized to GAPDH) for the indicated proteins, time points after TGFβ stimulation, and concentrations of MDL-28170 (n = 3 to 4). (B) Representative immunofluorescence images of TGFβ-induced EMT in NMuMG cells stained with E-cadherin (green), F-actin (red), and DAPI (blue). Dimethyl sulfoxide (DMSO) used as vehicle control. Scale bars, 100 μm. (C) Relative gene expression (normalized to Gapdh) in response to TGFβ with or without calpain inhibition (MDL) (n = 3). (D) Representative immunoblots (left) and quantification (right) for the indicated proteins, time points after TGFβ stimulation, and concentrations of calpeptin (n = 3). (E) Representative immunoblots (left) and quantification (right) for the indicated proteins, time points after TGFβ stimulation, and concentrations of the calcium channel blocker 2-APB (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, †P < 0.005, and ‡P < 0.001 by one-way ANOVA with Tukey’s post hoc test.

We observed that TGFβ induced calpain activity, as evidenced by production of a calpain-dependent filamin A C-terminal cleavage product (FLNA-C) (36). TGFβ stimulation induced FLNA-C accumulation in NMuMG cells that was attenuated by MDL-28170 in a dose-dependent manner (Fig. 1A). We confirmed that accumulation of αSMA and FLNA-C related to canonical, SMAD-dependent TGFβ signaling in this system, because treatment with SB431542, a TGFβ receptor kinase inhibitor that precludes TGFβ receptor-dependent SMAD2 and SMAD3 phosphorylation (fig. S1B), attenuated both.

Transition from an epithelial to a mesenchymal state was associated with down-regulation of cell surface expression of E-cadherin, reorganization of F-actin from a cortical to a stress fiber distribution, and enhanced mRNA expression of type I collagen (Col1a1), vimentin (Vim), Mmp2, and Mmp9 (Fig. 1, B and C). Treatment of NMuMG cells with MDL-28170 before administration of TGFβ attenuated each of these events.

A second broad-spectrum calpain inhibitor, calpeptin (37), also inhibited TGFβ-induced EMT in NMuMG cells in association with decreased FLNA cleavage (Fig. 1D). Similar results were obtained with 2-APB (38), a potent inhibitor of calcium channels and the calcium influx required for calpains to adopt a catalytically active conformation (Fig. 1E) (39, 40). Calpeptin, MDL-28170, and other available broad-spectrum calpain inhibitors also antagonize cathepsins, another class of calcium-dependent proteases. Evidence for the specific relevance of calpains included failure of CA-074-OMe, a cathepsin B and L inhibitor that does not cross-react with calpains, to suppress TGFβ-induced EMT in NMuMG cells (fig. S1C) (41, 42).

As reported previously, mesenchymal differentiation of NMuMG cells is reversed when TGFβ is removed (34). Here, we found that myofibroblasts derived from NMuMG cells showed a decline in αSMA expression, reorganization of F-actin, and reemergence of epithelial marker expression (E-cadherin) upon addition of MDL-28170 despite the continued presence of TGFβ (fig. S2).

Fig. 2 Dimeric calpain isoforms CAPN9 and CAPNS2 mediate TGFβ-induced EMT in NMuMG cells.

(A) Representative immunoblots (left, bracket indicates identical gel) and quantification (right, normalized to GAPDH) for indicated proteins and time points after TGFβ stimulation with or without CAST-IRES-GFP overexpression (n = 3). Representative αSMA immunoblots (left) for siRNA knockdown of dimeric calpain subunits. (B) CAPN1 (n = 4), (C) CAPN2 (n = 3), (D) CAPNS1 (n = 3), (E) CAPN9 (n = 5), and (F) CAPNS2 (n = 3) and quantification (right, normalized to GAPDH) with or without TGFβ stimulation. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, †P < 0.005, and ‡P < 0.001 by one-way ANOVA with Tukey’s post hoc test.

CAST-mediated inhibition of mesenchymal transition implicates dimeric calpain isoforms

We overexpressed CAST in NMuMG cells by transfecting with a CAST-IRES-GFP construct. The expression of CAST, as monitored by green fluorescent protein (GFP) abundance, was not influenced by the addition of TGFβ. Overexpression of CAST in TGFβ-stimulated NMuMG cells blocked the expression of αSMA and the cleavage of FLNA, but it did not alter SMAD2 phosphorylation (Fig. 2A).

The suppressive activity of CAST suggested involvement of a dimeric calpain isoform in TGFβ-induced EMT. Given the potential for EMT and fibrosis to occur in many cell types and tissues, we initially suspected involvement of CAPN1, CAPN2, or CAPNS1 given their ubiquitous expression. However, small interfering RNA (siRNA)–mediated suppression of any of these calpain subunits in TGFβ-stimulated NMuMG cells failed to attenuate EMT, as indicated by the quantity of αSMA expression (Fig. 2, B to D). This observation was initially perplexing as the alternative dimeric calpain large subunit, CAPN9, is reportedly predominantly expressed in the gastrointestinal tract (43), whereas the remaining small subunit, CAPNS2, has been less well studied but reportedly also shows highly restricted expression (28). In keeping with these data, our survey showed predominant expression of Capn9 and Capns2 message in the gastrointestinal tract and skin, respectively (fig. S3A). Neither CAPN9 nor CAPNS2 protein is expressed in NMuMG cells at baseline, but both showed potent induction upon treatment with TGFβ (Fig. 2, E and F). Suppression of this effect using siRNA strongly attenuated EMT, as shown by reduced αSMA protein abundance (Fig. 2, E and F) but did not affect TGFβ-dependent induction of EMT transcription factors (fig. S3B). These findings suggest that calpain activity is required for a distal event in TGFβ-mediated myofibroblast differentiation and/or performance.

Calpain inhibition in multiple cell types diminishes TGFβ-induced mesenchymal transition

Considering the critical role played by calpains in TGFβ-induced EMT in the NMuMG epithelial cell line, we sought to explore the relevance of this mechanism in additional cell types. Consistent with our findings in NMuMG cells, pharmacologic inhibition of calpains with MDL-28170 in a second epithelial cell line, Madin-Darby canine kidney (MDCK) cells, attenuated mesenchymal transition in a dose-dependent manner (fig. S4A). Treatment of normal human lung fibroblasts (NHLFs) with calpeptin had previously been shown to decrease the expression of TGFB1 and COL1A1 message in response to TGFβ (26). Here, calpain inhibition attenuated the expression of αSMA in NHLFs stimulated with TGFβ (fig. S4B).

Primary porcine aortic valve endothelial cells (PAVECs) undergo EnMT when stimulated with TGFβ, as evidenced by a mesenchymal transcriptional signature including expression of ACTA2 (encoding αSMA), VIM, MMP2, and MMP9 and down-regulation of CDH1 (encoding E-cadherin) (fig. S3A). Each of these events abated upon either CAST overexpression or siRNA-mediated knockdown of CAPNS2 expression (Fig. 3B). Inhibition of mesenchymal transition by PAVECs was associated with preservation of expression of E-cadherin and prevention of induction of vimentin (Fig. 3C).

Fig. 3 Dimeric calpain inhibition suppresses TGFβ-induced mesenchymal transition in primary PAVECs.

(A) Relative gene expression of PAVECs stimulated with TGFβ1 for 48 hours with CAST overexpression normalized to GAPDH (n = 3). (B) Relative gene expression (normalized to GAPDH) in response to TGFβ with or without siRNA knockdown of CAPNS2 (n = 3). (C) Representative immunofluorescence images of TGFβ-induced EnMT in PAVECs stained with E-cadherin (green), vimentin (red), and DAPI (blue). Scale bars, 100 μm. All quantitative data integrate each experiment performed in biologic quadruplicate. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, †P < 0.005, and ‡P < 0.001 by one-way ANOVA with Holm-Sidak post hoc test.

TGFβ induces CAPN9 expression by promoting translation with coordinated translation-dependent mRNA decay in NMuMG cells

We next sought to determine whether TGFβ-dependent induction of CAPN9 expression occurred at the level of transcription, translation, or both. We observed that upon TGFβ stimulation, Capn9 message diminished over time, whereas CAPN9 protein accumulated (fig. S5, A and B). Furthermore, cycloheximide-mediated translation inhibition attenuated TGFβ-dependent Capn9 message decay (fig. S5C); together, these data suggest translational regulation of Capn9 with coordinated translation-dependent mRNA decay. These findings parallel the regulation of expression of other EMT-inducing factors that have been demonstrated in NMuMG cells. For example, previous work showed that TGFβ induced the activation of AKT2, which, in turn, phosphorylated hnRNPE1, a factor that binds to hairpin structures in the 3′ untranslated region of Ile1 and Dab2 (44). Upon phosphorylation, hnRNPE1 is displaced from these cis-regulatory elements, allowing release of translation. To test the hypothesis that a similar mechanism is responsible for the regulation of CAPN9 expression in our system, we inhibited AKT2 phosphorylation with the competitive inhibitor CCT128930 in TGFβ-stimulated NMuMG cells. Contrary to our hypothesis, neither mRNA decay nor protein accumulation was prevented by AKT2 inhibition in the presence of TGFβ (fig. S5, D and E). Curiously, there appears to be heterogeneity in the cellular mechanisms regulating Capn9 expression, as TGFβ induced Capn9 message in MDCK cells (fig. S5F). These phenomena are likely specific to TGFβ exposure, as Wnt activation via WNT3A exposure failed to promote EMT or CAPN9 protein expression in NMuMG cells (fig. S5G).

Capn9−/− mice are resistant to bleomycin-induced lung fibrosis

We next sought to define the therapeutic potential of calpain inhibition in animal models of fibrosis. We used mice lacking CAPN9 function that were generated by deleting Capn9 exon 3, which contains the catalytic cysteine in the peptidase domain (32). Deletion of the 119 nucleotides corresponding to exon 3 is predicted to create a frameshift and hence a premature termination codon (PTC) a short distance (six codons) into exon 4 in mature mRNA. We confirmed these predictions using reverse transcription polymerase chain reaction (RT-PCR) and sequencing of complementary DNA (cDNA) (fig. S6A). Contrary to the prediction, the PTC did not initiate degradation of the mutant transcript via nonsense-mediated mRNA decay (Fig. 6B). Loss of expression of CAPN9 protein was confirmed using one commercially available antibody (Abnova) but not using a different reagent (Sigma), which detected a protein of the predicated mass for all dimeric calpains in both the stomach and lung of wild-type and Capn9−/− animals, indicating lack of specificity for CAPN9. The Abnova antibody appears to be specific for CAPN9 but cross-reacts with a slightly smaller protein in the lung, highlighting the limitations of these reagents for certain applications (fig. S6C). Last, we surveyed the expression of the dimeric calpain subunits Capn1/2/s1/s2 and the endogenous calpain inhibitor, Cast, in Capn9−/− animals. These data document that Capn9−/− animals can show a compensatory increase but are not deficient in any of these calpain family members (fig. S6D).

Experimentally induced lung fibrosis is commonly achieved via exposure to the chemotherapeutic agent bleomycin. Systemic delivery of bleomycin by a subcutaneously implanted osmotic pump results in penetrant subpleural fibrosis (45). Compared to wild-type mice, Capn9−/− mice exposed to bleomycin were protected from fibrosis, as assessed histologically by Masson trichrome staining (Fig. 4A), blinded histological observation (Fig. 4B), and total lung collagen content (Fig. 4C). Bleomycin lung injury in wild-type mice was associated with a dramatic mortality increase; however, Capn9−/− mice showed no mortality despite systemic delivery of bleomycin (Fig. 4D) and exhibited preserved lung function tests, as monitored by respiratory resistance, total lung capacity, and lung compliance (Fig. 4, E to G). Fibrosis in wild-type animals exposed to bleomycin was accompanied by an increase in the number of αSMA-positive myofibroblasts in the lung interstitium, whereas αSMA immunoreactivity was restricted to airways and blood vessels in vehicle-treated wild-type or bleomycin-treated Capn9−/− animals (Fig. 4H). Robust up-regulation of Capn9 message, but not that for other dimeric calpains, accompanied myofibroblast accumulation in wild-type animals as detected by RT-PCR and in situ RNA hybridization (RNA-ISH) (Fig. 4I). Cells that expressed Capn9 in bleomycin-treated lungs also expressed Acta2, indicating up-regulation of Capn9 expression in myofibroblasts (Fig. 4J).

Fig. 4 Capn9−/− mice are protected from bleomycin-induced lung fibrosis.

(A) Representative low-power Masson trichrome–stained lung from wild-type mice treated with saline or bleomycin and Capn9−/− mice treated with bleomycin. Scale bar, 500 μm. (B) Fibrosis scores of mouse lung stained with Masson trichrome (n = 3 to 8). (C) Collagen content of lungs from Capn9−/− or wild-type control mice treated with saline or bleomycin (Bleo) (n = 4 to 6). (D) Survival curves of Capn9−/− and wild-type control mice with saline or bleomycin treatment. Results are combined from three independent experiments. (E) Respiratory resistance (n = 3 to 6). (F) Total lung capacity (n = 3 to 4) and (G) lung compliance (n = 3 to 6) of saline (Sal) and bleomycin-treated wild-type and Capn9−/− mice. (H) Representative immunofluorescence of mouse lung stained for αSMA (red) or DAPI (blue). Scale bar, 100 μm. (I) Calpain mRNA normalized to Gapdh in wild-type mouse lung with saline (S) or bleomycin (B) treatment (C1/2/9/s1/s1 are Capn1/2/9/s1/s2, respectively, n = 3 to 5), and (J) representative Capn9 (top: scale bar, 20 μm, insets are ×1.5 magnifications) and Acta2 and Capn9 (bottom: scale bar, 10 μm) RNA-ISH of mouse lungs. Dot plot data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, †P < 0.005, and ‡P < 0.001 by one-way ANOVA with Holm-Sidak post hoc test (B, C, and E to G), log-rank test (D), and Student’s t test (I).

To ensure that the protection from lung fibrosis afforded by CAPN9 deficiency is not due to relative immunosuppression of Capn9−/− animals, we administered intratracheal bleomycin to induce a robust inflammatory response to injury. One week after insult, both wild-type and Capn9−/− lungs showed intense inflammatory infiltrates (fig. S7A). Flow cytometric analyses demonstrated no differences in the proportions of CD45+ cell subsets in response to bleomycin injury in Capn9−/− compared to wild-type animals (fig. S7B).

Capn9−/− mice are protected from carbon tetrachloride–induced liver fibrosis

Chronic carbon tetrachloride (CCl4)–induced liver injury drives periportal inflammation and fibrosis in mice (46). Wild-type mice treated with CCl4 for 4 weeks developed bridging fibrosis, whereas Capn9−/− mice were protected from fibrosis, as shown by low-power bright-field picrosirius red staining (Fig. 5A), birefringence of mature collagen fibrils under polarized light (Fig. 5B), blinded histologic scoring (Fig. 5C), and total liver collagen content (Fig. 5D). Robust up-regulation of Capn9 message was detectable adjacent to fibrotic tracks in CCl4-treated wild-type animals by RNA-ISH (Fig. 5E). These histologic changes were accompanied by markers of liver damage such as elevated serum concentration of the liver enzymes alanine transaminase and aspartate transaminase. Wild-type and Capn9−/− mice receiving injections of CCl4 had equal injury as indicated by liver function tests (Fig. 5F); however, unlike wild-type controls, Capn9−/− animals developed minimal fibrosis.

Fig. 5 Capn9−/− mice are protected from CCl4-induced liver fibrosis.

(A) Representative low-power bright-field images of picrosirius red–stained slides of mouse liver. Scale bar, 400 μm. (B) Representative picrosirius red–stained livers under polarized light. Scale bar, 200 μm. (C) Fibrosis scores of mouse liver stained with picrosirius red (n = 4 to 5). (D) Collagen content in livers from Capn9−/− or wild-type control mice treated with corn oil or CCl4 (n = 4 to 5). (E) Representative RNA-ISH of Capn9 in wild-type control mouse liver from mice treated with corn oil or CCl4 (red; scale bar, 100 μm). (F) Serum liver function tests of mice with or without injury (n = 4 to 5). Data are expressed as mean ± SEM. ns, not significant (P > 0.05), †P < 0.005, and ‡P < 0.001 by one-way ANOVA with Holm-Sidak post hoc test (C and D) and two-way ANOVA with Tukey’s post hoc test (F). Treatment effect was significant (P < 0.0001).

Capn9−/− mice are protected from angiotensin-2–induced cardiac fibrosis

Angiotensin-2 (Ang II) is capable of inducing a fibrotic synthetic repertoire in cardiac fibroblasts in vitro and in vivo (4750). In wild-type animals, Ang II infusion results in cardiac arterial intimal proliferation and perivascular collagen accumulation with collagen fibers extending into the cardiac interstitium (51). Here, we showed that Capn9−/− mice treated with Ang II were protected from perivascular and interstitial fibrosis, as shown by Masson trichrome staining (Fig. 6, A and B), total heart collagen content (Fig. 6C), and αSMA immunoreactivity (Fig. 6D). Despite systolic blood pressures equivalent to wild-type animals receiving Ang II (Fig. 6E), Capn9-deficient animals showed preservation of Ang II–induced left ventricular cardiac function as monitored by echocardiography (Fig. 6F).

Fig. 6 Capn9−/− mice are protected from Ang II–induced cardiac fibrosis.

(A) Representative Masson trichrome–stained slides of wild-type and Capn9-targeted mouse hearts from mice treated with saline or chronic Ang II infusion. Scale bars, 100 μm. (B) Quantification of blue-stained collagen on trichrome slides by ImageJ (n = 4 to 6). (C) Collagen content in hearts from Capn9−/− or wild-type control mice treated with saline (Sal) or Ang II (n = 5 to 8). (D) Representative images of mouse hearts from groups as in (A), stained for SMA (red) or DAPI (blue). Scale bars, 100 μm. (E) Systolic blood pressure of wild-type and Capn9−/− (C9−/−) animals receiving Ang II infusion (n = 4 to 5). (F) Measures of left ventricular function including ejection fraction and fractional shortening in mice treated with Ang II (n = 5 to 8). Data are expressed as mean ± SEM with statistical comparison between Ang II–treated wild-type mice and all other conditions. ns, not significant (P > 0.05), *P < 0.05, **P < 0.01, †P < 0.005, and ‡P < 0.001 by one-way ANOVA with Holm-Sidak post hoc test (B, D, and F) and two-way ANOVA with Tukey’s post hoc test (E). Treatment effect was significant (P < 0.0001).

Calpain inhibition reverses established lung fibrosis

To determine the therapeutic utility of calpain inhibition in clinically relevant disease models, we explored the ability of calpain inhibitors to suppress an established predisposition for lung fibrosis. Intratracheal delivery of bleomycin results in persistent lung fibrosis and physiologic defects for at least 6 months after the injury (52). Wild-type mice receiving therapeutic dosing of MDL-28170 1 week after intratracheal delivery of bleomycin showed full normalization of Col1a1 message expression, suggesting an overt arrest of ongoing fibrotic potential (Fig. 7, A and B).

Fig. 7 Therapeutic dosing of MDL-28170 ameliorates early established lung fibrosis.

(A) Representative Col1a1 RNA-ISH slides of mouse lungs 3 weeks after saline or bleomycin treatment and therapeutic vehicle or MDL dosing (delivered 1 week after bleomycin). Scale bars, 500 μm. (B) Quantification of red Col1a1 RNA-ISH staining (n = 14 to 15). Data are expressed as mean ± SEM. ns, not significant (P > 0.05) and ‡P < 0.001 by one-way ANOVA with Holm-Sidak post hoc test.

Human genetics suggests tolerance for CAPN9 deficiency

In humans, loss-of-function CAPN9 variants do not associate with a signal for intolerance (probability for loss-of-function intolerance, pLI = 0.00) (53). A polymorphic variant (rs28359631) that substitutes the obligate A at the −2 position of the splice acceptor for exon 4 is predicted to cause exon skipping and a consequent frameshift, leading to nonsense-mediated mRNA decay. This allele is common in the Genome Aggregation Database with a collective allele frequency of 1.0%. It is enriched in populations of East and South Asian ancestry (8.3 and 3.2% allele frequency, respectively) with homozygosity observed at rates compatible with Hardy-Weinberg equilibrium (0.8 and 0.1%, P = 0.47 and 0.43, respectively).

DISCUSSION

Despite the substantial contribution of fibrosis to global disease burden, few therapies target the accumulation or function of the cell type primarily responsible for pathologic ECM production, the myofibroblast (9). The lack of effective therapies reflects the tremendous difficulty in targeting fibrosis, a pathology that develops from the co-optation of complex biological processes involved in tissue development and injury response, including EMT (54). Moreover, fibrosis is a common—and indolent—pathologic outcome of a group of highly heterogeneous disorders, compounding the challenge to achieve a comprehensive understanding of disease pathogenesis and vulnerabilities. Nevertheless, the recent observation that curative hepatitis treatment culminates in reversal of liver fibrosis establishes that a fibrotic ECM is a modifiable consequence of disease (5557).

Here, we used in vitro assays of TGFβ-induced mesenchymal transition to investigate the role of calpains in the differentiation of myofibroblasts from various cell types. Our data show that TGFβ induces calpain activity, whereas inhibition of calpains prevents the induction of a mesenchymal phenotype. In the setting of calpain inhibition, early TGFβ signaling events, such as pSMAD2 nuclear accumulation and the induction of EMT transcriptions factors like Snai1, are preserved. These findings suggest that the relevant calpain-dependent events mediating TGFβ-induced EMT are distal in the differentiation or maturation of myofibroblasts. We were initially surprised to observe that TGFβ can cause myofibroblast differentiation using calpain isoforms that are not normally expressed in the progenitor cell but are rapidly and potently induced by TGFβ. In retrospect, this appears to be an elegant strategy to tightly regulate a process that is necessary and productive in development and wound healing but can prove deleterious if left unchecked. Restriction of this myofibroblast-promoting activity to calpains that are recruited after a fibrogenic stimulus, such as TGFβ, may prevent initiation of this pathway in response to physiological stimuli that normally result in activation of the constitutively expressed calpains. Prior work has demonstrated TGFβ-induced translational up-regulation of other EMT-inducing factors in NMuMG cells including DAB2 and ILE1, albeit by a different mechanism than observed for CAPN9 (44). Our data do not preclude the involvement or sufficiency of other calpains or calpain-independent mechanisms in mesenchymal transition in other cell types and contexts; this appears to be required, given the observation of normal development and tissue homeostasis in Capn9-deficient animals (32). Moreover, a predicted loss-of-function mutation in humans is relatively common, found in homozygosity, and appears to be in Hardy-Weinberg equilibrium, suggesting tolerance for CAPN9 deficiency. Although congenital loss of a gene is an imperfect model of therapeutic antagonism, these observations suggest the potential for a broad therapeutic window.

Our data build upon previous studies that show protection from bleomycin-induced lung fibrosis (26, 58) and Ang II–induced cardiac fibrosis (22) upon broad-spectrum calpain inhibition with calpeptin treatment or CAST overexpression, respectively. Both calpeptin and CAST are antagonists of the ubiquitous dimeric calpains, as well as CAPN9, and calpeptin inhibits other cysteine proteases including cathepsins (43, 59). Our demonstration of a specific role for Capn9 and Capns2 in fibrosis and in TGFβ-induced myofibroblast differentiation expands upon the known function of these dimeric calpain subunits. Existing literature describes CAPN9 function as largely restricted to the gastrointestinal tract, where coexpression of CAPN9 with CAPN8 in gastric pit cells forms a complex, termed G-calpain, that is involved in gastric mucosal injury response (32, 43). The tissue-restricted alternative small subunit, CAPNS2, appears to be largely redundant with CAPNS1 in vitro. However, CAPNS2 binds to the large catalytic subunits with lower affinity, does not undergo activation-mediated autolysis, and conveys some substrate specificity differences in vivo (28, 60).

The development of clinically useful calpain small-molecule inhibitors has been hampered by the challenge of developing agents with specificity for calpains over other cysteine proteases, such as some cathepsins. Despite these challenges, the existence of CAST, a highly specific inhibitor of dimeric calpains, indicates that the design of calpain-specific inhibitors is possible. Crystal structures of CAPN2-CAST complexes reveal that although CAST is an unstructured protein in solution, in the presence of calcium, it binds to calpain and wraps over the active site (31, 61). Calpain amino acid residues that interact with CAST near the active site are highly conserved in each of the CAST-inhibited isoforms, whereas CAPN3, which escapes CAST inhibition, is divergent, suggesting that calpain specificity may be achieved by inhibitors that display affinity for amino acids adjacent to the active site (62). Furthermore, crystal structures of the protease cores of CAPN1 and CAPN9 point to a number of differences that may be exploited for the development of CAPN9-specific antagonists (61).

Despite this progress, a number of limitations to this study should be considered. First, the observation that CAPN9-deficient NMuMG cells fail to support efficient TGFβ-induced EMT despite abundant expression of CAPN1, CAPN2, and CAPNS1 suggests the importance of a CAPN9 cleavage event that remains to be identified. Although many calpain substrates have been recognized, the identification of calpain isoform–specific substrates has proved challenging (59). Knowledge regarding the precise function of CAPN9 that influences fibrosis in model systems will inform the clinical utility of strategies aimed at its specific antagonism. As stated above, the lack of small-molecule antagonists that display high bioavailability, potency, and specificity for CAPN9—or all dimeric calpains—limits the immediate clinical application of these findings. Last, our observations are largely based on prophylactic modulation of chemically induced models of fibrosis in small animals, which may have a limited ability to predict efficacy in human presentations of disease. Such issues will be best addressed using pharmacologic manipulations in both preclinical models and human clinical trials.

MATERIALS AND METHODS

Study design

The purpose of this study was to identify and explore a pathway in fibrosis that may be amenable to pharmacologic antagonism. We used validated in vitro assays with complementary antagonism strategies to provide strong evidence for the role of dimeric calpains in TGFβ-induced mesenchymal transition. To implicate a specific calpain isoform, we used siRNA knockdown to find evidence that CAPN9 and CAPNS2 were required for the adoption of a mesenchymal phenotype in vitro. Because we found strong evidence for the importance of CAPN9 in in vitro assays in multiple cell types, we acquired animals lacking CAPN9 function and evaluated their response to experimentally induced lung, liver, and cardiac fibrosis.

Sample sizes were determined on the basis of prior experience with similar studies and pilot experiments. All mice used were males to eliminate the potential confounding influence of differences due to sex in response to fibrogenic agents. All mice were randomly assigned to treatment groups, and all analysis was performed blinded to genotype and treatment condition. Dosing and treatment duration for bleomycin, CCl4, and Ang II were determined on the basis of prior literature. No surviving animals were excluded from analysis. No outliers were excluded. The number of biologic replicates is specified in the figure legends or represented by dot plots.

Mice

All mice were cared for in compliance with approved protocols from the Animal Care and Use Committee of the Johns Hopkins University School of Medicine. Capn9tm1Hiso mice were derived and backcrossed to C57BL/6J previously (32). Murine Capn9-targeted sperm was acquired from the RIKEN BRC through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan (RBRC04790), and targeted mice were generated by the Johns Hopkins Transgenics Core (Baltimore, MD) and maintained by sibling crosses. C57BL/6J male control mice were acquired from the Jackson Laboratory (Bar Harbor, ME). Male mice were used for all experiments. All mice were kept in a specific pathogen–free facility on a 12-hour light/12-hour dark cycle and provided food and water ad libitum.

Cell culture

All cells were cultured at 37°C and 5% CO2. NMuMG cells [CRL-1636, the American Type Culture Collection (ATCC)] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (11965-118, Gibco), supplemented with 10% fetal bovine serum (FBS; F4135, Sigma), 2 mM l-glutamine (25030081, Thermo Fisher Scientific), and insulin (10 μg/ml) (12585014, Thermo Fisher Scientific). NHLFs (CC-2512, Lonza) were cultured with fibroblast growth media-2 and supplements (C-3132, Lonza). MDCK cells (CCL-34, ATCC) were cultured in minimum essential media (11095098, Thermo Fisher Scientific) supplemented with 10% FBS, 2 mM l-glutamine, and penicillin-streptomycin (1000 U/ml). PAVECs were isolated as previously described (63), grown in flasks coated with rat tail collagen I (50 μg/ml) (354236, BD Biosciences), and cultured in DMEM supplemented with 10% FBS, penicillin-streptomycin (1000 U/ml), and heparin (50 U/ml) (H3393, Sigma-Aldrich).

Small-molecule inhibitors

MDL-28170 (BML-PI130, Enzo Life Sciences), calpeptin (0448, Tocris), 2-APB (D9754, Sigma), CA-074-OMe (C5857, Sigma), SB431542 (S4317, Sigma), CCT128930 (S2635, Selleckchem), and cycloheximide [2112S, Cell Signaling Technologies (CST)] were prepared in stock solutions according to the manufacturer’s instructions.

In vitro EMT assays

NMuMG cells and MDCK cells were cultured and grown to confluency as described above. To induce EMT, cells were starved in media containing 0.5% FBS and pretreated with pharmacological inhibitors 24 hours before treatment with TGFβ1 at stated concentrations (240-B-010, R&D Systems). Culture media were replaced every 24 hours during all experiments. For some experiments, recombinant mouse Wnt3a (ab81484, Abcam) was used to stimulate serum-starved NMuMG cells as above. For reversal, NMuMG cells were starved for 24 hours, stimulated with TGFβ1 for 72 hours, and then incubated with MDL-28170 at 10 or 20 μM in starvation media for an additional 72 hours.

In vitro fibroblast-to-myofibroblast assay

NHLFs were subcultured as described above and then starved for 24 hours in fibroblast growth media containing no supplements with MDL-28170 at indicated concentrations. Cells were stimulated with TGFβ1 (10 ng/ml) with or without the presence of calpain inhibitor for 24 hours.

Transfection

NMuMG cells were transfected with full-length mouse CAST (MGC 3710078) inserted into pCMV-IRES-AcGFP (632435, Clontech) using Lipofectamine 2000 (11668019, Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were transfected for 5 days, split, and selected for stably integrated clones using geneticin (400 μg/ml) (10131027, Gibco). A polyclonal cell line was confirmed to express AcGFP by live cell fluorescent microscopy. For siRNA knockdown, NMuMG cells were transfected with a SMARTpool mixture of four siRNAs (GE Healthcare), targeting Capn1 (M-062006-01-0010), Capn2 (M-043027-01-0010), Capns1 (M-048840-01-0010), and Capns2 (M-014858-00-0010), or nontargeting control (D-001206-13-50). Two siRNAs targeting Capn9 were custom synthesized using previously validated sequences (59). Cells were transfected using Dharmafect 4 (T-2004-03, GE Healthcare) according to the manufacturer’s instructions at a final siRNA concentration of 100 nM. PAVECs were electroporated in solution using the Neon Transfection System (MPK10025, Invitrogen) as previously described (64). For CAPNS2 knockdown, siRNA targeting CAPNS2 (M-014858-00-0010, GE Healthcare) or nontargeting control (D-001206-13-50) were used. For CAST overexpression, PAVECs were electroporated in solution with plasmids encoding for either mouse CAST (pEF1α-Cast-IRES-IRFP) or empty vector.

Western blots

Cultured cells were harvested using radioimmunoprecipitation assay buffer (R0278, Sigma) and sonicated, and lysate protein concentration was quantified by bicinchoninic acid assay (23225, Pierce). Equal quantity of protein was submitted to SDS–polyacrylamide gel electrophoresis on 10% bis-tris gels (3450113, Bio-Rad) in Mops running buffer (NP0001-02, Thermo Fisher Scientific) and transferred onto a methanol-activated Immobilon-FL polyvinylidene fluoride membrane (IPFL00010, Millipore) in NuPage transfer buffer (NP0006, Thermo Fisher Scientific). Membranes were blocked (927-40000, LI-COR Biosciences) and probed overnight at 4°C. Antibodies used were as follows: mouse anti-αSMA (MAB 1420, R&D clone 1A4) at 1:1000, goat anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (sc-20457, Santa Cruz Biotechnology) at 1:5000, mouse anti-phosphoSMAD2 (04-953, Millipore clone A5S) at 1:500, rabbit anti–filamin A C-terminal fragment (2242-1, Epitomics) at 1:15,000, mouse anti-AcGFP (632380, Clontech clone JL-8) at 1:1000, rabbit anti-CAPN1 (2556, CST) at 1:1000, rabbit anti-CAPN2 (2539, CST) at 1:1000, mouse anti-CAPN9 (H00010753-M02, Abnova) at 1:1000, rabbit anti-CAPN9 (HPA020398, Sigma) at 1:1000, rabbit anti-CAPNS1 (ab92356, Abcam) at 1:1000, rabbit anti-CAPNS2 (LS-C133503, Lifespan Biosciences) at 1:1000, and rabbit anti–phospho–β-catenin (Ser552) (9566, CST). Species-appropriate secondary antibodies conjugated to IRdye 700 or IRdye 800 (LI-COR Biosciences) were used according to the manufacturer’s guidelines and analyzed using the LI-COR Odyssey system. Quantification was performed on each blot by normalizing to GAPDH loading control.

Immunofluorescence

NMuMG cells were plated and grown on sterilized glass coverslips and submitted to EMT assays as described above. Coverslips were fixed with 4% paraformaldehyde (PFA, EMS 15710) in phosphate-buffered saline (PBS) at room temperature and blocked in buffer (5% donkey serum and 0.1% Triton X-100 in PBS) at room temperature. Slides were incubated with mouse anti–E cadherin (610405, BD Biosciences) 1:500 in blocking buffer overnight at 4°C and then probed with donkey anti-mouse Alexa 488 (A-21202, Molecular Probes) 1:500, rhodamine-phalloidin (R415, Molecular Probes) 1:500, and 4′,6-diamidino-2-phenylindole (DAPI) (D1306, Molecular Probes) 1:25,000 for 1 hour at room temperature in the dark. After washing with PBS, coverslips were mounted on slides with ProLong Gold Mountant (P10144, Molecular Probes). Images were obtained with a Zeiss LSM710. For pSMAD2, NMuMG cells were plated, incubated in starvation media with 20 μM MDL-28170 pretreatment for 24 hours, stimulated with TGFβ1 (10 ng/ml) for 1 hour, and then washed and fixed in 4% PFA for 15 min at room temperature. Cells were permeabilized by incubating in 0.1% Triton X-100 for 5 min, blocked for 20 min, and incubated with a rabbit anti–phospho-SMAD2 antibody (04-953, 1:1000; MilliporeSigma) overnight in buffer [5% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS]. The cells were then probed with donkey anti-rabbit immunoglobulin G Alexa Fluor 594 (A-21207, Thermo Fisher Scientific) and mounted using VECTASHIELD with DAPI (H-1200, Vector Laboratories). Images were obtained with a Zeiss LSM 780. PAVECs grown on sterilized glass coverslips were fixed in 4% PFA overnight at 4°C, permeabilized with 0.2% Triton X-100 for 10 min, and incubated overnight at 4°C in 1% BSA, followed by another 4°C overnight incubation with either rabbit antihuman E-cadherin 1:100 (ab53033, Abcam) or mouse antihuman vimentin 1:100 (V9, Invitrogen). Samples were exposed to species-appropriate secondary antibodies conjugated to Alexa Fluor 488 or 568 (Invitrogen) at 1:100 in 1% BSA for 2 hours at room temperature and then mounted with media containing DAPI (H-1500, Vector Laboratories). Formalin-fixed paraffin-embedded tissue was sectioned at 5 μm, deparaffinized in xylene, rehydrated, and blocked for 1 hour at room temperature (PBS, 0.1% Tween 20, and 5% normal goat serum). Blocked samples were incubated with mouse anti–αSMA-Cy3 (C6198, Sigma clone 1A4) 1:1000 overnight at 4°C. Slides were counterstained with DAPI and mounted. Images were acquired on a Zeiss LSM710 (lung) or a Leica DMi8 (heart). Post-acquisition processing was performed equally for all representative images in a figure panel.

RNA analysis

RNA was harvested from cells using the RNeasy Kit (74106, Qiagen) with deoxyribonuclease (DNase) digestion (79254, Qiagen). RNA was harvested from dissected mouse organs after homogenization in an automatic bead homogenizer (FastPrep-24, MP Biomedicals) in TRIzol (15596018, Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was generated using a high capacity RNA to cDNA kit (4387406, Applied Biosystems). Quantitative PCR was done using TaqMan probes for mouse Col1A1 (Mm00801555_g1), Mmp2 (Mm00439498_m1), Mmp9 (Mm00442991_m1), Gapdh (Mm99999915_g1), Snai1 (Mm00441533_g1), Snai2 (Mm00441531_m1), Twist1 (Mm00442036_m1), Twist2 (Mm00492147_m1), Zeb1 (Mm00495564_m1), Zeb2 (Mm00497193_m1), porcine ACTA2 (Ss04245588_m1), CDH1 (Ss03377287_u1), VIM (Ss04330801_gH), MMP2 (Ss03394318_m1), MMP9 (Ss03392092_g1), and GAPDH (Ss03375629_u1). Canine quantitative PCR was performed using SYBR Green (4309155, Thermo Fisher Scientific), and canine CAPN9 primers were as follows: sense (5′-GCAGAGACCTTCGCAACTAA-3′) and antisense (5′-GCTGCATTTCTGGTATCAATGG-3′); canine GAPDH primers were as follows: sense (5′-AACATCATCCCTGCTTCCAC-3′) and antisense (5′-GACCACCTGGTCCTCAGTGT-3′). Samples were run on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher Scientific). Threshold cycle (Ct) values were corrected for loading and calculated using the 2−ΔCt method.

cDNA gel electrophoresis of Capn9 amplicons was performed by RNA isolation and total cDNA synthesis (as above). Amplification of mouse Capn9 cDNA was performed with Flash Phusion (F548L, Thermo Fisher Scientific) for 40 cycles with the following primers: exon 1 sense (5′-CTTTGTGTGGAAACGGCCAG-3′), exon 3 sense (5′-AGAAAGCACTGACCAGGGTG-3′), exon 5 sense (5′-TGGAAGACTTCACTGGGGGT-3′), exon 9 sense (5′-TGCAACCTCACACCTGATGC-3′), exon 5 antisense (5′-TCCATGGCTTCAATGGCACT-3′), exon 7 antisense (5′-CCCAAGGGTTACGGACTCTG-3′), exon 9 antisense (5′-AGCTTCCTTGGTGGATGGTC-3′), exon 11 antisense (5′-TCCCTACTCAGGTGTCCGTC-3′), and exon 13 antisense (5′-TGTGGAGTCGGCTTTGGAAG-3′). Mouse Gadph cDNA amplicons were generated with primers 5′-CAGGAGAGTGTTTCCTCGTCC-3′ and 5′-TTCCCATTCTCGGCCTTGAC-3′. Band intensities were measured with ImageJ [National Institutes of Health (NIH)] and normalized to Gapdh loading control.

RNA in situ hybridization

Formalin-fixed paraffin-embedded histology sections were pretreated with the RNAscope Target Retrieval Kit (322000, ACD) and then hybridized with the Mm-Capn9-O1 probe (487221, ACD), Mm-Acta2 (319531, ACD), or Mm-Col1a1 (319371, ACD). Immunofluorescent detection was performed with the RNAscope Fluorescent Multiplex Detection Kit (320851, ACD); chromogenic detection was performed with the RNAscope 2.5 HD Red Kit (322350, ACD) according to the manufacturer’s instructions. Fluorescent maximum intensity projection images were captured on a Zeiss LSM780 using a 40× objective. Image analysis of Col1a1-stained lung sections was performed by OracleBio using the Indica Labs HALO platform. After quality assessment, slides were scanned, and total lung tissue, excluding large bronchial and vascular structures, was selected for analysis. Data were output as Col1a1 message positivity (red staining) per total tissue area.

Histology

Tissue was fixed with 10% formalin for 24 hours before embedding in paraffin and sectioning for histological staining. Masson trichrome staining was conducted by AML Laboratories (St. Petersburg, FL) for the lung and liver histology and the Reference Histology Laboratory at Johns Hopkins University (Baltimore, MD) for the heart histology. Picrosirius red staining was performed using standard techniques. Lung and liver histology images were captured by whole-slide scan performed by HistoTox Labs (Boulder, CO). Histological scoring of lung and liver histology was performed by a blinded histopathologist (LNH ToxPath Consulting, LLC).

Bleomycin delivery and induced lung fibrosis

Bleomycin was delivered by osmotic pump as previously described (45). Briefly, osmotic minipumps (1007D, Alzet) designed to deliver their contents at 0.5 μl/hour for 7 days containing either 100 μl of saline vehicle or pharmaceutical grade bleomycin (100 U/kg; National Drug Code # 00703-3155-01, Teva Generics) were implanted. Pumps were removed on day 10 as recommended by the manufacturer, and mice were euthanized on day 35. Intratracheal bleomycin was delivered as previously described (65). Briefly, bleomycin suspended in PBS was delivered to anesthetized male mice 6 to 7 weeks of age at a dose of 1.5 U/kg. One week after bleomycin delivery, animals were administered therapeutic treatment with MDL-28170 (100 mg/kg) twice daily by oral gavage (200 mg/kg total daily dose) or vehicle (0.5% methylcellulose) for 2 weeks.

CCl4 induced hepatic fibrosis in mice

Male C57BL/6J and Capn9−/− mice, aged 10 to 12 weeks, received intraperitoneal injections twice a week with CCl4 (1 ml/kg) diluted 1:7 in corn oil for 4 weeks. Mice were euthanized 3 days after final injection and subjected to whole-body perfusion with PBS via cardiac puncture. Serum was isolated from whole blood using blood collection tubes (365967, BD Biosciences) as per the manufacturer’s protocol. Liver enzyme serum concentrations were determined by the Johns Hopkins Department of Comparative and Molecular Pathology Phenotyping Core Facility.

Ang II delivery and plethysmography

Wild-type mice 6 weeks of age were anesthetized using isoflurane and an osmotic pump (2004, Alzet) delivering Ang II (A9525, Sigma) at 1.2 μg kg−1 min−1, or saline was implanted beneath the midscapular loose skin. Pumps were left for 28 days, and mice were euthanized for tissue collection. Blood pressures were measured by tail cuff plethysmography in the week before sacrifice. Mice were habituated to the system, and, on the following day, at least three blood pressure readings were obtained per mouse and averaged.

Hydroxyproline assay

Total collagen content was determined in freshly harvested lung tissue using the Hydroxyproline Assay Kit (MAK008, Sigma) and normalized to total protein content (23225, Pierce). Liver and heart total collagen content was determined from formalin-fixed paraffin-embedded tissue samples (QZBtotcol1, QuickZyme Biosciences) and normalized to protein content (QZBtotprot1, QuickZyme Biosciences) according to the manufacturer’s protocols.

Pulmonary lung function testing

Pulmonary function testing was performed on a randomly selected subset of animals using a flexiVent ventilator (SCIREQ) as previously described (66). Tidal volume was set to 0.2 ml of 100% oxygen at a rate of 150 Hz with a positive end-expiratory pressure of 3 cmH2O.

Lung flow cytometry

Wild-type and Capn9-targeted male animals received intratracheal bleomycin as described above. Seven days after bleomycin administration, animals were euthanized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and perfused with 10-ml of cold PBS. The right lung was tied off and removed for histology; the left lung was inflated with digestion solution containing collagenase A (1.5 mg/ml) (10103586001, Sigma-Aldrich), DNaseI (0.4 mg/ml) (4716728001, Sigma-Aldrich), 5% FBS, and 10 mM Hepes in Hanks’ balanced salt solution. Cell suspensions were prepared, and single-cell analysis was performed as previously described (67).

Left ventricle cardiac function testing

Left ventricular heart function was determined as previously described (68). In brief, ventral hair was removed with Nair the day before echocardiography. Echocardiography was performed on unsedated mice using a Vevo 2100 (VisualSonics) equipped with a 40-MHz linear transducer. M-mode echocardiography was acquired from the parasternal short-axis view of the left ventricle.

Statistical analysis

Data are expressed as the mean ± SEM. Differences in measured variables were assessed by using a two-tailed Student’s t test for single comparisons or one-way or two-way analysis of variance (ANOVA) followed by indicated post hoc corrections for multiple-comparison testing. Given limited sample sizes, data were assumed to be normally distributed, but this was not statistically tested. No surviving animals were excluded from analysis. Hardy-Weinberg calculations were performed by comparing observed population frequencies with calculated expected population frequencies with a chi-square test with 1 degree of freedom. Results were considered statistically significant at P < 0.05. No statistical method was used to predetermine sample size; however sample sizes were based off of prior experience with similar studies. All measurements were performed blinded. Prism 7 (GraphPad Software) was used for all statistical analysis. Individual subject-level data are provided in data file S1.

SUPPLEMENTARY MATERIALS

stm.sciencemag.org/cgi/content/full/11/501/eaau2814/DC1

Fig. S1. TGFβ stimulation induced pSMAD2 nuclear localization, Alk5 inhibition prevented EMT, and cathepsin B and L inhibition did not alter EMT.

Fig. S2. Broad-spectrum calpain inhibition induced mesenchymal-to-epithelial transition in NMuMG cells.

Fig. S3. Expression of dimeric calpain message in mouse tissue and expression of EMT transcription factors in NMuMG cells.

Fig. S4. Calpain inhibition suppressed mesenchymal transition in multiple cell types.

Fig. S5. TGFβ induced expression of CAPN9 protein with coordinated translation-dependent mRNA decay in NMuMG cells.

Fig. S6. Capn9−/− mice expressed stable Capn9 message that lacks exon 3.

Fig. S7. Bleomycin induced an equal inflammatory response in wild-type and Capn9−/− animals.

Data file S1. Individual subject-level data.

REFERENCES AND NOTES

Acknowledgments: We are thankful to C. Schaefer and other individuals at Blade Therapeutics for contributions during the review process. We thank H. Sorimachi and S. Hata (Tokyo Metropolitan Institute of Medical Science, Japan) for providing Capn9tm1His mice through RIKEN BRC. Funding: This work was supported by the Howard Hughes Medical Institute, the Scleroderma Research Foundation, the NIH (R01AR068379 and R01HL128745), the Charles T. Bauer Foundation, the Johns Hopkins University School of Medicine Medical Scientist Training Program (T32GM007309), the Predoctoral Training Program in Human Genetics (T32GM007814), and the Johns Hopkins University School of Medicine Cellular and Molecular Medicine Program. Confocal microscopes from the JHU Microscope Facility were funded by the NIH (S10RR024550 and S10OD016374). Author contributions: H.C.D. oversaw all aspects of experimental design and interpretation of data. J.T.B. and R.A.G. carried out experiments and analysis on PAVECs. D.H.K. performed experiments on NMuMG, MDCK, and NHLF myofibroblast transition and performed cloning. Animal experiments were performed by D.H.K., T.J.C., V.N., J.D.B., and E.G.M., and Y.C. assisted in animal husbandry. V.N., D.B., and R.R. aided in mouse echocardiography and analysis. W.M. aided in lung function testing. M.A.S.-S. carried out experiments of TGFβ-induced translation-dependent Capn9 transcript decay. D.S.W. aided in experimental design and analysis. S.H. generated the Capn9tmHiso mouse. The paper was written by J.D.B., D.H.K., and H.C.D. Competing interests: H.C.D. and D.H.K. are coinventors on patent application PCT/US2015/048739 entitled “Targeting Capn9/Capns2 Activity as a Therapeutic Strategy for the Treatment of Myofibroblast Differentiation and Associated Pathologies.” H.C.D. is also a founder and scientific advisor to Blade Therapeutics Inc. and has equity in the company. D.H.K. is an employee of and owns share in Blade Therapeutics Inc. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials. Capn9-targeted sperm was obtained from RIKEN BRC under a material transfer agreement.
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