Dopamine D1 Receptor Stimulates Cathepsin K-Dependent Degradation and Resorption of Collagen I in Lung Fibroblasts Ana M

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RESEARCH ARTICLE Dopamine D1 receptor stimulates cathepsin K-dependent degradation and resorption of collagen I in lung fibroblasts Ana M. Diaz Espinosa, Patrick A. Link, Delphine Sicard, Ignasi Jorba, Daniel J. Tschumperlin and Andrew J. Haak*

ABSTRACT Haak et al., 2018; Jun and Lau, 2018). The most common rodent Matrix resorption is essential to the clearance of the extracellular matrix model for pulmonary fibrosis involves a single intratracheal (ECM) after normal wound healing. A disruption in these processes administration of bleomycin in healthy young mice, which induces constitutes a main component of fibrotic diseases, characterized by an inflammatory and a fibrotic phase characterized by overexpression ∼ – excess deposition and diminished clearance of fibrillar ECM proteins, of collagen I. At 30 60 days after bleomycin administration such as collagen type I. The mechanisms and stimuli regulating ECM resolution of the injury, clearance of fibrotic ECM and repair of the resorption in the lung remain poorly understood. Recently, agonism of lung architecture is observed (Della Latta et al., 2015; Moeller et al., dopamine receptor D1 (DRD1), which is predominantly expressed 2008; Williamson et al., 2015). The reversibility of fibrosis in model on fibroblasts in the lung, has been shown to accelerate tissue repair and systems is not limited to the lung; resolution of liver, kidney, skin and clearance of ECM following bleomycin injury in mice. Therefore, we heart fibrosis is also observed in experimental animal models of each investigated whether DRD1 receptor signaling promotes the degradation disease (Jeong et al., 2016; Jun and Lau, 2018; Kantari-Mimoun of collagen type I by lung fibroblasts. For cultured fibroblasts, we found et al., 2015; Kim et al., 2013; Lemaire et al., 2016; Schuppan, 2015; that DRD1 agonism enhances extracellular cleavage, internalization and Weiskirchen et al., 2019). In humans, multiple medications, including lysosomal degradation of collagen I mediated by cathepsin K, which chemotherapies, promote interstitial lung scarring, collagen results in reduced stiffness of cell-derived matrices, as measured by deposition, and reduced pulmonary function that resolves upon atomic force microscopy. In vivo agonism of DRD1 similarly enhanced cessation of drug exposure (Schwaiblmair et al., 2012). Clinical cases fibrillar collagen degradation by fibroblasts, as assessed by tissue of liver, kidney and heart fibrosis have also been observed to be labeling with a collagen-hybridizing peptide. Together, these results reversible (Duffield, 2014; Friedman et al., 2013; Gourdie et al., implicate DRD1 agonism in fibroblast-mediated collagen clearance, 2016; Jun and Lau, 2018; Zoubek et al., 2017). Taken together, these suggesting an important role for this mechanism in fibrosis resolution. observations suggest that the functional pathology of overabundant ECM deposition in fibrosis, even in IPF, may not be irreversible, and This article has an associated First Person interview with the first author thus understanding the mechanisms that promote fibrosis resolution of the paper. may lead to new and effective therapies for this disease. Pulmonary ECM is a complex structure composed of fibrous KEY WORDS: Fibrosis, Resorption, Cathepsin K, Dopamine proteins, glycoproteins and proteoglycans, which together sustain the receptor, Resolution structural and functional integrity of the tissue (Haak et al., 2018; Zhou et al., 2018). Matrix homeostasis is maintained by the balance in INTRODUCTION synthesis and degradation of the ECM proteins by resident cells Approximately 3 million people worldwide are affected by idiopathic (Chang et al., 2020; Humphrey et al., 2014). Following injury, pulmonary fibrosis (IPF) (Martinez et al., 2017), a progressive fibroblasts deposit collagen I, among other proteins of the ECM, interstitial lung disorder with a median survival from 2 to 4 years after and then, with resident macrophages, participate in repairing and diagnosis (Richeldi et al., 2017). A hallmark of this disease is the remodeling the lung architecture (Frantz et al., 2010; Wynn and excessive and uncontrolled deposition of the extracellular matrix Vannella, 2016), a process guided by the coordinated secretion and (ECM) fibrillar proteins, primarily collagen type I, by activated activation of matrix metalloproteases (MMPs) and cysteine cathepsins, fibroblasts that contributes to scarring of the tissue, disruption of and internalization of collagen through macropinocytosis, endocytosis alveolar architecture and loss of respiratory function (Haak et al., or phagocytosis. Once internalized, collagen I is further degraded in 2018; Martinez et al., 2017; McKleroy et al., 2013; Richeldi et al., acidic lysosomal compartments by cathepsin proteases (Bonnans et al., 2017). Intriguingly, evidence from experimental models and clinical 2014; Fonovićand Turk, 2014; Pakshir and Hinz, 2018). A substantial case studies suggests that the pathological deposition of fibrotic portion of collagen is degraded by cells before ever being deposited in scarring is reversible (Chitra et al., 2013; Della Latta et al., 2015; the lung, a process that can be controlled by cAMP signaling (Rennard et al., 1982). Whether cAMP signaling also plays a role in clearance of already deposited ECM remains unclear. Department of Physiology & Biomedical Engineering, Mayo Clinic, Rochester, MN The dopamine receptor D1 (DRD1) is a Gαs coupled G-protein 55905, USA. coupled receptor (GPCR) that, in the lung, is expressed predominantly *Author for correspondence ([email protected]) in fibroblasts and upon activation increases cAMP (Haak et al., 2020). Treatment with dihydrexidine (DHX), a DRD1 agonist, inactivates I.J., 0000-0001-6434-1021; D.J.T., 0000-0002-5115-9025; A.J.H., 0000-0002- YAP/TAZ leading to reduced expression of ECM and ECM cross- 5323-253X linking genes, fibroblast contraction, proliferation and TGFβ-stimulated in vitro Handling Editor: John Heath collagen I accumulation , and increased expression of cathepsin

Received 28 April 2020; Accepted 2 November 2020 K (Haak et al., 2019), a gene associated with collagen I degradation and Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs248278. doi:10.1242/jcs.248278 clearance (Bühling et al., 2004; Vidak et al., 2019). Furthermore, during RESULTS the fibrotic ECM deposition phase of the bleomycin mouse model, DRD1 signaling promotes degradation of cell-deposited transgenic mice overexpressing cathepsin K present reduced collagen collagen type I deposition (Srivastava et al., 2008) and treatment with DHX accelerates To assess the capacity of fibroblasts to deposit and degrade ECM, we the resolution of lung fibrosis (Haak et al., 2019). adapted methods for studying cell-derived matrices using fibroblasts Here, we aimed to determine whether signaling through DRD1 seeded at high density (Cukierman, 2002; Franco-Barraza et al., actively promotes the in vitro and in vivo extracellular clearance and 2016). We stimulated collagen deposition by treating normal human degradation of collagen I by lung fibroblasts. We observed lung fibroblasts (NHLFs) with TGFβ for 3 days and collected total that treatment with DHX promotes the degradation of cell- protein (both cellular and extracellular) to observe collagen deposited collagen I through the activity of cathepsin K and its abundance at this time point (Fig. 1A). At day 3, we then treated internalization into lysosomal compartments, resulting in the reduced the cells with DHX, a dopamine D1 receptor agonist, for an additional stiffness of cell-derived ECM and enhanced fibroblast-mediated 24 h and again collected total protein (both cellular and extracellular) collagen degradation in vivo. Our results demonstrate that dopamine at day 4. In prior work, we confirmed the specificity of DHX effects D1 agonism switches fibroblasts from a state of matrix deposition to a acting via D1 receptor using both receptor-specific antagonists as state of matrix resorption, highlighting the dopamine D1 receptor as a well as D1 receptor siRNAs (Haak et al., 2019). Here, we observed an target for reversing ECM accumulation in pulmonary fibrosis. increase in collagen I deposition between control and TGFβ day 3,

Fig. 1. DRD1 signaling promotes degradation of cell-deposited collagen type I. (A,B) Protein expression of collagen I and Col1α1 telopeptide, and degradation products of Col1α1 telopeptide, from primary lung fibroblasts stimulated with 2 ng/ml TGFβ for 3 days, and subsequently treated for 24 h with 10 µM DHX (A) or 10 µM forskolin (B) in medium containing 0.1% FBS. Protein lysates were collected at day 3 and 4. A representative blot and the quantification performed via densitometry are shown. Results are expressed as a fold change relative to TGFβ. n=4 independent experiments. Statistical validation by RM one-way ANOVA with Dunnett’s multiple comparison test. (C) Protein expression of collagen I and Col1α1 telopeptide, from primary lung fibroblasts grown without TGFβ and treated for 24 h with or without 10 µM DHX. n=3 independent experiments. Statistical validation by paired t-test. (D) Collagenolytic activity of primary lung fibroblasts plated in wells pre-coated with DQ Collagen, then stimulated with 2 ng/ml TGFβ and 10 µM DHX for 24 h. Images were taken using a Cytation 5 Cell Imaging Reader. Scale bars: 1 mm (4× objective), 20 µm (zoom). n=4 independent experiments. Statistical validation by paired t-test. Results are expressed as the mean± s.e.m., with statistical significance represented by P-value. All conditions contain a final concentration of 0.1% DMSO. Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs248278. doi:10.1242/jcs.248278 and no significant change between TGFβ at day 3 and 4, verifying the The cells were then treated with DHX or vehicle control for 24 h. presence of a cell-derived collagen-rich matrix prior to cell culture At day 4, decellularization was performed just before AFM treatment with the DRD1 agonist. However, treatment with DHX microindentation. The mean and individual Young’s modulus for reduced the collagen I content previously deposited by day 3, all 50 measurements performed per independent experiment is shown indicative of loss or degradation, not simply halting of production in Fig. 2B (bottom left), and the average Young’s modulus value (Fig. 1A). determined for each independent experiment is also shown (bottom Collagen I is most commonly found as a triple helix formed by two right). Consistent with our prior observations of DHX-stimulated α1 chains and one α2 chain that undergo several post-translational collagen I degradation (Fig. 1), we found that DHX stimulation alters modifications prior to their assembly for fibril formation (Kadler the stiffness of the cell-derived ECM after 24 h of treatment. While et al., 1996). After leaving the intracellular compartment as pro- the magnitude of the changes in stiffness we observed are relatively collagen, propeptide cleavage takes place at both ends of the protein, modest, they span a large fraction of the difference in normal and mediated by procollagen proteinase, resulting in formation of the fibrotic lung ECM stiffness previously observed in experimental and telopeptide (Kadler et al., 1996). The resulting collagen peptide human lung fibrosis (Liu et al., 2015, 2010), consistent with a undergoes fibril formation and cross-linking to form mature fibers physiologically relevant effect size. To support that these results were that build the extracellular matrix (McKleroy et al., 2013). As a way to an effect on ECM degradation and not altered collagen cross-linking, specifically assess the extracellular collagen content in our system, we we measured lysyl oxidase (LOX) activity from cell culture used an antibody that binds to the telopeptide and analyzed the same conditioned medium treated with TGFβ and with or without DHX. experimental time course (Fig. 1A). The changes in the collagen I α1 Consistent with previous findings (Wei et al., 2017), we observed an (Col1α1) telopeptide at ∼150 kDa strongly correlated with the increase in LOX activity with TGFβ treatment, but D1 agonism had observed changes in total collagen I at ∼150 kDa, confirming that the no effect on LOX enzymatic activity (Fig. S1). changes we observe with DHX and TGF-β are consistently observed for both total and extracellular deposited collagen I. In addition, DHX promotes collagen I degradation in vivo enhanced degradation products were also observed in telopeptide Our prior work demonstrated significantly increased resolution of western blots for samples treated with DHX for 24 h, consistent with experimental lung fibrosis with prolonged treatment with DHX collagenolytic activity induced by activation of DRD1 (Fig. 1A). We (Haak et al., 2019). However, whether acute treatment specifically also treated cells on day 3 with forskolin to directly activate adenylyl increases fibroblast-mediated collagen degradation was not assessed. cyclase, one of the downstream effectors of D1 receptor activation, In order to determine whether DHX treatment can acutely promote and observed similar reductions in collagen I (Fig. 1B), consistent fibroblast-mediated collagen I degradation in vivo we studied tissue with a role for cAMP in these observations. We have previously from prior experiments in which bleomycin was administered shown that DHX promotes resolution of bleomycin-induced lung intratracheally to Col1α1–GFP+ mice at day 0 to initiate lung fibrosis; however, DHX treatment has no measurable effect on lung injury. On day 10 and 11, these mice were then treated during collagen content in healthy mice (Haak et al., 2019), suggesting that D1 ongoing fibrosis with two doses of DHX (5 mg/kg of body weight agonism effects are dependent on the activation state of lung fibroblasts. intranasal) or vehicle control, 24 and 2 h prior to collecting lungs for Here, we treated unstimulated (no TGFβ) cultured lung fibroblasts with sectioning and staining (Haak et al., 2019). Collagen-hybridizing DHX for 24 h and observed no reduction in total or extracellular peptide (CHP) is a synthetic peptide designed to identify cleaved collagen I (Fig. 1C). These results are consistent with DHX promoting collagen products. It contains a repeating sequence of glycine, proline collagen I degradation selectively in TGFβ-activated cells. and hydroxyproline that specifically binds to unfolded collagen To functionally confirm the collagenolytic activity following DHX chains denatured by proteolytic activity, thereby reconstructing treatment, we used DQ Collagen, a fluorescein-labeled collagen the triple-helical structure. In previous studies, CHP was shown to substrate that generates a fluorescent signal upon cleavage (Della abundantly stain lung sections during the resolution phase of Porta et al., 1999; Sameni et al., 2009). Treatment of fibroblasts with bleomycin-induced fibrosis in young mice, consistent with the DHX for 24 h stimulated the collagenolytic activity of lung hypothesis that collagen proteolysis is an endogenous component of fibroblasts, generating a ∼2.5-fold increase of the fluorescent signal lung fibrosis resolution (Hwang et al., 2017). Based on these findings, (Fig. 1B). This finding further supports the capacity for DRD1 we incubated the lung slices from our acute DHX study with CHP agonism in fibroblasts to promote cleavage of extracellular type I (Fig. 3A). In these mice collagen type I-producing cells express GFP, collagen. a convenient means for predominantly identifying fibroblasts in situ. We observed a statistically significant enhancement in colocalization DHX alters the stiffness of cell-derived ECM of CHP with GFP+ cells in DHX-treated compared to vehicle-treated To analyze the functional outcome of D1 receptor signaling on cell- groups (Fig. 3C) (no CHP control staining is shown in Fig. S2A). derived matrices, we adapted a decellularizing protocol from Thus at day 10 post bleomycin administration, acute treatment with previously published methods (Cukierman, 2002). In order to assess DHX appears to mimic the previous results where CHP staining was the integrity of the ECM and the effectiveness of the de-cellularization increased in mice during the spontaneous resolution phase of methodology, immunofluorescence labeling of collagen type I was bleomycin-induced fibrosis at day 28 post bleomycin (Hwang et al., performed on intact fixed cultures and cell-derived matrices after 2017). Interestingly, we did not observe an overall enhanced staining decellularization (Fig. 2A). Image analysis confirmed the absence of of CHP in the lung sections in the DHX-treated group (Fig. 3B), cell nuclei and presence of collagen I with preserved fiber alignment consistent with the selective expression of the D1 receptor on lung and no loss of staining intensity after decellularization (Fig. 2A). fibroblasts and DHX specifically enhancing fibroblast-mediated Using atomic force microscopy (AFM), we extended a previously collagen degradation in response to D1 receptor activation. Equally, developed assay for characterizing in vitro cell-derived matrix we did not observe enhanced colocalization of CHP with GFP+ cells elasticity (Haak et al., 2019). First, cell-derived matrices were in sham-treated healthy mice (Fig. S2B), consistent with DHX not produced by stimulating lung fibroblasts for 3 days with TGFβ and promoting collagen degradation in unstimulated cultured fibroblasts ascorbic acid, as previously described (Franco-Barraza et al., 2016). (Fig. 1C). 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Fig. 2. DHX alters the stiffness of cell-derived matrices. (A) Collagen I intensity of intact (cell containing) and decellularized cell-derived matrices from NHLFs treated for 6 days with 2 ng/ml TGFβ and 20 µg/ml ascorbic acid. Images were obtained using a confocal microscope at 20× magnification. n=5 independent experiments. Scale bar: 200 µm. (B) Stiffness of cell-derived matrices from normal human lung fibroblast plated at confluency in gelatin-coated dishes and stimulated with 2 ng/ml TGFβ and 20 µg/ml ascorbic acid for 3 days, prior to treatment with 10 µM DHX for 24 h. On day 4, cell-derived matrices were decellularized by incubating with 20 mM ammonium hydroxide and 0.5% Triton X-100, and analyzed by AFM. For each experiment, the Young’s modulus was characterized by AFM microindentation from two unique areas, with 25 indentations per area. Phase-contrast images were obtained using AFM at 200× magnification. n=4 independent experiments. Statistical validation by Mann–Whitney test (decellularized ECM) and paired t-test (Averages: Decellularized ECM). Scale bars: 200 µm (phase-contrast images), 50 µm (AFM tip). Quantitative results are expressed as the mean±s.e.m., with statistical significance represented by P-value.

DHX promotes lysosomal internalization of collagen I labeling collagen I degradation with CHP and observed similar Canonical ECM resorption by fibroblasts involves internalization into colocalization between LAMP1 and CHP in cells treated with DHX lysosomes (Bonnans et al., 2014). To test whether DRD1 agonism (Fig. S3). These findings confirm that D1 receptor agonism mediates stimulates this canonical resorption pathway, we used super-resolution internalization of extracellular collagen into lysosomes. microscopy to image primary lung fibroblasts immuno-labeled for Col1α1 telopeptide and lysosomal membrane protein 1 (LAMP1), DHX-mediated degradation of collagen I is dependent a major transmembrane protein in the lysosomal compartment on cathepsin K (Fig. 4A). DHX enhanced uptake of extracellular Col1α1 One of the main effector proteases involved in lysosomal collagen I telopeptide, and, as assessed from 3D reconstruction of z-stacks, degradation is cathepsin K (Bühling et al., 2004; Sprangers and compartmentalized consistently and significantly with LAMP1- Everts, 2019). Based on our findings of DHX promoting lysosomal labeled lysosomes (Fig. 4B). We also repeated this experiment compartmentalization of collagen I , we investigated the effects of D1 Journal of Cell Science

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Fig. 3. DHX promotes collagen degradation in vivo. Col1α1–GFP mice injured through intratracheal administration of bleomycin (50 μl at 1.2 U/kg body weight) or DPBS control on day 0 and on day 10 were treated intranasally with DHX (5 mg/kg body weight) or vehicle control, at both 24 h and 2 h prior to lung harvest. (A) Representative images of lung slices (8 bleomycin treated lungs and 9 bleomycin+DHX treated lungs) stained with b-CHP and imaged using an LSM 780. Scale bar: 30 µm. (B) CHP images from lung slices were analyzed to determine total amount of CHP per image field, a representation of collagen degradation. Using ImageJ, thresholds were set for each individual image by two investigators that were blind to the sample identification; each point represents the average of two thresholds. n=31 images from 8 bleomycin treated lungs, n=25 images from 9 Bleomycin+DHX treated lungs. (C) CHP and Col1α1–GFP images from lung slices were acquired using a Cytation 5 Cell Imaging Reader with a 10× magnification objective. Using ImageJ, a threshold in each image was set to the top 5% of pixels with greatest intensity for both channels. CHP pixels colocalized with GFP pixels were measured and quantified as the percentage of colocalized pixels per image field. n=31 images from 8 bleomycin-treated lungs, n=24 images from 9 bleomycin+DHX-treated lungs. Statistical validation by unpaired t-test. Results are expressed as the mean±s.e.m., with statistical significance represented by P-value. agonism on cathepsin K expression, maturation and its functional role TIMP4 using an MMP array. We did not observe any appreciable in ECM degradation. Stimulation of lung fibroblasts with TGFβ changes in MMPs or TIMPs after DHX treatment (Fig. S4), reduced transcript levels of the CTSK gene. However, dopamine enhancing our focus on CTSK. receptor activation promoted enhanced transcript levels of the CTSK GPCR signaling through cAMP has previously been shown to gene (Fig. 5A). At the protein level, cathepsin K is synthesized and enhance acidification of lysosomal compartments in a variety of cell secreted in a pro-format that is ∼43 kDa in size and upon cleavage for types (Coffey et al., 2014; Guha et al., 2012; Liu et al., 2008), and activation, the molecular mass is reduced to ∼29 kDa (Brömme, cathepsin K is known to be spontaneously activated at low pH 2013). Treatment with DHX enhanced the expression of the low within lysosomes (Fonovićand Turk, 2014; McQueney et al., molecular mass active form of cathepsin K in cells stimulated with 1997). We measured lysosomal pH using LysoSensor Yellow/Blue TGFβ (Fig. 5B). Aside from cathepsin K, the major proteases which DND-160, which exhibits predominantly yellow fluorescence in degrade triple-helical collagen I are MMPs. To observe changes in acidic environments and blue fluorescence in less-acidic MMP protein expression after D1 receptor agonism, we measured environments. Dual-emission measurements with this probe allow expression of MMP1, MMP2, MMP3, MMP8, MMP 9, MMP10 and ratiometric quantification of lysosomal pH (Liu et al., 2012, 2008).

MMP13, and their endogenous inhibitors TIMP1, TIMP2 and We validated the methodology using compounds known to increase Journal of Cell Science

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Fig. 4. DHX promotes in vitro lysosomal internalization of collagen I. Human lung fibroblasts were grown on a BioCoat™ eight-well culture slide in the presence of 2 ng/ml TGFβ and 20 µg/ml ascorbic acid for 6 days. At 24 h and 2 h prior to fixation, cells were treated with 10 µM DHX. Cells were fixed at day 6 and stained for LAMP1 and Col1α1 telopeptide. Z-stack images were taken on a super-resolution microscope with a 63× magnification objective (n=4, independent experiments). (A) Representative images and (B) quantification of colocalization from optically sectioned, 3D images performed with Imaris 8. Results were normalized to account for changes in cell volume or number of cells per image field, and are expressed as the fold change relative to TGFβ and as the mean±s.e.m. Statistical analysis by paired t-test. All conditions contain a final concentration of 0.1% DMSO. Scale bar: 30 µm. lysosomal pH, namely tamoxifen and chloroquine (Fig. S5), and There is strong experimental support for collagen degradation being then confirmed that both DHX and forskolin acidify the lysosomal an essential component of resolution in patients with liver, kidney and environment (Fig. 5C). heart fibrosis, as well as in the animal models for each disease To assess the effect of cathepsin K on collagen I degradation, (Duffield, 2014; Friedman et al., 2013; Gourdie et al., 2016; Iredale we inhibited cathepsin K activity with odanacatib (ODN), a highly et al., 2013; Jeong et al., 2016; Jun and Lau, 2018; Kantari-Mimoun selective and potent inhibitor developed for the treatment of et al., 2015; Lemaire et al., 2016; Weiskirchen et al., 2019; Zoubek postmenopausal osteoporosis (Chapurlat, 2015). As observed et al., 2017). Recently, we have identified DHX, acting through previously, TGFβ enhanced collagen I expression, which was DRD1 expressed predominantly on fibroblasts, to have beneficial reduced by treatment with DHX. However, when we inhibited effects in mouse models of lung and liver fibrosis, resulting in cathepsin K activity with ODN, the effect of DHX on reducing the reduced levels of collagen I in vitro and in vivo (Haak et al., 2019). collagen I levels was blocked, and the amount of collagen I present Here, we found that agonism of DRD1 receptor through DHX was greater than that observed in fibroblasts stimulated only with promotes fibroblast-mediated degradation of cell-derived collagen I TGFβ (Fig. 5C). A similar trend was observed for Col1α1 telopeptide, through the activity of cathepsin K, a major cysteine protease demonstrating that ODN also prevented the DHX-mediated decrease responsible for extracellular and intracellular collagen I degradation. in the extracellular collagen I content. Furthermore, ODN prevented DHX enhanced both expression and activation of cathepsin K, the appearance of collagen I degradation products induced by DRD1 consistent with coordinated engagement of a matrix resorption agonism with DHX (Fig. 5D). ODN treatment of cells in the absence program stimulated by DRD1 agonism (Fig. 6). of DHX did not enhance collagen I deposition (Fig. S6). These data Degradation of mature collagen is challenging due to its tight are consistent with cathepsin K playing a primary effector role in the triple-helical structure and crosslinking with other ECM proteins, collagen I degradation stimulated by DRD1 receptor agonism in which obstructs access to proteolytic sites (Kafienah et al., 1998; lung fibroblasts. Finally, we repeated the DQ collagen analysis and Sprangers and Everts, 2019). ECM degradation involves membrane- confirmed ODN largely prevented DHX mediated collagen bound and secreted enzymes; cathepsin K and members of the MMP degradation (Fig. 5E). family have been identified as proteases capable of degrading fibrillar Taken together, our results thus demonstrate that DRD1 agonism collagen (Sprangers and Everts, 2019). A recent finding directly stimulates extracellular degradation and intracellular resorption of implicated cathepsin K in fibroblast-mediated maintenance of collagen I by fibroblasts, representing a potentially important collagen homeostasis in ex vivo mice tendons (Chang et al., 2020). fibroblast-mediated mechanism for clearance of fibrillar ECM In addition, protein expression of cathepsin K in tendon fibroblasts during injury repair and fibrosis resolution. was associated with increased collagen α1andα2 protein expression (Chang et al., 2020), suggesting a feedback mechanism between DISCUSSION collagen deposition and degradation that maintains homeostasis of Fibrosis progression is associated with an increased deposition of the extracellular compartment. Our results support previous findings fibrillar collagens, with collagen I representing 80% of the collagen that fibroblasts synthesize and deposit collagen I in the extracellular content (McKleroy et al., 2013). As it is consolidated in the ECM, compartment (Chang et al., 2020) and demonstrate that acute this protein transforms the extracellular compartment into a denser stimulation of the dopamine D1 receptor with DHX rapidly engages and more-rigid structure (Karsdal et al., 2017). In IPF, treatment cathepsin K activity to shift fibroblasts toward net degradation of options that halt matrix deposition have been developed, but collagen I, a potentially important step in resorption of excess ECM. mechanisms that promote matrix resorption have not been fully Conventionally, cathepsin K is thought to degrade intracellular explored. In addition, degradation of collagen I without disrupting collagen I in the lysosomal compartment. However, it has also been tissue homeostasis is an important challenge that must be considered. suggested to play a major role in the degradation of fibrillar collagen Journal of Cell Science

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Fig. 5. DRD1-stimulated degradation of cell-deposited collagen I is mediated by cathepsin K. (A) CTSK expression from primary lung fibroblasts plated at confluency and stimulated with 2 ng/ml TGFβ and 10 µM DHX for 24 h, prior to RNA isolation. Data are expressed as a fold change relative to control, with GAPDH as the housekeeping gene. n=3 independent experiments. (B) Protein expression of cathepsin K from primary lung fibroblasts plated at 80% confluency and stimulated with 2 ng/ml TGFβ for 2 days, prior to treatment for 24 h with 10 µM DHX. Protein lysates were collected at day 3. Quantification performed via densitometry; results expressed relative to control. n=4 independent experiments. (C) Lysosomal pH measured using LysoSensor dual excitation/emission dye. Cells were treated with 10 µM DHX and 10 µM forskolin and incubated 20 min prior to fluorescence quantification. (D) Protein expression of collagen I, Col1α1 telopeptide, and Col1α1 degradation products from primary lung fibroblasts plated at 80% confluency and stimulated with 2 ng/ml TGFβ for 3 days, prior to treatment for an additional 24 h with 10 µM DHX and 3 µM ODN. Protein lysates were collected at day 4. A representative blot and the quantification performed via densitometry are shown; results are expressed relative to TGFβ. n=4 independent experiments. In A–D, statistical validation is by RM one-way ANOVA with Dunnett’s multiple comparison test. Results are expressed as the mean±s.e.m., with statistical significance represented by P-value. All conditions contain a final concentration of 0.1% DMSO. (E) Collagenolytic activity of primary lung fibroblasts plated in wells pre-coated with DQ Collagen, then stimulated with 2 ng/ml TGFβ, 10 µM DHX, and 3 µM ODN for 24 h. Images were taken using a Cytation 5 Cell Imaging Reader. Scale bars: 1 mm (4× objective), 20 µm (zoom). n=4 independent experiments. Statistical validation by paired t-test. Results are expressed as the mean±s.e.m., with statistical significance represented by P-value. All conditions contain a final concentration of 0.1% DMSO. in the extracellular compartment (Bonnans et al., 2014; Chang et al., exerts a collagenolytic effect through cAMP elevation, we treated 2020; Haak et al., 2018; Kafienah et al., 1998; Sprangers and Everts, fibroblasts with forskolin, a positive stimulator of cAMP production 2019). Transgenic mice globally overexpressing CTSK are protected and observed similar effects to those seen upon treatment with against collagen deposition and fibrosis in the lung following DHX. Furthermore, GPCR signaling through cAMP has previously bleomycin injury (Srivastava et al., 2008), in line with the beneficial been shown to enhance acidification of lysosomes in a variety of effect of treating bleomycin-injured mice with DHX (Haak et al., cells (Coffey et al., 2014; Guha et al., 2012; Liu et al., 2008), and 2019). Interestingly, transcript levels of CTSK are highly expressed cathepsin K is known to be spontaneously activated at low pH in fibroblasts from patients with IPF (Bühling et al., 2004), within lysosomes (Fonovićand Turk, 2014; McQueney et al., suggesting that, potentially, mechanisms that regulate maturation 1997), providing a plausible mechanism linking DRD1 agonism to and activation of cathepsin K might be crucially important to the enhanced collagenolytic activity observed. Here, we report regulate collagen degradation and resorption in IPF. While we have reversal of the collagenolytic effects of a D1 receptor agonist when not studied the link between DHX treatment and enhanced we co-treated the cells with a cathepsin K inhibitor, both in total cathepsin K activity in detail, our previous work has demonstrated collagen I and in extracellular Col1α1 telopeptide, indicating a role that DHX, acting through DRD1, stimulates cAMP signaling in for cathepsin K in extracellular collagen degradation stimulated by lung fibroblasts (Haak et al., 2019). To investigate whether DHX DRD1 agonism. We also observed colocalization between collagen Journal of Cell Science

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Fig. 6. Schematic of D1 receptor agonism promoting collagen I degradation. Dihydrexidine (DHX) agonism of dopamine receptor D1 stimulates adenylate cyclase (AC) synthesis of cAMP. AC can also be directly activated by forskolin (FOR). cAMP promotes both expression of the CTSK gene and activation of pro-CTSK protein by acidification of the lysosomal compartment. Active CTSK cleaves collagen I in the extracellular space and in the lysosome.

telopeptide and LAMP1 in lysosomal compartments, a finding that MATERIALS AND METHODS could be explained by two mechanisms, phagocytosis of collagen Chemicals and reagents fibrils and degradation in the lysosomes, or degradation of collagen Dimethyl sulfoxide (DMSO, BP231-100) and L-ascorbic acid (BP351-500) I in the extracellular compartment (by either cathepsin K alone or were purchased from Thermo Fisher Scientific (Waltham, MA, USA). with other enzymes) and uptake into the lysosomal compartment. Ammonium hydroxide (LC11080-2) was acquired from LabChem Inc Extracellular cathepsin K might be activated in situ, or transported (Zelienople, PA, USA). Formalin solution (HT501128-4L), Triton X-100 (T8787) and tamoxifen (T5648-5G) were obtained from Sigma-Aldrich in active form from lysosomal compartments to the ECM. These (St Louis, MO, USA). Dihydrexidine (884) was purchased from Tocris mechanisms are not mutually exclusive, and future work will need Bioscience (Bristol, UK). Forskolin was purchased from Tocris Bioscience to further delineate their relative importance. (1099) and Cayman Chemical (11018, Ann Arbor, MI, USA). Odanacatib In addition, our results do not rule out important contributions from (24166) and chloroquine (14194) were acquired from Cayman Chemical. other collagenolytic pathways, which likely play important roles in TGFβ1 was purchased from eBioscience (14-8348-62; San Diego, CA, USA) unfolding the triple-helix and cleaving collagen peptides, notably and PreproTech (100-21C; Rocky Hill, NJ, USA). Biotin–CHP (b-CHP, MMPs and plasminogen activators. Interestingly, urokinase Bio60) was acquired from 3Helix (Salt Lake City, UT, USA). plasminogen activator (uPA), which itself is activated extracellularly by multiple cathepsin proteins (Vidak et al., 2019), was recently Cell culture shown to promote degradation of established lung fibrosis (Horowitz Primary normal human lung fibroblasts (NHLFs) were purchased from et al., 2019). An important extension of the current work will be to Lonza (Allendale, NJ, USA) or provided by Peter Bitterman and Craig further demonstrate the in vivo role of fibroblasts in collagen resorption Henke from the University of Minnesota (Minneapolis, MN, USA); NHLFs during fibrosis resolution. Prior work has demonstrated that depletion were isolated by explant culture from donors whose organs were rejected for transplantation, under a protocol approved by the University of Minnesota of macrophages during the resolution phase of mouse models of liver Institutional Review Board. NHLFs were cultured in Eagle’s minimal and lung fibrosis delays the clearance of collagen I (Duffield et al., essential medium (EMEM) (ATCC, Manassas, VA, USA) containing 10% 2005; Gibbons et al., 2011). However, work in dermal and lung tissues fetal bovine serum (FBS) and antibiotic-antimycotic (Thermo Fisher also suggests an important role for fibroblasts in proteolytic Scientific) unless otherwise noted. Cells were routinely characterized by degradation of collagen to maintain tissue homeostasis (Podolsky PCR or western blotting using specific fibroblast markers including et al., 2020; Zigrino et al., 2016). In the in vivo study performed here, PDGFRα and vimentin. Mycoplasma contamination was monitored we only treated mice for 24 h with DHX, which we had previously routinely by PCR. Experiments were performed with cells between shown selectively targets lung fibroblasts (Haak et al., 2019). We passages four and eight. found enhanced colocalization of CHP, indicative of collagen degradation, with Col1a1–GFP+ cells, supporting a contribution qPCR analysis from fibroblasts to collagen degradation and resorption. Importantly, NHLFs were plated on tissue culture dishes (Thermo Fisher Scientific, future work will need to ascertain the relative contributions of 60×15 mm) in EMEM containing 10% FBS and allowed to attach and grow for 48 h. Cells were stimulated with 2 ng/ml TGF-β and 10 µM DHX in fibroblasts, through cathepsin K and other collagenolytic enzymes, to EMEM containing 0.1% FBS for 24 h prior to RNA isolation using the maintenance of ECM homeostasis and resolution of fibrosis in the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the lung. However, our work strongly suggests that fibroblasts stimulated manufacturer’s instructions. Isolated RNA (165 ng) was then used to by D1 agonists play an important direct role in collagen degradation synthesize cDNA using SuperScript VILO (Invitrogen, Carlsbad, CA, and fibrosis resolution, emphasizing the potential utility of targeting USA). Quantitative PCR (qPCR) was performed using FastStart Essential this pathway therapeutically in IPF. DNA Green Master and analyzed with LightCycler 96 (Roche, Basel, Journal of Cell Science

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Switzerland). Data are expressed as a fold change by ΔΔCt relative to the AFM microindentation level of the GAPDH housekeeping gene, and normalized to control. All Measurements were performed on decellularized NHLFs cell-derived wells were treated with a final concentration of 0.1% DMSO. Primer matrices by a BioScope Catalyst atomic force microscope (Bruker, sequences were: GAPDH (forward, 5′-ACATCGCTCAGACACCATG-3′; Billerica, MA, USA) using a 2.5 µm radius sphere-tipped probe (Novascan, reverse, 5′-TGTAGTTGAGGTCAATGAAGGG-3′), CTSK (forward, 5′- Boone, IA, USA) with a spring constant determined at ∼110 pN/nm by the CTCCTTCCAGTTTTACAGCAAAG-3′; reverse, 5′-TTTCCCCAGTTT- thermal fluctuation method (Thundat et al., 1994). Force curves were acquired TCTCCCC-3′). with MIRO 2.0 (NanoScope 9.1, Bruker) at an indentation rate of 20 μm/s and a ramp size of 10 μm. The elastic modulus (E, Young’s modulus) was then DQ collagen extracted from each force curve by fitting with the Hertz contact model for A clear-bottom 96-well plate (Thermo Fisher Scientific) was coated with spherical indenter with NanoScope Analysis software v1.8 (Bruker) (Hertz, DQ™ Collagen (Thermo Fisher Scientific) diluted to 50 µg/ml in 1881; Sicard et al., 2017), and following the relationship: PureCol® (Advanced Biomatrix, Carlsbad, CA, USA) and incubated for 3 ð1 n2Þ 1 h at 37°C and 5% CO2. The coating solution was then removed, and E ¼ F; wells were gently washed with PBS. NHLFs were plated in EMEM 4 R1=2d3=2 containing 10% FBS and allowed to attach for 6 h. Medium was removed with R as the tip radius, δ the sample indentation, and ν as the Poisson’sratio, and cells were treated for 24 h with 2 ng/ml TGFβ, 10 µM DHX and 3 µM defined at 0.5 for cells (Mahaffy et al., 2004). For each dish, two different odanacatib in EMEM containing 0.1% FBS; all wells were treated with a areas (area size of 150×150 µm2) were analyzed with 25 force curves final concentration of 0.1% DMSO. After 24 h, cells were fixed with 10% randomly performed into each area. formalin and incubated for 1 h at room temperature with DAPI solution (Thermo Fisher Scientific) diluted at 1:1000 in DPBS (Life Technologies, Carlsbad, CA, USA). Images were taken and analyzed using a Cytation 5 Mice Col1a1–GFP transgenic mice were generated as previously described (Yata imager (BioTek, Winooski, VT, USA). Results are expressed as a fold et al., 2003). All animal experiments were carried out under protocols approved change relative to a control group. by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). Western blotting NHLFs were plated at 80% confluency in six-well plates (Thermo Fisher Bleomycin mouse studies Scientific) in EMEM containing 10% FBS, and allowed to attach for 24 h. The CHP analysis performed here made use of previously generated lung Medium was then exchanged with EMEM containing 0.1% FBS, and cells samples collected in our initial study of dopamine signaling in the context of – α + were stimulated for 72 h with 2 ng/ml TGFβ, prior to treatment with 10 µM pulmonary fibrosis (Haak et al., 2019). Briefly, 6 8-week-old Col1 1-GFP DHX or 10 µM forskolin for 24 h. All wells were treated with a final mice were anesthetized with ketamine/xylazine, and then treated with μ concentration of 0.1% DMSO. Total protein was isolated using RIPA buffer bleomycin (50 l at 1.2 U/kg body weight) or PBS control on day 0, as pH 7.6 containing Pierce phosphatase inhibitor and Halt™ protease and described previously (Haak et al., 2019). Mice were subsequently treated with phosphatase inhibitor cocktail (Thermo Fisher Scientific). Lysate protein DHX (5 mg/kg body weight) twice more, at 24 h and 2 h prior to lung harvest. concentration was determined using the Pierce™ BCA protein assay kit We fixed the right-side lung with 4% paraformaldehyde for 24 h before (Thermo Fisher Scientific) and samples were run on 4–15% Mini- rinsing with PBS and adding optical tissue compound (OCT Tissue Plus, μ PROTEAN® TGX™ gels (Bio-Rad, Hercules, CA, USA) at 100 V for 4585, Fisher Scientific); 6 m sections were cut for staining and imaging. 80 min. Proteins were transferred onto a PVDF membrane using the Trans- Blot® Turbo™ Transfer System (Bio-Rad). Membranes were incubated Laser scanning microscopy overnight with primary antibodies against the following proteins: collagen I For the in vitro studies, adapting from previously published methods (Novus Biologicals Inc., Centennial, CO, USA; NB600-408) diluted (Cukierman, 2002; Franco-Barraza et al., 2016), NHLFs were plated to 1:1000, cathepsin K (Santa Cruz Biotechnology, Dallas, TX, USA; sc- confluence onto gelatin (Cell Biologics, Chicago, IL, USA)-coated tissue 48353) diluted 1:200, Col1α1 telopeptide (Invitrogen; PA5-35380) diluted culture dishes (Thermo Fisher Scientific, 60×15 mm) in EMEM containing 1:1000 and GAPDH (Cell Signaling, Danvers, MA, USA; 14C10) diluted 10% FBS and allowed to attach for 24 h. Medium was then exchanged with 1:1000 in 5% nonfat dry milk (Bio-Rad). Membranes were washed with EMEM containing 0.1% FBS, 2 ng/ml TGFβ and 20 µg/ml ascorbic acid to TBS with 0.1% Tween 20 before 60 min incubation with HRP-conjugated enhance the ECM deposition, and cells were cultured for 6 days. At day 6, anti-rabbit IgG or anti-mouse IgG (Promega, Madison, WI) diluted 1:3000 medium was removed and cell-derived matrices were decellularized as in 5% nonfat dry milk. Membranes were imaged via a ChemiDoc™ Imaging previously described here. Subsequently, the collagen I intensity of intact System (Bio-Rad) and protein quantification was performed via (cell containing) and decellularized cell-derived matrices, were obtained densitometry using Image Lab v6.0 (Bio-Rad). Raw values were using a confocal microscope at 20× magnification. Fluorescence imaging normalized against housekeeping gene GAPDH and are presented as a was performed for five independent experiments, acquiring between 3 and 5 fold change relative to a control group. images per condition. Images were processed using ImageJ; for each experiment, the color channels are split and the mean gray value for each Preparation and de-cellularization of cell-derived matrices color is measured, for each image acquired per experiment. The data is Adapting from previously published methods (Cukierman, 2002; Franco- expressed as the average of the mean gray values for each condition. For the Barraza et al., 2016), NHLFs were plated to confluence onto gelatin (Cell in vivo studies, Col1α1–GFP lung slices were permeabilized using 0.1% Biologics, Chicago, IL, USA)-coated tissue culture dishes (Thermo Triton X-100 (X100, Millipore Sigma) and stained with biotin-conjugated Fisher Scientific, 60×15 mm) in EMEM containing 10% FBS and CHP (b-CHP) according to the manufacturer’s instructions, to investigate allowed to attach for 24 h. Medium was then exchanged with EMEM collagen resorption adjacent to fibroblasts. Secondary antibody conjugated containing 0.1% FBS, 2 ng/ml TGFβ and 20 µg/ml ascorbic acid to to Alexa Fluor 555 (S32355, Thermo Fisher) and Hoechst (33258, Tocris enhance the ECM deposition, and cells were cultured for 3 days prior to Bioscience), were used for visualization. Z-stack micrographs were taken treatment with 10 µM DHX. After 24 h, medium was removed and cell- using an LSM 780 with Zen software (Zeiss). Images were taken at the same derived matrices were decellularized by incubating with 20 mM laser power, gain and offset. Using ImageJ (Schindelin et al., 2012), images ammonium hydroxide for 5 min at room temperature. Subsequently, from each channel were processed together for brightness and contrast matrices were washed three times (1 min each time) with DPBS and modifications, with the γ-values adjusted to 2. incubated with 0.5% Triton X-100 for 30 s at room temperature. Finally, matrices were washed three times (1 min each time) with DPBS and CHP and colocalization quantification maintained in EMEM containing 0.1% FBS for AFM microindentation Lung slices stained with b-CHP were imaged using a Cytation 5 Cell experiment. Imaging Reader (BioTek Instruments) at 10× magnification. Images were Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs248278. doi:10.1242/jcs.248278 taken to avoid tissue folding or lung edges, within the entire imaging field 60 min, in a microplate reader at a dual Ex and Em of 329 and 440 nm, and containing tissue. For quantifying CHP and Col1α1–GFP colocalization, 384 and 540 nm. Data are presented as the ratio of 440 nm to 540 nm using ImageJ, thresholds were set to the top 5% of pixels for images taken in emission. the b-CHP and Col1α1–GFP channels. CHP pixels colocalized with GFP pixels were measured and quantified as the percentage of colocalized pixels MMP array per image field. In addition, CHP images were analyzed to determine total NHLFs were plated to confluence onto tissue culture dishes (Thermo Fisher amount of CHP per image field, as a representation of collagen degradation. Scientific, 60×15 mm) in EMEM containing 10% FBS, and allowed to attach Using ImageJ, thresholds were set for each individual image by two and grow for 48 h. Medium was then exchanged with EMEM containing investigators that were blind to the sample identification; each point 0.1% FBS and cells were stimulated with 2 ng/ml TGFβ, and treated with represents the average of two thresholds. 10 µM DHX for 24 h. The activity of human MMPs was measured from culture medium by using the Human MMP Array C1 (AAH-MMP-1-8, Super-resolution microscopy RayBiotech, Peachtree Corners, GA, USA) as per the manufacturer’s NHLFs were grown on a BioCoat™ eight-well culture slide in the presence instructions. of 2 ng/ml TGFβ and 20 µg/ml ascorbic acid for 5 days. At 24 h and 2 h μ prior to fixation, cells were treated with 10 M DHX. At day 6, cells were Statistical analysis fixed and stained (including antigen retrieval) for LAMP1 (NBP2-52721SS, Data analysis and plotting were performed using Prism 8.0 (GraphPad Novus Biologics), and with biotin-conjugated collagen hybridizing peptide Software, La Jolla, CA, USA). In vitro experimental results follow a normal ’ α (b-CHP) according to the manufacturer s instructions, and anti-Col1 1 distribution according to the Shapiro Wilk test, except for the graph telopeptide antibody (PA5-35380, Thermo Fisher Scientific). Secondary ‘decellularized ECM’ in Fig. 2B. Data sets derived from in vivo experiments antibody Alexa Fluor 647 (A31573, Thermo Fisher Scientific), Alexa Fluor (Fig. 3) have a normal distribution according to the D’Agostino and Pearson’s 488 (21202, Thermo Fisher Scientific), Alexa Fluor 555 (S32355, test. Statistical comparison between two groups was performed by paired and Invitrogen), and Hoechst (33258, Tocris Bioscience), were used for unpaired t-test, or Mann–Whitney test, according to the parametric or non- z visualization. We took -stack super resolution images on a Zeiss Elyra parametric behavior of the data, respectively. A statistical analysis of three or PS.1 super-resolution microscope using a 63× oil immersion objective (NA more groups was performed by repeated measures (RM) one-way ANOVA 1.4). Images were batch processed using Structured Illumination from Zen with Dunnett’s multiple comparison test, where the mean value of each group (Zeiss). The output files were imported into Imaris 8 (Bitplane, version was compared against a control group. Results are expressed as the mean± 8.2.0) for 3D reconstruction and we used the colocalization tool with s.e.m. unless otherwise indicated in the figure legends, with statistical P default settings (automatic thresholding based on -value) to quantify significance represented by P-value. The sample number (n) indicates the colocalization. To account for volumetric changes in cell size or number of number of independent samples in each experiment. images per field of view, the normalized number of voxels (volumetric pixels) of colocalized LAMP1 and collagen telopeptide were used to create Competing interests plots. Results are expressed as the fold change relative to TGFβ. A.J.H. and D.J.T. are co-inventors of a patent application (‘Methods of Treating Representative images were produced by using the maximum intensity Fibrotic Pathologies’ PCT/US2019/016178) related to the findings described in this projections obtained from Zen (Zeiss). Images were imported into ImageJ, article. and channels were batch processed together using brightness and contrast adjustments for visualization, with the γ-value set to 2. Colocalization Author contributions images were produced by determining pixels containing both LAMP1 and Conceptualization: A.M.D.E., D.J.T., A.J.H.; Methodology: A.M.D.E., P.A.L., D.S., telopeptide staining, or LAMP-1 and bCHP staining, using the ‘AND’ I.J., D.J.T., A.J.H.; Formal analysis: D.J.T., A.J.H.; Investigation: A.M.D.E., P.A.L., procedure of the image calculator. D.S., I.J.; Resources: D.J.T., A.J.H.; Data curation: A.M.D.E., P.A.L., D.S., I.J.; Writing - original draft: A.M.D.E., D.S.; Writing - review & editing: A.M.D.E., P.A.L., I.J., D.J.T., A.J.H.; Visualization: A.M.D.E., D.S., I.J.; Supervision: A.M.D.E., D.J.T., LOX activity assay A.J.H.; Funding acquisition: D.J.T., A.J.H. NHLFs were plated at 80% confluency in six-well plates (Thermo Fisher Scientific) in EMEM containing 10% FBS, and allowed to attach for 24 h. Funding Medium was then exchanged with EMEM containing 0.1% FBS, and cells This publication was supported by the National Institutes of Health (HL133320, were stimulated for 72 h with 2 ng/ml TGFβ and 20 µg/ml of ascorbic acid, HL105355 and HL092961), US Department of Defense (PR181132), the prior to treatment with 10 µM DHX for 24 h. After 24 h, medium from each Boehringer Ingelheim Discovery Award in Interstitial Lung Disease, the American condition was collected, and LOX activity was determined using the Lung Association Catalyst Award, and the Pulmonary Fibrosis Foundation Scholars fluorometric LOX activity assay kit (ab112139, Abcam, Cambridge, UK), as Award. Deposited in PMC for release after 12 months. per the manufacturer’s instructions. Briefly, cell culture supernatant was collected and centrifuged at 13,000 g for 5 min at 4°C. Then, in a clear- Supplementary information Supplementary information available online at bottom 96-well plate, reaction wells were set in duplicates with 50 μlofcell https://jcs.biologists.org/lookup/doi/10.1242/jcs.248278.supplemental culture medium and 50 μl of LOX reaction mix; 50 μl of assay buffer was used for the blank control. Plates were incubated at 37°C and 5% CO2 for Peer review history 40 min. Fluorescence was read on a microplate reader at excitation (Ex) and The peer review history is available online at emission (Em) of 540 nm and 590 nm, respectively, with a cut off at 570 nm. https://jcs.biologists.org/lookup/doi/10.1242/jcs.248278.reviewer-comments.pdf Background subtraction from the fluorescence intensity was performed by subtracting the blank control from the fluorescence intensity collected for each References sample well. Data is presented as the average of the fluorescent intensity. Bonnans, C., Chou, J. and Werb, Z. (2014). 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