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

© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs248278. doi:10.1242/jcs.248278 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

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