Meprin metalloproteases: Molecular regulation and function in inflammation and fibrosis

    Meprin Metalloproteases: Molecular Regulation and Function in Inflammation and Fibrosis Philipp Arnold, Anna Otte, Christoph Becker-Pauly PII: DOI: Reference:

S0167-4889(17)30122-2 doi:10.1016/j.bbamcr.2017.05.011 BBAMCR 18097

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

18 April 2017 5 May 2017 9 May 2017

Please cite this article as: Philipp Arnold, Anna Otte, Christoph Becker-Pauly, Meprin Metalloproteases: Molecular Regulation and Function in Inflammation and Fibrosis, BBA - Molecular Cell Research (2017), doi:10.1016/j.bbamcr.2017.05.011

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ACCEPTED MANUSCRIPT Meprin Metalloproteases: Molecular Regulation and Function in Inflammation and Fibrosis

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Philipp Arnold1, Anna Otte2, Christoph Becker-Pauly2#

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Institute of Anatomy, University of Kiel, Germany

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Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, Germany

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Correspondence: [email protected]

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Abstract

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The zinc-endopeptidases meprin α and meprin β are extracellular proteases involved in connective tissue homeostasis, intestinal barrier function and immunological processes. Meprins are unique amongst other extracellular proteases with regard to cleavage specificity

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and structure. Meprin α and meprin β have a strong preference for negatively charged amino acids around the scissile bond, reflected by cleavage sites identified in procollagen I, the amyloid precursor protein (APP) and the interleukin-6 receptor (IL-6R). In this review we report

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on recent findings that summarize the complex molecular regulation of meprins, particular folding, activation and shedding. Dysregulation of meprin α and meprin β is often associated with pathological conditions such as neurodegeneration, inflammatory bowel disease and fibrosis. Based on mouse models and patient data we suggest meprins as possible key regulators in the onset and progression of fibrotic disorders, leading to severe diseases such as pulmonary hypertension.

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ACCEPTED MANUSCRIPT Introduction Chronic Inflammation is often associated with additional pathological conditions, such as

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cancer and fibrosis (Coulouarn and Clement, 2014). Many genes have been identified to either

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contribute to the onset or progression of these diseases. Some of these may represent key

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regulators, thereby connecting chronic inflammation and tissue remodeling that finally results in a pathological phenotype. Metalloproteases meprin α and meprin β represent such regulatory factors. For instance, both enzymes were shown to have pro-inflammatory activity,

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they contribute to extracellular matrix (ECM) remodeling, and importantly their expression is upregulated in chronic inflammation, certain cancers, and fibrosis (Broder and Becker-Pauly,

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2013, Prox et al., 2015).

A well described physiological function of meprins is the maturation of fibrillar procollagens I

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and III through cleavage of the N- and C-terminal prodomains (Broder et al., 2013, Kronenberg

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et al., 2010). This proteolytic event is a prerequisite for collagen fibril assembly, and explains why decreased meprin expression leads to impaired connective tissue, whereas upregulation

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of the proteases is associated with fibrosis (Prox et al., 2015). Hence, meprins may be considered as therapeutic targets in future studies. However, it is not yet clear whether meprins are solely involved in ECM remodeling that triggers

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the symptoms of the disease, or additionally in the preceding inflammatory step that induces the onset of fibrosis. Several studies revealed pro- as well as anti-inflammatory activity of meprins (Broder and Becker-Pauly, 2013). The function of meprin metalloproteases clearly depends on their cellular localization. One example for a biological function of meprin β on the apical side of intestinal epithelial cells is the constitutive mucus detachment that prevents bacterial overgrowth (Schutte et al., 2014). Hence, meprin β activity in the intestinal lumen is important for barrier function and prevents bacterial infection. However, when inflammation occurs, meprin β at the mesenchymal site can induce pro-inflammatory stimulus, e.g. by cleaving the IL-6R to induce IL-6 trans-signaling (Arnold et al., 2017a). Several factors that regulate localization, activation and inhibition of meprins have been discovered over the last years. We have learned that not only transcriptional regulation, as 2

ACCEPTED MANUSCRIPT shown for AP-1 and meprin β (Biasin et al., 2014), is important to characterize protease function. Different proteases are expressed in very diverse amounts, but this does not equally correlate with proteolytic activity towards certain substrates. For instance, BACE-1, the well-

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known β-secretase for APP, is highly expressed in neurons and additionally up-regulated in Alzheimer’s disease (Vassar et al., 2014). Meprin β, also reported to generate Aβ peptides, is

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present at much lower concentrations in the brain. However, in vitro kinetic studies revealed that meprin β exhibited a 104- and 103-fold higher catalytic efficiency toward APP-peptide

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substrates when compared to BACE-1 (Bien et al., 2012). Additionally, APP is cleaved by meprin β already in the late Golgi and at the cell surface (Schonherr et al., 2016), whereas

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BACE-1 cleaves after internalization of APP in acidic endosomal compartments (Vassar et al., 2014). Thus, besides the transcriptional regulation of a protease, cleavage kinetics and subcellular localization of enzymes are also of importance for the proteolytic turnover of

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substrates. Additional factors that regulate substrate proteolysis are endogenously occurring

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activators and inhibitors of proteases. Taken together, this so called ‘protease web’ (Fortelny

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et al., 2014) builds the microenvironment that is important to understand proteolysis as a regulatory event in pathophysiology. In this review we summarize recent findings that explain how the metalloproteases meprin α

fibrosis.

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and meprin β are orchestrated in the protease web and how this influences inflammation and

Regulatory mechanisms influencing meprin activity The metalloproteases meprin α and meprin β have important physiological functions and can be dysregulated in certain associated pathologies. Hence, a detailed understanding of the regulation of these proteases may help to evaluate their therapeutic potential. In a recent study it was shown that the oncoprotein Reptin strongly regulates the expression of meprin α (Breig et al., 2017). For meprin β the AP-1 transcription factor complex was found to stimulate gene expression, which contributes to the onset and progression of fibrosis (Biasin et al., 2014). Over the last years, multiple factors that influence folding, cellular localization and activation 3

ACCEPTED MANUSCRIPT of meprins have been described. Meprin α is constitutively released into the extra cellular space as it is cleaved by furin within its inserted domain (Marchand et al., 1995) (Fig. 1A). This domain is absent in meprin β, which makes the protease primarily membrane-bound. However,

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it was shown that meprin β can be shed from the cell surface by ADAM 10/17, resulting in a soluble form of meprin β (Jefferson et al., 2013, Hahn et al., 2003). In the following, three new

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major findings on meprin β regulation will be addressed: i) activation of membrane-bound meprin β by the membrane-bound serine protease matriptase-2, ii) the interaction with

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tetraspanin 8 and iii) the influence of calcium ions on protein folding and activity. Shedding and activation of meprin α and meprin β

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Like most proteolytic enzymes meprin α and meprin β are expressed as zymogens and require activation through other proteases. Several tryptic serine proteases can activate both pro-

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forms by cleaving human meprin α between arginine 65 and asparagine 66 and meprin β

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between arginine 61 and asparagine 62 (Ohler et al., 2010) (Fig. 1B). Large parts of the meprin

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α and meprin β ectodomain are composed of identical domains. For meprin β these were crystalized and atomically resolved structures are available for the latent and active protease (Arolas et al., 2012). The crystal structures contain the protease domain (with or without the

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pro-peptide), the MAM (meprin A5 protein tyrosine phosphatase μ) domain and the TRAF (tumour-necrosis-factor-receptor-associated factor) domain. The only extracellular region missing in the crystal structure is the EGF-like domain, which is localized C-terminal to the TRAF domain and connects the extracellular part of meprin β to the transmembrane helix (Fig. 1A). Interestingly, meprins are the only proteins containing an extracellular TRAF domain. The crystal structures helped to elucidate the general orientation of membrane-bound meprin β at the cell surface and it became evident that the activation site is in close proximity to the membrane (Arolas et al., 2012) (Fig. 1B). Despite the almost identical domain composition there is one decisive difference between meprin α and meprin β. Meprin α contains an additional inserted domain between the TRAF and the EGF-like domain that is cleaved by furin (or other proprotein convertases) on the secretory pathway (Marchand et al., 1995). Thus, 4

ACCEPTED MANUSCRIPT meprin α is constitutively released into the extracellular space as a soluble protease. Additionally, meprin α forms large, non-covalently linked oligomers up to 6.4 MDa, by a so far unknown mechanism (Bertenshaw et al., 2003, Becker et al., 2003). Of note, meprin α is the

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largest extracellular protease known, concentrating high proteolytic activity in its oligomeric state.

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Different soluble serine proteases have been described for shed meprin α and meprin β that cleave off the pro-peptide and thereby induce proteolytic activity. Pancreatic trypsin and tissue

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kallikrein related peptidase (KLK) 5 activate both proteases (Ohler et al., 2010). Meprin α is exclusively activated by plasmin and meprin β by KLKs 4 and 8 (Rosmann et al., 2002). This

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might be of physiological importance in the small intestine as it was shown that the release of meprin β by its sheddases ADAM10/17 is important for proteolytic processing of the mucus protein mucine 2 (Muc2) (Schutte et al., 2014). Interestingly, substrates are differentially

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cleaved by membrane-bound and soluble meprin β. For the APP it was shown that only

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membrane-bound meprin β cleaves at the β-secretase site but not the shed protease (Bien et

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al., 2012). The same holds true for the IL-6R where only membrane-bound meprin β acts as a sheddase, whereas soluble meprin β exerts no proteolytic activity towards this substrate (Arnold et al., 2017a). Interestingly, the constitutive soluble meprin α can cleave the IL-6R and

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generates, as the membrane-bound meprin β, soluble IL-6R capable of trans-signaling. To exert proteolytic activity, activation of meprin β at the cell surface has to occur. Therefore, potential membrane-bound activators have been investigated. With the membrane-tethered serine protease matriptase-2 (MT-2), such an activator could be identified (Jackle et al., 2015). The favored maturation of meprin β by MT-2 was observed in immunoblot analysis and in activity measurements employing a specific fluorogenic peptide cleavage assay. Additionally, co-transfection of meprin β with MT-2 resulted in an appreciable decrease in soluble APPα, which correlates with increased β-secretase activity. In vivo, both proteases co-localize at the cell surface and were found on immune cells in the lamina propria of the small intestine. Activation of meprin β by MT-2 is a specific event as several other membrane-bound tryptic proteases tested did not activate the membrane-bound meprin β. 5

ACCEPTED MANUSCRIPT Meprin β is localized in tetraspanin enriched microdomains At the cell surface, the localization of protease and substrate is of critical importance. We have

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learned that meprins can exist as membrane-bound or soluble proteins, which are then

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differentially activated and exhibit distinctive substrate repertoires. To regulate localization and

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activity of a protease, interaction with other proteins, which are not necessarily proteolytic targets, can be important. One example is the regulation of ADAM17 by inactive rhomboids 1 and 2 (iRhom1/2) (McIlwain et al., 2012, Adrain et al., 2012). It was shown that iRhoms interact

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with ADAM17 in the endoplasmic reticulum and facilitate its transport via the Golgi compartment to the cell surface. Additionally, several tetraspanins (TSPANs) were shown to

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interact with ADAM proteases, building clusters of TSPAN enriched microdomains (TEMs) (Matthews et al., 2016). Identification of such non-proteolytic interaction partners helps to

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understand protease transport, cellular localization and substrate cleavage. Meprin β is highly

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expressed in the small intestine and is exclusively localized at the apical side of enterocytes. For the identification of non-proteolytic interaction partners we employed a yeast two-hybrid

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screen using an intestinal cDNA library. Here, TSPAN8 was found to specifically interact with meprin β in TEMs (Schmidt et al., 2016). Interestingly, APP was additionally detected in these

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TEMs demonstrating a likely interaction of protease and substrate in well-organized cell surface microdomains. This is in contrast to the major β-secretase BACE1, which cleaves APP in early endosomes (Vassar et al., 2014). It will be important to decipher additional non-proteolytic interaction partners of meprin α and meprin β that determine cellular localization, substrate interaction and half-life at the cell membrane. Folding and activity of meprin β is influenced by calcium Cleavage is abrogated in the presence of high calcium concentrations for different substrates of meprin β, such as Muc2 (Schutte et al., 2014) and the dentin sialo phosphoprotein (DSPP) (Arnold et al., 2017b, Tsuchiya et al., 2011). In these studies, the influence of calcium was attributed to substrate modification. In 2015 we showed that there may be a direct influence of 6

ACCEPTED MANUSCRIPT calcium on the proteolytic activity of meprin β (Arnold et al., 2015). Here, in the presence of calcium, a decreased activity of meprin β towards a fluorogenic peptide substrate and APP was observed. Through analysis of the structural properties of meprin β a potential calcium

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binding site was detected, which is composed of amino acids aspartate 204 (D204), aspartate 245 (D245), glutamate 163 (E163) and serine 242 (S242) (Fig. 1C). Mutation of D204 and

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D245 to alanine led to largely misfolded proteins that could not exit the ER or were found in large intracellular vesicles, respectively. Thus, we concluded that meprin β might require

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calcium for proper folding. This is further supported by molecular dynamics simulation where active meprin β is gradually transferred into a pro-meprin β-like conformation under increasing

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calcium concentrations (Schneppenheim et al., 2017). Further increase of calcium resulted in an unfolded meprin β with two calcium ions bound in the proposed binding motif. Indeed, meprin β can be inhibited by calcium, with an inhibition constant (Ki) of 11.5 mM CaCl2. The

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physiological extracellular calcium concentration however, is between 2.1-2.8 mM, which is

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not sufficient to inhibit meprin β. Local calcium concentrations can eventually be higher as

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found for example in bone or teeth (Somlyo et al., 1985). As bone and tooth dentin are both composed of a mineralized collagen I, meprins were discussed to play an important role there for pro-collagen maturation. For bone it was already shown that another pro-collagen

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converting enzyme, the bone morphogenetic protein 1 (BMP-1), seems to be of critical importance (Muir et al., 2014). Here a postnatal knockout of BMP-1 in mice resulted in the development of an osteogenesis imperfecta-like phenotype with deficient mineralization of bone tissue. The tendon and connective tissue of these mice however appeared not to be affected, despite the fact that they consist of large amounts of fibrillar collagens. Mice deficient for meprin α or meprin β on the other hand exhibit diminished collagen deposition and tensile strength in the skin (Broder et al., 2013). Again, in the presence of high calcium concentrations, there is no phenotype observed in meprin α or meprin β knock-out animals in tooth development and maintenance. In these mice developing or mature dentin was found to be normal (Arnold et al., 2017b). However, a strong phenotype could be described in mice deficient for BMP-1 and its close relative tolloid-like 1 (TLL1). Here, a severe defect in postnatal 7

ACCEPTED MANUSCRIPT root dentin formation and a partially impaired periodontal ligament homeostasis was detected (Wang et al., 2017). However, there was still a certain amount of maturated collagen, thus indicating that other proteases may partially compensate for the loss of BMP-1 and TLL1.

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In conclusion meprin β is directly influenced by calcium ions, most likely through binding to the catalytic domain complexed by a cluster of charged amino acid residues. Consequently,

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meprin β plays no significant role in collagen maturation in calcium-rich tissues such as bone or dentin, but has important physiological functions in other compartments. Characterization

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of meprins’ substrate specificity, structural properties and other regulatory elements is

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important to understand their role in the onset and/or progression of disease.

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Figure 1: Structural properties of meprin metalloproteases. A) Domain structure of meprin α and meprin β. Both consist of a pro-peptide that shields the active site of the protease domain. The protease domain is followed by a MAM-, a TRAF-, and an EGF-like domain. The proteases are membrane anchored via a transmembrane domain and contain a small C-terminal tail. Meprin α contains an additional inserted-domain that is cleaved by furin on the secretory pathway and leads to the release of meprin α into the extracellular space. By an unknown process meprin α forms large oligomers that can be observed by transmission electron microscopy. B) Meprin β is a membrane-bound dimer and the activation site is located between arginine 61 (Arg61) and asparagine 62. Additionally, tetraspanin 8 is shown to interact with meprin β in tetraspanin enriched microdomains. Furthermore, matriptase-2, the first known membrane-bound activator of meprin β, is shown. C) Structure of the active meprin β

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ACCEPTED MANUSCRIPT protease domain with the active site oriented to the left. The Zinc ion (orange) and the Zinc ion coordinating histidine residues (cyan) are displayed. Additionally, asparagine 62 (N62), glutamate 163 (E163), aspartate 204 and 245 (D204 and D245), and lysine 248 (K248) are shown in magnification. These residues were shown to be involved in correct protein folding and activation after pro-peptide

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cleavage.

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Meprins in inflammation and fibrotic diseases

Today it is widely accepted that excessive ECM turnover and deposition is tightly connected

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with chronic inflammatory disease. Initiation of an innate immune response is a necessary physiological process that can be induced by infections, mechanical stress or reactive oxygen

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species. Normally, this inflammatory process is resolved over time and the tissue returns to normal homeostasis. Under certain conditions however, the inflammation persists resulting in

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a pathological chronic inflammatory response. Most chronic inflammation is accompanied by

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the deposition of excess amounts of ECM, including large portions of collagen I, referred to as fibrosis (Trojanowska et al., 1998, Bhogal et al., 2005). Initiation of fibrosis is marked by an

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imbalance of pro- and anti-fibrotic cytokine profiles following the infiltration of mononuclear cells upon inflammation. Additionally, excessive ECM turnover by different proteases further drives inflammatory responses, which illustrates the interdependency of inflammation, tissue

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remodeling and matrix deposition. Sustained persistence of these processes and subsequent tissue scaring impairs proper organ function and may lead to complete organ failure. Over the last years, an array of proteins was described as substrates for meprins which are known to be involved in inflammatory disease and ECM remodeling (Tab. 1). Here, we highlight central events during inflammation and fibrosis that may be directly influenced by meprins.

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ACCEPTED MANUSCRIPT Table 1: In vivo substrates of meprin α and meprin β and potential biological functions. Substrates

Meprin α/β

Function

Literature

Meprin α Meprin β

Fibrillar collagen deposition in connective tissue of the skin and lung

Broder et al., 2013, PNAS Biasin et al., 2016, Sci Rep

Mucin 2

Meprin β

Main component of the intestinal mucus layer, important for host barrier function

Schütte et al., 2014, PNAS

APP

Meprin β

APP-derived Aβ peptides are associated with the onset and progression of Alzheimer’s disease

Schönherr et al., 2016, Mol Neurodegen Jefferson et al., 2011, J Biol Chem

IL-6 receptor

Meprin α Meprin β

Mediator of pro-inflammatory IL6 trans-signaling

IL-6

Meprin α Meprin β

Cleavage of IL-6 and possible inactivation; increased IL-6 serum levels in meprin-deficient mice

Banerjee et al., 2011, Am J Physiol Gastrointest Liver Physiol Keiffer & Bond, 2014, J Biol Chem

IL-18

Meprin β

Activation of IL-18

Banerjee & Bond, 2008, J Biol Chem

CD99

Meprin β

Cleavage of CD99 induces transendothelial cell migration (TEM)

Bedau et al., 2017, FASEB J

Nidogen 1

Meprin α Meprin β

Cleavage of nidogen-1 in nephrotoxicity

Herzog et al., 2015, Toxicol Lett

Thymosin-β4

Meprin α

Release of anti-inflammatory peptide Ac-SDKP

Kumar et al., 2016, Am J Physiol Renal Physiol

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Collagen I

Arnold et al., 2017, Sci Rep

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Meprins in physiological ECM remodeling One of the best evaluated functions of meprins in the ECM is the regulation of collagen assembly, whereby meprins are the only known proteases to cleave off the N- and the Cterminal pro-domains of collagen type I and III (Broder et al., 2013) (Fig. 2). This enables fibril formation without additional factors and distinguishes meprins from other collagen processing enzymes which are restricted to either C- or N-terminal cleavage. This role in collagen maturation is supported by the finding that meprin α and β were found upregulated in keloids, fibrotic skin tumors, where ECM is excessively accumulated. In contrast, mice deficient for meprins show decreased collagen deposition in the skin. Collagen type IV was also shown to be cleaved by meprins in vitro (Kruse et al., 2004). This could lead to promotion of immune cell infiltration across the basement membrane upon inflammatory processes, as shown for MMPs 11

ACCEPTED MANUSCRIPT and elastase (Delclaux et al., 1996, Monaco et al., 2006). Other substrates of meprin α and β related to the ECM network are nidogen-1, tenascin-C and fibronectin (Ambort et al., 2010, Kohler et al., 2000, Kruse et al., 2004, Walker et al., 1998). Nidogen-1 and tenascin-C are

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important basement membrane glycoproteins mediating binding to the ECM. Cleavage of these networks by proteases results in disruption of ECM-binding and often leads to the

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generation of pro-inflammatory fragments. Short-term meprin inhibitor (actinonin) application correlated with decreased excretion of fragmented nidogen-1 upon induced nephrotoxicity in

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vivo, which was verified in meprin β-deficient mice (Herzog et al., 2015). Tenascin-C was shown to be cleaved within its anti-adhesive domain by meprin β leading to increased cell

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spreading (Ambort et al., 2010). Fibronectin as well as collagen IV were described to not only be degraded by proteases but their cleavage fragments accomplish chemotactic and receptor activating or blocking properties, again underlining the cross-linkage between ECM remodeling

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and the innate immune system (Adair-Kirk and Senior, 2008). SPARC and fibulin were identified as additional ECM substrates for meprins based on a proteomics approach (Becker-

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Pauly et al., 2011). Here, a meprin mediated cleavage might lead to remodeling via disruption of ECM component binding and/or to the promotion of inflammation. SPARC was suggested to translocate into the nucleus via a pore complex (Yan et al., 2005). Interestingly, distinct

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functions of SPARC were identified after proteolytic cleavage into defined polypeptides (Sage et al., 2003) and proteolytic fragments of fibulin-3 serve as clinical markers in osteoarthritis (Runhaar et al., 2016). Meprins cleave inflammatory cytokines and contribute to immune cell extravasation Proteolytic cleavage of ECM and particularly membrane associated proteins is involved in tissue remodeling upon injury, in growth and homeostasis, but can also drive the local infiltration of immune cells. Shedding of adhesion molecules can result in the loss of cell-cell contacts and thereby promotes trans-endothelial migration. Upon inflammation, neutrophils initiate a proteolytic cascade that results in the cleavage of endothelial junction proteins (Pham, 2008). Matrix metalloprotease 2 (MMP-2) was shown to inactivate β1-integrin thereby decreasing cell adhesion during invasion or wound healing (Kryczka et al., 2012). In this line, 12

ACCEPTED MANUSCRIPT we discovered the adhesion molecule CD99 on leukocytes as meprin β substrate (Bedau et al., 2017). Processing of CD99 by meprin β induced cell migration in vitro, thus strongly suggesting a role of this protease in inflammatory processes upon upregulation (Fig. 2).

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Indeed, full length CD99 accumulated upon meprin inhibition in vivo.

Induction of inflammation by resident immune cells, i.e. macrophages, is followed by the

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release of inflammatory mediators. Meprin β was shown to induce the production of proinflammatory cytokines, including interleukin-1β (IL-1β), interleukin-18 and interleukin-6 (IL-6)

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in macrophages (Li et al., 2014). Additionally, interleukin-8 induction was described to be mediated by meprin β in human bronchial epithelial cells (Bergin et al., 2008). Cytokines of the

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cytokine family 1 are directly activated by proteases released by neutrophils and mast cells during inflammation (Afonina et al., 2015). Despite the well-described intracellular maturation, IL-1β and IL-18 were proposed to be directly activated by meprin α (Herzog et al., 2009) or

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meprin β (Banerjee and Bond, 2008), respectively, in vitro. On the other side, cytokines can also be inactivated by proteases. IL-6, a key regulator in inflammation, loses activity in the

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presence of proteases released by activated neutrophils at inflammatory sites (Bank et al., 1999, McGreal et al., 2010). Meprin α and meprin β were also shown to cleave IL-6, thereby abolishing cytokine activity in vitro (Keiffer and Bond, 2014). This is in line with significantly

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increased IL-6 protein levels, among other cytokines, in meprin α and meprin β double deficient mice when challenged with dextran sodium sulfate (Banerjee et al., 2011). Shedding of transforming growth factor alpha α (TGF α) and epidermal growth factor (EGF) by meprin α was described to induce EGF receptor/ mitogen-activated protein kinase (EGFR/MAPK) signaling or toll like receptor (TLR) responses (Bergin et al., 2008, Minder et al., 2012). The like-wise pro-inflammatory extracellular signal-regulated kinases (ERK1/2) mediated pathway was described to be indirectly activated by meprin. Here, activation of ADAM10 by meprin β led to ERK1/2-signaling in macrophages (Li et al., 2014). Furthermore, we recently identified the IL-6 cytokine receptor as substrate of meprins. Cleavage of the receptor by soluble meprin α or membrane-bound meprin β at the cell surface leads to proinflammatory trans-signaling of IL-6 (Arnold et al., 2017a) (Fig. 2). 13

ACCEPTED MANUSCRIPT The above described pathways influenced by meprin proteases are known to induce inflammation but also cell proliferation and migration. Hence, evaluating the molecular mechanisms that regulate meprins, described earlier in this review, is crucial to assess

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eventual therapeutic approaches.

Figure 2: Secreted and membrane-tethered substrates of meprins. Shedding of the IL-6 receptor (IL-6R) by meprins can induce pro-inflammatory trans-signaling. Cleavage of CD99 by meprin β promotes transendothelial cell migration. Meprin β can be activated by matriptase-2 (MT2) at the cell surface. Metalloproteases ADAM10/17 are sheddases of meprin β. Soluble meprins are activated by soluble tryptic serine proteases.

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ACCEPTED MANUSCRIPT Meprins in fibrosis Meprin metalloproteases were found to be upregulated in keloids, which are fibrotic skin tumors characterized by pathological ECM formation (Kronenberg et al., 2010). Vice versa, mice

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deficient for meprin α or meprin β display decreased collagen deposition in the skin correlating with impaired tensile strength (Broder et al., 2013). This resembles the phenotype of certain

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Ehlers-Danlos syndrome patients, with a defect in collagen maturation leading to increased skin extensibility (Byers and Murray, 2014). In a mouse model of systemic inflammation,

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induced by bleomycin administration, meprin β was described to support collagen I maturation in the lung (Biasin et al., 2017). Hence, lung fibrosis upon acute lung injury is proposed to be

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triggered by alterations in meprin expression. Meprin β was further described as the most upregulated gene in the lung of transgenic mice that serve as a model of idiopathic pulmonary arterial hypertension (IPAH), namely Fra2 mice (Biasin et al., 2014). In IPAH pathology

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inflammatory processes in the lung of patients correlate with excess deposition of ECM around lung arteries which consequently leads to organ failure within a few years after diagnosis

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(Ahmed and Palevsky, 2014, Montani et al., 2014). Hallmarks of the vascular remodeling in pulmonary hypertension pathology are inflammation as well as increased cell proliferation and ECM turnover (Humbert et al., 2004). Therapies are only effective upon early diagnosis and

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mainly target further progression of disease (McLaughlin et al., 2004, Fujimoto et al., 2015, Proudman et al., 2007). In the Fra2-transgenic mouse model, the Fos-related antigen 2 (Fra2) protein is overexpressed which is part of the activator protein-1 (AP-1) heterodimeric transcription complex (Eferl et al., 2008). The AP-1 complex, which is comprised of Fra2 and a Jun protein family member, is described to control transcription of genes involved in vascular lung and connective tissue formation (Eferl et al., 2008). Biasin and colleagues revealed transcriptional regulation of meprin β by Fra2 in primary human smooth muscle cells (PASMCs), to be regulated by TGFβ pro-fibrotic signaling (Biasin et al., 2014). Indeed, Fra2 overexpression was also detected in lung samples of IPAH patients. Additionally, it was shown that underlying systemic sclerosis, an autoimmune disease with fibrotic growth of connective tissues, results in worse prognosis in pulmonary hypertension patients (Chaisson and 15

ACCEPTED MANUSCRIPT Hassoun, 2013, Mathai and Hassoun, 2011) and also the Fra2-transgenic mice resemble this systemic sclerotic phenotype.

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Meprins in chronic inflammation

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Since leukocyte infiltration and the production of pro-inflammatory mediators are promoted by

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meprin activity, this can additionally drive the inflammatory response not only in IPAH patients. Subsequently, cytokine mediated immune response by meprins was connected with other chronic inflammatory diseases, like vasculitis, inflammatory bowel disease (IBD) or renal and

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urogenital injury (Banerjee and Bond, 2008, Crisman et al., 2004, Kentsis et al., 2013, Yura et al., 2009). High abundance of meprin α in urine is described as a valuable clinical marker of

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Kawasaki disease (KD), an acute vasculitis in children, leading to severe morbidity and without early treatment eventually leading to death (Kentsis et al., 2013). Furthermore, meprin β

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cleavage of tenascin-C, an adhesion modulating and signal transducing glycoprotein as

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described above, correlated with increased cell spreading in vitro und was proposed to have regulatory effects in chronic inflammatory diseases (Ambort et al., 2010). Meprins are

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abundantly expressed in kidney, and high meprin levels in human urine have been correlated with urinary tract infections (Bond et al., 2005). Nephrotoxicity mediated by meprins was also shown in renal tubular cells (Carmago et al., 2002) and in induced acute kidney injury in mice

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(Herzog et al., 2015, Yura et al., 2009). These data propose meprins as promising therapeutic targets to restore the balance between inflammation and ECM deposition in severe pathologies (Fig. 3). The above mentioned mouse models for inflammatory diseases represent valuable models for therapeutic studies, particularly Fra2-transgenic mice, since here chronic inflammation is combined with systemic sclerosis.

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Figure 3: Meprins are important proteolytic regulators of inflammatory processes and ECM remodeling. Upon certain stimuli, meprins can induce pro-inflammatory signals that lead to rearrangement of the mesenchymal environment. Invasion of immune cells in such tissues and differentiation of ECM producing cells can result in pathological collagen deposition and fibrosis development.

Therapeutic strategies to inhibit meprin activity General anti-inflammatory therapies have a broad range of applications and are constantly optimized and renewed. Fibrotic diseases, on the other hand, are often treated by targeting growth factors like TGF β (Hawinkels and Ten Dijke, 2011) or administering collagenases directly to keloids and wounds (Alipour et al., 2016, Bae-Harboe et al., 2014). Inhibition of 17

ACCEPTED MANUSCRIPT meprins may be a strategy to not only target the symptoms but additionally prevent the onset of fibrosis. Several inhibitors have been developed against metalloproteases, based on different chemical

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moieties. Hydroxamate-based inhibitors chelating the zinc-ion are frequently used in research. Among those, actinonin, a naturally occurring compound with antibacterial activity was

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identified as a potent inhibitor for meprin α, but also to a lesser extent for meprin β (Kruse et al., 2004). However, actinonin is rather unspecific, which is shown by its inhibitory capacity

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towards leucyl-aminopeptidases (Xu et al., 1998). Although hydroxamate inhibitors possess strong binding-affinities to the catalytic zinc, their stability is metabolically labile and clinical

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trials have been disappointing in the past (Vandenbroucke and Libert, 2014). Another class of inhibitors also targeting the zinc within the active site of metalloproteases are for example thiolbased compounds. Selective inhibitors targeting MMP-13 have been developed for

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osteoarthritis treatment with sulfonate-groups forming salts with the zinc-ion (Hu et al., 2005).

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Until now, the most potent inhibitors for meprin β are based on sulfonate-moieties,

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unfortunately still lacking high specificity (Madoux et al., 2014). Phosphorous-based inhibitors in contrast were demonstrated to be highly specific for metalloproteases, mimicking natural substrates in their transition states (Dive et al., 2004). Although they contain peptide bonds in

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their chemical structure, phosphinic peptides were shown to be fully stable and non-toxic in vivo (Johnson et al., 2011). Moreover, when compared to other zinc-metalloproteinase inhibitors (thiol, carboxylate or hydroxamates), the whole active site cleft of the proteinase (both primed and unprimed site) can be probed, allowing better inhibitor selectivity. One example is the use of phosphinic peptides directed against MMP-12 that have been shown to reduce inflammation in rheumatoid arthritis in vivo (Marchant et al., 2014). Due to their striking cleavage specificity (Becker-Pauly et al., 2011) meprin metalloproteases are promising candidates for the successful development of specific phosphinic inhibitors. Based on recent findings reviewed in this article, using biochemical and cellular approaches, employing mouse models as well as analysis of human tissues, meprins could indeed be important factors for the onset and progression of chronic inflammation and associated fibrotic 18

ACCEPTED MANUSCRIPT diseases. Future studies will show if the metalloproteases meprin α and meprin β are suitable targets for medical treatment of such conditions.

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Acknowledgements

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We thank Dr. Michelle Rothaug for proofreading. This work was supported by the Deutsche

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Forschungsgemeinschaft (DFG) SFB 877 (Proteolysis as a Regulatory Event in

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Pathophysiology, project A9).

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ACCEPTED MANUSCRIPT Highlights - Review of latest research on the metalloproteases meprin α and meprin β in health and disease.

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- New aspects of molecular regulation of meprins that influence proteolytic activity and

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cellular localization.

- Summary of physiological functions of meprins in extracellular matrix remodeling and

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inflammation.

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- Dysregulation of meprins is associated with chronic inflammation and fibrosis.

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