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Metalloproteinases: A parade of functions in matrix biology and an outlook for the future

Suneel S. Apte, William C. Parks

PII: S0945-053X(15)00088-8 DOI: doi: 10.1016/j.matbio.2015.04.005 Reference: MATBIO 1165

To appear in: Matrix Biology

Received date: 16 April 2015 Accepted date: 17 April 2015

Please cite this article as: Apte, Suneel S., Parks, William C., : A parade of functions in matrix biology and an outlook for the future, Matrix Biology (2015), doi: 10.1016/j.matbio.2015.04.005

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Metalloproteinases:

a parade of functions in matrix biology and an outlook

for the future

Suneel S. Apte1* and William C. Parks2*

1Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA, and 2Cedars-Sinai Medical Center,

Los Angeles, CA USA

*Correspondences to:

Suneel S. Apte, MBBS, D.Phil Department of Biomedical Engineering (ND20), Cleveland Clinic Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195, United States. Phone: 216 445 3278 Fax: 1 216 444 9198 Email: [email protected]

William C. Parks, PhD Cedars-Sinai Medical ACCEPTEDCenter MANUSCRIPT 8700 Beverly Blvd., A9403 Los Angeles, CA 90048 USA Phone: 424-315-4307 Fax: 310-967-8370 Email: [email protected]

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Abstract:

This issue of Matrix Biology is devoted to exploring how metalloproteinases – here inclusive of related families of extracellular proteinases – act on extracellular matrix (ECM) proteins to influence an astonishing diversity of biological systems and diseases. Since their discovery in the 1960’s, matrix metalloproteinases (MMPs) have oft and widely been considered as the principal mediators of ECM destruction. However, as becomes clear from several articles in this issue, MMPs affect processes that both promote and limit ECM assembly, structure, and quantity. Furthermore, it has become increasingly apparent that ECM proteolysis is neither the exclusive function of MMPs nor their only sphere of influence. Thus, other may be important participants in ECM proteolysis, and indeed they are. The ADAMTS (a -like and domain with type 1 repeat) proteinases, BMP/tolloid , and meprins have all emerged as major mechanisms of ECM proteolysis. An aggregate view of proteolysis as an exquisitely specific and crucial post-translational modification of secreted proteins emerges from these reviews. The cumulative evidence strongly suggests that although some MMPs can and do cleave ECM components, notably fibrillar collagens, the majority of these proteinases are not key physiological participants in morphogenesis nor in control of matrix metabolism in homeostasis or disease. In contrast, deficiency of ADAMTS proteases leads to a remarkableACCEPTED array of morphogenetic MANUSCRIPT defects and connective tissue disorders consistent with a specialized role in turnover of the embryonic provisional ECM and in ECM assembly. -related proteases emerge into crucial positions in ECM assembly and turnover, although they also have numerous roles related to morphogen and growth factor regulation. To further turn the traditional view on its head, it is clear that many MMPs are key participants in many, diverse immune and inflammation processes rather than ECM proteolysis. The overlap in the activities within and between these families leads to the view that ECM proteolysis, which is indispensable for life, was over-engineered to an ACCEPTED MANUSCRIPT

extraordinary extent during vertebrate evolution. That these proteinases, which likely evolved within networks regulating morphogenesis, immunity and regeneration, also participate in diseases is a side effect of human longevity. Attempts to inhibit metalloproteinases in human diseases thus require continuing appraisal of their biological roles and cautious evaluation of potential new therapeutic opportunities.

Key Words: metalloproteinase, extracellular matrix, ADAMTS, merpin

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In this Special Issue of Matrix Biology, thirty minireviews present some of the diversity of metalloproteinase functions in the context of ECM. Although this issue could not be all-inclusive, it attempts to balance physiological and disease roles and basic and translational research and features a geographic diversity of authors. This Issue represents an astonishing range of research interests. In this prefatory article, we attempt to provide an overview of the relevant historical, fundamental, and translational setting for the Issue and seek to predict where the field may be headed in the future.

1. The metalloproteinases presented in this special issue. In this issue, the articles focus on three subfamilies of metalloproteinases with demonstrated functions in ECM metabolism: the matrix metalloproteinases (MMPs, matrixins), ADAMTS proteinases, and (BMP/tolloid proteases and meprins in mammals) [1]. These groups comprise numerous endopeptidases that each contain an Zn2+ ion (hence the prefix “metallo”) and are among nearly 200 Zn-dependent metalloproteinases found in mammals

[2]. They all belong to the metzincin clan, which is characterized by a 3-histidine (His) zinc- binding catalytic motif (His-Glu-Xaa-Xaa-His + His) and a conserved methionine (Met) following the active site [3]. A glutamate (Glu) residue within the catalytic motif activates a zinc-bound H2O molecule providing the nucleophile that cleaves peptide bonds. The metzincins are eitherACCEPTED secreted or membrane MANUSCRIPT-associated (i.e., transmembrane or membrane linked via glycolipid anchors) and are synthesized as pre-pro-polypeptides. Generally speaking, although there are a few exceptions in the ADAMTS family, the propeptides inhibit proteolytic activity and must be removed from the zymogen to elicit proteolytic activity, a process termed activation [4]. Many metalloproteinase zymogens – including all and about a third of the MMPs – contain a furin-recognition sequence between the pro- and catalytic domains and are activated intracellularly before secretion or at the cell surface. Although these metalloproteinases share a similar catalytic domain topology, ACCEPTED MANUSCRIPT

the domains immediately downstream differ radically among the metzincin families. These structural distinctions are the defining features of the respective families [1].

Because the focus of this Issue is ECM proteolysis, we have excluded the ADAM proteinases (a disintegrin-like and metalloproteinase), which have emerged as the major force in protein ectodomain shedding from the cell surface, thereby controlling a wide range of cell signaling processes [5-7]. That said, ADAM proteinases can still interface with

ECM metabolism, albeit via indirect mechanisms, such as by shedding the ectodomain of ECM receptors or influencing signaling pathways upstream of ECM homeostasis [8, 9]. Most articles in this issue focus on matrix metalloproteinases (MMPs), reflecting both their historic and current high profile in laboratory investigations. The issue features reviews on the roles for specific MMPs in acting on ECM components to control processes in stem cell biology [10, 11], muscle biology [12], central nervous system homeostasis and disease [13, 14], angiogenesis (cite: MATBIO-D-14-00118), tissue repair in skin [15] and liver [16], inflammation [17], vascular disease [18], destructive lung disease [19], and cancer [11, 20]. As stated, MMPs can affect these and other processes by proteolysis of non-ECM proteins, and discussion of these other functions and mechanisms can be found elsewhere [21-24]. Reviews by Itoh [25] and Gaffney et al. [26] reflect the considerable emphasis given to membrane-type (MT)ACCEPTED MMPs by investigators MANUSCRIPT in the MMP field. The article by Wells et al. [17] discusses the emerging role for byproducts of ECM proteolysis by MMPs – matrikines – which is also a function for several ADAMTS proteases [9].

The other MMP articles focus on a range of topics related to proteinase activity, specificity, and regulation. The importance of defining MMP-substrate interactions and inhibitory strategies is reflected in the articles by Steve van Doren [27] and Gregg Fields [28]. Key to understanding MMP function – indeed, to understanding the function of any proteinase - is identification and validation of its physiologic substrate (s), and Schlage and ACCEPTED MANUSCRIPT

auf dem Keller [29] illustrate how proteomics provides a powerful, unbiased approach toward this goal. Furthermore, two articles discuss how MMPs are regulated by TIMPs [30] and by other cellular mechanisms [31].

2. The lexicon of proteolysis. Before we continue with our overview of this Issue, we wish to discuss the commonly used terms applied to the activity of proteinases and how their meaning implies distinct biologic functions. Proteolysis is a widely used mechanisms for post-translational modification of proteins. Three words are often loosely used when discussing ECM proteolysis: turnover, remodeling, and degradation. Although each term intends to define a process that is, in essence, proteolysis, these terms do convey discrete biologic outcomes. Turnover (or accretion) is the normal, physiologic breakdown and replacement of a protein or protein complex. For example, laminins are deposited in basement membranes, and with time, degraded and replaced with new laminins. Many proteins, especially intracellular proteins, have short half-lives, on the order of 48 h or less.

In contrast, some ECM proteins, especially large, insoluble polymers, such as elastin, and interstitial collagens, may have extremely long half-lives ranging from months to years. Thus, turnover of many - but not all - ECM proteins is a relatively slow process. Remodeling could be used more narrowly for the breakdown and clearance of ECM in tissues undergoingACCEPTED architectural changes, MANUSCRIPT such as during digit web regression in developing limbs, involution of the postpartum uterus, and reduction of scar tissue in healed wounds. Unlike turnover, remodeling may not include systematic replacement of the ECM that was cleared. We propose that the term degradation be used to indicate the untimely, unregulated, or excessive matrix destruction seen in disease, typically in chronic conditions such as arthropathies, destructive lung diseases, cancer/metastatic tissue invasion and others. ACCEPTED MANUSCRIPT

The three terms defined above – turnover, degradation, and remodeling – are all indicative of ECM catabolism. However, proteolysis does not always spell destruction, and two other terms emphasize the anabolic functions of proteinases. Processing is the proteolytic modification of (typically) latent proteins that results in their maturation. It controls the activity or generation of numerous, diverse proteins, including those of the complement and coagulation cascades, digestive enzymes, peptide hormones, neuropeptides, ECM precursors (e.g., procollagen), and others. Cleavage is a similar term indicating proteolysis at a single site (or more) generating two (or more) discreet protein fragments. Cleavage can have loss-of-function effects, such as when fibrillar collagen proteolysis by leads to thermal instability of the triple helix, which in turn potentiates proteolysis by other enzymes, or gain-of-function proteolysis, such as the removal of a propeptide, release of functional ectodomains, and generation of bioactive fragments, such as matrikines.

3. Which metalloproteinases degrade ECM? Although MMPs have long been considered to be the prototypic matrix-degrading proteases, in recent years and with the application of engineered genetic models, they have been shown to function more as processing enzymes that control numerous, diverse cell processes. Indeed, via cleavage of cytokines, chemokines, antimicrobialACCEPTED peptides, and cell MANUSCRIPT surface proteins, MMP proteolysis affects critical regulatory functions related to immunity, tissue repair, differentiation, and cell transformation [20, 21, 23, 24, 32]. Furthermore, some MMPs may have functions that are independent of their catalytic activity. For example, the hemopexin domain of MMP12 has intrinsic antimicrobial activity [33] and this is internalized and functions as a transcription factor controlling host responses to viral infection [34]. It has also become clear that, as a family, MMPs are not fully responsible for bulk or homeostatic turnover of ECM. Notable exceptions to this generalization are MMP13 and two MT-MMPs, MMP14 ACCEPTED MANUSCRIPT

(MT1-MMP) and MMP16 (MT3-MMP), which have essential roles in turnover of the collagen matrix of bone [35-42].

In contrast, other metalloproteinase families have emerged as notable effectors of ECM maturation, assembly, and turnover. Meprins and BMP/tolloid metalloproteinases, the mammalian metalloproteinases related to crayfish astacins, are the topic of two chapters. One article covers the emerging roles for meprins, which have an unusual subunit structure [43], in procollagen processing and their relevance to fibrosis [44], whereas the second reviews the diverse roles of BMP1/tolloid-like proteinases in extracellular matrix assembly and growth factor activation [45].

The ADAMTS proteinases have taken their place among the leading participants in ECM turnover, especially in morphogenesis, where dramatic roles for MMPs have been elusive. ADAMTS proteases have a similar reprolysin-like active site motif (His-Glu-Xaa-

Xaa-His + His-Asp) as ADAMs, but the similarities end there. ADAMTS proteases are not transmembrane proteins, but all are secreted. Like other secreted metalloproteinases, such as several MMPs [4], it is likely that ADAMTS proteases operate quite close to the cell surface through interactions with pericellular matrix or cell-surface molecules. Among the seven chapters devotedACCEPTED to ADAMTS proteases, MANUSCRIPT two are of a broad nature, one discussing the insights currently available from mammalian genetic studies [46], and the other on the complex emerging associations between ADAMTS proteinases and cancer [47]. Four, more specialized reviews, examine their functions in angiogenesis [48], fertility [49], collagen biosynthesis [50], and nervous system disorders [51]. A review on nematode ADAMTS mutants identifies the ECM networks that these proteases participate in [52] and will be useful in seeking a parallel with similar mammalian networks. ACCEPTED MANUSCRIPT

4. Upending the traditional view of matrix metalloproteinases. The world view of MMPs had become somewhat one-dimensional over time. However, through the 30 solicited reviews, this special issue seeks to re-evaluate this view and to provide an update on new developments and concepts. It is probably fair to say that to many biologists, matrix-degrading metalloproteinases were a monolith comprising just MMPs. Whenever discussed in many reviews, chapters, and indeed original research articles, MMPs are assumed to operate only in the catabolism, typically large scale destructive catabolism of

ECM in tissue remodeling and disease. Based on these generally held assumptions, it would seem that MMPs functioned only to destroy tissue. Such views have been further fueled by the inordinate amount of attention given to MMPs as real or aspirational targets in cancer, inflammation, and arthritis [53, 54]

Two lines of thought ought to have mitigated against this rather simplistic view some time ago. The first vertebrate matrix-degrading metalloproteinase to be discovered was identified in a crucial morphogenetic process, namely removal of the vestigial tail and fins in tadpoles that allowed transition to terrestrial life [55]. Without this enzyme, the tadpole would have been, both colloquially and literally, dead in the water. Since then, accumulating evidence indicates that many, perhaps all, morphogenetic events require ECM proteolysis. AsACCEPTED all developing tissues contain MANUSCRIPT a variety of ECM proteins, a requirement for extensive proteolytic remodeling during development seems intuitive.

But are MMPs really required for morphogenesis and organogenesis? Observations with knock-out mice have demonstrated rather clearly that with very few exceptions, such as the MT-MMP roles mentioned above, MMPs do not play critical roles in development. On the other hand, a requirement for MMP-mediated proteolysis may not be evident from the traditional approach of analyzing single knockouts if embryogenesis involves coordination of multiple proteinases. Cooperative roles have been shown for ADAMTS ACCEPTED MANUSCRIPT

proteinases [46] and some MT-MMPs [56] in vivo. Furthermore, as this issue highlights, a number of metalloproteinases are required not only for ECM turnover but for its assembly as well. Typically, the metalloproteinases that facilitate ECM assembly, such as some BMP/tolloid proteases and ADAMTS, as discussed in the issue [44-46, 52], do so through molecular activation or maturation of matrix precursor proteins.

The second line of thought concerns the observed expansion of during vertebrate evolution [57]. It was counterintuitive to have assumed that enzymes that have evolved over epochs arose only to cause disease in the host and lead to its eventual demise. That metalloproteinases are indeed involved in disease is irrefutable, but their primacy is in physiology, not disease and is best reconciled with Theodosius Dobzhansky’s view that “Nothing makes sense except in the light of evolution” .

5. What does the future hold for the field? For much of the past decade, the MMP field experienced a void resulting from the disillusionment of cancer researchers and the pharmaceutical industry with MMPs as cancer targets [23, 53]. Because these failed trails used drugs that likely inhibited all metalloproteinases, the disappointing outcomes were not a surprise to many. On the other hand, it is clear that several individual MMPs do contribute to cancerACCEPTED progression and poor outcomesMANUSCRIPT in many other diseases. Hence, as discussed above and in the thoughtful review in this Issue by Shay et al. [20], now is the time to regroup and reassess molecular targets and pathways in cancer using more specific therapeutic reagents than small molecule inhibitors. Additionally, fundamental investigations into the nature of cancer and the role of proteinases therein should continue, because our knowledge, to understate the fact, is imperfect.

A surge of translational interest resulted from the finding that two ADAMTS proteinases, ADAMTS4 and ADAMTS5 were the crucial enzymes involved in aggrecan ACCEPTED MANUSCRIPT

degradation in osteoarthritic cartilage. However, this interest failed to sustain sufficient momentum owing to limitations imposed by the intrinsic challenges of osteoarthritis, namely, its long latency, protracted clinical course, paucity of sensitive biomarkers, and potential side effects from long-term inhibition of ADAMTS4 and ADAMTS5. The hazards of an imperfect state of knowledge get a salutary reminder from the observation that ADAMTS proteases were first discovered 17 years ago, yet next to nothing is known about many members of this family. This is not to say that all attempts at therapeutic design should be halted, but rather they should be restrained while delving deeper into the fundamental nature of selected target metalloproteinases.

There are a bright lights on the horizon, however, since new technologies, such as mass- spectrometry based proteomics, gene editing, and the ability to make and manipulate stem cells, will together expedite knowledge acquisition and provide tools for designing new drugs. As the biology of a protease is really the biology of its substrates, thought should be given to approaches that identify the substrate rather than the explore the protease. For example, an antibody inhibiting substrate cleavage such as recently described to prevent versican cleavage [58] could obviate side effects resulting from broad-spectrum inhibition by small molecule inhibitors, or effects on other substrates of the target protease whose cleavage is desirableACCEPTED. In these endeavors, the MANUSCRIPT field ought to be enormously cheered by the many exciting discoveries yet to come, and the promise that lies ahead.

Acknowledgements: The authors thank all the contributors to this Special Issue for their thoughtful, comprehensive, and provocative reviews. Special thanks are due to Renato V. Iozzo and the publisher Elsevier for proposing the creation of the Special Issue and for supportive and constructive help throughout. Work in the authors’ laboratories is supported by National Institutes of Health awards HL107147, EY021151 and EY024943 (to

SA) and HL089455 and HL098067 to (WCP). ACCEPTED MANUSCRIPT

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