The Role of Timps in Regulation of Extracellular Matrix Proteolysis

Review

The role of TIMPs in regulation of extracellular matrix proteolysis

Valerie Arpino a,d, Michael Brock a,d and Sean E. Gill a,b,c,d a - Centre for Critical Illness Research, Lawson Health Research Institute, London Health Sciences Center, London, Ontario, Canada b - Division of Respirology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada c - Department of Medicine, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada d - Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada

Correspondence to Sean E. Gill: 800 Commissioners Road East, Centre for Critical Illness Research, Victoria Research Labs, A6-134, London, ON, N6A 5W9, Canada. [email protected] http://dx.doi.org/10.1016/j.matbio.2015.03.005 Edited by W.C. Parks and S. Apte

Abstract

Tissue inhibitors of metalloproteinases (TIMPs), which inhibit matrix metalloproteinases (MMPs) as well as the closely related, a disintegrin and metalloproteinases (ADAMs) and ADAMs with thrombospondin motifs (ADAMTSs), were traditionally thought to control extracellular matrix (ECM) proteolysis through direct inhibition of MMP-dependent ECM proteolysis. This classical role for TIMPs suggests that increased TIMP levels results in ECM accumulation (or fibrosis), whereas loss of TIMPs leads to enhanced matrix proteolysis. Mice lacking TIMP family members have provided support for such a role; however, studies with these TIMP deficient mice have also demonstrated that loss of TIMPs can often be associated with an accumulation of ECM. Collectively, these studies suggest that the divergent roles of TIMPs in matrix accumulation and proteolysis, which together can be referred to as ECM turnover, are dependent on the TIMP, specific tissue, and local tissue environment (i.e. health vs. injury/disease). Ultimately, these combined factors dictate the specific metalloproteinases being regulated by a given TIMP, and it is likely the diversity of metalloproteinases and their physiological substrates that determines whether TIMPs inhibit matrix proteolysis or accumulation. In this review, we discuss the evidence for the dichotomous roles of TIMPs in ECM turnover highlighting some of the common findings between different TIMP family members. Importantly, while we now have a better understanding of the role of TIMPs in regulating ECM turnover, much remains to be determined. Data on the specific metalloproteinases inhibited by different TIMPs in vivo remains limited and must be the focus of future studies. © 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction the current paper and can be found in a number of excellent reviews [4–7]. There are four homo- The tissue inhibitors of metalloproteinases logous members of the TIMP family [1–3]. General- (TIMPs) are tissue specific, endogenous inhibitors ly, all TIMPs are capable of inhibiting all known of metalloproteinases, including the matrix metallo- MMPs; however, the efficacy of MMP inhibition proteinases (MMPs) as well as the closely related, varies with each TIMP. For example, TIMP1 is a a disintegrin and metalloproteinases (ADAMs) strong inhibitor of many MMPs except for some and ADAMs with thrombospondin motifs (ADAMTSs) of the membrane type (MT)-MMPs, including [1–3]. In the current paper, various MMPs and ADAMs MMP14, -15, -16, -19, and -24 [1,2]. As well, both will be mentioned as it pertains to the role of the TIMP1 and -3 associate with latent or pro-MMP9 different TIMPs; however, complete descriptions of while TIMP2, -3 and -4 are capable of interaction these metalloproteinases is beyond the scope of with pro-MMP2 [1]. 0022-2836/© 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Matrix Biol. (2015) 44–46, 247–254 248 The role of TIMPs in regulation of extracellular matrix proteolysis

TIMPs also inhibit ADAMs and ADAMTSs, deposition, and all of these process have been although, this inhibition is primarily accomplished found to be controlled by TIMPs (Fig. 1). by TIMP3. The proteolytic activity of ADAM10 can be Collectively, these findings suggest that TIMPs inhibited by TIMP1 and -3, whereas TIMP3 alone is can both directly inhibit ECM proteolysis as well as an efficient inhibitor of ADAMTS4 and -TS5, which indirectly control ECM turnover and that the are postulated to cleave aggrecan, a component specific roles for each TIMP is likely dependent of cartilage matrix [8–10]. TIMP3 can also inhibit on the metalloproteinases inhibited by the given ADAM12S mediated cleavage of insulin-like growth TIMP within a specific, local tissue environment factor binding protein (IGFBP) 3 and -5 [11], and (i.e. health vs. injury/disease; Table 1; Fig. 1). The TIMP3, or the N-terminal domain of TIMP3, subsequent sections will describe the evidence for specifically inhibits ADAM17 [12,13]. each of the TIMPs in direct inhibition of ECM Localization of TIMPs is important in the control proteolysis as well as indirect regulation of ECM of TIMP function. TIMP1, -2, and -4 were initially turnover. thought to be soluble inhibitors, whereas TIMP3 was localized to the extracellular matrix (ECM) TIMP1 through interaction with heparan sulfate and other sulfated proteoglycans [14]. More recently TIMPs have been shown to localize with cell surface Direct Inhibition of ECM proteolysis by TIMP1 proteins; however, this interaction has primarily been associated with metalloproteinase-indepen- Many pathological conditions, including the forma- dent functions for the various TIMPs and as yet, tion of scar tissue, pulmonary fibrosis and diabetic has not been found to impact ECM turnover [15– nephropathy, are associated with excessive deposi- 19]. For instance, TIMP1 has been shown to tion of ECM components leading to fibrosis [22–24]. mediate angiogenesis through interaction with β1 Interestingly, TIMP1 expression is increased within integrin and CD63 [16,17]. Additionally, TIMP3 has these profibrotic environments, which suggests a been shown to antagonize vascular endothelial role for TIMP1 in restricting ECM proteolysis. For growth factor (VEGF) signaling by binding directly example, TIMP1 expression is increased in mesan- to the VEGF receptor [18,19]. gial cells treated with recombinant human connec- Initial evidence for the role of TIMPs in ECM tive tissue growth factor in conjunction with high proteolysis was the ability of TIMPs to inhibit various glucose concentrations, which replicates the condi- MMPs in vitro and the association of increased tions causing diabetic nephropathy [24]. This in- expression of TIMPs with an accumulation of matrix crease in TIMP1 is associated with a significant (i.e. lung fibrosis) [20]. Ultimately, this belief led to reduction in the matrix degradative capacity of the generally accepted idea that the balance mesangial cells, and the addition of a TIMP1 between MMPs and TIMPs was responsible for neutralizing antibody abolishes this reduction in the ECM proteolysis, and that a shift in the balance matrix degradative capacity [24]. in favor of MMPs resulted in increased ECM Mice lacking TIMP1 (Timp1−/− mice) have provid- proteolysis, whereas a shift in the balance in favor ed additional evidence supporting the role of TIMP1 of TIMPs resulted in protection of the ECM and in control of ECM proteolysis within the cardiovas- decreased proteolysis. Importantly, examination of cular system. Timp1−/− mice exhibit increased left the phenotypes of mice lacking different TIMPs ventricular wall stress due to decreased myocardial demonstrated that TIMPs have many different roles, fibrillar collagen content as discovered by compar- one of which is directly regulating ECM proteolysis ative endocardiographic studies, suggesting TIMP1 (Fig. 1). plays a role in the preservation of normal myocardial It has become apparent, however, that the structure and function [25]. Furthermore, when fed balance between metalloproteinases and TIMPs an atherogenic Western-type diet, Timp1−/− mice controls much more than simply ECM proteolysis. In crossed with apolipoprotein E knockout (apoEO) fact, studies using mice genetically deficient for mice exhibited a greater number of abdominal aortic different metalloproteinases or TIMPs have demon- aneurysms, characterized by infiltrated macro- strated that metalloproteinases process many bio- phages and extensively fragmented elastic lamellae, logically active proteins, including cytokines, compared to that of apoEO mice alone [26]. Such chemokines, and cell surface proteins [7,21].Fur- gelatinolytic activity present at the site of the aortic thermore, regulation of these biologically active rupture was eliminated with the addition of recombi- proteins by the metalloproteinase/TIMP axis has nant TIMP1 [26]. Collectively, both the expression an indirect effect on matrix turnover in many studies as well as the functional studies in Timp1−/− instances. For example, altered transforming mice strongly suggest that TIMP1 has the ability to growth factor (TGF) β signaling, inflammation, or attenuate degradation of the extracellular matrix in the number of myofibroblast-like cells all potentially healthy tissues as well as under pathological alter ECM turnover leading to increased ECM conditions. The role of TIMPs in regulation of extracellular matrix proteolysis 249

Fig. 1. The diverse functions of TIMPs in regulating matrix turnover are dependent on the TIMP, the specific tissue and local tissue environment, as well as the specific metalloproteinases being regulated by a given TIMP. (A) TIMPs are known to regulate metalloproteinase activity within the ECM thereby directly inhibiting ECM proteolysis. (B) TGFβ is sequestered within the ECM and can be released and activated by metalloproteinase activity. This release of TGFβ subsequently leads to increased ECM deposition by stromal cells leading to fibrosis. Inhibition of metalloproteinases by TIMPs restricts TGFβ activation thereby decreasing ECM deposition. (C) Modulation of cell-surface cytokines and cytokine receptors by metalloproteinases can lead to increased inflammation (i.e. neutrophil chemotaxis). TIMP activity can inhibit shedding of these cell surface proteins, and as a result, indirectly regulate ECM turnover through control of inflammation.

Indirect regulation of ECM turnover by TIMP1 TIMP2 Based on the increased expression of Timp1 in pulmonary fibrosis, one would predict that TIMP1 Direct inhibition of ECM proteolysis by TIMP2 attenuates ECM turnover [27]. However, induction of lung fibrosis in mice lacking TIMP1 (Timp1−/− mice) Multiple studies provide evidence supporting a role reveals that Timp1−/− mice have similar severity of for TIMP2 in inhibition of ECM proteolysis in several fibrosis as wild type mice following lung injury [28]. tissues, although many of these studies are Interestingly, while fibrosis was similar between correlative in nature. For instance, correlative data wild type and Timp1−/− mice, inflammation was supports a role for TIMP2 in protecting the ECM from significantly increased in Timp1−/− mice suggesting proteolysis in fracture healing [30], keloids [31], liver that TIMP1 has a key role in restricting inflammation [32,33], kidneys [34,35], Dupuytren's contracture following lung injury [28]. This enhanced inflamma- [36,37], and in heart tissues [38–42]. As a specific tion may have contributed to the fibrosis observed in example, patients suffering from Dupuytren's these mice. contracture, a disease caused by excessive ECM Examination of liver fibrosis following injury revealed deposition leading to fixed flexion of joints in the a similar role for TIMP1 in the liver. Timp1−/− mice hand, have a disruption in the balance between had significantly increased injury, inflammation, and TIMP2 and MMP2 in favor of TIMP2, which suggests fibrosis following carbon tetrachloride-induced liver that increased TIMP2 is associated with pathophys- injury compared to wild type mice [29]. Thus, these iological ECM accumulation [36,37]. studies support a role for TIMP1 in restricting both Deposition of excess ECM is also a key patho- inflammation and ECM accumulation/fibrosis follow- physiological feature of heart disease. Interestingly, ing injury. the ECM sparing role for TIMP2 appears to be 250 The role of TIMPs in regulation of extracellular matrix proteolysis

dependent on the model of injury with TIMP2 having distinct roles in different models of heart disease. Following transverse aortic constriction, Timp2 mRNA levels decreased in mice lacking the tran-

is)orindirect scriptional regulator p8, and this decrease was associated with reduced collagen deposition [38]. Furthermore, following induction of myocardial infarction in mice lacking TIMP2 (Timp2−/− mice), heart tissue was found to have less dense and more Indirect disorganized fibrillar collagen [43]. This adverse ECM remodeling was found to be due to greater MMP14 activity, which supports a role for TIMP2 in regulation of ECM proteolysis through inhibition of Direct MMP14 [43].

Indirect regulation of ECM turnover by TIMP2

TIMP2 directly inhibits metalloproteinases, Indirect including MMP2, but TIMP2 is also known to be required for activation of MMP2 through association with MMP14 [44]. While MMP2 is thought to have a role in degrading the ECM, increased MMP2 activity

Direct has also been associated with increased fibrosis due to inflammation in models of recurrent lung injury [45]. Furthermore, studies by Kandalam and colleagues found that cardiac repair following transverse aortic −/− Indirect constriction was impaired in Timp2 mice [46]. This persistent injury in mice lacking TIMP2 was associated with increased fibrosis due to elevated collagen ebr aeoie as categorized members either direct inhibition (direct of ECM proteolys 1–2 papers 3–4 papersfiber stability or more papers 5 resulting from higher levels of SPARC (secreted protein acidic and rich in cysteine) [46]. Direct Collectively, these studies provide evidence that TIMP2 may act to indirectly control ECM abundance, in some cases through activation of Published effects of TIMPs (from TIMP deficient mice) MMP2. Indirect TIMP3 TIMP1 TIMP2 TIMP3 TIMP4

Direct Inhibition of ECM proteolysis by TIMP3 Direct TIMP3 has been demonstrated to have many different functions including regulation of inflammation through inhibition of ADAM17 [47]. There is strong correlative and functional evidence, however, suggesting that TIMP3 is a key inhibitor of ECM remodeling. For example, the highly controlled process of embryo implantation relies heavily on ECM degradation by MMPs [48]. Specifically, trophoblast cells, which form the outer layer of the implanting blastocyst, express Mmp9, while mater- Tissue Cardiac Respiratory Renal Other nal uterine cells express Timp3 [48]. The presence of various amounts of exogenous TIMP3 within an extracellular matrix gel incubated with blastocysts

Matrix turnover phenotypes observed in mice lacking different TIMP family resulted in a dose-dependent reduction in extracellular matrix degradation, illustrating the critical role of TIMP3 in regulating uterine ECM degradation during Table 1. (indirect regulation of ECM turnover) effects embryo implantation [48]. The role of TIMPs in regulation of extracellular matrix proteolysis 251

In addition to embryo implantation, TIMP3 acts as a proteolysis. Interestingly, following injury, the role major facilitator of extracellular matrix sparing in several for TIMP3 with respect to the ECM becomes other homeostatic processes, including lung and bone more complex. For example, in mice lacking development and remodeling as well as mammary TIMP3, fibrosis is enhanced following bleomycin-in- gland involution. In mice lacking TIMP3 (Timp3−/− duced injury, suggesting that the presence of TIMP3 mice), metalloproteinase activity is increased in the would actually restrict ECM deposition [58]. While lungs during development leading to a significant the mechanism leading to this fibrosis remains to be reduction in fibronectin abundance and consequently, determined, Timp3−/− mice have impaired resolution impaired branching of the bronchiole tree compared to of inflammation following lung injury, which could lungs from wild type mice. Importantly, treatment with contribute to the enhanced fibrotic response [58,59]. GM6001 (both in whole organ culture or in vivo in Studies have revealed similar roles for TIMP3 in pregnant mice) rescues the enhanced degradation of both the injured heart and kidney [60–62]. Aortic fibronectin and the impaired bronchiole branching in banding leading to cardiac mechanical stress Timp3−/− lungs [49,50]. Metalloproteinase activity is resulted in increased fibrosis in Timp3−/− hearts increased in the lungs of adult Timp3−/− mice as well, compared to wild type hearts due to enhanced and this increase is also associated with increased TGFβ1 activation [60]. Additionally, kidney fibrosis is ECM degradation, specifically collagen and fibronectin increased following injury in Timp3−/− vs. wild type degradation [51,52]. Furthermore, the increased me- mice due to altered activation of pericytes leading to talloproteinase activity and enhanced ECM remodeling a shift in pericyte-phenotype to a myofibroblast-like observed in the lungs of Timp3−/− mice is also cell [61,62]. Collectively, these studies suggest that associated with decreased lung function and increased TIMP3 potentially mediates fibrosis through multiple lung compliance, and is exacerbated in models of pathways, from controlling inflammation to regula- sepsis [52,53]. tion of TGFβ1 activation and pericyte phenotype. The hind-limb joints of Timp3−/− mice also possess significantly reduced amounts of articular cartilage exhibited by extensive cleavage of collagen and TIMP4 aggrecan due to increased MMP and ADAMTS activity, which suggests that decreased expression of TIMP3 may play a pathophysiologic role in the Direct inhibition of ECM proteolysis by TIMP4 development of osteoarthritis [54]. Additionally, TIMP4 appears to be the least studied of the TIMPs studies have shown that mice lacking TIMP3 experience accelerated mammary gland involution and as such, there are limited publications discussing the role of TIMP4 in regulation of ECM proteolysis. characterized by earlier fragmentation of fibronectin There is, however, correlative data in an array of due to increased gelatinase activity [55]. These tissues suggesting that TIMP4 restricts ECM proteol- events, however, are rescued with the biochemical ysis in the developing lung [63], female reproductive reconstitution of recombinant TIMP3 [55]. tract [64],eyes[65], and transplanted tissues [66]. Finally, TIMP3 deficiency has been shown to lead For example, vaginal tissue is a site of constant to maladaptive cardiovascular ECM remodeling ECM remodeling due to the shedding and subse- following models of myocardial infarction or hyper- quent thickening of the endometrial lining during the tension [56,57]. Following ligation of the coronary −/− menstrual cycle. Pelvic organ prolapse (POP) is artery, Timp3 mice exhibit accelerated left ventricular systolic dysfunction compared to wild thought to be dependent on a lack of ECM deposition and increased ECM destruction leading to a weak- type littermates, coupled with increased gelatinase ening of the pelvic floor [64]. Timp4 expression is activity and reduced myocardial collagen content significantly lower in patients suffering from POP, [56]. Following angiotensin-II-induced hypertension, −/− which supports a role for TIMP4 in protection of the mesenteric and carotid arteries from Timp3 mice ECM from increased proteolysis [64]. have increased gelatinolytic and elastolytic activity as well as suppressed collagen and elastin content vs. arteries from wild type mice, which are rescued Indirect regulation of ECM turnover by TIMP4 by treatment with doxycycline, an MMP inhibitor [57]. Collectively, these studies support a key role for While studies examining TIMP4 are limited, there TIMP3 in regulating ECM proteolysis in both healthy is convincing evidence that, similar to other TIMP tissue and disease, thereby controlling tissue struc- family members, TIMP4 also acts to restrict fibrosis. ture and function. Following myocardial ischemia/reperfusion, ECM remodeling often occurs with an initial fibrotic Indirect regulation of ECM turnover by TIMP3 response followed by ECM turnover. In mice lacking TIMP4, interstitial fibrosis was enhanced one week In many healthy tissues, such as the lung, heart following injury compared to wild type mice [67]. and bone, TIMP3 has been found to restrict ECM Interestingly, this augmented fibrosis was 252 The role of TIMPs in regulation of extracellular matrix proteolysis associated with greater MMP14 activity as well as Received 19 December 2014; increased inflammation, suggesting that TIMP4 Received in revised form 9 March 2015; regulates ECM deposition through inhibition of Accepted 11 March 2015 MMP14 and restriction of inflammation [67]. Available online 21 March 2015

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