Review

BMP-1/tolloid-like proteinases synchronize matrix assembly with activation to promote morphogenesis and tissue remodeling

Sandrine Vadon-Le Goff, David J.S. Hulmes and Catherine Moali

UMR 5305, Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique CNRS / University of Lyon, 69367 Lyon, France

Correspondence to Catherine Moali: Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, 7 Passage du Vercors, 69367 Lyon, France. [email protected] http://dx.doi.org/10.1016/j.matbio.2015.02.006 Edited by W.C. Parks and S. Apte

Abstract

Bone morphogenetic -1 (BMP-1)/tolloid-like proteinases, here called BTPs, include the proteases originally identified for their roles in the C-terminal maturation of fibrillar procollagens (“procollagen C-proteinase”). Though numerous other substrates have since been discovered, the BTPs remain the main proteases involved in extracellular matrix assembly with little or no implication in matrix degradation. During the same period however, the BTPs have also become established as important proteases in the activation of growth factors, including TGF-β1, BMP-2/-4, GDF-8/-11 and IGFs, as well as the release of anti-angiogenic fragments from parent . The BTPs are therefore key players in many pathophysiological processes such as morphogenesis, tissue repair and tumor progression. This mini-review summarizes our current knowledge of the functions of BTPs, their substrates and unusual mechanisms of regulation, and discusses their potential as new targets for future therapies. © 2015 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 [4]. With the identification of two further human , encoding mammalian tolloid-like 1 and 2 (mTLL-1, Bone morphogenetic protein-1 (BMP-1) was origi- mTLL-2), the entire BMP-1/tolloid-like proteinase nally identified in bone extracts capable of inducing subfamily (called here BTPs) was born [5] (Fig. 1A). bone formation at ectopic sites [1]. Unlike the other Bmp1-andTll1-null mice are perinatal or embryonic BMPs found in these extracts however, all of which lethal, respectively [6,7], involving multiple develop- are members of the TGF-β growth factor superfamily, mental defects (ventral wall closure, skeletal and heart BMP-1 is a zinc-dependent metalloproteinase (a development) and pointing to specific roles for each frequent cause of confusion in the literature). The proteinase. In contrast, in vitro, there is considerable BMP-1 molecule consists of an -like catalytic overlap in the activities of the different BTPs, which domain followed by several CUB (for complement suggests that the in vivo observations are most C1r/C1s, uEGF, BMP-1) and EGF domains (Fig. 1A). probably explained by spatial and temporal variations As a member of the astacin family, which also includes in expression between the different isoforms [7]. the meprins, BMP-1 belongs to the metzincin clan [2], It is generally agreed that BMP-1 is the most active comprising (in humans) the , the matrix form, which may reflect the roles of the non-catalytic metalloproteinases (MMPs), and the ADAMs/ domains. In this regard, the isolated catalytic domain ADAMTSs. Human BMP-1 was found to resemble has been found to be relatively promiscuous, showing Drosophila tolloid, a proteinase involved in dorso- specificity to be conferred by the non-catalytic domains ventral patterning with additional CUB and EGF [8]. In terms of three-dimensional structures, only those domains [3], and later a mammalian homolog of tolloid of the catalytic domains of BMP-1 and mTLL-1 are was identified (mTLD) which turned out to be a known [9] (Fig. 1B). A particular feature of the active splice-variant encoded by the same as BMP-1 site, found only in BTPs and not in other metzincins, is

0022-2836/© 2015 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) 44–46,14–23 Bone morphogenetic protein-1/tolloid-like proteinases 15

Fig. 1. Structures of BTPs and associated proteins. (A) Domain structures of BMP-1, mTLD, mTLL-1 and mTLL-2 as well as the PCPEs. (B) Close-up of the active site in the astacin domain of BMP-1 [9] showing Zn2+ ions (pink), key residues and also Phe128 which is mutated to Leu in a recessive form of OI [100]. Numbering begins at the start of the mature protein, after cleavage of the propeptide region. (C) Diagram of the horseshoe shape of BMP-1 deduced by Berry et al. [10]. (D) Interaction of the CUB1CUB2 region of PCPE-1 with the stalk region of the procollagen III C-propeptide trimer (CPIII) [57]. the presence of a highly unusual disulfide bond aspects of matrix assembly, as well as more recently a between two adjacent cysteines. These occur in a large number of substrates involved in growth factor relatively flexible cysteine-rich loop that is thought to activation and . Another feature of the regulate substrate access through conformational BTPs, unlike the MMPs which are controlled by changes [9]. Concerning the full-length proteins, low TIMPs, is the relative scarcity of specific endogenous resolution structures have been determined for BMP-1, inhibitors. Instead, BTP activity seems to be controlled mTLD and mTLL-1 [10,11]. They reveal BMP-1 to be a by substrate-specific enhancer molecules. Here we monomer in solution, with the C-terminal CUB domain review the substrate repertoire of the BTPs, their folded back towards the catalytic domain (Fig. 1C). In regulation by other extracellular matrix components contrast, both mTLD and mTLL-1 form dimers, in such and their implications as potential therapeutic targets a way that the catalytic domain is relatively hidden, in different forms of disease. suggesting a substrate-exclusion mechanism for the control of substrate specificity. A distinguishing feature of the BTPs, in the context Functions of BTPs: shaping organs of ECM (extracellular matrix), is their exclusive and tissues involvement in biological processes other than just degradation of matrix proteins per se. Originally called procollagen C-proteinases (PCPs) by matrix biolo- Matrix assembly gists for their role in the control of fibril assembly by removal of the procollagen C-propeptides, PCP-1 and Fibrillar (I, II, III, V, XI) are synthesized as PCP-2 were subsequently identified, respectively, as soluble precursor forms (procollagens) which must BMP-1 and mTLD [12,13]. Since then, numerous undergo proteolytic maturation to assemble into fibrils substrates have been identified regulating various (Fig. 2). While ADAMTS-2, -3 and -14 are known to 16 Bone morphogenetic protein-1/tolloid-like proteinases

Fig. 2. Schematic representation of known BTP substrates, related functions and associated regulatory proteins. See text for details. Question marks indicate a controversial activity or the absence of proof of a similar mechanism in mammals.

remove the N-terminal extensions of procollagens I–III minor fibrillar procollagens V and XI, both at the N- (N-propeptides; see the review by Colige & and C-terminus [19–22], together with subtilisin-like Bekhouche in this special issue), BTPs are the main proprotein convertases (SPCs) and ADAMTS-2. enzymes in charge of the rate-limiting step of fibril Noteworthy, BTPs cleave procollagens V and XI formation, that is the removal of the C-terminal within their N-propeptide, leading to the retention of propeptides. Very little is known about the molecular the variable domain and short triple helix which can details governing the C-proteinase activity except that project outside the fibril surface and limit fibril growth aggregated forms of procollagens are more efficiently (Fig. 2; [23]). Another mechanism limiting the lateral cleaved than soluble forms [14] and that, in contrast to growth of fibrils, with major consequences what is known for the N-proteinase activity, both on fibril diameter and spacing, is the interaction trimeric forms and individual procollagen chains can between fibrillar collagens and small leucine-rich be processed by BTPs [15]. Interestingly, the long- proteoglycans (SLRPs). Interestingly, three mem- lived dogma that ADAMTSs and BTPs were the sole bers of the SLRP family (decorin, biglycan and proteases involved in fibrillar procollagen processing mimecan/osteoglycin) have been shown to undergo was recently challenged by the discovery that meprins proteolytic maturation by BTPs in their N-terminal can also act as N- and C-proteinases of procollagens I region [24–26]. and III ([16,17]; see the review by Prox et al. in this Finally, BTPs have been shown to control the special issue). However, compensatory mechanisms assembly of basement membranes (BM) through the must be restricted to specific situations/tissues since maturation of collagen VII [27], the main component mice deficient for the Bmp1 gene have largely of anchoring fibrils which ensure the cohesion of the abnormal collagen fibrils [6,18]. BM with the underlying stroma, and also laminin 332, In addition to the maturation of procollagens I–III, an important BM molecule which can also bind cell BTPs also participate in the complex maturation of receptors on epithelial cells (Fig. 2). BTPs can Bone morphogenetic protein-1/tolloid-like proteinases 17 cleave both the α3 and γ2 chains of laminin 332 [42]), other proteinases such as MMPs and plasmin [28,29], thereby regulating its ability to induce cell can perform the subsequent steps which consist in the migration [30] or to be stably incorporated into the degradation of LAP and in the release of active BM. TGF-β1. More recently, we have shown that BTPs could also contribute to maintaining high levels of Matrix mechanical properties active TGF-β1 in tissues by promoting the degradation of two TGF-β antagonists (soluble betaglycan and Connective tissues differ by their tensile strength, CD109; [43]). their elasticity and their resistance to compressive BTPs can also activate several other members of loads. These properties are controlled by matrix the TGF-β or IGF superfamilies. This occurs either by composition and organization but also by the level of degradation of propeptides, as for GDF-8/11 [44,45], cross-links that stabilize the three-dimensional net- or of antagonists, as for BMP-2/4 (; [46,47]) works formed by ECM proteins. Very important in and IGF-1/2 (IGFBP3; [48]). These BTP targets affect this context are the lysyl oxidases, a family of a large number of pathophysiological processes copper-dependent amine oxidases that oxidatively ranging from dorso-ventral patterning and morpho- deaminate specific lysines in collagen and elastin to genesis to muscle growth or tumor progression. trigger the spontaneous formation of cross-links Finally, BTPs cleave several proteins endowed (Fig. 2; [31]). Given their implication in the activation with strong angiogenic properties. First, endorepel- of two isoforms of lysyl oxidases (LOX and LOXL; lin, the anti-angiogenic fragment of the large BM [32–34]), BTPs are also in the front line in the proteoglycan perlecan, is cleaved by BTPs to regulation of tissue stiffness and stiffness-dependent release the C-terminal LG3 domain which provides cell responses [35]. most of its anti-angiogenic potential [49]. Similarly, In hard tissues such as bone and dentin, a further BTPs can turn prolactin and growth hormone into degree of stiffness can be achieved through mineral- potent anti-angiogenic molecules [50,51] and abol- ization. Proteins of the SIBLING (small integrin-binding ish the pro-metastatic potential of angiopoietin-like N-linked glycoproteins) family, which bear an protein 2 [52]. unusually high number of glycosylation and phosphor- ylation sites and can bind calcium, are known to play a Other functions major role in the initiation of this process. The acidic domains of some SIBLING proteins can be released Two more atypical substrates of the BTP family by proteolysis, thereby probably enhancing their ability have been recently described. The first is gliomedin, a to bind calcium. This is the case for two of these, dentin transmembrane protein containing a collagen-like matrix protein-1 (DMP-1) and dentin sialophospho- domain within its extracellular moiety, which forms protein (DSPP), which are well-described substrates insoluble aggregates to stabilize the nodes of Ranvier of BTPs [36–38]. in the peripheral nervous system after two subsequent cleavages by SPCs and BTPs [53]. The second is Activation of signaling molecules apolipoprotein A1 which becomes the major protein component of HDL (high density lipoprotein) after TGF-β1 can induce the up-regulation of several removal of only 6 residues at the N-terminus of the important structural ECM proteins such as collagens protein [54]. The latter substrates point to unexpected and fibronectin and, in general, can modulate nega- roles for the BTP family which suggest that we are just tively or positively the expression of a large variety of at the beginning of deciphering their multifaceted matrix proteins, proteinases (among which BMP-1 functions. and mTLD [39,40]), proteinase inhibitors and growth factors. Even more strikingly, TGF-β1 can drastically modify the nature of the proteins expressed by a given Modulators of BTP action cell type, for example through the induction of EMT (epithelial to mesenchymal transition) [41].TGF-β1is As mentioned above, BTPs are somewhat pecu- secreted as a latent complex, in which the growth liar in the protease landscape in that their activities factor remains bound to its propeptide (LAP) and is are controlled by activators rather than inhibitors. targeted to the extracellular matrix through the However, most of these activators are substrate- covalent association of LAP with LTBPs (latent specific suggesting that their use is restricted to TGF-β binding proteins). When needed, TGF-β1 well-defined situations. activation can proceed through several mechanisms, both non-proteolytic and proteolytic, in a context-de- Activators pendent manner. The proteolytic pathway is initiated by the cleavage of LTBPs which releases the complex Procollagen C-proteinase enhancers (PCPEs, also formed by TGF-β1 and LAP from the ECM. While known as PCOLCEs) were the first BTP activators to BMP-1 is known to play a major role in this step (Fig. 2; be discovered. Initially co-purified with BMP-1 from 18 Bone morphogenetic protein-1/tolloid-like proteinases mouse fibroblasts [55], PCPE-1 is a 55 kDa ubiqui- be a “broad-spectrum” activator of BTPs. This tous glycoprotein devoid of catalytic activity but able to enhancement is weak in vitro but can be potentiated enhance C-propeptide removal from procollagens I-III in cellulo [76]. up to 10 fold [55–57]. Of the three domains forming Finally, the BMP antagonist chordin also has its PCPE-1 (Fig. 1A; CUB1-CUB2-NTR), only the con- dedicated activators [77,78]. These act by forming tiguous CUB domain region is necessary and ternary complexes with both chordin and BMPs and sufficient for its activity [58–60]; the NTR domain, promote the enhanced cleavage of chordin by BTPs which is homologous to TIMPs, is however able to and the modulation of BMP signaling. They include interact with heparan sulfate proteoglycans (HSPGs), twisted gastrulation, a small cysteine-rich glycoprotein thus mediating PCPE-1 anchoring to the cell surface expressed during development (but also found in and allowing an even greater stimulation of BMP-1 adult mouse brain [79]), and olfactomedin noelin tiarin activity [61,62]. Recent biochemical and structural factor-1 (ONT1; known as olfactomedin-like 3 or studies [57,59,63] have dissected the mechanism of OLFML3 in mammals) found in Xenopus (although action of PCPE-1 and shown that it functions via direct this last mechanism was not yet confirmed in and tight binding to the stalk region of the C-propep- mammals [80]). tide trimer, close to the cleavage site, with a 1:1 stoichiometry (Fig. 1D). This strong interaction with Inhibitors the procollagen substrate gives a structural basis to the striking specificity of PCPE-1 for this type of To date, α2-macroglobulin, a broad-spectrum pro- substrate [38,64,65]. However, this specificity might teinase inhibitor present in high amounts in serum, is not be restricted to the C-terminal region of procolla- the only endogenous inhibitor of mammalian BTPs gens since PCPE-1 was recently shown to enhance unambiguously reported [81]. This inhibitor irrevers- the N-terminal cleavage of procollagen V [66] through ibly inactivates BMP-1 and may regulate its circulating a completely unknown mechanism. concentration [82]. A second isoform, PCPE-2, while similar in structure In Xenopus, a very potent inhibitor of xolloid- and activity to PCPE-1 [67],hasamuchmorelimited related (a Xenopus BTP) was discovered in 2006 tissue expression. Its major site of expression in the [83] and shown to interfere with chordin cleavage. developing mouse embryo is non ossified cartilage This inhibitor named sizzled is a member of the where PCPE-1 is not found while, in adult tissues, secreted frizzled-related protein (sFRP) family and both PCPE-1 and -2 are found at high levels in the regulates dorso-ventral patterning as well as Wnt heart [68], suggesting both complementary and signaling. Although shared by zebrafish [84] and at redundant functions of the two isoforms. Intriguingly least one other Xenopus sFRP [85], the transposition also, PCPE-2 was recently found to be associated of this regulatory mechanism to mammals is a matter with serum HDL and to stimulate apolipoprotein A1 of controversy. Among the five mammalian sFRPs, cleavage by BMP-1 [69,70], an activity which might be only sFRP-2 has been reported to affect BTP activity specific to this isoform. and the results are highly contradictory, including Another substrate-specific enhancer of BTPs is inhibition [83,86], activation [87] or no effect [38,88]. periostin, a matricellular protein strongly expressed These discrepancies probably reflect the high con- during development and matrix remodeling [71,72], text-dependence of this regulation and/or the exis- which promotes proteolytic activation of lysyl oxidase tence of multiple conformational states for sFRPs. by BMP-1. Periostin-null mice show aberrant collagen The most recently reported, though equally con- fibrillogenesis with reduced cross-links [73], and troversial, inhibitor of BTPs is BMP-4, which has overexpression of periostin in calvarial osteoblasts been described as modulating its own activation, leads to increased LOX processing [74]. Direct through interaction with BMP-1 [89] or its prodomain interaction of periostin with BMP-1 has been demon- [90], creating in both cases a negative feedback loop strated, together with triggering of BMP-1 deposition for BMP signaling. to fibronectin ECM but the precise mechanism of this enhancement remains to be established. Periostin could act as a scaffolding protein mediating BMP-1, New targets for therapy LOX and fibronectin interactions or have a more active role such as induction of a conformational change in one of the partners. Fibrosis Interestingly, fibronectin itself appears to behave as an activator of BTPs. Though first described as an Since the discovery of the procollagen C-protein- enhancer of LOX cleavage [75], fibronectin is also ases (BTPs), and subsequently the procollagen capable of enhancing the cleavage of other BTP C-proteinase enhancers (PCPEs), there has been substrates such as chordin, biglycan, procollagen I considerable interest in these proteins as possible or IGFBP-3 [48,76]. As such, and in contrast to the therapeutic targets for the prevention of excess proteins described above, fibronectin is thought to collagen deposition in fibrosis. For example, Bone morphogenetic protein-1/tolloid-like proteinases 19 injection of a small molecule inhibitor of BTPs secretion and reduced glycosylation [99]. As a result, reduced scar elevation in a rabbit skin model of procollagen processing is defective, resulting in a hypertrophic scarring [91], presumably by reducing similar phenotype to the procollagen cleavage site collagen deposition following wounding. Consistent mutations [102]. The second mutation (p.Phe249Leu) with this, transcription of PCPE-1 was recently found is in the astacin domain [100] (Fig. 1), and therefore to be up-regulated in samples of hypertrophic and affects catalysis directly. This also perturbs fibril keloid scars [92], as was expression of both BMP-1 formation, but in this case bone mineral density is and PCPE-1 in a mouse model of corneal scarring relatively low [101]. In comparison, a conditional [93]. Following the discovery that mTLD was present in Bmp1/Tll1 knockout model induced at 4–6weeks human plasma, increasing the amount of circulating was recently studied and found to give rise to an mTLD in a rat model of chronic kidney disease was OI-type phenotype [103]. For PCPE-1, knockout mice found to exacerbate renal fibrosis, while the opposite are viable, but collagen fibrils are irregular in shape effect followed treatment with an anti-mTLD antibody and bones have impaired material properties that are [82]. In addition, in a rat model of liver fibrosis, PCPE-1 compensated by increased bone thickness [18]. was found to be up-regulated at both the transcriptional and protein level [94]. Cancer Cardiac fibrosis has been subject to a number of studies regarding BTPs and associated proteins. In By proteomic analysis of tissue samples, BMP-1 a mouse model induced by coronary artery ligation, has recently been shown to be one of a group of there was an increase in Bmp1 expression [87] signature extracellular matrix proteins associated which was accompanied by an even greater with tumor metastasis from colon to liver [104].Ina increase in Sfrp2 expression. These data were similar vein, both BMP-1 and PCPE-1 were found to confirmed at the protein level using a similar model be among a list of desmoplastic markers secreted by in rats [86]. There the similarities ended however, as cancer associated fibroblasts in colorectal cancer in the first study reduced cardiac fibrosis was found [105]. Furthermore, microRNA miR-194 downregu- −/− using Sfrp2 mice, while, in the second study, lation of Bmp1 expression was found to reduce lung fibrosis was reduced by increasing the amount of metastasis, apparently by reducing levels of TGF-β sFRP-2. These results mirror the equally divergent activation and consequent expression of MMP-2/-9 data from in vitro studies (§‘Inhibitors’). With regard [106]. A key component of metastasis is angiogen- to PCPEs, induction of cardiac fibrosis in rats (also esis. In this regard, in addition to the roles of BTPs in by aortic ligation) was found to increase the angiogenesis discussed earlier (§‘Activation of expression of PCPE-1 [95]. Also, since PCPE-2 is signaling molecules’), PCPE-1 has been found to relatively highly expressed in heart, cardiac fibrosis be one of five proteins that are essential for −/− induced in Pcolce2 mice by transverse aortic endothelial cell lumen formation [107]. Together, constriction was studied and found to result in lower these results indicate that BMP-1 and PCPE-1 are collagen deposition, compared to wild-type, with important components of the tumor microenviron- reduced myocardial stiffness and increased survival ment. More detailed studies will tell us if the inhibition [96]. Finally, the first study of PCPE-1 expression of their activity can have a significant impact on conducted on human samples showed a significant tumor progression and metastasis. increase of protein levels in myocardial fibrosis linked to aortic valve stenosis [97]. These latter results support the potential of PCPEs as new Conclusions targets for anti-fibrotic therapies. In summary, BTPs can simultaneously trigger Bone disorders matrix assembly and boost the synthesis of matrix proteins via a direct effect on growth factors such as BTPs have also been associated with genetic TGF-β and IGFs. These functions seem particularly disorders of bone. Missense mutations in the BTP coherent in the contexts of development, growth and cleavage sites in both the procollagen α1(I) and α2(I) tissue repair. However, recent proteomic studies chains have been described in osteogenesis imper- aimed at discovering new BTP substrates [43] fecta (OI) [98], leading to delayed procollagen suggest that we are just beginning to uncover processing. As a result, delayed cleavage of the the full spectrum of their activities and that future C-propeptides disrupts fibril formation and surpris- studies will reveal new and unexpected roles. In this ingly leads to increased bone mineral density, along regard, the BTP regulatory proteins described above with the increased susceptibility to fracture. More may represent interesting alternatives to target recently, two homozygous missense mutations have specific functions without affecting other activities been described in BMP-1/mTLD in recessive forms of BTPs. Since this article was accepted, Syx et al of OI [99–101]. The first mutation (p.Gly12Arg) is in [108] have reported additional mutations in BTPs the signal sequence and leads to severely impaired associated with severe, progressive OI. 20 Bone morphogenetic protein-1/tolloid-like proteinases

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