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Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan in the extracellular matrix, and is expressed at high levels in tissues during development and remodeling in pathological conditions. Its core protein is cleaved at a region close to the N-terminal end of CSβ domain by several members of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, i.e., ADAMTS-1, 4, 5, 9, 15, and 20. Here, using a CRISPR/Cas9 system, we generated knock-in mice (V1R), which express an ADAMTS cleavage-resistant versican. Some V1R homozygote mice, termed R/R, exhibit syndactyly and organ hemorrhage. In wound healing experiments, R/R wound shows accumulation of versican and activated TGFβ-signaling in the early stage, leading to faster healing than wild type wound. Immunostaining for Ki67, CD31, smooth muscle α-actin, periostin demonstrates higher levels of overall cell proliferation and an increased number of endothelial cells and myofibroblasts. Immunostaining for CD11b and qRT-PCR for macrophage markers revealed increased levels of inflammatory cell infiltration, especially those of M1 macrophages. Cultured R/R dermal fibroblasts revealed increased deposition of versican, type I and III collagens, and hyaluronan, and upregulation of Smad2/3 signaling. Taken together, these results demonstrate that the cleavage site determines versican turnover and that versican plays a central role in the provisional matrix during the wound repair.

Thrombospondin-4 (TSP-4) belongs to the thrombospondin protein family that consists of five highly homologous members. A number of novel functions have been recently assigned to TSP-4 in cardiovascular and nervous systems, inflammation, cancer, and the motor unit, which have attracted attention to this extracellular matrix (ECM) protein. These newly discovered functions set TSP-4 apart from other thrombospondins. For example, TSP-4 promotes angiogenesis while other TSPs either prevent it or have no effect on new blood vessel growth; TSP-4 reduces fibrosis and collagen production while TSP-1 and TSP-2 promote fibrosis in several organs; unlike other TSPs, TSP-4 appears to have some structural functions in ECM. The current information about TSP-4 functions in different organs and physiological systems suggests that this evolutionary conserved protein is a major regulator of the extracellular matrix (ECM) organization and production and tissue remodeling during the embryonic development and response to injury. In this review article, we summarize the properties and functions of TSP-4 and discuss its role in tissue remodeling.

The members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of secreted proteins, MIG-17 and GON-1, play essential roles in Caenorhabditis elegans gonadogenesis. The genetic and molecular analyses of these proteinases uncovered novel molecular interactions regulating the basement membrane (BM) during the migration of the gonadal leader cells. MIG-17, which is localized to the gonadal BM recruits or activates fibulin-1 and type IV collagen, which then recruits nidogen, thereby inducing the remodeling of the BM that is required for directional control of leader cell migration. GON-1 acts antagonistically with fibulin-1 to regulate the levels of type IV collagen accumulation in the gonadal BM, which facilitates active migration of the leader cells. The cooperative action of MIG-17 and GON-1 represents an excellent model for understanding the mechanisms of organogenesis mediated by ADAMTS proteinases.

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.

Available online xxxxKeywords:Matricellular proteinsIntervertebral discDisc degenerationExtracellular matrixMicroenvironment Theso-called“matricellular”proteinshaverecentlyemergedasimportantregulatorsofcell–extracellularmatrix(ECM) interactions. These proteins modulate a variety of cell functions through a range of interactions withcell-surface receptors, hormones, proteases and structural components of the ECM. As such, matricellularproteinsarecrucialregulatorsofcellphenotype,andconsequentlytissuefunction.Thedistinctcelltypesandmi-croenvironments that together form the IVD provide an excellent paradigm to study how matricellular proteinsmediate communication within and between adjacent tissue types. In recent years, the role of severalmatricellular proteins in the intervertebral disc has been explored in vivo using mutant mouse models inwhich the expression of target matricellular proteins was deleted from either one or all compartments of theintervertebraldisc.Thecurrentreviewoutlineswhatispresentlyknownabouttherolesofthematricellularpro-teins belonging to the CCN family, SPARC (Secreted Protein, Acidic, and Rich in Cysteine), and thrombospondin(TSP) 2 in regulating intervertebral disc cell–ECM interactions, ECM synthesis and disc tissue homeostasisusing geneticallymodifiedmousemodels.Furthermore,weprovidea briefoverview ofrecent preliminarystud-iesofothermatricellularproteinsincluding,periostin(POSTN)andtenascin(TN).EachspecifictissuetypeoftheIVDcontainsadifferentmatricellularproteinsignature,whichvariesbasedonthespecificstageofdevelopment,maturityordisease.AgrowingbodyofdirectgeneticevidencelinksIVDdevelopment,maintenanceandrepairtothe coordinate interaction of matricellular proteins within their respective niches and suggests that several ofthese signaling modulators hold promise in the development of diagnostics and/or therapeutics targetingintervertebral disc aging and/or degeneration.© 2014 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/3.0/).Contents1. Introduction 02. CCNproteins:keymediatorsofnucleuspulposusECMcompositionandtissuehomeostasis 02.1. CCN2 02.2. CCN1&CCN3 03. SPARC and TSP2: crucial mediators of collagen organization and annulus fibrosusintegrity 03.1. SPARC 03.2. TSP2 04. Newhorizons:othermatricellularproteins 05. Conclusion 0Authorcontributions 0Roleoffundingsource 0Competingintereststatement 0References 0

The thrombospondins (TSPs) are a family of matricellular proteins that regulate cellular phenotype through interactions with a myriad of other proteins and proteoglycans. We have identified a novel interaction of the members of the TSP gene family with stromal interaction molecule 1 (STIM1). This association is robust since it is preserved in Triton X-100, can be detected with multiple anti-TSP-1 and anti-STIM1 antibodies, and is detected in a wide range of cell types. We have also found that STIM1 co-immunoprecipitates with TSP-4 and cartilage oligomeric matrix protein (COMP), and that a recombinant version of the N-terminal domain of STIM1 binds to the signature domain of TSP-1 and COMP. The association of the TSPs with STIM1 is observed in both the presence and absence of calcium indicating that the calcium-dependent conformation of the signature domain of TSPs is not required for binding. Thus, this interaction could occur in the ER under conditions of normal or low calcium concentration. Furthermore, we observed that the expression of COMP in HEK 293 cells decreases STIM1-mediated calcium release activated calcium (CRAC) channel currents and increases arachidonic acid calcium (ARC) channel currents. These data indicate that the TSPs regulate STIM1 function and participate in the reciprocal regulation of two channels that mediate calcium entry into the cell.

Increasing evidence suggests critical functions of thrombospondins (TSPs) in a variety of physiological and pathological processes. With the growing understanding of the importance of these matricellular proteins, the need to understand the mechanisms of regulation of their expression and potential approaches to modulate their levels is also increasing. The regulation of TSPs expression is multi-leveled, cell- and tissue-specific, and very precise. However, the knowledge of mechanisms modulating the levels of TSPs is fragmented and incomplete. This review discusses the known mechanisms of regulation of TSPs levels and the gaps in our knowledge that prevent us from developing strategies to modulate the expression of these physiologically important proteins.

Pseudoachondroplasia (PSACH) is a skeletal dysplasia characterized by disproportionate short stature, small hands and feet, abnormal joints and early onset osteoarthritis. PSACH is caused by mutations in thrombospondin-5 (TSP-5, also known as cartilage oligomeric matrix protein or COMP), a pentameric extracellular matrix protein primarily expressed in chondrocytes and musculoskeletal tissues. The thrombospondin gene family is composed of matricellular proteins that associate with the extracellular matrix (ECM) and regulate processes in the matrix. Mutations in COMP interfere with calcium-binding, protein conformation and export to the extracellular matrix, resulting in inappropriate intracellular COMP retention. This accumulation of misfolded protein is cytotoxic and triggers premature death of chondrocytes during linear bone growth, leading to shortened long bones. Both in vitro and in vivo models have been employed to study the molecular processes underlying development of the PSACH pathology. Here, we compare the strengths and weaknesses of current mouse models of PSACH and discuss how the resulting phenotypes may be translated to clinical therapies.

R-spondins are a family of four matricellular proteins produced by a variety of cell-types. Structurally, R-spondins contain a TSR1 domain that retains the tryptophan structure and a modified cysteine-rich CSVCTG region. In addition, the R-spondins contain two furin repeats implicated in canonical Wnt signaling. R-spondins positively regulate canonical Wnt signaling by reducing Wnt receptor turnover and thereby increasing beta-catenin stabilization. R-spondins are prominently expressed in the developing skeleton and contribute to limb formation, particularly of the distal digit. Additionally, results suggest that R-spondins may contribute to the maintenance of adult bone mass by regulating osteoblastogenesis and bone formation.

ADAMTS-like proteins are related to ADAMTS metalloproteases by their similarity to ADAMTS ancillary domains. Here, we have characterized ADAMTSL5, a novel member of the superfamily with a unique modular organization that includes a single C-terminal netrin-like (NTR) module. Alternative splicing of ADAMTSL5 at its 5′ end generates two transcripts that encode different signal peptides, but the same mature protein. These transcripts differ in their translational efficiency. Recombinant ADAMTSL5 is a secreted, N-glycosylated 60 kDa glycoprotein located in the subcellular matrix, on the cell-surface, and in the medium of transfected cells. RT-PCR and western blot analysis of adult mouse tissues showed broad expression. Western blot analysis suggested proteolytic release of the NTR module in transfected cells as well as in some mouse tissues. Immunostaining during mouse organogenesis identified ADAMTSL5 in musculoskeletal tissues such as skeletal muscle, cartilage and bone, as well as in many epithelia. Affinity-chromatography demonstrated heparin-binding of ADAMTSL5 through its NTR-module. Recombinant ADAMTSL5 bound to both fibrillin-1 and fibrillin-2, and co-localized with fibrillin microfibrils in the extracellular matrix of cultured fibroblasts, but without discernible effect on microfibril assembly. ADAMTSL5 is the first family member shown to bind both fibrillin-1 and fibrillin-2. Like other ADAMTS proteins implicated in microfibril biology through identification of human and animal mutations, ADAMTSL5 could have a role in modulating microfibril functions.

Udgivelsesdato: 2009-Oct We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better K(i) value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3.

The ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family includes 19 secreted proteinases in man. ADAMTS16 is a recently cloned gene expressed at high levels in fetal lung and kidney and adult brain and ovary. The ADAMTS-16 protein currently has no known function. ADAMTS16 is also expressed in human cartilage and synovium where its expression is increased in tissues from osteoarthritis patients compared to normal tissues. In this study, we ascertained that the full length ADAMTS16 mRNA was expressed in chondrocytes and cloned the appropriate cDNA. Stable over-expression of ADAMTS16 in chondrosarcoma cells led to a decrease in cell proliferation and migration, though not adhesion, as well as a decrease in the expression of matrix metalloproteinase-13 (MMP13). The transcription start point of the human ADAMTS16 gene was experimentally identified as 138 bp upstream of the translation start ATG and the basal promoter was mapped out to -1802 bp. Overexpression of Egr1 induced ADAMTS16 promoter constructs of -157/+138 or longer whilst Sp1 induced all ADAMTS16 promoter constructs. Transforming growth factor beta (TGFbeta) stimulated expression of endogenous ADAMTS16 gene expression in chondrocyte cell lines.

The inhibitor of angiogenesis, thrombospondin 2 (TSP2), belongs to a group of matricellular proteins that are induced in response to injury and modulate the healing of dermal wounds. Thus, TSP-2-null mice display abnormal connective tissue architecture and increased angiogenesis in the dermis, and heal wounds at an accelerated rate. In this study, we report that the content of TSP2 is increased in the uninjured skin of aged mice. Furthermore, in primary dermal fibroblasts, TSP2 expression is increased both as a function of the age of the donor and days in culture. To determine the significance of the increased TSP2 in aged mice (two years or older), we performed full-thickness excisional wounds and compared their healing in aged and young (3–4 months) wild-type and TSP2-null mice. Gross morphological examination of wounds indicated that aged TSP2-null mice healed faster than their aged wild-type counterparts, but healing in aged mice was always sub-optimal in comparison to that in young animals. Surprisingly, despite the increase in TSP2, a potent inhibitor of angiogenesis, in wounds in aged mice, the vascular density of these wounds was not reduced in comparison to that in young animals. However, immunohistochemical analysis of healing wounds revealed a shift in the peak content of TSP2, from day 10 in young mice to day 14 or later in aged mice, and there was a corresponding delay in the expected increase in matrix metalloproteinase (MMP) 2 levels in aged TSP2-null mice. We suggest that the delay in expression of TSP2 and MMP2 in the wounds of aged mice could contribute to their impaired rate of wound healing.

The complete primary structure of ADAMTSL-3/punctin-2, a novel member of the family designated ADAMTSL (a disintegrin-like and metalloprotease domain with thrombospondin type I motifs-like), was determined by cDNA cloning from a human placenta library. The predicted open reading frame encodes a protein of 1690 amino acids that has considerable similarity to ADAMTSL-1/punctin-1. These multi-domain proteins lack both a protease domain and a disintegrin-like domain but are remarkably similar in their domain organization to the ADAMTS proteases, hence the name ADAMTS-like. Punctin-2 contains thrombospondin type 1 repeats (TSRs), a cysteine-rich domain and a cysteine-free spacer domain in the precise order in which they occur in the ADAMTS proteases. However, the number and organization of the TSRs in punctin-2 is unique with respect to the ADAMTS proteases. Punctin-2 contains 13 TSRs arranged in two arrays separated by a region containing three immunoglobulin-like repeats. Northern blot analysis of RNA from human adult tissues demonstrated that ADAMTSL3 is widely expressed, with highest expression in liver, kidney, heart and skeletal muscle, whereas it is expressed at low levels in mouse embryos. We characterized two punctin-2 polyclonal antisera. Using these and a monoclonal antibody to a C-terminal myc tag, we show that in transfected COS-7 cells, punctin-2 is expressed as a 210-kDa glycoprotein that is located in the extracellular matrix. The domain structure of punctin-2 and its matrix localization suggest that it might play a role in cell–matrix interactions or in assembly of specific extracellular matrices.

Thrombospondins (TSPs) belong to a family of five, secreted, modular glycoproteins encoded by separate genes (Adams and Lawler, 2004, 2011). Group A TSPs (TSP1 and TSP2) are homotrimers, whereas group B TSPs (TSP3, -4, and -5/COMP) are homopentamers (Adams, 2001), and all are secreted as disulfide-bonded complexes. Although the five members of the TSP family differ with respect to structure, cell type, and temporal expression, all TSPs bind to components of the extracellular matrix as well as to a number of cell surface receptors, enabling TSPs to modulate cellular behaviors in a wide array of tissue contexts. TSPs are the prototype of the matricellular protein (Bornstein, 1995) and have been the subject of intense study for four decades. First identified in 1971 as protein released from thrombin-stimulated platelets, TSP1 (thrombin sensitive protein) was initially studied for its role in platelet aggregation and related hemostatic functions (Baenziger et al., 1971; Gartner and Dockter, 1984; Lahav et al., 1982; Lawler et al., 1978; Phillips et al., 1980). The eponym thrombospondin was proposed by Lawler and co-workers (Lawler et al., 1978) who isolated intact thrombospondin complexes from thrombin-treated platelets in physiological saline. Thrombospondin is a filamentous glycoprotein ~70 nm long and binds to heparin-affinity columns, and thus, to heparan sulfate proteoglycans in vivo (Fig. 1). Over the next two decades, it was discovered that TSP1 is synthesized and secreted by endothelial cells as well as other cell types in culture (Asch et al., 1986; Jaffe et al., 1982; Jaffe et al., 1985; Jaffe et al., 1983; McPherson et al., 1981; Mosher et al., 1982; Raugi and Lovett, 1987). The expression of TSP1 by diverse cell types, identification of interacting proteins, and the subsequent recognition that TSP1 binds to specific cell surface molecules in a saturable manner (Asch et al., 1987; McKeown-Longo et al., 1984; Murphy-Ullrich and Mosher, 1987; Roberts et al., 1985; Silverstein et al., 1984) provided the first evidence that TSPs might regulate cell behavior through direct interactions with cell surface receptors to activate intracellular signaling pathways: these first known receptors turned out to be CD36 (GP88) and heparan sulfate proteolgycans (syndecans) and established precedents for identification of other TSP receptors (Asch et al., 1987; Asch et al., 1992; Chung et al., 1999; Godyna et al., 1995; Goicoechea et al., 2000; Lawler and Hynes, 1989; Lawler et al., 1988; Murphy-Ullrich et al., 1988; Sun et al., 1989; Tuszynski et al., 1993; Wang and Frazier, 1998). Subsequent work showed that TSP1 expression is regulated by growth factors (PDGF, TGF-β), that TSP1 can enhance cellular responses to growth factors such as EGF, antagonize VEGF signaling, or variably regulate FGF family signaling (Gupta et al., 1999; Iruela-Arispe et al., 1999; Majack et al., 1985, 1986; Orr et al., 2003; Penttinen et al., 1988; Taraboletti et al., 1992). Elegant biochemistry, protein structural studies, and cloning and sequencing of the TSP1 gene further elucidated relationships between discrete modular domains of TSPs and specific cellular functions as well as regulators of gene expression (Bornstein et al., 1990; Dixit et al., 1986; Frazier et al., 1987; Lawler, 1986; Slane et al., 1988; Sun and Mosher, 1991). Together these early findings stimulated many diverse and fruitful avenues of investigation which have yielded surprising insights into the diverse functions of this remarkable family of proteins. Fig. 1 Model of TSP1 monomer showing the N-terminal laminin G-like domain, the oligomerization region of interchain disulfide bonding, the procolllagen-like, von Willebrand factor_C module, the properdin-like type 1 (TSR) repeats, the EGF-like repeats, and the ... This thematic series of interrelated mini-reviews will focus on recent additions to the armamentarium of TSP functions. The review by Mosher and Adams (2012, this issue) employs both evolutionary and structural perspectives to discern the functions of thrombospondins as well as other matricellular proteins, especially in the context of the complex network of intermolecular interactions occurring in the extracellular matrix and at the cell surface. The review by Roberts and co-workers (2012, this issue) highlights the role of TSP1 as a regulator of local and systemic physiology through its ability to attenuate nitric oxide signaling. The identification of TSP1 as a regulator of nitric oxide signaling has significantly broadened our understanding of the role of TSP1, especially in vascular physiology and pathology. The review by Risher and Eroglu (2012, this issue) discusses exciting, novel findings documenting a role for TSP in synaptogenesis through interactions between astrocyte-secreted TSPs and their neuronal receptor, calcium channel subunit α2δ-1, also a receptor for gabapentin. Collectively, these new findings have implications for neuronal development and responses to CNS injury. Finally, Sweetwyne and Murphy-Ullrich (2012, this issue) focus on distinct roles for TSP1 mediated by different domains which impact basic cellular processes key for wound healing, tissue repair, and fibrosis. These entail stimulation of collagen expression through the N-terminal domain calreticulin-binding sequence, transactivation of EGF receptor by the EGF-like domains, and regulation of latent TGF-β activation by the TSR type 1 repeats. Many of these findings were initially presented at the 2010 FASEB Summer Research Conference on Thrombospondins and Other Matricellular Proteins in Tissue Organization and Homeostasis. Clearly, despite several decades of research, the thrombospondins continue to surprise us with their broad and clinically-significant roles in physiology and disease. The field of matrix biology has been enriched by the often unexpected roles of thrombospondins in normal development and physiology and in the pathophysiology of diverse disease processes.

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.

Thrombospondin-1 (TSP-1) is a matricellular protein that regulates cellular phenotype during tissue genesis and repair. It acts as a molecular facilitator by bringing together cytokines, growth factors, matrix components, membrane receptors and extracellular proteases. TSP-1 binds to a wide variety of integrin and non-integrin cell surface receptors. The binding sites for these receptors on TSP-1 are dispersed throughout the molecule, with most domains binding multiple receptors. In some cases, TSP-1 binds to multiple receptors concurrently, and recent data indicate that there is cross-talk between the receptor systems. Thus, TSP-1 may function to direct the clustering of receptors to specialized domains for adhesion and signal transduction.

Thrombospondin (TSP) 2 is a close relative of TSP1 but differs in its temporal and spatial distribution in the mouse. This difference in expression undoubtedly reflects the marked disparity in the DNA sequences of the promoters in the genes encoding the two proteins. The synthesis of TSP2 occurs primarily in connective tissues of the developing and growing mouse. In the adult animal the protein is again produced in response to tissue injury and in association with the growth of tumors. Despite the abnormalities in collagen fibrillogenesis, fragility of skin, and laxity of tendons and ligaments observed in the TSP2-null mouse, TSP2 does not appear to contribute directly to the structural integrity of connective tissue elements. Instead, emerging evidence supports a mode of action of TSP2 'at a distance', i.e. by modulating the activity and bioavailability of proteases and growth factors in the pericellular environment and, very likely, by interaction with cell-surface receptors. Thus, TSP2 qualifies as a matricellular protein, as defined in the introduction to this minireview series. The phenotype of TSP2-null mice has been very helpful in providing clues to the functions of TSP2. In addition to histological and functional abnormalities in connective tissues, these mice display an increased vascularity of the dermis and subdermal tissues, increased endosteal bone growth, a bleeding defect, and a marked adhesive defect of dermal fibroblasts. Our laboratory has established that TSP2 binds matrix metalloproteinase 2 (MMP2) and that the adhesive defect in TSP2-null fibroblasts results from increased MMP2 activity. The investigation of the basis for the other defects in the TSP2-null mouse is likely to yield equally interesting results.

Thrombospondin-1 and -2 (TSP1 and TSP2) are multifunctional, multimodular extracellular matrix proteins encoded by separate genes. We compared the distributions of TSP1 and TSP2 in mouse embryos (day 10 and later) by immunohistochemistry. TSP1 was detected on day 10 in the heart and intestinal epithelium, on day 11 in megakaryocytes, and on day 14 in the lung. TSP2 was not detected until day 14, with strongest staining in mesenchymal condensation that gives rise to cartilage and bone. The distribution of TSP2 was different from but overlapped with the distribution of TSP1. TSP1 was found in cartilage proper with diminished staining around chondrocytes undergoing differentiation and hypertrophy, whereas TSP2 was restricted to the matrix surrounding chondrocytes of the growth zone cartilage. TSP2 and TSP1 were both expressed in centers of intramembranous ossification that form the skull bones, in reticular dermis, on the apical surface of nasal epithelium, in skeletal muscle, and in the sheath surrounding vibrissae. Areas of exclusive staining for TSP2 included the perichondrium surrounding the cartilage of the nasal cavities, developing bone of the lower mandible, and adrenal gland. The distinct localizations of TSP1 and TSP2 indicate that the two proteins have specific functions during mouse embryogenesis.

As soon as procollagen precursors of fibrillar collagens were discovered in the early 1970s, it became apparent that connective tissues must contain proteolytic activities that cleave the N-propeptides and the C-propeptides from procollagens. Isolation and characterization of the enzymic activities, however, proved to be unexpectedly difficult. Both proteinases are large and are synthesized in several different forms with polypeptide chains ranging in size from 70 kDa to about 130 kDa. The N-proteinase has the unusual property of cleaving the N-propeptides from type I and type II procollagens if the proteins are in a native conformation, but not if the proteins are partially unfolded so that the N-telopeptides are no longer in a hair-pin configuration. The C-proteinase specifically cleaves native and denatured types I, II and III procollagens. It also specifically cleaves a precursor of lysyl oxidase and laminin 5. Both enzymes and their variants have structures that place them in a large and expanding super-family of over 200 zinc-binding metalloproteinases. The larger of two forms of the N-proteinase contains an RGD sequence for binding through integrins and properdin repeats similar to those found in thrombospondin. The shorter 70 kDa form of the C-proteinase is identical to the protein that was previously identified as bone morphogenic protein-1. Both the 70 kDa C-proteinase and two larger forms are homologous to proteins that are expressed early in development in a variety of organisms, including Drosophila, sea urchin, and fish. Therefore, the data suggest that both the N- and C-proteinases have important biological functions in addition to the roles in the processing of procollagens.