Targeting the extracellular matrix: Matricellular proteins regulate cell–extracellular matrix communication within distinct niches of the intervertebral disc

The so-called “matricellular” proteins have recently emerged as important regulators of cell–extracellular matrix (ECM) interactions. These proteins modulate a variety of cell functions through a range of interactions with cell-surface receptors, hormones, proteases and structural components of the ECM. As such, matricellular proteins are crucial regulators of cell phenotype, and consequently tissue function. The distinct cell types and microenvironments that together form the IVD provide an excellent paradigm to study how matricellular proteins mediate communication within and between adjacent tissue types. In recent years, the role of several matricellular proteins in the intervertebral disc has been explored in vivo using mutant mouse models in which the expression of target matricellular proteins was deleted from either one or all compartments of the intervertebral disc. The current review outlines what is presently known about the roles of the matricellular proteins 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 homeostasis using genetically modified mouse models. Furthermore, we provide a brief overview of recent preliminary studies of other matricellular proteins including, periostin (POSTN) and tenascin (TN). Each specific tissue type of the IVD contains a different matricellular protein signature, which varies based on the specific stage of development, maturity or disease. A growing body of direct genetic evidence links IVD development, maintenance and repair to the coordinate interaction of matricellular proteins within their respective niches and suggests that several of these signaling modulators hold promise in the development of diagnostics and/or therapeutics targeting intervertebral disc aging and/or degeneration.     

Matricellular proteins in cancer: a focus on secreted Frizzled-related proteins

Tumours are complex entities, wherein cancer cells interact with myriad soluble, insoluble and cell associated factors. These microenvironmental mediators regulate tumour growth, progression and metastasis, and are produced by cancer cells and by stromal components such as fibroblast, adipocytes and immune cells. Through their ability to bind to extracellular matrix proteins, cell surface receptors and growth factors, matricellular proteins enable a dynamic reciprocity between cancer cells and their microenvironment. Hence, matricellular proteins play a critical role in tumour progression by regulating where and when cancer cells are exposed to key growth factors and regulatory proteins. Recent studies suggest that, in addition to altering Wingless (Wnt) signalling, certain members of the Secreted Frizzled Related Protein (sFRP) family are matricellular in nature. In this review, we outline the importance of matricellular proteins in cancer, and discuss how sFRPs may function to both inhibit and promote cancer progression in a context-dependent manner. By considering the matricellular functionality of sFRPs, we may better understand their apparently paradoxical roles in cancers.     

Evaluation of matricellular proteins in systemic and local immune response to Mycobacterium tuberculosis infection

Matricellular proteins such as osteopontin (OPN), galectin‐9 (Gal‐9), and tenascin‐C (TN‐C) are expressed not only under normal physiological conditions, but also during infection, inflammation and tumorigenesis. Plasma concentrations of matricellular proteins were studied to determine their diagnostic value as potential markers of tuberculosis (TB) activity. It was found that concentrations of OPN and TN‐C were higher in patients with active TB than in healthy controls and individuals with latent infection. Moreover, LTBI patients had higher concentrations of OPN than did healthy controls. Gal‐9 concentrations did not differ significantly between groups. Concentrations of matricellular proteins were higher in pleural fluid than in the plasma of patients with TB. Expression of matricellular proteins was also investigated in TB granulomas and other granulomatous diseases. Positive OPN and Gal‐9 staining was observed in TB and sarcoidosis granulomas, but not in Crohn disease granulomas. The fibrotic ring around granulomas stained positive for TN‐C in TB and sarcoidosis, but not in Crohn disease. Of the three matricellular proteins studied, OPN and TN‐C may serve as reliable plasma markers for monitoring TB activity, whereas Gal‐9 seems to be expressed more at the site of infection than in the systemic circulation.     

Tracheal Aspirate Levels of the Matricellular Protein SPARC Predict Development of Bronchopulmonary Dysplasia

Background

Isolation of tracheal aspirate mesenchymal stromal cells (MSCs) from premature infants has been associated with increased risk of bronchopulmonary dysplasia (BPD). MSCs show high levels of mRNAs encoding matricellular proteins, non-structural extracellular proteins that regulate cell-matrix interactions and participate in tissue remodeling. We hypothesized that lung matricellular protein expression predicts BPD development.

Methods

We collected tracheal aspirates and MSCs from mechanically-ventilated premature infants during the first week of life. Tracheal aspirate and MSC-conditioned media were analyzed for seven matricellular proteins including SPARC (for Secreted Protein, Acidic, Rich in Cysteine, also called osteonectin) and normalized to secretory component of IgA. A multiple logistic regression model was used to determine whether tracheal aspirate matricellular protein levels were independent predictors of BPD or death, controlling for gestational age (GA) and birth weight (BW).

Results

We collected aspirates from 89 babies (38 developed BPD, 16 died before 36 wks post-conceptual age). MSC-conditioned media showed no differences in matricellular protein abundance between cells from patients developing BPD and cells from patients who did not. However, SPARC levels were higher in tracheal aspirates from babies with an outcome of BPD or death (p<0.01). Further, our logistic model showed that tracheal aspirate SPARC (p<0.02) was an independent predictor of BPD/death. SPARC deposition was increased in the lungs of patients with BPD.

Conclusions

In mechanically-ventilated premature infants, tracheal aspirate SPARC levels predicted development of BPD or death. Further study is needed to determine the value of SPARC as a biomarker or therapeutic target in BPD.     

Matricellular proteins and inflammatory cells: A task force to promote or defeat cancer?

In the last years it became clear that the tumor microenvironment plays a major role in neoplastic growth. Proteins secreted either by the malignant cells or by the tumor-associated stromal cells act as extracellular signal transductors, orchestrating tumor progression. Sentinel cells of the innate immune system patrol the different organs and have proven either to promote tumor growth or induce tumor suppression. In recent years, members of the matricellular family of extracellular proteins were shown to be involved in different aspects of the inflammatory response during tumor development, although in contradictory ways. In this review we discuss the evidence available up to date that relates matricellular proteins with the regulation of the inflammatory response and tumor progression.     

Matricellular proteins in development: Perspectives from the Drosophila heart

The Drosophila model represents an attractive system in which to study the functional contribution of specific genes to organ development. Within the embryo, the heart tube serves as an informative developmental paradigm to analyze functional aspects of matricellular proteins. Here, we describe two essential extracellular matricellular proteins, Multiplexin (Mp) and Lonely heart (Loh). Each of these proteins contributes to the development and morphogenesis of the heart tube by regulating the activity/localization of essential extracellular proteins. Mp, which is secreted by heart cardioblasts and is specifically distributed in the lumen of the heart tube, binds to the signaling protein Slit, and facilitates its local signaling at the heart's luminal domain. Loh is an ADAMTS-like protein, which serves as an adapter protein to Pericardin (a collagen-like protein), promoting its specific localization at the abluminal domain of the heart tube. We also introduce the Drosophila orthologues of matricellular proteins present in mammals, including Thrombospondin, and SPARC, and discuss a possible role for Teneurins (Ten-A and Ten-M) in the heart. Understanding the role of these proteins provides a novel developmental perspective into the functional contribution of matricellular proteins to organ development.     

Functional role of periostin in development and wound repair: implications for connective tissue disease

Integrity of the extracellular matrix (ECM) is essential for maintaining the normal structure and function of connective tissues. ECM is secreted locally by cells and organized into a complex meshwork providing physical support to cells, tissues, and organs. Initially thought to act only as a scaffold, the ECM is now known to provide a myriad of signals to cells regulating all aspects of their phenotype from morphology to differentiation. Matricellular proteins are a class of ECM related molecules defined through their ability to modulate cell-matrix interactions. Matricellular proteins are expressed at high levels during development, but typically only appear in postnatal tissue in wound repair or disease, where their levels increase substantially. Members of the CCN family, tenascin-C, osteopontin, secreted protein acidic rich in cysteine (SPARC), bone sialoprotein, thrombospondins, and galectins have all been classed as matricellular proteins. Periostin, a 90 kDa secreted homophilic cell adhesion protein, was recently added to matricellular class of proteins based on its expression pattern and function during development as well as in wound repair. Periostin is expressed in connective tissues including the periodontal ligament, tendons, skin and bone, and is also prominent in neoplastic tissues, cardiovascular disease, as well as in connective tissue wound repair. This review will focus on the functional role of periostin in tissue physiology. Fundamentally, it appears that periostin influences cell behaviour as well as collagen fibrillogenesis, and therefore exerts control over the structural and functional properties of connective tissues in both health and disease. Periostin is a novel matricellular protein with close homology to Drosophila fasciclin 1. In this review, the functional role of periostin is discussed in the context of connective tissue physiology, in development, disease, and wound repair.     

SPARC and tumor growth: Where the seed meets the soil?

Matricellular proteins mediate interactions between cells and their extracellular environment. This functional protein family includes several structurally unrelated members, such as SPARC, thrombospondin 1, tenascin C, and osteopontin, as well as some homologs of these proteins, such as thrombospondin 2 and tensascin X. SPARC, a prototypic matricellular protein, and its homolog hevin, have deadhesive effects on cultured cells and have been characterized as antiproliferative factors in some cellular contexts. Both proteins are produced at high levels in many types of cancers, especially by cells associated with tumor stroma and vasculature. In this Prospect article we summarize evidence for SPARC and hevin in the regulation of tumor cell growth, differentiation, and metastasis, and we propose that matricellular proteins such as these perform critical functions in desmoplastic responses of tumors that culminate in their dissemination and eventual colonization of other sites.     

Lipocalin-7 is a matricellular regulator of angiogenesis

Background

Matricellular proteins are extracellular regulators of cellular adhesion, signaling and performing a variety of physiological behaviors such as proliferation, migration and differentiation. Within vascular microenvironments, matricellular proteins exert both positive and negative regulatory cues to vascular endothelium. The relative balance of these matricellular cues is believed to be critical for vascular homeostasis, angiogenesis activation or angiogenesis resolution. However, our knowledge of matricellular proteins within vascular microenvironments and the mechanisms by which these proteins impact vascular function remain largely undefined. The matricellular protein lipocalin-7 (LCN7) is found throughout vascular microenvironments, and circumstantial evidence suggests that LCN7 may be an important regulator of angiogenesis. Therefore, we hypothesized that LCN7 may be an important regulator of vascular function.

Methodology and Principal Findings

To test this hypothesis, we examined the effect of LCN7 overexpression, recombinant protein and gene knockdown in a series of in vitro and in vivo models of angiogenesis. We found that overexpression of LCN7 in MB114 and SVEC murine endothelial cell lines or administration of highly purified recombinant LCN7 protein increased endothelial cell invasion. Similarly, LCN7 increased angiogenic sprouting from quiescent endothelial cell monolayers and ex vivo aortic rings. Moreover, LCN7 increased endothelial cell sensitivity to TGF-β but did not affect sensitivity to other pro-angiogenic growth factors including bFGF and VEGF. Finally, morpholino based knockdown of LCN7 in zebrafish embryos specifically inhibited angiogenic sprouting but did not affect vasculogenesis within injected embryos.

Conclusions and Significance

No functional analysis has previously been performed to elucidate the function of LCN7 in vascular or other cellular processes. Collectively, our results show for the first time that LCN7 is an important pro-angiogenic matricellular protein of vascular microenvironments.     

Role of matricellular proteins in cardiac tissue remodeling after myocardial infarction

After onset of myocardial infarction (MI), the left ventricle (LV) undergoes a continuum of molecular, cellular, and extracellular responses that result in LV wall thinning, dilatation, and dysfunction. These dynamic changes in LV shape, size, and function are termed cardiac remodeling. If the cardiac healing after MI does not proceed properly, it could lead to cardiac rupture or maladaptive cardiac remodeling, such as further LV dilatation and dysfunction, and ultimately death. Although the precise molecular mechanisms in this cardiac healing process have not been fully elucidated, this process is strictly coordinated by the interaction of cells with their surrounding extracellular matrix (ECM) proteins. The components of ECM include basic structural proteins such as collagen, elastin and specialized proteins such as fibronectin, proteoglycans and matricellular proteins. Matricellular proteins are a class of non-structural and secreted proteins that probably exert regulatory functions through direct binding to cell surface receptors, other matrix proteins, and soluble extracellular factors such as growth factors and cytokines. This small group of proteins, which includes osteopontin, thrombospondin-1/2, tenascin, periostin, and secreted protein, acidic and rich in cysteine, shows a low level of expression in normal adult tissue, but is markedly upregulated during wound healing and tissue remodeling, including MI. In this review, we focus on the regulatory functions of matricellular proteins during cardiac tissue healing and remodeling after MI.     

The CCN family of genes: a perspective on CCN biology and therapeutic potential

The CCN family of genes currently comprises six secreted proteins (designated CCN1-6 after Cyr61/CCN1; ctgf/CCN2; Nov/CCN3; WISP1/CCN4; WISP2/CCN5, WISP3/CCN6) with a similar mosaic primary structure. It is now well accepted that CCN proteins are not growth factors but matricellular proteins that modify signaling of other molecules, in particular those associated with the extracellular matrix. CCN proteins are involved in mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types. Since their first identification as matricellular factors, the CCN proteins now figure prominently in a variety of major diseases and are now considered valid candidates for therapeutic targeting. Dissection of the molecular mechanisms governing the biological properties of these proteins is being actively pursued by an expanding network of scientists around the globe who will meet this year at the 5th International Workshop on the CCN family of Genes, organized by the International CCN Society ( http://ccnsociety.com ), home for an international cadre of collaborators working in the CCN field.     

CCN proteins in the musculoskeletal system: current understanding and challenges in physiology and pathology

The acronym for the CCN family was recently revised to represent “cellular communication network”. These six, small, cysteine-enriched and evolutionarily conserved proteins are secreted matricellular proteins, that convey and modulate intercellular communication by interacting with structural proteins, signalling factors and cell surface receptors. Their role in the development and physiology of musculoskeletal system, constituted by connective tissues where cells are interspersed in the cellular matrix, has been broadly studied. Previous research has highlighted a crucial balance of CCN proteins in mesenchymal stem cell commitment and a pivotal role for CCN1, CCN2 and their alter ego CCN3 in chondrogenesis and osteogenesis; CCN4 plays a minor role and the role of CCN5 and CCN6 is still unclear. CCN proteins also participate in osteoclastogenesis and myogenesis. In adult life, CCN proteins serve as mechanosensory proteins in the musculoskeletal system providing a steady response to environmental stimuli and participating in fracture healing. Substantial evidence also supports the involvement of CCN proteins in inflammatory pathologies, such as osteoarthritis and rheumatoid arthritis, as well as in cancers affecting the musculoskeletal system and bone metastasis. These matricellular proteins indeed show involvement in inflammation and cancer, thus representing intriguing therapeutic targets. This review discusses the current understanding of CCN proteins in the musculoskeletal system as well as the controversies and challenges associated with their multiple and complex roles, and it aims to link the dispersed knowledge in an effort to stimulate and guide readers to an area that the writers consider to have significant impact and relevant potentialities.     

Transglutaminase-mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein

Tissue transglutaminase (TG2) is a widely distributed, protein-crosslinking enzyme having a prominent role in cell adhesion as a β1 integrin co-receptor for fibronectin. In bone and teeth, its substrates include the matricellular proteins osteopontin (OPN) and bone sialoprotein (BSP). The aim of this study was to examine effects of TG2-mediated crosslinking and oligomerization of OPN and BSP on osteoblast cell adhesion. We show that surfaces coated with oligomerized OPN and BSP promote MC3T3-E1/C4 osteoblastic cell adhesion significantly better than surfaces coated with the monomeric form of the proteins. Both OPN and BSP oligomer-adherent cells showed more cytoplasmic extensions than those cells grown on the monomer-coated surfaces indicative of increased cell connectivity. Our study suggests a role for TG2 in promoting the cell adhesion function of two matricellular substrate proteins prominent in bone, tooth cementum and certain tumors.     

Yin and Yang Part Deux: CCN5 inhibits the pro-fibrotic effects of CCN2

There is no treatment for fibrotic disease is a significant cause of mortality. CCN2 Members of the CCN family of matricellular proteins have a characteristic four domain structure. CCN2 (connective tissue growth factor) is believed to play an essential role in fibrogenesis. In a recent paper, data are provided that CCN5 (wisp2), which lacks the carboxy-terminal heparin-binding domain shared by the other CCN proteins, may act as a dominant-negative protein to suppress CCN2-mediated fibrogenesis. These data are consistent with the notion that different CCN proteins may enhance or suppress each other's action and also suggest that CCN5, may be used as a novel anti-fibrotic therapy.     

When there’s smoke there’s.....CCN2

There is no treatment for fibrotic disease is a significant cause of mortality. CCN2 Members of the CCN family of matricellular proteins have a characteristic four domain structure. CCN2 (connective tissue growth factor) is believed to play an essential role in fibrogenesis. In a recent paper, data are provided that CCN5 (wisp2), which lacks the carboxy-terminal heparin-binding domain shared by the other CCN proteins, may act as a dominant-negative protein to suppress CCN2-mediated fibrogenesis. These data are consistent with the notion that different CCN proteins may enhance or suppress each other’s action and also suggest that CCN5, may be used as a novel anti-fibrotic therapy.     

Transglutaminase-mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein

Tissue transglutaminase (TG2) is a widely distributed, protein-crosslinking enzyme having a prominent role in cell adhesion as a β1 integrin co-receptor for fibronectin. In bone and teeth, its substrates include the matricellular proteins osteopontin (OPN) and bone sialoprotein (BSP). The aim of this study was to examine effects of TG2-mediated crosslinking and oligomerization of OPN and BSP on osteoblast cell adhesion. We show that surfaces coated with oligomerized OPN and BSP promote MC3T3-E1/C4 osteoblastic cell adhesion significantly better than surfaces coated with the monomeric form of the proteins. Both OPN and BSP oligomer-adherent cells showed more cytoplasmic extensions than those cells grown on the monomer-coated surfaces indicative of increased cell connectivity. Our study suggests a role for TG2 in promoting the cell adhesion function of two matricellular substrate proteins prominent in bone, tooth cementum and certain tumors.     

CCN3 and calcium signaling

The CCN family of genes consists presently of six members in human (CCN1-6) also known as Cyr61 (Cystein rich 61), CTGF (Connective Tissue Growth Factor), NOV (Nephroblastoma Overexpressed gene), WISP-1, 2 and 3 (Wnt-1 Induced Secreted Proteins). Results obtained over the past decade have indicated that CCN proteins are matricellular proteins, which are involved in the regulation of various cellular functions, such as proliferation, differentiation, survival, adhesion and migration. The CCN proteins have recently emerged as regulatory factors involved in both internal and external cell signaling. CCN3 was reported to physically interact with fibulin-1C, integrins, Notch and S100A4. Considering that, the conformation and biological activity of these proteins are dependent upon calcium binding, we hypothesized that CCN3 might be involved in signaling pathways mediated by calcium ions.     

Thrombospondin-2 and SPARC/osteonectin are critical regulators of bone remodeling

Thrombospondin-2 (TSP2) and osteonectin/BM-40/SPARC are matricellular proteins that are highly expressed by bone cells. Mice deficient in either of these proteins show phenotypic alterations in the skeleton, and these phenotypes are most pronounced under conditions of altered bone remodeling. For example, TSP2-null mice have higher cortical bone volume and are resistant to bone loss associated with ovariectomy, whereas SPARC-null mice have decreased trabecular bone volume and fail to demonstrate an increase in bone mineral density in response to a bone-anabolic parathyroid hormone treatment regimen. In vitro, marrow stromal cell (MSC) osteoprogenitors from TSP2-null mice have increased proliferation but delayed formation of mineralized matrix. Similarly, in cultures of SPARC-null MSCs, osteoblastic differentiation and mineralized matrix formation are decreased. Overall, both TSP2 and SPARC positively influence osteoblastic differentiation. Intriguingly, both of these matricellular proteins appear to impact MSC fate through mechanisms that could involve the Notch signaling system. This review provides an overview of the role of TSP2 and SPARC in regulating bone structure, function, and remodeling, as determined by both in vitro and in vivo studies.     

Periostin is required for matricellular localization of CCN3 in periodontal ligament of mice

CCN3 is a matricellular protein that belongs to the CCN family. CCN3 consists of 4 domains: insulin-like growth factor-binding protein-like domain (IGFBP), von Willebrand type C-like domain (VWC), thrombospondin type 1-like domain (TSP1), and the C-terminal domain (CT) having a cysteine knot motif. Periostin is a secretory protein that binds to extracellular matrix proteins such as fibronectin and collagen. In this study, we found that CCN3 interacted with periostin. Immunoprecipitation analysis revealed that the TSP1-CT interacted with the 4 repeats of the Fas 1 domain of periostin. Immunofluorescence analysis showed co-localization of CCN3 and periostin in the periodontal ligament of mice. In addition, targeted disruption of the periostin gene in mice decreased the matricellular localization of CCN3 in the periodontal ligament. Thus, these results indicate that periostin was required for the matricellular localization of CCN3 in the periodontal ligament, suggesting that periostin mediated an interaction between CCN3 and the extracellular matrix.     

SPARC, a matricellular protein: at the crossroads of cell–matrix communication: [Matrix Biology (2000) 569–580]

The publisher regrets that the above article was published with several typographical errors. The corrected version appears on the following pages. SPARC is a multifunctional glycoprotein that belongs to the matricellular group of proteins. It modulates cellular interaction with the extracellular matrix (ECM) by its binding to structural matrix proteins, such as collagen and vitronectin, and by its abrogation of focal adhesions, features contributing to a counteradhesive effect on cells. SPARC inhibits cellular proliferation by an arrest of cells in the G1 phase of the cell cycle. It also regulates the activity of growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-2, and vascular endothelial growth factor (VEGF). The expression of SPARC in adult animals is limited largely to remodeling tissue, such as bone, gut mucosa, and healing wounds, and it is prominent in tumors and in disorders associated with fibrosis. The crystal structure of two of the three domains of the protein has revealed a novel follistatin-like module and an extracellular calcium-binding (EC) module containing two EF-hand motifs. The follistatin-like module and the EC module are shared by at least four other proteins that comprise a family of SPARC-related genes. Targeted disruption of the SPARC locus in mice has shown that SPARC is important for lens transparency, as SPARC-null mice develop cataracts shortly after birth. SPARC is a prototypical matricellular protein that functions to regulate cell–matrix interactions and thereby influences many important physiological and pathological processes.