Altered integration of matrilin-3 into cartilage extracellular matrix in the absence of collagen IX

The matrilins are a family of four noncollagenous oligomeric extracellular matrix proteins with a modular structure. Matrilins can act as adapters which bridge different macromolecular networks. We therefore investigated the effect of collagen IX deficiency on matrilin-3 integration into cartilage tissues. Mice harboring a deleted Col9a1 gene lack synthesis of a functional protein and produce cartilage fibrils completely devoid of collagen IX. Newborn collagen IX knockout mice exhibited significantly decreased matrilin-3 and cartilage oligomeric matrix protein (COMP) signals, particularly in the cartilage primordium of vertebral bodies and ribs. In the absence of collagen IX, a substantial amount of matrilin-3 is released into the medium of cultured chondrocytes instead of being integrated into the cell layer as in wild-type and COMP-deficient cells. Gene expression of matrilin-3 is not affected in the absence of collagen IX, but protein extraction from cartilage is greatly facilitated. Matrilin-3 interacts with collagen IX-containing cartilage fibrils, while fibrils from collagen IX knockout mice lack matrilin-3, and COMP-deficient fibrils exhibit an intermediate integration. In summary, the integration of matrilin-3 into cartilage fibrils occurs both by a direct interaction with collagen IX and indirectly with COMP serving as an adapter. Matrilin-3 can be considered as an interface component, capable of interconnecting macromolecular networks and mediating interactions between cartilage fibrils and the extrafibrillar matrix.     

TIMP-3 binds to sulfated glycosaminoglycans of the extracellular matrix

Of the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B beta-strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases.     

Targeting angiogenesis with compounds from the extracellular matrix

The extracellular matrix (ECM) is the central element of a pericellular network of bioactive molecules. It orchestrates molecular interactions, availability and activity, acting as a key regulator of cell functions and complex biological processes, including physiological and pathological angiogenesis. The ECM serves as a source of both stimulatory and inhibitory angiogenesis regulatory factors. The observation that several endogenous inhibitors of angiogenesis derive from the ECM proves its importance in physiological angiogenesis, and point to the ECM as a precious source of therapeutic agents for angiogenesis-driven diseases, including cancer growth and metastatic dissemination. This review focuses on the different approaches to exploit ECM molecules for designing tools for therapeutic inhibition or monitoring of pathological angiogenesis, with particular focus on antineoplastic therapy, and emphasis on peptides of ECM moieties and mimetic small molecules.     

Binding of plasminogen to extracellular matrix

We have previously demonstrated that plasminogen immobilized on various surfaces forms a substrate for efficient conversion to plasmin by tissue plasminogen activator (t-PA) (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, R. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human plasminogen to the extracellular matrix synthesized in vitro by cultured endothelial cell monolayers. The binding was specific, saturable at plasma plasminogen concentrations, reversible, and lysine-binding site-dependent. Functional studies demonstrated that matrix immobilized plasminogen was a much better substrate for t-PA than was fluid phase plasminogen as shown by a 100-fold decrease in Km. Activation of plasminogen by t-PA and urokinase on the matrix was equally efficient. The plasmin generated on the matrix, in marked contrast to fluid phase, was protected from its fast-acting inhibitor, alpha 2-plasmin inhibitor. Matrix-associated plasmin converted bound Glu- into Lys-plasminogen, which in turn is more rapidly activated to plasmin by t-PA. The extracellular matrix not only binds and localizes plasminogen but also improves plasminogen activation kinetics and prolongs plasmin activity in the subendothelial microenvironment.     

Bacterial proteins binding to the mammalian extracellular matrix

Pathogenic bacteria frequently express surface proteins with affinity for components of the mammalian extracellular matrix, i.e. collagens, laminin, fibronectin or proteoglycans. This review summarizes our current knowledge on the mechanisms of bacterial adherence to extracellular matrices and on the biological significance of these interactions. The best-characterized bacterial proteins active in these interactions are the mycobacterial fibronectin-binding proteins, the fibronectin- and the collagen-binding proteins of staphylococci and streptococci, specific enterobacterial fimbrial types, as well as the polymeric surface proteins YadA of yersinias and the A-protein of Aeromonas. Some of these bacterial proteins are highly specific for an extracellular matrix protein, some are multifunctional and express binding activities towards a number of target proteins. The interactions can be based on a protein-protein or on a protein-carbohydrate interaction, or on a bridging mechanism mediated by a bivalent soluble target protein. Many of the interactions have also been demonstrated on tissue sections or in vivo, and adherence to the extracellular matrix has been shown to promote bacterial colonization of damaged tissues.     

Release of the extracellular matrix from conidia ofBlumeria graminis in relation to germination

The release of extracellular matrix (ECM) and the emergence of germ tubes from conidia ofBlumeria graminis were studied by light microscopy and micromanipulation. More prompt and frequent ECM release was confirmed on an artificial hydrophobic substratum than on an artificial hydrophilic substratum. Conidia initially incubated on the hydrophilic substratum were transferred by micromanipulation to either the hydrophobic or the hydrophilic substrata. Immediately after transfer onto the hydrophobic substratum, 75% of conidia released ECM, whereas only 16% did so upon transfer to the hydrophilic substratum. Conidia transferred onto the hydrophobic substratum produced a primary germ tube (PGT) more promptly and frequently than those transferred to the hydrophilic substratum. Thus, conidia recognize and respond to substratum hydrophobicity perhaps immediately after contact. When inoculated onto either isolated barley cuticle or the hydrophobic artificial substratum, 2/3 of the conidia produced a PGT from their polar regions. By contrast, on the hydrophilic substratum 2/3 of conidia did so from the side region. These results show that substratum hydrophobicity affects the location of PGT emergence from conidia. Furthermore, the study indicates that very rapid recognition of surface hydrophobicity by conidia promotes ECM release and this in turn may influence the location of PGT emergence.     

Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-beta3 and fibronectin

Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children. Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as an important agent in symptomatic rotavirus infection. To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used. NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7. An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4. Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization. After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids [aa] 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884). Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145. Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4. Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation. NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction. In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4. The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established.     

Morphogenetic messages are in the extracellular matrix: biotechnology from bench to bedside

The origin and evolution of multicellular metazoa was accompanied by the appearance of extracellular matrix. The demineralized extracellular matrix of bone is enriched in morphogenetic proteins that induce bone. Bone morphogenetic proteins (BMPs) are intimately bound to collagens. BMP-4 has high affinity for type-IV collagen, and other binding proteins such as noggin and chordin. Soluble morphogens are kept in the solid state by extracellular matrix. In this sense Nature used the principles of affinity matrices long before humans patented the principle of affinity chromatography.     

Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix

The conjunctive presence of mechanical stress and active transforming growth factor β1 (TGF-β1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-β1 from self-generated stores in the ECM. Contraction of myofibroblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-β1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-β1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-β1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-β1 and its receptor. We propose activation of TGF-β1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM.     

Matrilin-3 activates the expression of osteoarthritis-associated genes in primary human chondrocytes

Here, we tested the matrilin-3-dependent induction of osteoarthritis-associated genes in primary human chondrocytes. Matrilin stimulation leads to the induction of MMP1, MMP3, MMP13, COX-2, iNOS, IL-1β, TNFα, IL-6 and IL-8. Furthermore, we show the participation of ADAMTS4 and ADAMTS5 in the in vitro degradation of matrilin-3. We provide evidence for a matrilin-3-dependent feed-forward mechanism of matrix degradation, whereby proteolytically-released matrilin-3 induces pro-inflammatory cytokines as well as ADAMTS4 and -5 indirectly via IL-1β. ADAMTS4 and ADAMTS5, in turn, cleave matrilin-3 and may release more matrilin-3 from the matrix, which could lead to further release of pro-inflammatory cytokines and proteases in cartilage.     

Plasmin-mediated release of the guidance molecule F-spondin from the extracellular matrix

Serine proteases are implicated in a variety of processes during neurogenesis, including cell migration, axon outgrowth, and synapse elimination. Tissue-type plasminogen activator and urokinase-type activator are expressed in the floor plate during embryonic development. F-spondin, a gene also expressed in the floor plate, encodes a secreted, extracellular matrix-attached protein that promotes outgrowth of commissural axons and inhibits outgrowth of motor axons. F-spondin is processed in vivo to yield an amino half protein that contains regions of homology to reelin and mindin, and a carboxyl half protein that contains either six or four thrombospondin type I repeats (TSRs). We have tested F-spondin to see whether it is subjected to processing by plasmin and to determine whether the processing modulates its biological activity. Plasmin cleaves F-spondin at its carboxyl terminus. By using nested deletion proteins and mutating potential plasmin cleavage sites, we have identified two cleavage sites, the first between the fifth and sixth TSRs, and the second at the fifth TSR. Analysis of the extracellular matrix (ECM) attachment properties of the TSRs revealed that the fifth and sixth TSRs bind to the ECM, but repeats 1-4 do not. Structural functional experiments revealed that two basic motives are required to elicit binding of TSR module to the ECM. We demonstrate further that plasmin releases the ECM-bound F-spondin protein.     

Effect of microtubule-specific drugs upon spatial organization of extracellular matrix in fibroblastic cultures.

The aim of this investigation was to study the effects of microtubule-specific drugs, taxol and colcemid, on the distribution of cell-associated extracellular matrix in dense cultures of fibroblasts. Immunomorphological examination of human seven-day cultures revealed a dense network of fibronectin and tenascin matrix filaments preferentially oriented in parallel with the long axes of cell bodies. Depolymerization of the microtubular system by colcemid and its disorganization by taxol led to rapid and drastic changes in the organization of matrix network: fibronectin and tenascin filaments became disordered and, in particular, lost any orientation. These data show that the microtubular system controls the morphological organization, not only of intracellular cytoskeletal systems, but also of extracellular matrix structures.     

Hydrogels as extracellular matrix mimics for 3D cell culture

Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two‐dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three‐dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three‐dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three‐dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic–biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism. Biotechnol. Bioeng. 2009;103: 655–663. © 2009 Wiley Periodicals, Inc.     

A new collagen from the extracellular matrix of Sepia officinalis cartilage

Guanidinium chloride treatment of Sepia officinalis cartilage solubilized a component that contained hydroxyproline. Electron-microscopy observation of rotary-shadowed preparations of this component revealed it to consist of rod-like units themselves consisting of filaments. Dialysis of an acetic acid solution against ATP afforded polymeric aggregates consisting of a succession of two or three thick sections showing transverse electron-opaque banding, separated by thinner sections without banding. Electrophoresis produced a main band of about 140 kDa sensitive to bacterial collagenase. After reduction with mercaptoethanol, electrophoresis afforded a 40-kDa band. Pepsin digestion resulted in additional electrophoretic bands. These data suggest the presence of a collagen in Sepia cartilage with characteristics unlike those of any known collagen.     

Intrinsic disorder of the extracellular matrix

The extracellular matrix is very well organized at the supramolecular and tissue levels and little is known on the potential role of intrinsic disorder in promoting its organization. We predicted the amount of disorder and identified disordered regions in the human extracellular proteome with established computational tools. The extracellular proteome is significantly enriched in proteins comprising more than 50% of disorder compared to the complete human proteome. The enrichment is mostly due to long disordered regions containing at least 100 consecutive disordered residues. The amount of intrinsic disorder is heterogeneous in the extracellular protein families, with the most disordered being collagens and the small integrin-binding ligand N-linked glycoproteins. Although most domains found in extracellular proteins are structured, the fibronectin III domains contain a variable amount of disordered residues (up to 92%). Binding sites for heparin and integrins are found in disordered sequences of extracellular proteins. Intrinsic disorder is evenly distributed in hubs and ends in the interaction network of extracellular proteins with their extracellular partners. In contrast, extracellular hubs are significantly enriched in disorder in the network of extracellular proteins with their extracellular, membrane and intracellular partners. Disorder could thus provide the structural plasticity required for the hubs to interact with membrane and intracellular proteins. Organization and assembly of the extracellular matrix, development of mineralized tissues and cell-matrix adhesion are the biological processes overrepresented in the most disordered extracellular proteins. Extracellular disorder is associated with binding to growth factors, glycosaminoglycans and integrins at the molecular level.