A crucial sequence for transglutaminase type 2 extracellular trafficking in renal tubular epithelial cells lies in its N-terminal beta-sandwich domain

Transglutaminase type 2 (TG2) catalyzes the formation of an ε-(γ-glutamyl)-lysine isopeptide bond between adjacent peptides or proteins including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlie tissue scarring and fibrosis. The extracellular trafficking of TG2 is crucial to its role in ECM homeostasis; however, the mechanism by which TG2 escapes the cell is unknown as it has no signal leader peptide and therefore cannot be transported classically. Understanding TG2 transport may highlight novel mechanisms to interfere with the extracellular function of TG2 as isoform-specific TG2 inhibitors remain elusive. Mammalian expression vectors were constructed containing domain deletions of TG2. These were transfected into three kidney tubular epithelial cell lines, and TG2 export was assessed to identify critical domains. Point mutation was then used to highlight specific sequences within the domain required for TG2 export. The removal of β-sandwich domain prevented all TG2 export. Mutations of Asp(94) and Asp(97) within the N-terminal β-sandwich domain were identified as crucial for TG2 externalization. These form part of a previously identified fibronectin binding domain ((88)WTATVVDQQDCTLSLQLTT(106)). However, siRNA knockdown of fibronectin failed to affect TG2 export. The sequence (88)WTATVVDQQDCTLSLQLTT(106) within the β-sandwich domain of TG2 is critical to its export in tubular epithelial cell lines. The extracellular trafficking of TG2 is independent of fibronectin.     

Toward single cell traction microscopy within 3D collagen matrices

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels.     

Alteration of Cellular Behavior and Response to PI3K Pathway Inhibition by Culture in 3D Collagen Gels

Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.     

Overexpression of lumican affects the migration of human colon cancer cells through up-regulation of gelsolin and filamentous actin reorganization

Cell migration is a multistep process initiated by extracellular matrix components that leads to cytoskeletal changes and formation of different protrusive structures at the cell periphery. Lumican, a small extracellular matrix leucine-rich proteoglycan, has been shown to inhibit human melanoma cell migration by binding to α2β1 integrin and affecting actin cytoskeleton organization. The aim of this study was to determine the effect of lumican overexpression on the migration ability of human colon adenocarcinoma LS180 cells. The cells stably transfected with plasmid containing lumican cDNA were characterized by the increased chemotactic migration measured on Transwell filters. Lumican-overexpressing cells presented the elevated filamentous to monomeric actin ratio and gelsolin up-regulation. This was accompanied by a distinct cytoskeletal actin rearrangement and gelsolin subcellular relocation, as observed under laser scaning confocal microscope. Moreover, LS180 cells overexpressing lumican tend to form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin at the submembrane region of the cells. In conclusion, the elevated level of lumican secretion to extracellular space leads to actin cytoskeletal remodeling followed by an increase in migration capacity of human colon LS180 cells. These data suggest that lumican expression and its presence in ECM has an impact on colon cancer cells motility and may modulate invasiveness of colon cancer.     

Integrins stimulate phosphorylation of neurofilament NF-M subunit KSP repeats through activation of extracellular regulated-kinases (Erk1/Erk2) in cultured motoneurons and transfected NIH 3T3 cells

Integrin-mediated interactions of cells with components of the extracellular matrix (ECM) regulate cell survival, cell proliferation, cell differentiation and cell migration through activation of multiple intracellular signal transduction pathways. In this study, we have demonstrated that integrin-matrix interactions promote KSP tail-domain phosphorylation of neurofilament medium molecular weight subunits (NF-M) in cultured rat spinal cord motoneurons and NF-M transfected NIH 3T3 cells. We found that laminin and fibronectin induce NF-M tail-domain phosphorylation in motoneurons and NIH 3T3 cells transfected with NF-M, respectively. This phosphorylation was selectively inhibited by PD98059, a specific MEK1 inhibitor. This suggests that laminin and fibronectin-induced MEK1 activation and the downstream targets Erk1 and Erk2 are involved in NF-M KSP tail-domain phosphorylation. This pathway appears to represent one of the mechanisms whereby integrin-extracellular matrix interactions are involved in phosphorylation of the NF-M KSP tail domain.     

Effect of extracellular matrix on prolactin secretion and mRNA accumulation in GH3 cells

The matrix upon which cells grow affects their morphology, growth rate, response to external stimuli, and protein synthesis. GH3 cells, a well-characterized rat pituitary tumor cell line, synthesize and secrete growth hormone and prolactin (Prl). These cells are rounded, attach loosely, and form clumps when plated on plastic. GH3 cells plated on an extracellular matrix (ECM) from bovine corneal endothelial cells become flattened and strongly adherent to the culture dish, and have an initial increased rate of proliferation. Cells cultured on plastic have a 48-hr lag period before the start of cell division; this can be shortened by increasing the concentration of serum in the medium. Since GH3 cells store little Prl, hormone release is a good index of Prl synthesis. Prl secretion from cells cultured on extracellular matrix is twice as great as from cells cultured on plastic. The increase in Prl secretion from cells grown on extracellular matrix paralleled by a concomitant increase in the accumulation of prolactin mRNA. Cells cultured on plastic secrete more Prl in response to TRH stimulation than do cells cultured on ECM. Cells grown on either surface were unresponsive to dopamine. Thus, culturing cells on ECM may change their morphology and affect the synthesis and regulation of specific cellular proteins and their mRNAs.     

Matrix loading: assembly of extracellular matrix collagen fibrils during embryogenesis

Nothing in biology stimulates the imagination like the development of a single fertilized egg into a newborn child. Consequently, a major focus of biomedical research is aimed at understanding cell differentiation, proliferation, and specialization during child health and human development. However, the fact that the increase in size and shape of the growing embryo has as much to do with the extracellular matrix (ECM) as with the cells themselves, is largely overlooked. Cells in developing tissues are surrounded by a fiber-composite ECM that transmits mechanical stimuli, maintains the shape of developing tissues, and functions as a scaffold for cell migration and attachment. The major structural element of the ECM is the collagen fibril. The fibrils, which are indeterminate in length, are arranged in different tissues in exquisite supramolecular architectures, including parallel bundles, orthogonal lamellae, and concentric weaves. This article reviews our current understanding of the synthesis and assembly of collagen fibrils, and discusses challenging questions about how cells assemble an organized ECM during embryogenesis.     

The Del1 deposition domain can immobilize 3α-hydroxysteroid dehydrogenase in the extracellular matrix without interfering with enzymatic activity

Developing methods that result in targeting of therapeutic molecules in gene therapies to target tissues has importance, as targeting can increase efficacy and decrease off target-side-effects. Work from my laboratory previously showed that the extracellular matrix protein Del1 is organized in the extracellular matrix (ECM) via the Del1 deposition domain (DDD). In this work, a fusion protein with DDD was made to assay the ability to immobilize an enzyme without disrupting enzymatic function. A prostatic cancer-derived cell line LNCap that grows in an androgen-dependent manner was used with 3α-hydroxysteroid dehydrogenase (3 αHD), which catalyzes dihydrotestosterone (DHT). Plasmids encoding a 3αHD:DDD fusion were generated and transfected into cultured cells. The effects of 3αHD immobilized in the ECM by the DDD were evaluated by monitoring growth of LNCap cells and DHT concentrations. It was demonstrated that the DDD could immobilize an enzyme in the ECM without interfering with function.     

Effects of relaxin on matrix remodeling enzyme activity of cultured equine ovarian stromal cells

Relaxin participates in extracellular matrix (ECM) remodeling in many reproductive organs, including the ovary, by regulating proteolytic enzyme activity. Accumulated evidence indicates this action of relaxin is involved in ovarian follicle development and ovulation. Equine follicles are embedded in cortex that is at the center of the ovary and they must expand/emigrate to the fossa, the only site in the ovary for ovulation. Due to the tremendous expansion of the follicle in this species, we hypothesized that ovarian stromal remodeling would be extensive. Therefore, cultured equine ovarian stromal cell (EOSC) lines were obtained from stroma at the apex of large follicles and the effects of relaxin on gelatinases A and B, tissue inhibitors of matrix metalloproteinases (TIMPs), plasminogen activators (PAs) and PA inhibitor-1 (PAI-1) activities were assessed. Our results showed that equine relaxin increased the activity of total gelatinase A (both pro forms and mature forms) and latent progelatinase B present in conditioned medium, latent progelatinase A present in cell extracts, and TIMP-1 and TIMP-2 present in conditioned medium. This study also revealed that equine relaxin increased the urokinase-type PA activity in conditioned medium and cell extracts, tissue-type PA activity in ECM and PAI-1 activity in conditioned medium. These results suggest that relaxin may contribute to equine follicle growth and migration, and facilitate ovulation by modulating the degradation of ECM in ovarian stromal tissue.     

Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms

To study possible functional differences of the 18-kD and high molecular weight forms of basic fibroblast growth factor (bFGF), we have examined the effect of endogenous production of different bFGF forms on the phenotype of NIH 3T3 cells. Cells transfected with cDNAs coding for either 18-kD bFGF (18-kD bFGF) or all four molecular forms (18, 22, 22.5, 24 kD; wild type [WT] bFGF) exhibit increased migration and decreased FGF receptor number compared to parental cells. However, migration and FGF receptor number of cells transfected with a cDNA coding only for high molecular weight bFGF (22, 22.5, and 24 kD; HMW bFGF) were similar to that of parental cells transfected with vector alone. Cells expressing HMW, 18 kD, or WT bFGF grew to high saturation densities in 10% serum. However, only cells expressing HMW or WT bFGF grew in low serum. Cell surface or metabolic labeling of the different cell types followed by immunoprecipitation with anti-bFGF antibody showed primarily cell surface-associated 18-kD bFGF. In addition, when cells expressing exclusively HMW bFGF were transfected with a cDNA coding for 18-kD bFGF, migration was increased, bFGF receptors were down-regulated, and 18-kD bFGF was found on the cell surface. Cells expressing 18-kD bFGF transfected with a cDNA encoding FGF receptor-2 lacking the COOH-terminal domain (dominant negative bFGF receptor) exhibited a flat morphology and decreases in migration and saturation density. Cells expressing HMW bFGF transfected with the dominant negative bFGF receptor continued to grow to a high saturation density, proliferated in low serum, and exhibited no morphological changes. These results indicate that increased cell migration and FGF receptor down-regulation are mediated by the extracellular interaction of 18-kD bFGF with its cell surface receptor. Growth in low serum may be stimulated by the intracellular action of HMW bFGF through mechanisms independent of the presence of a cell surface receptor. Thus, the different molecular forms of bFGF may act through distinct but convergent pathways.     

Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization

Cells generate mechanical force to organize the extracellular matrix (ECM) and drive important developmental and reparative processes. Likewise, tumor cells invading into three-dimensional (3D) matrices remodel the ECM microenvironment. Importantly, we previously reported a distinct radial reorganization of the collagen matrix surrounding tumors that facilitates local invasion. Here we describe a mechanism by which cells utilize contractility events to reorganize the ECM to provide contact guidance that facilitates 3D migration. Using novel assays to differentially organize the collagen matrix we show that alignment of collagen perpendicular to the tumor-explant boundary promotes local invasion of both human and mouse mammary epithelial cells. In contrast, organizing the collagen matrix to mimic the ECM organization associated with noninvading regions of tumors suppresses 3D migration/invasion. Moreover, we demonstrate that matrix reorganization is contractility-dependent and that the Rho/Rho kinase pathway is necessary for collagen alignment to provide contact guidance. Yet, if matrices are prealigned, inhibiting neither Rho nor Rho kinase inhibits 3D migration, which supports our conclusion that Rho-mediated matrix alignment is an early step in the invasion process, preceding and subsequently facilitating 3D migration.     

PC12 Cells Overexpressing Tissue Plasminogen Activator Regenerate Neurites to a Greater Extent and Migrate Faster than Control Cells in Complex Extracellular Matrix

Abstract: PC12 cells were stably transfected with expression vectors containing rat tissue plasminogen activator (tPA) under control of either a cytomegalovirus or rous sarcoma virus promoter. Cell lines were characterized using protease assays, ELISAs, immunoblots, northern blots, and Southern blots. Control PC12 cells or cells containing vectors alone released about 1 pg tPA/cell/24 h, whereas cells stably transfected with a tPA cDNA released 2–5 pg tPA/cell/24 h. A strong correlation existed between the amount of tPA released and the ability of cells to degrade extracellular matrix. Experiments with protease inhibitors and antibodies against tPA and plasminogen indicated that degradation of matrix involved tPA-generated plasmin and that the amount of matrix degraded was dependent on the amount of tPA released. Cells expressing high levels of tPA migrated on a three-dimensional matrix about twice as fast as control cells and regenerated neurites within three-dimensional gels of Matrigel to a greater extent than control cells. Antibodies that inhibited tPA and plasminogen decreased migration and neurite regeneration, indicating that tPA was involved in both events. PC12 cells overexpressing tPA should provide a useful model system for investigating neural functions of tPA including its role in migration and regeneration.     

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.     

Factor XIIIA transglutaminase expression and secretion by osteoblasts is regulated by extracellular matrix collagen and the MAP kinase signaling pathway

Osteoblast differentiation is regulated by the presence of collagen type I (COL I) extracellular matrix (ECM). We have recently demonstrated that Factor XIIIA (FXIIIA) transglutaminase (TG) is required by osteoblasts for COL I secretion and extracellular deposition, and thus also for osteoblast differentiation. In this study we have further investigated the link between COL I and FXIIIA, and demonstrate that COL I matrix increases FXIIIA levels in osteoblast cultures and that FXIIIA is found as cellular (cFXIIIA) and extacellular matrix (ecmFXIIIA) forms. FXIIIA mRNA, protein expression, cellular localization and secretion were enhanced by ascorbic acid (AA) treatment and blocked by dihydroxyproline (DHP) which inhibits COL I externalization. FXIIIA mRNA was regulated by the MAP kinase pathway. Secretion of ecmFXIIIA, and its enzymatic activity in conditioned medium, were also decreased in osteoblasts treated with the lysyl oxidase inhibitor β-aminopropionitrile, which resulted in a loosely packed COL I matrix. Osteoblasts secrete a latent, inactive dimeric ecmFXIIIA form which is activated upon binding to the matrix. Monodansyl cadaverine labeling of TG substrates in the cultures revealed that incorporation of the label occurred at sites where fibronectin co-localized with COL I, indicating that ecmFXIIIA secretion could function to stabilize newly deposited matrix. Our results suggest that FXIIIA is an integral part of the COL I deposition machinery, and also that it is part of the ECM-feedback loop, both of which regulate matrix deposition and osteoblast differentiation.     

The short arm of the laminin gamma2 chain plays a pivotal role in the incorporation of laminin 5 into the extracellular matrix and in cell adhesion

Laminin 5 is a basement membrane component that actively promotes adhesion and migration of epithelial cells. Laminin 5 undergoes extracellular proteolysis of the gamma2 chain that removes the NH(2)-terminal short arm of the polypeptide and reduces the size of laminin 5 from 440 to 400 kD. The functional consequence of this event remains obscure, although lines of evidence indicate that cleavage of the gamma2 chain potently stimulated scattering and migration of keratinocytes and cancer cells. To define the biological role of the gamma2 chain short arm, we expressed mutated gamma2 cDNAs into immortalized gamma2-null keratinocytes. By immunofluorescence and immunohistochemical studies, cell detachment, and adhesion assays, we found that the gamma2 short arm drives deposition of laminin 5 into the extracellular matrix (ECM) and sustains cell adhesion. Our results demonstrate that the unprocessed 440-kD form of laminin 5 is a biologically active adhesion ligand, and that the gamma2 globular domain IV is involved in intermolecular interactions that mediate integration of laminin 5 in the ECM and cell attachment.     

Tissue inhibitor of metalloproteinases-2 binding to membrane-type 1 matrix metalloproteinase induces MAPK activation and cell growth by a non-proteolytic mechanism

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.     

Fibroblast biology. Signals targeting the synovial fibroblast in arthritis

Fibroblast-like cells in the synovial lining (type B lining cells), stroma and pannus tissue are targeted by many signals, such as the following: ligands binding to cell surface receptors; lipid soluble, small molecular weight mediators (eg nitric oxide [NO], prostaglandins, carbon monoxide); extracellular matrix (ECM)-cell interactions; and direct cell-cell contacts, including gap junctional intercellular communication. Joints are subjected to cyclic mechanical loading and shear forces. Adherence and mechanical forces affect fibroblasts via the ECM (including the hyaluronan fluid phase matrix) and the pericellular matrix (eg extracellular matrix metalloproteinase inducer [EMMPRIN]) matrices, thus modulating fibroblast migration, adherence, proliferation, programmed cell death (including anoikis), synthesis or degradation of ECM, and production of various cytokines and other mediators [1]. Aggressive, transformed or transfected mesenchymal cells containing proto-oncogenes can act in the absence of lymphocytes, but whether these cells represent regressed fibroblasts, chondrocytes or bone marrow stem cells is unclear.     

An Inhibitory Antibody Blocks the First Step in the Dithiol/Disulfide Relay Mechanism of the Enzyme QSOX1

Quiescin sulfhydryl oxidase 1 (QSOX1) is a catalyst of disulfide bond formation that undergoes regulated secretion from fibroblasts and is over-produced in adenocarcinomas and other cancers. We have recently shown that QSOX1 is required for incorporation of particular laminin isoforms into the extracellular matrix (ECM) of cultured fibroblasts and, as a consequence, for tumor cell adhesion to and penetration of the ECM. The known role of laminins in integrin-mediated cell survival and motility suggests that controlling QSOX1 activity may provide a novel means of combating metastatic disease. With this motivation, we developed a monoclonal antibody that inhibits the activity of human QSOX1. Here, we present the biochemical and structural characterization of this antibody and demonstrate that it is a tight-binding inhibitor that blocks one of the redox-active sites in the enzyme, but not the site at which de novo disulfides are generated catalytically. Sulfhydryl oxidase activity is thus prevented without direct binding of the sulfhydryl oxidase domain, confirming the model for the interdomain QSOX1 electron transfer mechanism originally surmised based on mutagenesis and protein dissection. In addition, we developed a single-chain variant of the antibody and show that it is a potent QSOX1 inhibitor. The QSOX1 inhibitory antibody will be a valuable tool in studying the role of ECM composition and architecture in cell migration, and the recombinant version may be further developed for potential therapeutic applications based on manipulation of the tumor microenvironment.     

Amyloid precursor-like protein 2 promotes cell migration toward fibronectin and collagen IV.

Previous studies have established that in response to wounding, the expression of amyloid precursor-like protein 2 (APLP2) in the basal cells of migrating corneal epithelium is greatly up-regulated. To further our understanding of the functional significance of APLP2 in wound healing, we have measured the migratory response of transfected Chinese hamster ovary (CHO) cells expressing APLP2 isoforms to a variety of extracellular matrix components including laminin, collagen types I, IV, and VII, fibronectin, and heparan sulfate proteoglycans (HSPGs). CHO cells overexpressing either of two APLP2 variants, differing in chondroitin sulfate (CS) attachment, exhibit a marked increase in chemotaxis toward type IV collagen and fibronectin but not to laminin, collagen types I and VII, and HSPGs. Cells overexpressing APLP2-751 (CS-modified) exhibited a greater migratory response to fibronectin and type IV collagen than their non-CS-attached counterparts (APLP2-763), suggesting that CS modification enhanced APLP2 effects on cell migration. Moreover, in the presence of chondroitin sulfate, transfectants overexpressing APLP2-751 failed to exhibit this enhanced migration toward fibronectin. The APLP2-ECM interactions were also explored by solid phase adhesion assays. While overexpression of APLP2 isoforms moderately enhanced CHO adhesion to laminin, collagen types I and VII, and HSPGs lines, especially those overexpressing APLP2-751, exhibited greatly increased adhesion to type IV collagen and fibronectin. These observations suggest that APLP2 contributes to re-epithelialization during wound healing by supporting epithelial cell adhesion to fibronectin and collagen IV, thus influencing their capacity to migrate over the wound bed. Furthermore, APLP2 interactions with fibronectin and collagen IV appear to be potentiated by the addition of a CS chain to the core proteins.     

The developmental roles of the extracellular matrix: beyond structure to regulation

Cells in multicellular organisms are surrounded by a complex three-dimensional macromolecular extracellular matrix (ECM). This matrix, traditionally thought to serve a structural function providing support and strength to cells within tissues, is increasingly being recognized as having pleiotropic effects in development and growth. Elucidation of the role that the ECM plays in developmental processes has been significantly advanced by studying the phenotypic and developmental consequences of specific genetic alterations of ECM components in the mouse. These studies have revealed the enormous contribution of the ECM to the regulation of key processes in morphogenesis and organogenesis, such as cell adhesion, proliferation, specification, migration, survival, and differentiation. The ECM interacts with signaling molecules and morphogens thereby modulating their activities. This review considers these advances in our understanding of the function of ECM proteins during development, extending beyond their structural capacity, to embrace their new roles in intercellula signaling.