Wnt family proteins are secreted and associated with the cell surface.

Members of the Wnt gene family are proposed to function in both normal development and differentiation as well as in mammary tumorigenesis. To understand the function of Wnt proteins in these two processes, we present here a biochemical characterization of seven Wnt family members. For these studies, AtT-20 cells, a neuroendocrine cell line previously shown to efficiently process and secrete Wnt-1, was transfected with expression vectors encoding Wnt family members. All of the newly characterized Wnt proteins are glycosylated, secreted proteins that are tightly associated with the cell surface or extracellular matrix. We have also identified native Wnt proteins in retinoic acid-treated P19 embryonal carcinoma cells, and they exhibit the same biochemical characteristics as the recombinant proteins. These data suggest that Wnt family members function in cell to cell signaling in a fashion similar to Wnt-1.     

Processing of Laminin-5 and Its Functional Consequences: Role of Plasmin and Tissue-type Plasminogen Activator

The laminin-5 component of the extracellular matrices of certain cultured cells such as the rat epithelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell– matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5–rich matrices of pp126 epithelial cells fail to assemble hemidesmosomes and are motile. To understand these contradictory phenomena, we have compared the forms of heterotrimeric laminin-5 secreted by 804G and MCF-10A cells with those secreted by pp126 cells, using a panel of laminin-5 subunit-specific antibodies. The α3 subunit of laminin-5 secreted by pp126 cells migrates at 190 kD, whereas that secreted by 804G and MCF-10A cells migrates at 160 kD. The pp126 cell 190-kD α3 chain of laminin-5 can be specifically proteolyzed by plasmin to a 160-kD species at enzyme concentrations that do not apparently effect the laminin-5 β and γ chains. After plasmin treatment, pp126 cell laminin-5 not only impedes cell motility but also becomes competent to nucleate assembly of hemidesmosomes. The possibility that plasmin may play an important role in processing laminin-5 subunits is supported by immunofluorescence analyses that demonstrate colocalization of laminin-5 and plasminogen in the extracellular matrix of MCF-10A and pp126 cells. Whereas tissue-type plasminogen activator (tPA), which converts plasminogen to plasmin, codistributes with laminin-5 in MCF-10A matrix, tPA is not present in pp126 extracellular matrix. Treatment of pp126 laminin-5–rich extracellular matrix with exogenous tPA results in proteolysis of the laminin-5 α3 chain from 190 to 160 kD. In addition, plasminogen and tPA bind laminin-5 in vitro. In summary, we provide evidence that laminin-5 is a multifunctional protein that can act under certain circumstances as a motility and at other times as an adhesive factor. In cells in culture, this functional conversion appears dependent upon and is regulated by tPA and plasminogen.     

TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages

Transforming growth factor-beta1 (TGF-beta1), a key cytokine for control of cell growth, extracellular matrix formation, and inflammation control, is secreted by many cells present in the arteriosclerotic plaque. Lipid accumulation in the vessel wall is regarded as an early step in atherogenesis and depends on uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors and their transformation into foam cells. Prominent members of the scavenger receptor family are the class A type I and II receptors (ScR-A), the class B receptor CD36, and the recently detected lectin-like oxidized LDL receptor-1 (LOX-1), which, unlike the native LDL receptor (LDL-R), are not feedback controlled. CD36 is responsible for >50% of modified LDL uptake into human monocyte-derived macrophages. We therefore studied whether TGF-beta1 influences expression and function of ScR-A, CD36, and LOX-1 in monocytes using RT-PCR and flow cytometry. Total uptake of oxidized LDL by monocytoid cells, reflecting the combined function of all scavenger receptors, was significantly reduced by TGF-beta1. At initially low picomolar concentrations, TGF-beta1 decreased CD36 mRNA and protein surface expression and ScR-A mRNA levels in the human monocytic cell line THP-1 and in freshly isolated and cultivated human monocytes, whereas LOX-1 mRNA was increased. Expression of LDL-R and beta-actin was not affected by TGF-beta1. In conclusion, depression of scavenger receptor function in monocytes by TGF-beta1 in low concentrations reduces foam cell formation. Together with matrix control by TGF-beta1, this may be important for atherogenesis and plaque stabilization.     

Expression and structure of human SPARC transcripts: SPARC mRNA is expressed by human cells involved in extracellular matrix production and some of these cells show an unusual expression pattern

A mouse SPARC cDNA clone was used to elucidate the expression of SPARC mRNA in normal diploid human cells as well as in tumor cells. Among 40 cell lines examined, 19 showed expression. The mRNA transcribed by the majority of the expressors are 2.1 kb with a trace amount of 3 kb. However, three cell types, undifferentiated basal keratinocytes, their differentiated derivatives, and breast adenocarcinoma cells, showed an expression pattern distinct from the typical one, having abundant 3-kb mRNA but no detectable 2.1-kb mRNA. The mRNA was translated and the product secreted. This expression pattern was not observed before in human cells and was not found in tumor cells of keratinocytes, squamous carcinoma cells, or many other adenocarcinoma cells. We showed by Northern hybridization that the SPARC-expressing melanocytic melanoma cell lines produced laminin, a component of extracellular matrix. Other cell types expressing the SPARC mRNA were also reported to synthesize extracellular matrix components. Thus, our results indicate an association between SPARC gene expression and production of extracellular matrix. However, the opposite is not true since non-SPARC-producers may or may not produce extracellular matrix. For example, A431 cell line, which does not express SPARC mRNA, is known to produce extracellular matrix components while the normal diploid melanocytes and undifferentiated embryonal carcinoma cells, which do not express SPARC mRNA, do not produce extracellular matrix component.     

Alternative splicing generates a secreted form of N-CAM in muscle and brain

A number of different membrane associated isoforms of the neural cell adhesion molecule (N-CAM) have previously been identified. Here the structure of a novel secreted isoform of N-CAM is established by analysis of a cDNA corresponding to an N-CAM mRNA from human skeletal muscle. The mRNA incorporates a novel sequence block into the extracellular domain, which introduces an in-frame stop codon and thus prematurely terminates the coding sequence, generating a truncated N-CAM polypeptide. Analysis of genomic clones indicates that the inserted sequence is present as a discrete exon within the human N-CAM gene, and Northern analysis shows it to be associated specifically with a 5.2 kb mRNA species from skeletal muscle and brain. Stable transfectants expressing the secreted isoform accumulate it in the cytoplasm and release it to the culture medium. In contrast, cells transfected with cDNA encoding lipid-tailed N-CAM express it predominantly at the cell surface. The existence of a secreted isoform may further expand the spectrum of N-CAM function beyond its known involvement in intercellular adhesion to extracellular matrix interactions.     

Extracellular matrix and nuclear localization of beta ig-h3 in human bladder smooth muscle and fibroblast cells

The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions.     

Myocilin蛋白在小梁网组织中的作用

Myocilin基因是与原发性开角型青光眼成因有关的基因。其蛋白产物myocilin蛋白是一种分泌型糖蛋白,具有特征性区域：N端亮氨酸拉链区,中央链接区,C端类嗅质蛋白（嗅素）区。眼组织中,小梁网myocilin蛋白表达水平最高且在细胞内外均可检测到。细胞内myocilin蛋白由小梁网细胞以外泌体样囊泡形式释放至胞外,突变时分泌受阻并异常聚集,使细胞致敏诱发凋亡。细胞外myocilin蛋白通过与一种或多种细胞外基质蛋白相互作用影响细胞的形态、粘接、迁移活动,调节细胞外基质的成分和结构,从而影响房水流出系统。     

Subcellular distribution of Wnt-1 at adherens junctions and actin-rich densities in endothelial cells

The Wnt family of signaling proteins functions in embryonic development and mammalian oncogenesis. It is unknown whether these molecules have a role in normal, postdevelopmental, homeostatic processes. Possessing a putative signal sequence and potential glycosylation sites, Wnt-1 is believed to be secreted and remain associated with the cell surface and extracellular matrix. While it has been suggested that Wnt proteins may target cytoskeletal structures more directly, no definitive studies have identified an intracellular association and function for these molecules. Here, we report that Western blots of lysates from retinoic-acid-differentiated P19 cells and bovine endothelial cells indicate the presence of a 45-kDa Wnt-1 protein. In endothelium, Wnt-1 was present in both the Triton X soluble and the insoluble cell fractions. Immunocytochemical labeling localized Wnt-1 to adherens junctions, codistributing with beta-catenin. Wnt-1 also was detected at actin-rich densities (ARDs) within basal cell regions. In wounded monolayers, ARDs delineated the distal margins of cells undergoing directed migration. Transfection with antisense oligonucleotides to Wnt-1 resulted in reduced cohesion of wound edge cells, abnormal protrusive activity, and random movement. Our data indicate that Wnt-1 protein is present in postdevelopmental endothelial cells where it associates with cytoskeletal elements and may retain function as a tissue polarity gene.     

Coxsackievirus B3 infection induces cyr61 activation via JNK to mediate cell death

Coxsackievirus B3 (CVB3), an enterovirus in the Picornavirus family, is the most common human pathogen associated with myocarditis and idiopathic dilated cardiomyopathy. We found upregulation of the cysteine-rich protein gene (cyr61) after CVB3 infection in HeLa cells with a cDNA microarray approach, which is confirmed by Northern blot analysis. It is also revealed that the extracellular amount of Cyr61 protein was increased after CVB3 infection in HeLa cells. cyr61 is an early-transcribed gene, and the Cyr61 protein is secreted into the extracellular matrix. Its function is related to cell adhesion, migration, and neuronal cell death. Here, we show that activation of the cyr61 promoter by CVB3 infection is dependent on JNK activation induced by CVB3 replication and viral protein expression in infected cells. To explore the role of Cyr61 protein in infected HeLa cells, we transiently overexpressed cyr61 and infected HeLa cells with CVB3. This increased CVB3 growth in the cells and promoted host cell death by viral infection, whereas down-expression of cyr61 with short interfering RNA reduced CVB3 growth and showed resistance to cell death by CVB3 infection. In conclusion, we have demonstrated a new role for cyr61 in HeLa cells infected with CVB3, which is associated with the cell death induced by virus infection. These data thus expand our understanding of the physiological functions of cyr61 in virus-induced cell death and provide new insights into the cellular factors involved.     

Testican‐3: a brain‐specific proteoglycan member of the BM‐40/SPARC/osteonectin family

The testicans are a three‐member family of secreted proteoglycans structurally related to the BM‐40/secreted protein acidic and rich in cystein (SPARC) osteonectin family of extracellular calcium‐binding proteins. In vitro studies have indicated that testicans are involved in the regulation of extracellular protease cascades and in neuronal function. Here, we describe the biochemical characterization and tissue distribution of mouse testican‐3 as well as the inactivation of the corresponding gene. The expression of testican‐3 in adult mice is restricted to the brain, where it is located diffusely within the extracellular matrix, as well as associated with cells. Brain‐derived testican‐3 is a heparan sulphate proteoglycan. In cell culture, the core protein is detected in the supernatant and the extracellular matrix, whereas the proteoglycan form is restricted to the supernatant. This indicates possible interactions of the testican‐3 core protein with components of the extracellular matrix which are blocked by addition of the glycosaminoglycan chains. Mice deficient in testican‐3 are viable and fertile and do not show an obvious phenotype. This points to a functional redundancy among the different members of the testican family or between testican‐3 and other brain heparan sulphate proteoglycans.     

Latent transforming growth factor-beta binding proteins (LTBPs)--structural extracellular matrix proteins for targeting TGF-beta action

Growth factors of the transforming growth factor-beta family are potent regulators of the extracellular matrix formation, in addition to their immunomodulatory and regulatory roles for cell growth. TGF-beta s are secreted from cells as latent complexes containing TGF-beta and its propeptide, LAP (latency-associated peptide). In most cells LAP is covalently linked to an additional protein, latent TGF-beta binding protein (LTBP), forming the large latent complex. LTBPs are required for efficient secretion and correct folding of TGF-beta s. The secreted large latent complexes associate covalently with the extracellular matrix via the N-termini of the LTBPs. LTBPs belong to the fibrillin-LTBP family of extracellular matrix proteins, which have a typical repeated domain structure consisting mostly of epidermal growth factor (EGF)-like repeats and characteristic eight cysteine (8-Cys) repeats. Currently four different LTBPs and two fibrillins have been identified. LTBPs contain multiple proteinase sensitive sites, providing means to solubilize the large latent complex from the extracellular matrix structures. LTBPs are now known to exist both as soluble molecules and in association with the extracellular matrix. An important consequence of this is LTBP-mediated deposition and targeting of latent, activatable TGF-beta into extracellular matrices and connective tissues. LTBPs have a dual function, they are required both for the secretion of the small latent TGF-beta complex as well as directing bound latent TGF-beta to extracellular matrix microfibrils. However, it is not known at present whether LTBPs are capable of forming microfibrils independently, or whether they are a part of the fibrillin-containing fibrils. Most LTBPs possess RGD-sequences, which may have a role in their interactions with the cell surface. At least LTBP-1 is chemotactic to smooth muscle cells, and is involved in vascular remodelling. Analyses of the expressed LTBPs have revealed considerable variations throughout the molecules, generated both by alternative splicing and utilization of multiple promoter regions. The significance of this structural diversity is mostly unclear at present.     

A tetranectin-related protein is produced and deposited in extracellular matrix by human embryonal fibroblasts

Tetranectin is a tetrameric human plasma protein that binds to plasminogen kringle 4. Its amino acid sequence is homologous with the C-terminal parts of asialoglycoprotein receptors and proteoglycan core proteins. In the present study, we have demonstrated that the human embryonal fibroblast cell line WI-38 produce a tetranectin-related molecule, which might, by several criteria, be similar to tetranectin from plasma. These criteria include immunoblotting analysis of conditioned cell medium revealing a protein band with Mr 17,000, indistinguishable from the Mr of plasma tetranectin. A preparation obtained by purification of conditioned medium by affinity chromatography on an anti-(plasma tetranectin) IgG column also contained the Mr 17,000 protein. This protein (partly purified from the conditioned medium) was shown by crossed immunoelectrophoresis to bind to heparin, CaCl2 and plasminogen kringle 4, as previously described for tetranectin in plasma. Importantly, this tetranectin-related protein is not only present in conditioned culture medium, but the Mr 17,000 protein reacting with anti-(plasma tetranectin) IgG was also present in the extracellular material, remaining after removal of WI-38 cells from the culture dishes, as demonstrated by immunoblotting analysis and immunocytochemical staining. We conclude that WI-38 cells produce a tetranectin-related protein and secrete it into the extracellular matrix.     

Purification of the extracellular domain of the membrane protein GlialCAM expressed in HEK and CHO cells and comparison of the glycosylation

Adhesion molecules are essential for a wide range of biological and physiological functions, including cell-cell interactions, cell interactions with the extracellular matrix, cell migration, proliferation and survival. Defects in cell adhesion have been associated with pathological conditions such as neoplasia, and neurodegenerative diseases. We have identified a new adhesion molecule of the immunoglobulin family, GlialCAM. The same protein was recently published under the name hepaCAM and was suggested to be associated with hepatocellular carcinoma. Here we have expressed and purified the extracellular domain of this molecule in two mammalian expression systems, HEK and CHO cells. A three step purification protocol gave an over 95% pure protein. The extracellular domain of GlialCAM possesses several potential N- and O-glycosylation sites. Glycosylation is one of the most common post-translational modifications of secreted proteins and of the extracellular domains of membrane bound proteins. It can influence both the activity and the stability of the protein. The glycosylation pattern has been shown to depend on the cell type where the protein is expressed. We examined if differences in the glycosylation of this protein could be detected when it was expressed in the two commonly used mammalian expression systems, HEK and CHO. Differences in the glycosylation were detected.     

Cigarette smoke alters the secretome of lung epithelial cells

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label‐free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.     

Alpha-actinin interactions with syndecan-4 are integral to fibroblast–matrix adhesion and regulate cytoskeletal architecture

All cells of the musculoskeletal system possess transmembrane syndecan proteoglycans, notably syndecan-4. In fibroblasts it regulates integrin-mediated adhesion to the extracellular matrix. Syndecan-4 null mice have a complex wound repair phenotype while their fibroblasts have reduced focal adhesions and matrix contraction abilities. Signalling through syndecan-4 core protein to the actin cytoskeleton involves protein kinase Cα and Rho family G proteins but also direct interactions with α-actinin. The contribution of the latter interaction to cell–matrix adhesion is not defined but investigated here since manipulation of Rho GTPase and its downstream targets could not restore a wild type microfilament organisation to syndecan-4 null cells. Microarray and protein analysis revealed no significant alterations in mRNA or protein levels for actin- or α-actinin associated proteins when wild type and syndecan-4 knockout fibroblasts were compared. The binding site for syndecan-4 cytoplasmic domain was identified as spectrin repeat 4 of α-actinin while further experiments confirmed the importance of this interaction in stabilising cell–matrix junctions. However, α-actinin is also present in adherens junctions, these organelles not being disrupted in the absence of syndecan-4. Indeed, co-culture of wild type and knockout cells led to adherens junction-associated stress fibre formation in cells lacking syndecan-4, supporting the hypothesis that the proteoglycan regulates cell–matrix adhesion and its associated microfilament bundles at a post-translational level. These data provide an additional dimension to syndecan function related to tension at the cell–matrix interface, wound healing and potentially fibrosis.     

Expression of secreted frizzled-related protein 2 in a primary canine mammary tumor cell line: a candidate tumor marker for mammary tumor cells

Canine mammary tumors (CMTs) have been proposed to be a good animal model for human breast cancer. To provide a basis for the tumorigenic study of CMTs, cell lines were established using a modified cell culture technique. The epithelial morphology and immunostaining with cytokeratin 18 confirmed the epithelial origin of the cells. In an investigation of possible mammary tumorigenesis-related factors, the expression of Wnt signaling-related proteins was detected in cell lines. Secreted frizzled-related protein 2 (SFRP2) was abundantly expressed in CMT cells but not in normal canine mammary gland (MG) cells. Secreted frizzled-related protein 2 was secreted into the culture medium and was associated with the extracellular matrix. In addition, increased expressions of beta-catenin and cyclin D1 were observed in cells overexpressing SFRP2. The marked differential expression of SFRP2 reveals that this protein may be a potential candidate marker for CMTs. The CMT cell line established in this study provides a useful tool and experimental model for understanding both the tumorigenesis of CMTs and the role of Wnt signaling in cancers.     

A 60-kD protein mediates the binding of transforming growth factor-beta to cell surface and extracellular matrix proteoglycans

The biological activity of many cytokines is regulated by binding proteins present at the cell surface, in extracellular matrices or in soluble phase. We describe here a TGF-beta binding protein that is both an extracellular matrix and a cell surface protein. When intact extracellular matrices of HEP-G2 cells were affinity cross-linked with 125I-TGF-beta 1, two major binding components were seen: a 250-kD, proteoglycan-like molecule, presumed to be betaglycan, and a 60-kD protein. The 60-kD TGF-beta-binding protein was also present at the cell surface. It could be released from the cell surface by treating cells with high salt, heparin, chondroitin sulfate, heparitinase, or chondroitinase, indicating that it is bound to heparan sulfate and chondroitin sulfate proteoglycans. The 60-kD protein bound TGF-beta 1 with an apparent dissociation constant of 1.6 nM, and there were 30,000 binding sites per cell at the cell surface. In addition to the HEP-G2 cells and another hepatoma cell line, the 60-kD protein was also found in a human colon carcinoma (HT-29) cell line but not in rat kidney (NRK- 49F) or human fibroblast (HUT-12) cell lines. The 60-kD protein could be extracted from cells containing it and transferred to the surface of previously negative cells. The 60-kD protein may serve to regulate the binding of TGF-beta to its signal transducing receptors by targeting TGF-beta to appropriate locations in the microenvironment of cells.     

Matrix metalloproteinases and their inhibitors,modulators of neuro-immune interactions and of pathophysiological processes in the nervous system

The matrix metalloproteinases (MMPs) belong to a growing family of Zn2+-dependent endopeptidases, secreted or membrane-bound (MT-MMP), that regulate or degrade by proteolytic cleavage protein components of the extracellular matrix, cytokines, chemokines, cell adhesion molecules and a variety of membrane receptors. MMP activity is counterbalanced by their physiological inhibitors, the tissue inhibitors of MMPs (TIMPs), a family of 4 secreted multifunctional proteins that have growth promoting activities. In physiological conditions MMP activity is tightly regulated and altered MMP regulation is associated with pathological processes including inflammation, cell proliferation, cell death and tissue remodeling. The MMP/TIMP system is involved in the development and function of cells of the immune system by promoting their differentiation, activation, migration across basement membranes and tissues. In the last years, data has accumulated indicating that the MMP/TIMP system is expressed in the nervous system where it regulates neuro-immune interactions and plays a major role in pathophysiological processes. In this review, we present recent in vivo and in vitro studies that highlight the contribution of the MMP/TIMP system to various diseases of the nervous system, involving blood brain barrier breakdown, neuroinflammation, glial reactivity, neuronal death, reactive plasticity, and to developmental and physiological processes including cell migration, axonal sprouting and neuronal plasticity. This review also alludes to the beneficial effects of synthetic MMP inhibitors in different animal models of neuropathology. In all, a further understanding of the role of MMPs and TIMPs in the nervous system should contribute to unravel mechanisms of neuronal plasticity and pathology and set the basis of new therapeutic strategies in nervous system disorders based on the development of synthetic MMP inhibitors.     

Functional significance of metastasis-inducing S100A4(Mts1) in tumor-stroma interplay

Causal implication of S100A4 in inducing metastases was convincingly shown previously. However, the mechanisms that associate S100A4 with tumor progression are not well understood. S100A4 protein, as a typical member of the S100 family, exhibits dual, intracellular and extracellular, functions. This work is focused on the extracellular function of S100A4, in particular its involvement in tumor-stroma interplay in VMR (mouse adenocarcinoma cell line) tumor cells, which exhibit stroma-dependent metastatic phenotype. We demonstrated the reciprocal influence of tumor and stroma cells where tumor cells stimulate S100A4 secretion from fibroblasts in culture. In turn, extracellular S100A4 modifies the cytoskeleton and focal adhesions and triggers several other events in tumor cells. We found stabilization of the tumor suppressor protein p53 and modulation of its function. In particular, extracellular S100A4 down-regulates the pro-apoptotic bax and the angiogenesis inhibitor thrombospondin-1 genes. For the first time, we demonstrate here that the S100A4 protein added to the extracellular space strongly stimulates proteolytic activity of VMR cells. This activity most probably is associated with matrix metalloproteinases and, in particular, with matrix metalloproteinase-13. Finally, the application of the recombinant S100A4 protein confers stroma-independent metastatic phenotype on VMR tumor cells. In conclusion, our results indicate that metastasis-inducing S100A4 protein plays a pivotal role in the tumor-stroma environment. S100A4 released either by tumor or stroma cells triggers pro-metastatic cascades in tumor cells.