EMILIN1-α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation

EMILIN1 promotes α4β1 integrin-dependent cell adhesion and migration and reduces pro-transforming growth factor-β processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal keratinocyte layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to α4β1 and α9β1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-α4/α9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/2 pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/2 phosphorylation that in turn inhibited Smad2 signaling by phosphorylation of residues Ser245/250/255. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for keratinocyte α9β1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.     

EMILIN1/α9β1 Integrin Interaction Is Crucial in Lymphatic Valve Formation and Maintenance

Lymphatic vasculature plays a crucial role in the maintenance of tissue interstitial fluid balance. The role of functional collecting lymphatic vessels in lymph transport has been recently highlighted in pathologies leading to lymphedema, for which treatments are currently unavailable. Intraluminal valves are of paramount importance in this process. However, valve formation and maturation have not been entirely elucidated yet, in particular, the role played by the extracellular matrix (ECM). We hypothesized that EMILIN1, an ECM multidomain glycoprotein, regulates lymphatic valve formation and maintenance. Using a mouse knockout model, we show that in the absence of EMILIN1, mice exhibit defects in lymphatic valve structure and in lymph flow. By applying morphometric in vitro and in vivo functional assays, we conclude that this impaired phenotype depends on the lack of α9β1 integrin engagement, the specific lymphatic endothelial cell receptor for EMILIN1, and the ensuing derangement of cell proliferation and migration. Our data demonstrate a fundamental role for EMILIN1-integrin α9 interaction in lymphatic vasculature, especially in lymphatic valve formation and maintenance, and underline the importance of this ECM component in displaying a regulatory function in proliferation and acting as a “guiding” molecule in migration of lymphatic endothelial cells.     

Structural insights into the extra cellular segment of integrinβ5 and molecular interaction studies

Abstract

Primary tumor cells often spread to other organs by metastasis. Despite of it, primary tumor cells break their surrounding extra cellular matrix (ECM) proteins and reach the destination organ by the process of intravasation and extravasation. Metastasized tumor cells induce the process of angiogenesis, this highly regulated process involves several ECM proteins. However, integrins are primarily involved in the blood vessel growth and repair. Therefore, integrins are promising angiogenesis targets. Integrins are receptors on cell surface, involved in signal transduction and attachments in extra cellular matrix (ECM). IntegrinαVβ3 and αVβ5 are implicated in tumor angiogenesis, metastasis, inflammation and bone resorption. The crystal structure of integrinαvβ5 is not available in protein structural databases, therefore; molecular model of integrinβ5 structure was prepared and stereo chemical model quality was checked. Integrin β5 active sites were identified based on insilico analysis tools. Further, molecular level interactions between integrinβ5 and ECM proteins were predicted. In the present study ECM proteins such as focal adhesion kinase 1 (FAK1), annexin A5 and P21 activated kinase 4 (PAK4) were considered for protein-protein docking, to understand inter molecular interactions. The predicted model is conceived to be stereo chemically good and can be used for molecular interaction studies of angiogenic inhibitors.     

Dissection of laminin by cathepsin G into its long-arm and short-arm structures and localization of regions involved in calcium dependent stabilization and self-association

Native laminin-nidogen complex isolated from mouse Engelbreth-Holm-Swarm tumor was treated with purified cathepsin G or leucocyte elastase, two neutral serine proteases which play a role in inflammatory processes accompanied by degradation of basement membranes. Both enzymes were found to be more active than porcine pancreatic elastase. In the absence of Ca2+, laminin fragments produced by leucocyte elastase resembled those formed by the pancreatic enzyme but at physiological concentrations of Ca2+ cleavage by cathepsin G was much more selective. Initially laminin (900 kDa) was cleaved at two major sites only with similar rates leading to three fragments. Fragment C1-4 (about 550 kDa) comprises the intact three short arms of the molecule and fragment C8-9 (about 350 kDa) contains the entire triple-coiled region by which its three chains are assembled and the major part of the terminal globular domain of the long arm. The remaining C-terminal region of this domain was recovered as fragment C3 of about 50 kDa. Stabilization against proteolytic attack was restricted to the region of fragment C1-4 and only this fragment exhibited strong Ca2+ dependent self-association similar to that of intact laminin or of its complex with nidogen. The associative properties of fragment C1-4 were dramatically diminished upon removal of the tip of one of the short arms comprising fragment 4. In addition, this provides a clear assignment of the important laminin function to a distinct domain in one of its short arms. The new fragment C8-9 may be employed for exploring the properties and possible functions of the upper long-arm region which so far has not been available as a fragment.     

Regulation of the extrinsic apoptotic pathway by the extracellular matrix glycoprotein EMILIN2

Elastin microfibril interface-located proteins (EMILINs) constitute a family of extracellular matrix (ECM) glycoproteins characterized by the presence of an EMI domain at the N terminus and a gC1q domain at the C terminus. EMILIN1, the archetype molecule of the family, is involved in elastogenesis and hypertension etiology, whereas the function of EMILIN2 has not been resolved. Here, we provide evidence that the expression of EMILIN2 triggers the apoptosis of different cell lines. Cell death depends on the activation of the extrinsic apoptotic pathway following EMILIN2 binding to the TRAIL receptors DR4 and, to a lesser extent, DR5. Binding is followed by receptor clustering, colocalization with lipid rafts, death-inducing signaling complex assembly, and caspase activation. The direct activation of death receptors by an ECM molecule that mimics the activity of the known death receptor ligands is novel. The knockdown of EMILIN2 increases transformed cell survival, and overexpression impairs clonogenicity in soft agar and three-dimensional growth in natural matrices due to massive apoptosis. These data demonstrate an unexpected direct and functional interaction of an ECM constituent with death receptors and discloses an additional mechanism by which ECM cues can negatively affect cell survival.     

Effect of fibroblast activation protein and alpha2-antiplasmin cleaving enzyme on collagen types I, III, and IV

The circulating enzyme, α₂-antiplasmin cleaving enzyme (APCE), has very similar sequence homology and proteolytic specificity as fibroblast activation protein (FAP), a membrane-bound proteinase. FAP is expressed on activated fibroblasts associated with rapid tissue growth as in embryogenesis, wound healing, and epithelial-derived malignancies, but not in normal tissues. Its presence on stroma suggests that FAP functions to remodel extracellular matrix (ECM) during neoplastic growth. Precise biologic substrates have not been defined for FAP, although like APCE, it cleaves α₂-antiplasmin to a derivative more easily cross-linked to fibrin. While FAP has been shown to cleave gelatin, evidence for cleavage of native collagen, the major ECM component, remains indistinct. We examined the potential proteolytic effects of FAP or APCE alone and in concert with selected matrix metalloproteinases (MMPs) on collagens I, III, and IV. SDS-PAGE analyses demonstrated that neither FAP nor APCE cleaves collagen I. Following collagen I cleavage by MMP-1, however, FAP or APCE digested collagen I into smaller peptides. These peptides were analogous to, yet different from, those produced by MMP-9 following MMP-1 cleavage. Amino-terminal sequencing and mass spectrometry analyses of digestion mixtures identified several peptide fragments within the sequences of the two collagen chains. The proteolytic synergy of APCE in the cleavage of collagen I and III was not observed with collagen IV. We conclude that FAP works in synchrony with other proteinases to cleave partially degraded or denatured collagen I and III as ECM is excavated, and that derivative peptides might function to regulate malignant cell growth and motility.     

Emilin1 deficiency causes structural and functional defects of lymphatic vasculature

Lymphatic-vasculature function critically depends on extracellular matrix (ECM) and on its connections with lymphatic endothelial cells (LECs). However, the composition and the architecture of ECM have not been fully taken into consideration in studying the biology and the pathology of the lymphatic system. EMILIN1, an elastic microfibril-associated protein, is highly expressed by LECs in vitro and colocalizes with lymphatic vessels in several mouse tissues. A comparative study between WT and Emilin1-/- mice highlighted the fact that Emilin1 deficiency in both CD1 and C57BL/6 backgrounds results in hyperplasia, enlargement, and frequently an irregular pattern of superficial and visceral lymphatic vessels and in a significant reduction of anchoring filaments. Emilin1-deficient mice also develop larger lymphangiomas than WT mice. Lymphatic vascular morphological alterations are accompanied by functional defects, such as mild lymphedema, a highly significant drop in lymph drainage, and enhanced lymph leakage. Our findings demonstrate that EMILIN1 is involved in the regulation of the growth and in the maintenance of the integrity of lymphatic vessels, a fundamental requirement for efficient function. The phenotype displayed by Emilin1(-/-) mice is the first abnormal lymphatic phenotype associated with the deficiency of an ECM protein and identifies EMILIN1 as a novel local regulator of lymphangiogenesis.     

PS integrins and laminins: key regulators of cell migration during Drosophila embryogenesis

During embryonic development, there are numerous cases where organ or tissue formation depends upon the migration of primordial cells. In the Drosophila embryo, the visceral mesoderm (vm) acts as a substrate for the migration of several cell populations of epithelial origin, including the endoderm, the trachea and the salivary glands. These migratory processes require both integrins and laminins. The current model is that αPS1βPS (PS1) and/or αPS3βPS (PS3) integrins are required in migrating cells, whereas αPS2βPS (PS2) integrin is required in the vm, where it performs an as yet unidentified function. Here, we show that PS1 integrins are also required for the migration over the vm of cells of mesodermal origin, the caudal visceral mesoderm (CVM). These results support a model in which PS1 might have evolved to acquire the migratory function of integrins, irrespective of the origin of the tissue. This integrin function is highly specific and its specificity resides mainly in the extracellular domain. In addition, we have identified the Laminin α1,2 trimer, as the key extracellular matrix (ECM) component regulating CVM migration. Furthermore, we show that, as it is the case in vertebrates, integrins, and specifically PS2, contributes to CVM movement by participating in the correct assembly of the ECM that serves as tracks for migration.     

Integrins and chondrocyte–matrix interactions in articular cartilage

The integrin family of cell adhesion receptors plays a major role in mediating interactions between cells and the extracellular matrix. Normal adult articular chondrocytes express α1β1, α3β1, α5β1, α10β1, αVβ1, αVβ3, and αVβ5 integrins, while chondrocytes from osteoarthritic tissue also express α2β1, α4β1, α6β1. These integrins bind a host of cartilage extracellular matrix (ECM) proteins, most notably fibronectin and collagen types II and VI, which provide signals that regulate cell proliferation, survival, differentiation, and matrix remodeling. By initiating signals in response to mechanical forces, chondrocyte integrins also serve as mechanotransducers. When the cartilage matrix is damaged in osteoarthritis, fragments of fibronectin are generated that signal through the α5β1 integrin to activate a pro-inflammatory and pro-catabolic response which, if left unchecked, could contribute to progressive matrix degradation. The cell signaling pathways activated in response to excessive mechanical signals and to fibronectin fragments are being unraveled and may represent useful therapeutic targets for slowing or stopping progressive matrix destruction in arthritis.     

Matrix metalloproteinase-1 induces cleavage of exogenous alphaB-crystallin transduced by a cell-penetrating peptide

Cell-penetrating peptides (CPPs), including TAT-CPP, have been used to deliver exogenous proteins into living cells. Although a number of proteins fused to TAT-CPP can be delivered into various cells, little is known about the proteolytic cleavage of TAT-fusion proteins in cells. In this study, we demonstrate that a small heat shock protein (sHSP), alphaB-crystallin (αB-crystallin), delivered by TAT-CPP is susceptible to proteolytic cleavage by matrix metalloproteinase-1 (MMP-1) in cardiac myoblast H9c2 cells. Recombinant TAT-αB-crystallin was efficiently transduced into H9c2 cells. For a few hours following protein transduction, generation of a 14-kDa fragment, a cleavage band of TAT-αB-crystallin, increased in a time-dependent manner. This fragment was observed only in detergent-insoluble fractions. Interestingly, treatment with MMP inhibitors blocked the cleavage of TAT-αB-crystallin. In test tubes, recombinant MMP-1 processed TAT-αB-crystallin to generate the major cleavage fragment 14-kDa, as observed in the cells treated with TAT-αB-crystallin. The N-terminal sequences of the 14-kDa fragment were identified as Leu-Arg-Ala-Pro-Ser-Trp-Phe, indicating that this fragment is generated by cleavage at Phe54-Leu55 of αB-crystallin. The MMP-1-selective inhibitor abolished the production of 14-kDa fragments in cells. In addition, the cleaved fragment of TAT-αB-crystallin was significantly reduced in cells transfected with MMP-1 siRNA. Moreover, the enzymatic activity of MMP-1 was markedly increased in TAT-αB-crystallin-treated cells. TAT-αB-crystallin has a cytoprotective effect on H9c2 cells under hypoxic insult, moreover, MMP-1-selective inhibitor treatment led to even increased cell viability. These results suggest that MMP-1 is responsible for proteolytic cleavage of TAT-αB-crystallin during its intracellular transduction in H9c2 cells.     

Proteolysis of latent transforming growth factor-beta (TGF-beta )-binding protein-1 by osteoclasts. A cellular mechanism for release of TGF-beta from bone matrix

The binding of growth factors to the extracellular matrix (ECM) may be a key pathway for regulation of their activity. We have shown that a major mechanism for storage of transforming growth factor-beta (TGF-beta) in bone ECM is via its association with latent TGF-beta-binding protein-1 (LTBP1). Although proteolytic cleavage of LTBP1 has been reported, it remains unclear whether this represents a physiological mechanism for release of matrix-bound TGF-beta. Here we examined the role of LTBP1 in cell-mediated release of TGF-beta from bone ECM. We first characterized the soluble and ECM-bound forms of latent TGF-beta produced by primary osteoblasts. Next, we examined release of ECM-bound TGF-beta by bone resorbing cells. Isolated avian osteoclasts and rabbit bone marrow-derived osteoclasts released bone matrix-bound TGF-beta via LTBP1 cleavage. 1,25-Dihydroxyvitamin D3 enhanced LTBP1 cleavage, resulting in release of 90% of the ECM-bound LTBP1. In contrast, osteoblasts failed to cleave LTBP1 or release TGF-beta from bone ECM. Cleavage of LTBP1 by avian osteoclasts was inhibited by serine protease and metalloproteinase (MMP) inhibitors. Studies using purified proteases showed that plasmin, elastase, MMP2, and MMP9 were able to cleave LTBP1 to produce 125-165-kDa fragments. These studies identify LTBP1 as a novel substrate for MMPs and provide the first demonstration that LTBP1 proteolysis may be a physiological mechanism for release of TGF-beta from ECM-bound stores, potentially the first step in the pathway by which matrix-bound TGF-beta is rendered active.     

Selective integrin subunit reduction disrupts fibronectin extracellular matrix deposition and fibrillin 1 gene expression

Integrins are transmembrane receptors that can specifically bind extracellular matrix (ECM) proteins. Assembly of the ECM protein fibronectin into fibrils has been shown to be a cell-mediated process that requires integrins. Like fibronectin, fibrillin 1 is an ECM glycoprotein that can assemble into fibrils, but the role of integrins in fibril formation is not understood. To investigate the role of integrins in fibrillin 1 ECM deposition, cells that normally produce and assemble fibrillin 1 fibers in vitro were stably transfected with plasmid constructs encoding short interfering RNAs that target specific integrin subunits. Cells that were deficient in α2- and β3-integrin subunits produced and deposited fibronectin normally, but cells that were deficient for α5 and αV were unable to elaborate a fibronectin matrix, although they continued to produce and secrete the protein. Surprisingly, the cells that were unable to elaborate a fibronectin matrix also lost fibrillin 1 gene expression.     

Role of integrins in cancer: survey of expression patterns

Tumor cells are characterized by uncontrolled growth, invasion to surrounding tissues, and metastatic spread to distant sites. Mortality from cancer is often due to metastasis since surgical removal of tumors can enhance and prolong survival. The integrins constitute a family of transmembrane receptor proteins composed of heterodimeric complexes of noncovalently linked alpha and beta chains. Integrins function in cell-to-cell and cell-to-extracellular matrix (ECM) adhesive interactions and transduce signals from the ECM to the cell interior and vice versa. Hence, the integrins mediate the ECM influence on cell growth and differentiation. Since these properties implicate integrin involvement in cell migration, invasion, intra- and extra-vasation, and platelet interaction, a role for integrins in tumor growth and metastasis is obvious. These findings are underpinned by observations that the integrins are linked to the actin cytoskeleton involving talin, vinculin, and alpha-actinin as intermediaries. Such cytoskeletal changes can be manifested by rounded cell morphology, which is often coincident with tumor transformation via decreased or increased integrin expression patterns. For the various types of cancers, different changes in integrin expression are further associated with tumor growth and metastasis. Tumor progression leading to metastasis appears to involve equipping cancer cells with the appropriate adhesive (integrin) phenotype for interaction with the ECM. Therapies directed at influencing integrin cell expression and function are presently being explored for inhibition of tumor growth, metastasis, and angiogenesis. Such therapeutic strategies include anti-integrin monoclonal antibodies, peptidic inhibitors (cyclic and linear), calcium-binding protein antagonists, proline analogs, apoptosis promotors, and antisense oligonucleotides. Moreover, platelet aggregation induced by tumor cells, which facilitates metastatic spread, can be inhibited by the disintegrins, a family of viper venom-like peptides. Therefore, adhesion molecules from the integrin family and components of angiogenesis might be useful as tumor progression markers for prognostic and for diagnostic purposes. Development of integrin cell expression profiles for individual tumors may have further potential in identifying a cell surface signature for a specific tumor type and/or stage. Thus, recent advances in elucidating the structure, function, ECM binding, and signaling pathways of the integrins have led to new and exciting modalities for cancer therapeutics and diagnoses.     

Proteolytic cleavage of retinoblastoma protein upon DNA damage and Fas-mediated apoptosis

Proteolytic cleavage of key cellular proteins by caspases (ICE, CPP32, and Ich-1/Nedd2) may be crucial to the apoptotic process. The retinoblastoma tumor suppressor gene is a negative regulator of cell growth and the retinoblastoma protein (pRb) exhibits anti-apoptotic function. We show that pRb is cleaved during apoptosis induced by either UV irradiation or anti-Fas antibody. Our studies implicate CPP32-like activity in the proteolytic cleavage of pRb. The kinetics of proteolytic cleavage of pRb during apoptosis differ from that observed for other cellular proteins, suggesting that the specific cleavage of pRb during apoptosis may be an important event.     

Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture

Fibroblast feeder cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem cells (hESCs). The feeder cells secrete various growth factors and extracellular matrix (ECM) proteins into extracellular milieu. However, the roles of the feeder cell-secreted factors are largely unclear. Animal feeder cells and use of animal serum also make current feeder cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free feeder cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) feeder cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) feeder cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive feeder cells produce laminin-511 and express laminin-binding integrins α3ß1, α6ß1 and α7ß1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct feeder cells.     

Identification of protease-sensitive sites in Human Endothelial-Monocyte Activating Polypeptide II protein

The cleaved approximately 22-kDa form of Endothelial-Monocyte Activating Polypeptide [mature (m)EMAP II] functions as a potent inhibitor of tumor growth. Although the anti-tumor effect of mEMAP II has been described, little is known regarding the cleavage of mEMAP II from its precursor form (pEMAP II). We determined that pEMAP II is expressed at the cell membrane surface and proteinases MMP-9, elastase, and cathepsin L release protein fragments consistent with mEMAP II molecular mass. MMP-9 and elastase generate a approximately 25-26 kDa spanning fragments, while cathepsin L generates a approximately 22 kDa fragment. Although several fragments are processed from pEMAP II within a 44 AA residue stretch, cathepsin L cleaves pEMAP II within 4 amino acids of the determined N-terminal sequence, suggesting that this region is sensitive to proteinases.     

A novel regulatory function of CDKN1A/p21 in TNFα-induced matrix metalloproteinase 9-dependent migration and invasion of triple-negative breast cancer cells

Metastasis is the leading cause of mortality in patients with highly invasive cancers and, as such, is a major problem for medicine. It has been increasingly recognized that cancer-related inflammation plays an important role in promoting invasion and the metastatic process in which cell motility and upregulation of proteolytic enzymes are crucial events. TNFα is a proinflammatory cytokine known to stimulate synthesis of MMP9, a zinc- and calcium-dependent endopeptidase contributing to the regulation of ECM remodeling and cell signaling. However, the precise molecular mechanism of TNFα-induced MMP9 gene expression in cancers is still not fully understood. This study shows that TNFα-induced cell migration and invasion involve ERK1/2-dependent up-regulation of CDKN1A/p21 expression in highly aggressive breast cancer cells and that CDKN1A/p21 plays an important regulatory role in TNFα-induced MMP9 gene expression, indicating an unknown function of CDKN1A/p21 as a regulator of proteolytic activity in cancer cells.     

A neuronal cell line (PC12) expresses two beta 1-class integrins-alpha 1 beta 1 and alpha 3 beta 1-that recognize different neurite outgrowth-promoting domains in laminin

Integrins mediate neuronal process outgrowth on components of the ECM. Integrin alpha subunit-specific antibodies have been used to examine the roles of individual beta 1 integrins in attachment and neurite outgrowth by the neuronal cell line, PC12, in response to laminin and collagen. alpha 1 beta 1 and alpha 3 beta 1 were identified as the major beta 1 integrins expressed by PC12 cells. In functional assays, both alpha 1 beta 1 and alpha 3 beta 1 mediated PC12 cell interactions with laminin, whereas alpha 1 beta 1 alone mediated responses to collagen types I and IV. alpha 1 beta 1 and alpha 3 beta 1 were shown to recognize two different neurite-promoting sites in laminin: alpha 1 beta 1 interacted with the cross-region of laminin present in proteolytic fragments E1-4 and E1; alpha 3 beta 1 recognized a site in the long arm contained in laminin fragment E8. Thus, PC12 cells express two beta 1 integrins, which together function in attachment and neurite outgrowth on laminin and collagen. These integrins are candidates for mediating neurite outgrowth of sympathetic and other neurons in response to these ECM components.     

Target-derived matricryptins organize cerebellar synapse formation through α3β1 integrins

Trans-synaptic organizing cues must be passed between synaptic partners for synapses to properly form. Much of our understanding of this process stems from studies at the neuromuscular junction, where target-derived growth factors, extracellular matrix (ECM) molecules, and matricryptins (proteolytically released fragments of ECM molecules) are all essential for the formation and maintenance of motor nerve terminals. While growth factors and ECM molecules also contribute to the formation of brain synapses, it remains unclear whether synaptic roles exist for matricryptins in the mammalian brain. We report that collagen XVIII and its matricryptin endostatin are generated by cerebellar Purkinje cells and are necessary for the organization of climbing fiber terminals in these neurons. Moreover, endostatin is sufficient to induce climbing fiber terminal formation in vitro by binding and signaling through α3β1 integrins. Taken together, these studies reveal roles for both matricryptins and integrins in the organization of brain synapses.     

Proteolytic fragments of fibronectin function as matrikines driving the chemotactic affinity of prostate cancer cells to human bone marrow mesenchymal stromal cells via the α5β1 integrin

The haematopoietic niche is contributed to by bone marrow-resident mesenchymal stromal cells (BM-MSCs) and subverted by prostate cancer cells. To study mechanisms by which BM-MSCs and prostate cancer cells may interact, we assessed the migration, invasion, adhesion and proliferation of bone-derived prostate cancer cells (PC-3) in co-culture with pluripotent human BM-MSCs. We observed a strong adhesive, migratory and invasive phenotype of PC-3 cells with BM- MSC-co-culture and set out to isolate and characterize the bioactive principle. Initial studies indicated that chemotaxis was secondary to a protein residing in the >100kDa fraction. Size-exclusion chromatography (SEC) recovered peak activity in a high-molecular weight fraction containing thrombospondin-1 (TSP1). While TSP1 immunodepletion decreased activity, put-back with purified TSP1 did not reproduce bioactivity. Further purification of the TSP1-containing high-molecular weight fraction of the BM-MSC secretome with heparin-affinity chromatography recovered bioactivity with highly restricted bands on polyacrylamide gel electrophoresis, determined by mass spectroscopy to be proteolytic fragments of fibronectin (FN). Put-back experiments with full-length FN permitted adhesion but failed to induce migration. Monospecific antibodies to FN blocked adhesion. Proteolytic cleavage of FN generated FN fragments which now induced migration. Neutralizing monoclonal antibodies to FN receptors α5 and β1 integrins, and α5 knockdown specifically blocked migration and adhesion. Conclusion: Fibronectin fragments (FNFr) function as matrikines driving the chemotactic affinity of prostate cancer cells via the α5β1 integrin. Taken together with the high-frequency of α5β1 expression in disseminated prostate cancer cells in bone marrow aspirates from patients, the FNFr/FN-α5β1 interaction warrants further study as a therapeutic target.