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Scleroderma, the chronic autoimmune disease is a consequence of inflammation in the connective tissue. Prolonged duration affects formation of compact connective tissue strands (scarring) within the target organ. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndMT) are the source of fibroblast phenotype-resembling cells. EndMT contributes to reorganization of the focal adhesion proteins (FA), including integrins, and intensive extracellular matrix (ECM) remodelling. However, in endothelial cells, the relationship between EndMT and the interaction of integrin receptors with lumican - a component of ECM, is still unclear. Our findings indicate that at the early stages of EndMT caused by Snail-1 transcription factor overexpression, the level of the β1 integrin subunit and its phosphorylation are elevated. Simultaneously, the changes in the level of proteins that build FAs and promote activation of integrin receptors as well as a decrease in lumican quantity were observed. These modulations contributed to increased migration of human microvascular endothelial cells, HMEC-1. Our findings were achieved by WB, ELISA and wound healing assay. Taken altogether, transfection of HMEC-1 cells with Snail-1 plasmids inducing the early stages of EndMT results in the increase of total FAK and integrin β1 phosphorylation as well as cell migration: phenomena which are modulated by interaction with lumican. [ABSTRACT FROM AUTHOR]

Abstract: The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin β1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2β1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2β1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K d ≥200nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2β1 integrin. [ABSTRACT FROM AUTHOR]

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix. [Copyright &y& Elsevier]

Extracellular matrix assembly is a multistep process and the various steps in collagen fibrillogenesis are thought to be influenced by a number of factors, including other noncollagenous matrix molecules. The synthesis and deposition of extracellular matrix by corneal fibroblasts grown within three-dimensional collagen gel cultures were examined to elucidate the factors important in the establishment of tissue-specific matrix architecture. Corneal fibroblasts in collagen gel cultures form layers and deposit small-diameter collagen fibrils (approximately 25 nm) typical of the mature corneal stroma. The matrix synthesized contains type VI collagen in a filamentous network and type I and type V collagen assembled as heterotypic fibrils. The amount of type V collagen synthesized is relatively high and comparable to that seen in the corneal stroma. This matrix is deposited between cell layers in a manner reminiscent of the secondary corneal stroma, but is not deposited as densely or as organized as would be found in situ. No keratan sulfate proteoglycan, a proteoglycan found only in the corneal stroma, was synthesized by the fibroblasts in the collagen gel cultures. The assembly and deposition of small-diameter fibrils with a collagen composition and structure identical to that seen in the corneal stroma in the absence of proteoglycans typical of the secondary corneal stroma imply that although proteoglycan-collagen interactions may function in the establishment of interfibrillar spacing and lamellar organization, collagen-collagen interactions are the major parameter in the regulation of fibril diameter.