Soluble Integrin Ligands and Growth Factors Independently Rescue Neuroblastoma Cells from Apoptosis under Nonadherent Conditions

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (cromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 ± 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 ± 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca²⁺levels nor onde novoprotein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since ([1]) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to β₁and α₁subunits and ([2]) the antibody-induced clustering of α₁, α₃, α_v, β₁, and β₃integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 μg/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. Thede novoprotein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-β₁antibody was associated with the increased expression ofbcl-2.In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.     

Integrin α1β1 Mediates a Unique Collagen-dependent Proliferation Pathway In Vivo

Activation of integrins upon binding to extracellular matrix proteins is believed to be a crucial step for the regulation of cell survival and proliferation. We have used integrin α1-null mice to investigate the role of this collagen receptor in the regulation of cell growth and survival in vivo. α1-deficient animals, which are viable and fertile, have a hypocellular dermis and a deficiency in dermal fibroblast proliferation as embryos. In vitro analysis of α1-null embryonic fibroblasts has revealed that their proliferation rate is markedly reduced when plated on collagenous substrata, despite normal attachment and spreading. Moreover, on the same collagenous matrices, α1-null fibroblasts fail to recruit and activate the adaptor protein Shc. The failure to activate Shc is accompanied by a downstream deficiency in recruitment of Grb2 and subsequent mitogen-activated protein kinase activation. Taken together with the growth deficiency observed on collagens, this finding indicates that the α1β1 is the sole collagen receptor which can activate the Shc mediated growth pathway. Thus, integrin α1 has a unique role among the collagen receptors in regulating both in vivo and in vitro cell proliferation in collagenous matrices.     

The mesenchymal α11β1 integrin attenuates PDGF-BB-stimulated chemotaxis of embryonic fibroblasts on collagens

α11β1 constitutes the most recent addition to the integrin family and has been shown to display a binding preference for interstitial collagens found in mesenchymal tissues. We have previously observed that when α11β1 integrin is expressed in cells lacking endogenous collagen receptors, it can mediate PDGF-BB-dependent chemotaxis on collagen I in vitro. To determine in which cells PDGF and α11β1 might cooperate in regulating cell migration in vivo, we studied in detail the expression and distribution of α11 integrin chain in mouse embryos and tested the ability of PDGF isoforms to stimulate the α11β1-mediated cell migration of embryonic fibroblasts.Full-length mouse α11 cDNA was sequenced and antibodies were raised to deduced α11 integrin amino acid sequence. In the embryonic mouse head, α11 protein and RNA were localized to ectomesenchymally derived cells. In the periodontal ligament, α11β1 was expressed as the only detectable collagen-binding integrin, and α11β1 is thus a major receptor for cell migration and matrix organization in this cell population. In the remainder of the embryo, the α11 chain was expressed in a subset of mesenchymal cells including tendon/ligament fibroblasts, perichondrial cells, and intestinal villi fibroblasts. Most of the α11-expressing cells also expressed the α2 integrin chain, but no detectable overlap was found with the α1 integrin chain. In cells expressing multiple collagen receptors, these might function to promote a more stable cell adhesion and render the cells more resistant to chemotactic stimuli.Wild-type embryonic fibroblasts activated mainly the PDGF β receptor in response to PDGF-BB and migrated on collagens I, II, III, IV, V, and XI in response to PDGF-BB in vitro, whereas mutant fibroblasts that lacked α11β1 in their collagen receptor repertoire showed a stronger chemotactic response on collagens when stimulated with PDGF-BB. In the cellular context of embryonic fibroblasts, α11β1 is thus anti-migratory.We speculate that the PDGF BB-dependent cell migration of mesenchymal cells is tightly regulated by the collagen receptor repertoire, and disturbances of this repertoire might lead to unregulated cell migration that could affect normal embryonic development and tissue structure.     

Fibroblast α11β1 Integrin Regulates Tensional Homeostasis in Fibroblast/A549 Carcinoma Heterospheroids

We have previously shown that fibroblast expression of α11β1 integrin stimulates A549 carcinoma cell growth in a xenograft tumor model. To understand the molecular mechanisms whereby a collagen receptor on fibroblast can regulate tumor growth we have used a 3D heterospheroid system composed of A549 tumor cells and fibroblasts without (α11^+/+) or with a deletion (α11^-/-) in integrin α11 gene. Our data show that α11^-/-/A549 spheroids are larger than α11^+/+/A549 spheroids, and that A549 cell number, cell migration and cell invasion in a collagen I gel are decreased in α11^-/-/A549 spheroids. Gene expression profiling of differentially expressed genes in fibroblast/A549 spheroids identified CXCL5 as one molecule down-regulated in A549 cells in the absence of α11 on the fibroblasts. Blocking CXCL5 function with the CXCR2 inhibitor SB225002 reduced cell proliferation and cell migration of A549 cells within spheroids, demonstrating that the fibroblast integrin α11β1 in a 3D heterospheroid context affects carcinoma cell growth and invasion by stimulating autocrine secretion of CXCL5. We furthermore suggest that fibroblast α11β1 in fibroblast/A549 spheroids regulates interstitial fluid pressure by compacting the collagen matrix, in turn implying a role for stromal collagen receptors in regulating tensional hemostasis in tumors. In summary, blocking stromal α11β1 integrin function might thus be a stroma-targeted therapeutic strategy to increase the efficacy of chemotherapy.     

Soluble Collagen VI Induces Tyrosine Phosphorylation of Paxillin and Focal Adhesion Kinase and Activates the MAP Kinase Erk2 in Fibroblasts

Signals from the extracellular matrix can modulate cellular differentiation and gene expression. We have shown previously that in contrast to other extracellular matrix molecules pepsin-solubilized collagen VI (CVI) can stimulate DNA synthesis of various mesenchymal cell types, apparently independent of integrin-mediated signal transduction. In order to further elucidate collagen VI-induced signaling events, we exposed mouse 3T3 fibroblasts and human HT1080 fibrosarcoma cells to soluble CVI. CVI induced tyrosine phosphorylation of proteins that associate with focal adhesions, such as paxillin, focal adhesion kinase (FAK), and p130CAS. Furthermore, it activated the mitogen-activated protein kinase, erk2. Kinetic analysis showed that these phosphorylations were transient, reaching a maximum after 5 min for transformed HT1080 cells and 30 min for 3T3 fibroblasts. These effects were partly inhibited by a beta1-integrin function blocking antibody and by single chains of CVI. Our results indicate that soluble fragments of native collagen VI, a ubiquitous component of the interstitial extracellular matrix, can mediate stimulation of DNA synthesis via tyrosine phosphorylation of paxillin, FAK, p130CAS, and erk2 in the absence of classical growth factors. Thus, CVI may serve as a matrix-derived sensor that allows for rapid reconstitution of a tissue defect by activating nearby mesenchymal cells.     

��3 Integrin in Cardiac Fibroblast Is Critical for Extracellular Matrix Accumulation during Pressure Overload Hypertrophy in Mouse

The adhesion receptor β3 integrin regulates diverse cellular functions in various tissues. As β3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of β3 integrin in cardiac fibrosis by using wild type (WT) and β3 integrin null (β3−/−) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, β3−/− mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from β3−/− mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in β3−/− mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and β3−/− mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that β3−/− cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in β3−/− cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that β3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.     

Involvement of ligand occupancy in Insulin-like growth factor-I (IGF-I) induced cell growth in osteoblast like MC3T3-E1 cells

Growth factors and matrix proteins regulate the proliferation and differentiation of osteoblasts. The insulin-like growth factor (IGF) system comprises IGF-I, IGF-II, and six high-affinity IGF-binding proteins (IGFBPs). IGFs stimulate cell growth in many types of tissue; IGF-binding proteins regulate cellular actions and can affect cell growth. IGF-I is involved in differentiation, proliferation, and matrix formation in osteoblasts; IGFBP-5 is associated with the extracellular matrix (ECM) and can potentiate the actions of IGF-I. We investigated the effect of ECM proteins on the responses of MC3T3-E1 osteoblast cells to IGF-I and IGFBP-5. In addition, because extracellular signal-regulated kinases 1 and 2 (Erk 1/2) affect cell growth, we evaluated the effects of IGFBP-5 on Erk 1/2 phosphorylation in MC3T3-E1 cells. IGF-I caused an increase in IGFBP-5 expression in cultured MC3T3-E1 cells, and IGF-I plus IGFBP-5 significantly increased cell growth. Likewise, the addition of IGF-I and IGFBP-5 to cultured MC3T3-E1 cells increased the synthesis of the ECM proteins osteopontin (OPN) and thrombospondin-1 (TSP-1), which can bind to alphaVbeta3 integrin receptors on the cell surface. By contrast, the addition of an antibody against ECM proteins inhibited the effects of OPN and TSP-1 on IGFBP-5 expression. The stimulatory effect of IGFBP-5 was mediated via Erk 1/2 activation. These data suggest that IGFBP-5 regulates Erk 1/2 phosphorylation in cultured MC3T3-E1 cells via ECM proteins that may ultimately stimulate the growth of osteoblasts. We determined whether occupation of the alphaVbeta3 integrin receptor affects IGF-I receptor (IGF-IR)-mediated signaling and function in MC3T3-E1 osteoblast cells. Occupation of the alphaVbeta3 integrin receptor with ECM proteins induced IGF-I-stimulated IGF-IR phosphorylation. Conversely, in the presence of the alphaVbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-IR activation was inhibited. IGF-I-stimulated IGF-IR phosphorylation was accompanied by IRS-1 phosphorylation and MAPK activation. However, these effects were attenuated by echistatin. Thus, occupancy of the alphaVbeta3 disintegrin receptor modulates IGF-I-induced IGF-IR activation and IGF-IR-mediated function in MC 3T3-E1 osteoblasts.     

Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice

The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.     

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

β1 Integrin-mediated collagen gel contraction is stimulated by PDGF

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent β₁ integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-β1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin β₁-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-β₁ integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the β₁ integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.     

Identification of an Endogenously Generated Cryptic Collagen Epitope (XL313) That May Selectively Regulate Angiogenesis by an Integrin Yes-associated Protein (YAP) Mechano-transduction Pathway

Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin αvβ3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by αvβ3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting αvβ3 signaling, this collagen epitope promoted αvβ3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates αvβ3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control αvβ3 signaling by targeting a pro-angiogenic and inflammatory ligand of αvβ3 rather than the receptor itself.     

Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg²⁺ or Mn²⁺ but not Ca²⁺. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ~110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.     

A Cell Signal Pathway Involving Laminin-5, α3β1 Integrin, and Mitogen-activated Protein Kinase Can Regulate Epithelial Cell Proliferation

Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the α3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogen-activated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor α3β1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin α3β1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing α3 integrin antibody. In addition, antibody activation of β1 integrin restores the proliferation of epithelial cells treated with LN5 function-perturbing antibodies. These data indicate that a complex comprising LN5 and α3β1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation.     

Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells.     

Stromal Integrin α11β1 Affects RM11 Prostate and 4T1 Breast Xenograft Tumors Differently

Purpose

It has been implied that the collagen binding integrin α11β1 plays a role in carcinogenesis. As still relatively little is known about how the stromal integrin α11β1 affects different aspects of tumor development, we wanted to examine the direct effects on primary tumor growth, fibrosis, tumor interstitial fluid pressure (PIF) and metastasis in murine 4T1 mammary and RM11 prostate tumors, using an in vivo SCID integrin α11-deficient mouse model.

Methods

Tumor growth was measured using a caliper, PIF by the wick-in-needle technique, activated fibroblasts by α-SMA immunofluorescence staining and fibrosis by transmission electron microscopy and picrosirius-red staining. Metastases were evaluated using hematoxylin and eosin stained sections.

Results

RM11 tumor growth was significantly reduced in the SCID integrin α11-deficient (α11-KO) compared to in SCID integrin α11 wild type (WT) mice, whereas there was no similar effect in the 4T1 tumor model. The 4T1 model demonstrated an alteration in collagen fibril diameter in the integrin α11-KO mice compared to WT, which was not found in the RM11 model. There were no significant differences in the amount of activated fibroblasts, total collagen content, collagen organization or PIF in the tumors in integrin α11-deficient mice compared to WT mice. There was also no difference in lung metastases between the two groups.

Conclusion

Deficiency of stromal integrin α11β1 showed different effects on tumor growth and collagen fibril diameter depending on tumor type, but no effect on tumor PIF or development of lung metastasis.     

α2β1 Integrin Mediates Dermal Fibroblast Attachment to Type VII Collagen via a 158-Amino-Acid Segment of the NC1 Domain

Dermal fibroblasts are in apposition to type VII (anchoring fibril) collagen in both unwounded and wounded skin. The NC1 domain of type VII collagen contains multiple submodules with homology to known adhesive molecules, including fibronectin type III-like repeats and a potential RGD cell attachment site. We previously reported the structure and matrix binding properties of authentic and recombinant NC1. In this study, we examined the interaction between dermal fibroblasts and the NC1 domain of type VII collagen. We found that both recombinant and authentic NC1 vigorously promoted human fibroblast attachment. Adhesion of fibroblasts to NC1 was dose dependent, saturable, and abolished by both polyclonal and monoclonal antibodies to NC1. Cell adhesion to NC1 was divalent cation dependent and specifically inhibited by a monoclonal antibody directed against the α2 or β1 integrin subunits, but not by the presence of RGD peptides. Furthermore, the cell-binding activity of NC1 was not conformation dependent, since heat-denatured NC1 still promoted cell adhesion. Using a series of recombinant NC1 deletion mutant proteins, the cell binding site of NC1 was mapped to a 158-aa (residues 202–360) subdomain. We conclude that human dermal fibroblasts interact with the NC1 domain of type VII collagen and this cell–matrix interaction is mediated by the α2β1 integrin and is RGD independent.     

Syndecan-1-Induced ECM Fiber Alignment Requires Integrin αvβ3 and Syndecan-1 Ectodomain and Heparan Sulfate Chains

Expression of the cell surface proteoglycan syndecan-1 (Sdc1) is frequently induced in stromal fibroblasts of invasive breast carcinomas. We have recently identified a correlation between stromal Sdc1 expression and extracellular matrix (ECM) fiber alignment, both in vitro and in vivo. ECMs derived from Sdc1-positive human mammary fibroblasts (HMF) showed an aligned fiber architecture, which contrasted markedly with the more random fiber arrangement in the ECM produced by Sdc1-negative HMFs. We further demonstrated that aligned fiber architecture promotes the directional migration and invasion of breast carcinoma cells. To decipher the molecular mechanisms governing the formation of an aligned, invasion-permissive ECM, a series of Sdc1 mutants was introduced into HMF. We found that both the ectodomain and heparan sulfate chains of Sdc1 were required for full activity of Sdc1 in regulating ECM alignment, while transmembrane and cytoplasmic domains were dispensable. Sdc1 regulates the activities of several integrins via its ectodomain. Integrins are key players in the assembly of fibronectin-rich ECM. In addition, integrins are capable of regulating cell morphology and cell shape and orientation may affect ECM architecture. Therefore, we investigated the role of integrins in Sdc1-mediated ECM fiber alignment. Sdc1-overexpressing HMF gained an enhanced spindle-shaped morphology when cultured in an overconfluent state under conditions permissive for ECM production, which was partially reversed by siRNA-mediated silencing of β3 integrin expression. Moreover, suppression of αvβ3 integrin activity by a function-blocking antibody or β3 knockdown largely abolished the aligned ECM fiber architecture and consequently the invasion-permissive properties of the ECM induced by Sdc1. The results suggest that Sdc1 may modulate fibronectin fibrillogenesis and/or alter cell morphology during ECM production through αvβ3 integrin, thereby mediating ECM fiber alignment. Understanding the mechanisms governing ECM organization may lead to the development of novel stroma-targeted therapy for breast cancer, aiming at converting an invasion-permissive to an invasion-restrictive microenvironment.     

The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by alphaVbeta3 integrin in human breast cancer cells.

The influence of alphaVbeta3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing beta3 integrin status. Overexpression of beta3 integrin caused increased cell surface expression of alphaV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. beta3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, alphaVbeta3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of beta3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with beta3 integrin expression. Although our studies confirm important biological effects of alphaVbeta3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, beta3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by alphaVbeta3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.     

Clustering of Syndecan-4 and Integrin β1 by Laminin α3 Chain–derived Peptide Promotes Keratinocyte Migration

Syndecans function as receptors for extracellular matrix (ECM) with integrins in cell spreading. However, the molecular mechanism of their specific involvement in cell migration or in wound healing has not been elucidated yet. Here, we report that a synthetic peptide, PEP75, which contains the syndecan-binding sequence of the laminin α3LG4 module, induces keratinocyte migration in in vitro and in vivo. Soluble PEP75 induced the clustering of syndecan-4 and conformation-modified integrin β1 colocalized with syndecan-4 in soluble PEP75-induced clusters. Treatment of cells in solution with PEP75 resulted in the exposure of the P4G11 antibody epitope of integrin β1 in immunostaining as well as in flow cytometry and augmented integrin β1–dependent cell adhesion to ECM. Pulldown assays demonstrated that PEP75 bound to syndecan-4, but not to integrin β1. A siRNA study revealed a role for syndecan-4 in PEP75-induced up-regulation of P4G11 antibody binding and migration of HaCaT cells. We conclude that binding of soluble PEP75 to syndecan-4 induces the coupling of integrin β1, which is associated with integrin β1-conformational changes and activation, and leads to keratinocyte migration. To activate integrin function through syndecans could be a novel therapeutic approach for chronic wound.