Mediate Cell Attachment Research Articles

Hyaluronan coating improves liver engraftment of transplanted human biliary tree stem/progenitor cells

BackgroundCell therapy of liver diseases with human biliary tree stem cells (hBTSCs) is biased by low engraftment efficiency. Coating the hBTSCs with hyaluronans (HAs), the primary constituents of all stem cell niches, could facilitate cell survival, proliferation, and, specifically, liver engraftment given that HAs are cleared selectively by the liver.MethodsWe developed a fast and easy method to coat hBTSCs with HA and assessed the effects of HA-coating on cell properties in vitro and in vivo.ResultsThe HA coating markedly improved the viability, colony formation, and population doubling of hBTSCs in primary cultures, and resulted in a higher expression of integrins that mediate cell attachment to matrix components. When HA-coated hBTSCs were transplanted via the spleen into the liver of immunocompromised mice, the engraftment efficiency increased to 11% with respect to 3% of uncoated cells. Notably, HA-coated hBTSC transplantation in mice resulted in a 10-fold increase of human albumin gene expression in the liver and in a 2-fold increase of human albumin serum levels with respect to uncoated cells. Studies in distant organs showed minimal ectopic cell distribution without differences between HA-coated and uncoated hBTSCs and, specifically, cell seeding in the kidney was excluded.ConclusionsA ready and economical procedure of HA cell coating greatly enhanced the liver engraftment of transplanted hBTSCs and improved their differentiation toward mature hepatocytes. HA coating could improve outcomes of stem cell therapies of liver diseases and could be immediately translated into the clinic given that GMP-grade HAs are already available for clinical use.

KSHV gB associated RGD interactions promote attachment of cells by inhibiting the potential migratory signals induced by the disintegrin-like domain.

BackgroundKaposi’s sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is not only expressed on the envelope of mature virions but also on the surfaces of cells undergoing lytic replication. Among herpesviruses, KSHV gB is the only glycoprotein known to possess the RGD (Arg-Gly-Asp) binding integrin domain critical to mediating cell attachment. Recent studies described gB to also possess a disintegrin-like domain (DLD) said to interact with non-RGD binding integrins. We wanted to decipher the roles of two individually distinct integrin binding domains (RGD versus DLD) within KSHV gB in regulating attachment of cells over cell migration.MethodsWe established HeLa cells expressing recombinant full length gB, gB lacking a functional RGD (gBΔR), and gB lacking a functionally intact DLD (gBΔD) on their cell surfaces. These cells were tested in wound healing assay, Transwell migration assay, and adhesion assay to monitor the ability of the RGD and DLD integrin recognition motifs in gB to mediate migration and attachment of cells. We also used soluble forms of the respective gB recombinant proteins to analyze and confirm their effect on migration and attachment of cells. The results from the above studies were authenticated by the use of imaging, and standard biochemical approaches as Western blotting and RNA silencing using small interfering RNA.ResultsThe present report provides the following novel findings: (i) gB does not induce cell migration; (ii) RGD domain in KSHV gB is the switch that inhibits the ability of DLD to induce cellular migration thus promoting attachment of cells.ConclusionsIndependently, RGD interactions mediate attachment of cells while DLD interactions regulate migration of cells. However, when both RGD and DLD are functionally present in the same protein, gB, the RGD interaction-induced attachment of cells overshadows the ability of DLD mediated signaling to induce migration of cells. Furthering our understanding of the molecular mechanism of integrin engagement with RGD and DLD motifs within gB could identify promising new therapeutic avenues and research areas to explore.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2173-9) contains supplementary material, which is available to authorized users.

Molecular Tension Sensors for Probing the Mechanical and Chemical Roles of Distinct Integrin Classes

Integrins are heterodimeric transmembrane proteins that mediate cell attachment to the extracellular matrix (ECM). Mammalian cells express numerous α/β-integrin heterodimers, each with a unique combination of affinities for individual ECM components and distinct roles in intracellular signal transduction. Notably, αvβ3, αvβ5, and α5β1 integrins recognize structurally similar RGD-based binding motifs, but play distinct, and central, roles in cancer metastasis (αvβ3 integrin), septic shock (αvβ5 integrin), and angiogenic sprouting (α5β1 integrin). In this study, we used Forster resonance energy transfer (FRET)-based molecular tension sensors (MTSs) that report on the mechanical forces exerted by individual integrin heterodimers to probe the roles of αvβ3, αvβ5, and α5β1 integrins in cell adhesion and traction force generation. We developed MTS variants that incorporated a minimal RGD sequence derived from fibronectin (MTSRGD), an RGD sequence derived from vitronectin (MTSVN), or the complete ninth and tenth type-III domains from fibronectin (MTSFN). Using primary dermal fibroblasts, we found that both MTSRGD and MTSVN recruited αvβ3 and αvβ5 integrins to large, force-generating adhesions at the cell periphery. In contrast, MTSFN supported the recruitment of α5β1 integrin to force-producing adhesions. However, measurements at the single-molecule level revealed that the forces experienced by individual MTSRGD molecules were on average larger than those experienced by MTSFN. These and other data suggest that force production at the level of individual integrins is regulated by the interaction of the integrin with its extracellular ligand. More broadly, our results imply that αv-containing and α5β1 integrins play distinct roles within adhesions, and that ligand affinity and traction force generation are not necessarily coupled at the level of individual integrins.

Design and Purification of Subunit Vaccines for Prevention of Clostridium difficile Infection.

Clostridium difficile is a gram-positive bacterium responsible for a large proportion of nosocomial infections in the developed world. C. difficile secretes toxins A and B (TcdA and TcdB) and both toxins act synergistically to induce a spectrum of pathological responses in infected individuals ranging from pseudomembranous colitis to C. difficile-associated diarrhea. Toxins A and B have been actively investigated as components of prophylactic vaccine as well as targets for therapeutic intervention with antibodies. Expression of such toxins by recombinant technology is often difficult and may require special handling and adherence to strict safety regulations during the manufacturing process due to the inherent toxicity of the proteins. Both toxins are large proteins (308 kDa and 270 kDa, respectively) and contain distinct domains mediating cell attachment, cellular translocation, and enzymatic (glucosidase) activity. Here we describe methods to produce fragments of Toxin B for their subsequent evaluation as components of experimental C. difficile vaccines. Methods presented include selection of fragments encompassing distinct functional regions of Toxin B, purification methods to yield high quality proteins, and analytical evaluation techniques. The approach presented focuses on Toxin B but could be applied to the other component, Toxin A, and/or to any difficult to express or toxic protein.

Follicle-Stimulating Hormone Increases Gap Junctional Communication Between Somatic and Germ-Line Follicular Compartments During Murine Oogenesis.

Germ cells develop in intimate contact and communication with somatic cells of the gonad. In female mammals, oocyte development depends crucially on gap junctions that couple it to the surrounding somatic granulosa cells of the follicle, yet the mechanisms that regulate this essential intercellular communication remain incompletely understood. Follicle-stimulating hormone (FSH) drives the terminal stage of follicular development. We found that FSH increases the steady-state levels of mRNAs encoding the principal connexins that constitute gap junctions and cadherins that mediate cell attachment. This increase occurs both in granulosa cells, which express the FSH-receptor, and in oocytes, which do not. FSH also increased the number of transzonal projections that provide the sites of granulosa cell-oocyte contact. Consistent with increased connexin expression, FSH increased gap junctional communication between granulosa cells and between the oocyte and granulosa cells, and it accelerated oocyte development. These results demonstrate that FSH regulates communication between the female germ cell and its somatic microenvironment. We propose that FSH-regulated gap junctional communication ensures that differentiation processes occurring in distinct cellular compartments within the follicle are precisely coordinated to ensure production of a fertilizable egg.

Interrogating protonated/deuterated fibronectin fragment layers adsorbed to titania by neutron reflectivity and their concomitant control over cell adhesion

The fibronectin fragment, 9th-10th-type III domains (FIII9-10), mediates cell attachment and spreading and is commonly investigated as a bioadhesive interface for implant materials such as titania (TiO2). How the extent of the cell attachment-spreading response is related to the nature of the adsorbed protein layer is largely unknown. Here, the layer thickness and surface fraction of two FIII9-10 mutants (both protonated and deuterated) adsorbed to TiO2 were determined over concentrations used in cell adhesion assays. Unexpectedly, the isotopic forms had different adsorption behaviours. At solution concentrations of 10 mg l(-1), the surface fraction of the less conformationally stable mutant (FIII9'10) was 42% for the deuterated form and 19% for the protonated form (fitted to the same monolayer thickness). Similarly, the surface fraction of the more stable mutant (FIII9'10-H2P) was 34% and 18% for the deuterated and protonated forms, respectively. All proteins showed a transition from monolayer to bilayer between 30 and 100 mg l(-1), with the protein longitudinal orientation moving away from the plane of the TiO2 surface at high concentrations. Baby hamster kidney cells adherent to TiO2 surfaces coated with the proteins (100 mg l(-1)) showed a strong spreading response, irrespective of protein conformational stability. After surface washing, FIII9'10 and FIII9'10-H2P bilayer surface fractions were 30/25% and 42/39% for the lower/upper layers, respectively, implying that the cell spreading response requires only a partial protein surface fraction. Thus, we can use neutron reflectivity to inform the coating process for generating bioadhesive TiO2 surfaces.

Nutrition and immunity in newborns

Through fetal life, infancy and childhood, the immune system undergoes a process of functional maturation. The adequacy of this process is dependent on environmental factors, and there is strong evidence of the impact of preand post-natal nutrition in this regard [1]. The early postnatal period is a critical window for immune maturation of newborns. Exclusive human milk feeding for the first 6 months of life is recognized as the normative standard for infant feeding [2,3]. Human milk contains many hundreds to thousands of distinct bioactive molecules that protect against infection and inflammation as cytokines, nucleotides, hormones, and growth factors [2,3]. These specific protective components are so numerous, that science is just beginning to understand their functions. One of these is osteopontin. Human colostrum is very rich in osteopontin and mature human milk contains8 fold more osteopontin than cow’s milk. Recent studies suggest that human milk osteopontin might regulate inducible nitric oxide synthase and synthesis of nitric oxide, improve barrier function and reduce inflammation. Thereby mediating cell attachment, migration, chemotaxis and intracellular signaling, osteopontinhas important barrier functions, protecting the intestinalmucosa against drug-induced colitis in murine models, increasing the levels ofTGFbeta1 and decreasing pro-inflammatory cytokines [4]. Nutrition is a key component also for the composition of intestinal microbiota [5]. Several studies have provided conclusive evidence of the critical role of the intestinal microbiota in regulating both mucosal and systemic immunity [5]. Immediately after delivery, the human infant acquires a much complex microbiota. It is known that intestinal microbiome composition and diversity are affected by several factors as maternal clinical conditions, route of delivery, antibiotic administration and infant diet (breastversus formula-fed infants). In breast-fed babies, Bifidobacteria and Lactobacilli quickly become dominant, whereas in formula-fed babies, Bacteroides species are prevalent, alongside other bacteria known to be enteric pathogens. Furthermore, breast milkcontains high concentrations of oligosaccharides, which ferment in the bowel and promote the growth of Bifidobacterium gut commensals. Dysbiosis in early life has been associated with immune-mediated childhood disorders and obesity [5]. In the long-term, nutritional strategies might improve the development of the microbiome and intestine with the final aim to enhance the clinical care of high-risk infants as low birth weight and premature infants. However, nutritional research to date suggests that there will not be a simple or single nutritional intervention but multiple actions are needed to reverse the association between malnutrition and infection.

Western blot analysis of adhesive interactions under fluid shear conditions: the blot rolling assay.

Western blotting has proven to be an important technique in the analysis of receptor-ligand interactions (i.e., by ligand blotting) and for identifying molecules mediating cell attachment (i.e., by cell blotting). Conventional ligand blotting and cell blotting methods employ non-dynamic (static) incubation conditions, whereby molecules or cells of interest are placed in suspension and overlaid on membranes. However, many cell-cell and cell-matrix adhesive interactions occur under fluid shear conditions, and shear stress itself mediates and/or facilitates the engagement of these physiologically appropriate receptors and ligands. Notably, shear forces critically influence the adhesion of circulating cells and platelets to vessel walls in physiologic cell migration and hemostasis, as well as in inflammatory and thrombotic disorders, cancer metastasis, and atherosclerosis. Use of non-dynamic blotting conditions to analyze such interactions can introduce bias, overtly missing relevant effectors and/or exaggerating the relative role(s) of non-physiologic adhesion molecules. To address this shortfall, we have developed a new technique for identifying binding interactions under fluid shear conditions, the "blot rolling assay." Using this method, molecules in a complex mixture are resolved by gel electrophoresis, transferred to a membrane that is rendered semitransparent, and the membrane is then incorporated into a parallel-plate flow chamber apparatus. Under controlled flow conditions, cells or particles bearing adhesion proteins of interest are then introduced into the chamber and interactions with individual immobilized molecules (bands) can be visualized in real time. The substrate molecule(s) supporting adhesion under fluid shear can then be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. This method thus allows for the identification, within a complex mixture and without prior isolation or purification, of both known and previously uncharacterized adhesion molecules operational under dynamic conditions.

The interaction between uPAR and vitronectin triggers ligand-independent adhesion signalling by integrins.

The urokinase-type plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure-function analyses of uPAR, VN and integrins, we document that uPAR-mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin-matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non-canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non-integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch.

Poly(NaSS) functionalization modulates the conformation of fibronectin and collagen type I to enhance osteoblastic cell attachment onto Ti6Al4V.

Functionalization of surfaces with poly(sodium styrenesulfonate) (poly(NaSS)) has recently been found to enhance osteointegration of implantable materials. Radical polymerization of poly(NaSS) on titanium (Ti)-based substrates has been used to improve their long-term performance by preventing fibrosis and consequently implant loosening. However, the influence of the sulfonate groups on the early cell behavior and the associated molecular phenomena remains to be understood. In this work, we used quartz crystal microbalance with dissipation (QCM-D) to elucidate the role of poly(NaSS) in enhancing osteoblastic cell attachment. This was measured by following the cell attachment using the MC3T3-E1 cell line, on fetal bovine serum (FBS) preadsorbed surfaces and on substrates adsorbed with a series of relevant proteins, bovine serum albumin (BSA), fibronectin (Fn), and collagen type I (Col I). Comparison of the performance of poly(NaSS) with other clinically important substrates such as Ti alloy Ti6Al4V, gold, and poly(desamino-tyrosyl-tyrosine ethyl ester carbonate) (poly(DTEc)) indicates poly(NaSS) to be a superior substrate for MC3T3-E1 cells attachment. This attachment was found to be integrin mediated in the presence of Fn and Col I. Antibodies specific to the RGD peptide and the N- and C-terminal HB-binding domains reacted more intensively with Fn adsorbed on poly(NaSS). Fn adapts a conformation favorable to RGD mediated cell attachment when adsorbed onto poly(NaSS).

CAS proteins in health and disease: an update.

The CAS family of scaffolding proteins has increasingly attracted scrutiny as important for regulation of cancer-associated signaling. BCAR1 (also known as p130Cas), NEDD9 (HEF1, Cas-L), EFS (Sin), and CASS4 (HEPL) are regulated by and mediate cell attachment, growth factor, and chemokine signaling. Altered expression and activity of CAS proteins are now known to promote metastasis and drug resistance in cancer, influence normal development, and contribute to the pathogenesis of heart and pulmonary disease. In this article, we provide an update on recently published studies describing signals regulating and regulated by CAS proteins, and evidence for biological activity of CAS proteins in normal development, cancer, and other pathological conditions.

Single-Molecule Analysis of Pseudomonas fluorescens Footprints

Understanding the molecular mechanisms of bacterial adhesion and biofilm formation is an important topic in current microbiology and a key in nanomedicine for developing new antibacterial strategies. There is growing evidence that the production of extracellular polymeric substances at the cell-substrate interface plays a key role in strengthening bacterial adhesion. Yet, because these adhesive polymers are available in small amounts and are localized at interfaces, they are difficult to study using traditional techniques. Here, we use single-molecule atomic force microscopy (AFM) to functionally analyze the biophysical properties (distribution, adhesion, and extension) of bacterial footprints, that is, adhesive macromolecules left on substrate surfaces after removal of the attached cells. We focus on the large adhesin protein LapA from Pseudomonas fluorescens, which mediates cell attachment to a wide diversity of surfaces. Using AFM tips functionalized with specific antibodies, we demonstrate that adhesion of bacteria to hydrophobic substrates leads to the active accumulation of the LapA protein at the cell-substrate interface. We show that single LapA proteins left on the substrate after cell detachment localize into microscale domains corresponding to the bacterial size and exhibit multiple adhesion peaks reflecting the adhesion and extension of adsorbed LapA proteins. The mechanical behavior of LapA-based footprints makes them ideally suited to function as multipurpose bridging polymers, enabling P. fluorescens to attach to various surfaces. Our experiments show that single-molecule AFM offers promising prospects for characterizing the biophysics and dynamics of the cell-substrate interface in the context of bacterial adhesion, on a scale that was not accessible before.

Matrix gla protein binds to fibronectin and enhances cell attachment and spreading on fibronectin.

Background. Matrix Gla protein (MGP) is a vitamin K-dependent, extracellular matrix protein. MGP is a calcification inhibitor of arteries and cartilage. However MGP is synthesized in many tissues and is especially enriched in embryonic tissues and in cancer cells. The presence of MGP in those instances does not correlate well with the calcification inhibitory role. This study explores a potential mechanism for MGP to bind to matrix proteins and alter cell matrix interactions. Methods. To determine whether MGP influences cell behavior through interaction with fibronectin, we studied MGP binding to fibronectin, the effect of MGP on fibronectin mediated cell attachment and spreading and immunolocalized MGP and fibronectin. Results. First, MGP binds to fibronectin. The binding site for MGP is in a specific fibronectin fragment, called III1-C or anastellin. The binding site for fibronectin is in a MGP C-terminal peptide comprising amino acids 61–77. Second, MGP enhances cell attachment and cell spreading on fibronectin. MGP alone does not promote cell adhesion. Third, MGP is present in fibronectin-rich regions of tissue sections. Conclusions. MGP binds to fibronectin. The presence of MGP increased cell-fibronectin interactions.

Tuning of Cell–Biomaterial Anchorage for Tissue Regeneration

Which mechanisms mediate cell attachment to biomaterials? What role does the surface charge or wettability play on cell-material anchorage? What are the currently investigated strategies to modify cell-matrix adherence spatiotemporally? Considering the development of scaffolds made of biocompatible materials to temporarily replace the structure and/or function of the extracellular matrix, focus is given to the analysis of the specific (i.e., cell adhesive peptide sequences) and unspecific (i.e., surface charge, wettability) mechanisms mediating cell-matrix interactions. Furthermore, because natural tissue regeneration is characterized by the dynamic attachment/detachment of different cell populations, the design of advanced scaffolds for tissue engineering, based in the spatiotemporal tuning of cell-matrix anchorage is discussed.

Syndecan-1 Serves as the Major Receptor for Attachment of Hepatitis C Virus to the Surfaces of Hepatocytes

Our recent studies demonstrated that apolipoprotein E mediates cell attachment of hepatitis C virus (HCV) through interactions with the cell surface heparan sulfate (HS). HS is known to covalently attach to core proteins to form heparan sulfate proteoglycans (HSPGs) on the cell surface. The HSPG core proteins include the membrane-spanning syndecans (SDCs), the lycosylphosphatidylinositol-linked glypicans (GPCs), the basement membrane proteoglycan perlecan (HSPG2), and agrin. In the present study, we have profiled each of the HSPG core proteins in HCV attachment. Substantial evidence derived from our studies demonstrates that SDC1 is the major receptor protein for HCV attachment. The knockdown of SDC1 expression by small interfering RNA (siRNA)-induced gene silence resulted in a significant reduction of HCV attachment to Huh-7.5 cells and stem cell-differentiated human hepatocytes. The silence of SDC2 expression also caused a modest decrease of HCV attachment. In contrast, the siRNA-mediated knockdown of other SDCs, GPCs, HSPG2, and agrin had no effect on HCV attachment. More importantly, ectopic expression of SDC1 was able to completely restore HCV attachment to Huh-7.5 cells in which the endogenous SDC1 expression was silenced by specific siRNAs. Interestingly, mouse SDC1 is also fully functional in mediating HCV attachment when expressed in the SDC1-deficient cells, consistent with recent reports that mouse hepatocytes are also susceptible to HCV infection when expressing other key HCV receptors. Collectively, our findings demonstrate that SDC1 serves as the major receptor protein for HCV attachment to cells, providing another potential target for discovery and development of antiviral drugs against HCV.

Integrin β1 Is Required for Dermal Homeostasis

Integrins are heterodimers that mediate cell attachment to the extracellular matrix. Previously, we used fibroblast-specific knockout mice to show that the integrin β1 subunit was required in vivo for dermal fibrogenesis and cutaneous tissue repair. Here, we show that integrin β1 expression by fibroblasts is required for dermal homeostasis; at 8 weeks after deletion of integrin β1, the dermis is significantly thinner, expressing less collagen and displaying reduced generation of reactive oxygen species (ROS). Cultured integrin β1-deficient fibroblasts show reduced rac1 activation and ROS generation. Overexpressing rac1 in integrin β1-deficient fibroblasts restored ROS generation; adding hydrogen peroxide to integrin β1-deficient fibroblasts restored Col1a2 (collagen, type I, α2) and α-smooth muscle actin (α-SMA) mRNA expression and α-SMA protein expression and stress fiber formation. Thus integrin β1 expression by fibroblasts is required for the maintenance of dermal connective tissue via a rac/ROS pathway.

Invadopodia Detection and Gelatin Degradation Assay.

This protocol is designed to quantify invadopodia formation and activity. Invadopodia are protrusive structures elaborated by cancer cells that mediate cell attachment and remodeling of the extracellular matrix. These structures contribute to the ability of cancer cells to invade and metastasize. In this protocol, both the presence of invadopodia and their activity is simultaneously assessed and quantified by a fluorescent microscopy-based assay.

Discoidin Domain Receptors Promote α1β1- and α2β1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx’GEx”. The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state.

β1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival

Extracellular matrix (ECM) integrity in the central nervous system (CNS) is essential for neuronal homeostasis. Signals from the ECM are transmitted to neurons through integrins, a family of cell surface receptors that mediate cell attachment to ECM. We have previously established a causal link between the activation of the matrix metalloproteinase-9 (MMP-9), degradation of laminin in the ECM of retinal ganglion cells (RGCs), and RGC death in a mouse model of retinal ischemia-reperfusion injury (RIRI). Here we investigated the role of laminin-integrin signaling in RGC survival in vitro, and after ischemia in vivo. In purified primary rat RGCs, stimulation of the β1 integrin receptor with laminin, or agonist antibodies enhanced RGC survival in correlation with activation of β1 integrin’s major downstream regulator, focal adhesion kinase (FAK). Furthermore, β1 integrin binding and FAK activation were required for RGCs’ survival response to laminin. Finally, in vivo after RIRI, we observed an up-regulation of MMP-9, proteolytic degradation of laminin, decreased RGC expression of β1 integrin, FAK and Akt dephosphorylation, and reduced expression of the pro-survival molecule bcl-xL in the period preceding RGC apoptosis. RGC death was prevented, in the context of laminin degradation, by maintaining β1 integrin activation with agonist antibodies. Thus, disruption of homeostatic RGC-laminin interaction and signaling leads to cell death after retinal ischemia, and maintaining integrin activation may be a therapeutic approach to neuroprotection.

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.