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Embigin (gp70), a transmembrane glycoprotein, has been shown to regulate hematopoietic stem cell and progenitor cell niche. Still, little is known about its expression and function in other organ systems during development or adulthood. By combining immunofluorescence, RNA sequencing, and in vivo mouse models, we show that embigin is highly expressed during development and in adult lung, kidney, epididymis, skin, and testis. Adult Emb-/- mice have a normal lifespan and fertility without apparent pathologies. In contrast, the Emb-/- embryos are significantly smaller than their WT littermates. Markedly increased mortality of the Emb-/- embryos is seen especially during the neonatal period. Embigin is present in the placenta, but placental morphology and gene expression patterns stay unaltered. At E17.5, Emb-/- mice show defective morphogenesis of the lung, low alkaline phosphatase activity in amniotic fluid, and remarkable activation of genes involved in cell proliferation in the lungs. Thus, lung underdevelopment explains the high neonatal mortality. Our work demonstrates the crucial role of embigin during development, and it paves the way to further characterization of embigin in specific organ systems in development and homeostasis.Summary statementEmbigin is a basigin-group transmembrane glycoprotein. In vivo mouse model shows that embigin is crucial for embryonic lung development and neonatal survival.

Collagen XIII is a conserved transmembrane collagen mainly expressed in mesenchymal tissues. Previously, we have shown that collagen XIII modulates tissue development and homeostasis. Integrins are a family of receptors that mediate signals from the environment into the cells and vice versa. Integrin α11β1 is a collagen receptor known to recognize the GFOGER (O=hydroxyproline) sequence in collagens. Interestingly, collagen XIII and integrin α11β1 both have a role in the regulation of bone homeostasis. To study whether α11β1 is a receptor for collagen XIII, we utilized C2C12 cells transfected to express α11β1 as their only collagen receptor. The interaction between collagen XIII and integrin α11β1 was also confirmed by surface plasmon resonance and pull-down assays. We discovered that integrin α11β1 mediates cell adhesion to two collagenous motifs, namely GPKGER and GF(S)QGEK, that were shown to act as the recognition sites for the integrin α11-I domain. Furthermore, we studied the in vivo significance of the α11β1-collagen XIII interaction by crossbreeding α11 null mice (Itga11−/−) with mice overexpressing Col13a1 (Col13a1oe). When we evaluated the bone morphology by microcomputed tomography, Col13a1oe mice had a drastic bone overgrowth followed by severe osteoporosis, whereas the double mutant mouse line showed a much milder bone phenotype. To conclude, our data identifies integrin α11β1 as a new collagen XIII receptor and demonstrates that this ligand-receptor pair has a role in the maintenance of bone homeostasis.

Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of β1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1β1-, α2β1- and α10β1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-containing adhesion suprastructures to the non-collagenous fibril periphery but not to the collagenous fibril cores. The biological importance of the well-investigated recognition of collagen molecules by integrins is unknown. Possible scenarios may include fibrillogenesis, fibril degradation and/or phagocytosis, recruitment of cells to remodeling sites, or molecular signaling across cytoplasmic membranes. In these circumstances, collagen molecules may lack a fibrillar organization. However, other processes requiring robust biomechanical functions, such as fibril organization in tissues, cell division, adhesion, or migration, do not involve direct integrin-collagen interactions.

Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-β-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and β-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance., eLife digest Ovarian cancer is one of the deadliest types of cancer in women. There are two main reasons for the aggressiveness of this cancer. First, ovarian cancer cells can spread to other parts of a woman’s body before she has been diagnosed, where the cells grow as tiny clumps or spheres of tumor cells, also called tumorspheres. Second, in the majority of patients, some ovarian cancer cells will develop resistance to the chemotherapy used. It is not clear exactly how these tumor cells become resistant to therapy. One way in which cells could do this is by gaining extra copies of genes that remove toxic substances or repair DNA, which help them withstand the therapy. Here, Osterman, Ozmadenci, Keinschmidt, Taylor, Barrie, Jiang, Bean, Sulzmaier et al. set up a new experimental method to study how some ovarian cancer cells resist chemotherapy. Comparing ovarian cancer cells from mice at early and late stages of the disease showed that the later-stage, more aggressive cells had more genetic changes. One of these changes affected the gene for a protein called FAK, which was found to have more copies than normal. The FAK protein is an enzyme that helps cancer cells move around. In cells from mice with late-stage cancer, FAK was over-active and present at high levels. When these cells grew as tumorspheres, the tumors were more resistant to chemotherapy than their early-stage counterparts. In patients who have received chemotherapy, surviving tumor cells also exhibit high levels of FAK activity. Human ovarian cancer cells that are resistant to chemotherapy can be grown into tumors in mice, where they retain their resistance to chemotherapy. However, if chemotherapy is combined with a drug that targets the FAK enzyme, the tumors shrink. This experiment highlights a possible weak spot of these tumor cells. To understand how FAK makes ovarian cancer cells resistant to chemotherapy, Osterman et al. deleted the gene for FAK from the cells and then looked at how this changed the levels of activation of different genes. They found that, in addition to its effects on cell movement, FAK also activated a group of genes that increase resistance to chemotherapy and repair damaged DNA. This better understanding of how ovarian cancer cells resist chemotherapy could lead to new therapies. In particular, there is now a clinical trial for women with chemo-resistant ovarian cancer in which standard chemotherapy is combined with an inhibitor of the FAK protein.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. The molecular basis of cSCC progression to invasive and metastatic disease is still incompletely understood. Here, we show that fibroblasts and transforming growth factor-β (TGF-β) signaling promote laminin-332 synthesis in cancer cells in an activated H-Ras-dependent manner, which in turn promotes cancer cell invasion. Immunohistochemical analysis of sporadic UV-induced invasive human cSCCs (n = 208) revealed prominent cSCC cell specific immunostaining for laminin-332 γ2 chain, located in the majority of cases (90%, n = 173) in the invasive edge of the tumors. To mimic the progression of cSCC we established 3D spheroid cocultures using primary skin fibroblasts and HaCaT/ras-HaCaT human keratinocytes. Our results indicate that in 3D spheroids, unlike in monolayer cultures, TGF-β upregulates laminin-332 production, but only in cells that harbour oncogenic H-Ras. Accumulation of laminin-332 was prevented by both H-Ras knock down and inhibition of TGF-β signaling by SB431542 or RAdKD-ALK5 kinase-defective adenovirus. Furthermore, fibroblasts accelerated the invasion of ras-HaCaT cells through collagen I gels in a Ras/TGF-β signaling dependent manner. In conclusion, we demonstrate the presence of laminin-332 in the invasive front of cSCC tumors and report a new Ras/TGF-β-dependent mechanism that promotes laminin-332 accumulation and cancer cell invasion.

Long non-coding RNAs (lncRNAs) regulate various cellular processes, and they have emerged as potential biomarkers and therapeutic targets in cancer. We have previously characterized the oncogenic role of lncRNA PICSAR (p38 inhibited cutaneous squamous cell carcinoma associated lincRNA) in cutaneous squamous cell carcinoma (cSCC), the most common metastatic skin cancer. In this study, we show that knockdown of PICSAR in cSCC cells upregulates expression of α2, α5 and β1 integrins, resulting in increased cell adhesion and decreased cell migration on collagen I and fibronectin. In contrast, overexpression of PICSAR in cSCC cells downregulates expression of α2, α5 and β1 integrins on cell surface, resulting in decreased cell adhesion on collagen I and fibronectin and increased cell migration. These results demonstrate a novel mechanism for regulation of the expression of collagen and fibronectin binding integrins by lncRNA PICSAR, leading to altered adhesion and migration of cSCC cells. This article has an associated First Person interview with the first author of the paper., Summary: Long non-coding RNA PICSAR decreases adhesion and promotes migration of cutaneous squamous cell carcinoma cells by regulating the expression of collagen and fibronectin binding α2β1 and α5β1 integrins.

Microvesicles (MVs) are extracellular vesicles secreted by various cell types that are involved in intercellular communication. We hypothesized that in human periodontal disease, the pocket epithelium releases MVs, which then modulate gene expression in the underlying fibroblasts to control periodontal inflammation. MVs were isolated from culture medium of gingival epithelial cells (GECs) treated with oral bacterial biofilm extract or left untreated. Biofilm treatment significantly increased MV release from the GECs. Mass spectrometry of GEC-MVs identified a total of 2,173 proteins, of which about 80% were detected in MVs from both control and biofilm-treated GECs. Among 80 signature genes of human gingival fibroblasts, 20 were significantly regulated ( P < 0.05) by MVs from control and biofilm-treated GECs in a similar manner. Matrix metalloproteinase 1 and 3 and interleukin 6 and 8 showed the strongest regulation at the mRNA and protein levels. Several cellular signaling pathways were activated by GEC-MVs in human gingival fibroblasts, including Smad and mitogen-activated protein kinase–associated pathways ERK1/2, JNK, and p38. However, ERK1/2 signaling dominated in the MV-induced gene expression changes. The results demonstrate that GEC-MVs have a strong regulatory effect on the expression of fibroblast genes associated with inflammation and matrix degradation and that bacterial biofilm stimulates the generation of GEC-MVs. This suggests that bacterial biofilms can contribute to the initiation and progression of periodontal disease by promoting a tissue-destructive phenotype in gingival fibroblasts via the enhanced secretion of epithelial MVs.

In inflammatory arthritis peptidyl arginine deiminase (PAD) enzymes can citrullinate arginine residues in extracellular matrix (ECM) proteins, such as collagens and fibronectin. This may lead to the generation of anti-citrullinated protein antibodies, important diagnostic markers in rheumatoid arthritis. In addition, the citrullination may directly affect protein function. Based on structural analysis, we found that most ECM-associated growth factors (GFs) have arginine residues in their receptor recognition sites. Thus, they are potential functional targets of extracellular citrullination. To examine this further, we focused on the citrullination of transforming growth factor-βs (TGF-β), well-known ECM-associated GFs. PAD-treatment of CHO-LTBP1 cell derived matrix, rich with TGF-β, decreased the level of TGF-β activity as detected by HaCaT and MLEC-PAI-1/Lu reporter cells. Additional experiments indicated that PAD-treatment inhibits the integrin-mediated TGF-β activation since PAD-treatment decreased the binding of integrin αVβ6 ectodomain as well as integrin-mediated spreading of MG-63 and HaCaT cells to β1-latency associated peptide (TGF-β1 LAP). The citrullination of the RGD site, an important integrin recognition motif, was confirmed by mass spectrometry. Furthermore, the citrullination of active TGF-β1 inhibited its binding to recombinant TGF-β receptor II, and prevented its ability to activate TGF-β signaling. Thus, extracellular PAD activity can affect the function of ECM-associated growth factors by different mechanisms. Importantly, the citrullination of both latent and active TGF-β has the potency to regulate the inflammatory process.

Phorbol diester PMA (phorbol 12-myristate 13-acetate) is a well-known promoter of tumor progression. PMA also regulates cell adhesion by several mechanisms including conformational activation of integrins and integrin clustering. Here, PMA was shown to induce lamellipodia formation and reorganization of the adhesion sites as well as actin and vimentin filaments independently of integrin preactivation. To further analyze the mechanism of PMA action, the protein composition in the α1β1 integrin/collagen IV adhesion sites was analyzed by mass spectrometry and proteomics. In four independent experiments we observed the reduced recruitment of vimentin in relation to integrin α1 subunit. This was in full agreement with the fact that we also detected the retraction of vimentin from cell adhesions by confocal microscopy. Furthermore, the accumulation of kindlin-2 into cell adhesions was significantly increased after PMA treatment. Kindlin-2 siRNA inhibited cell spreading as well as the formation of actin fibrils and cell adhesions, but did not prevent the effect of PMA on lamellipodia formation. Thus, kindlin-2 recruitment was considered to be a consequence rather than the primary cause for the loss of connection between vimentin and the adhesion sites.

Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal keratinocyte microvesicles (KC-MVs) in epithelial–stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing–associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from keratinocyte-like cells (HaCaT) were added to fibroblast cultures, expression of 21 genes was significantly regulated (P

The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the αV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding α2-, α3- and α6-containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to α6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41. Originally included in thesis in manuscript form.

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC–MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1β1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix.

Background The composition and organization of extracellular matrix (ECM) are important regulators of cell behavior. In particular in the prostate, this central role of the ECM is further stressed by the fact that several potential markers of prostate stem cells are matrix receptors. Methods We established 12 fibroblastic cell lines from cancerous and non-cancerous areas of six prostates and allowed the cells to produce ECM under cell culture conditions. We also performed a proteome wide analysis of the ECM components by mass spectrometry. To study the in vitro activation of fibroblastic cells we compared the differences between the ECM produced in cell culture by six non-cancerous-tissue-derived fibroblasts and the in vivo matrisome from the corresponding non-cancerous tissue of prostate. Results Our results suggest that at tissue level the ECM is mainly produced by fibroblastic cells and that it contains standard collagen I fibrils and fibril-associated proteins. Beaded-filament forming collagen VI is also abundant and basement membranes potentially contain five laminin subtypes and collagens XV and XVIII. As the main finding, we also detected differences when in vivo and in vitro matrisomes were compared. Only 65 out of 206 proteins were found to be common for both in vivo and in vitro samples. Majority of the 55 proteins, which were solely detected in in vivo samples, were considered to be plasma derived. Eighty-six proteins were solely found from in vitro fibroblast-derived ECM, and most of them were related to matrix remodeling or growth factor action, proposing that the activation of fibroblasts in cell culture may remarkably modify their gene expression profile. Finally, in comparison to traditional 2D in vitro cell culture, the ECM composition of 3D spheroid culture was analyzed. The matrisome in spheroid culture did not resemble the in vivo ECM more closely than in monolayer culture. Conclusions Artificial activation of ECM remodeling seems to be a distinctive feature in in vitro models. In conclusion the constitution of ECM produced by prostate derived fibroblasts in vitro is similar, but not identical to the prostate ECM in vivo as shown here by mass spectrometric analysis.

Targeting nanoparticles to desired intracellular compartments is a major challenge. Integrin-type adhesion receptors are connected to different endocytosis routes in a receptor-specific manner. According to our previous observations, the internalization of an α2β1-integrin-echovirus-1 complex takes place via a macropinocytosis-like mechanism, suggesting that the receptor could be used to target nanoparticles to this specific entry route. Here, silica-based nanoparticles, carrying monoclonal antibodies against the α2β1 integrin as address labels, were synthesized. Studies with flow cytometry, atomic force microscopy and confocal microscopy showed the particles to attach to the cell surface via the α2β1 integrin. Furthermore, quantitative analysis of nanoparticle trafficking inside the cell performed with the BioImageXD software indicated that the particles enter cells via a macropinocytosis-like process and end up in caveolin-1 positive structures. Thus, we suggest that different integrins can guide particles to distinct endocytosis routes and, subsequently, also to specific intracellular compartments. In addition, we show that with the BioImageXD software it is possible to conduct sensitive and complex analyses of the behavior of small fluorescent particles inside cells, using basic confocal microscopy images.

High expression of the collagen receptor, α2β1 integrin, on platelets of ITGA2 807T-allele carriers has been identified as a risk factor for thromboembolic conditions, and α2β1 inhibitors are considered to be potential therapeutic agents. In 59 genotyped individuals, we measured α2 expression levels on platelets and analyzed platelet adhesion to collagen under flow conditions. A sulfonamide-type small-molecule inhibitor of α2β1 integrin decreased average platelet adhesion in individuals with the C/T807T genotype but not in those harboring C807C. Thus, genotype can be used to select a human subpopulation that has the highest probability of showing a positive response to α2β1 inhibitors.

Cell surface integrin receptors mediate cell adhesion, migration and cellular signaling in all nucleated cells. They are activated by binding to extracellular ligands or by intracellular proteins, such as kindlins that engage with their cytoplasmic tails. Cells in the periodontal tissues express several integrins with overlapping ligand-binding capabilities. A distinct phenotype in the periodontium has only been described for knockouts or mutations of three integrin subunits, α11, β6 and β2. Integrin α11β1 appears to have some regulatory function in the periodontal ligament of continuously erupting incisors in mice. Integrin αvβ6 is expressed in the junctional epithelium (JE) of the gingiva. Animals deficient in this receptor develop classical signs of periodontal disease, including inflammation, apical migration of the JE and bone loss, suggesting that it plays a role in the regulation of periodontal inflmmation, likely through activation of transforming growth factor-β1. Lack of integrin activation in the JE is also associated with periodontitis. Patients with kindlin-1 mutations have severe early-onset periodontal disease. Finally, patients with mutations in the leukocyte-specific β2 integrin subunit have severe periodontal problems due to lack of transiting neutrophils in the periodontal tissues.

Integrins α L β 2 , α M β 2 and α X β 2 are expressed on leukocytes. Their primary ligands are counter transmembrane receptors or plasma proteins, such as intercellular cell adhesion molecule-1 (ICAM-1) or components of complement system (iC3b, iC4b), respectively. Function blocking antibodies for these integrins may also reduce cell adhesion to collagens. To make the first systematical comparison of human α L β 2 , α M β 2 and α X β 2 as collagen receptors, we produced the corresponding integrin αI domains both in wild-type and activated form and measured their binding to collagens I–VI. In the “closed” (wild-type) conformation, the α L I and α M I domains bound with low avidity to their primary ligands, and the interaction with collagens was also very weak. Gain-of-function mutations α L I306G, α L K287C/K294C and α M I316G are considered to mimic “open”, activated αI domains. The binding of these activated αI domains to the primary ligands was clearly stronger and they also recognized collagens with moderate avidity ( K d L I domain favored collagen I ( K d ≈ 80 nM) when compared to collagen IV. The integrin α X I domain acted in a very different manner since already in native, wild-type form it bound to collagen IV and iC3b ( K d ≈ 200–400 nM). Antibodies against α X β 2 and α M β 2 blocked promyelocytic leukemia cell adhesion to the collagenous GFOGER motif, a binding site for the β 1 integrin containing collagen receptors. In brief, leukocyte β 2 integrins may act as collagen receptors in a heterodimer specific manner.

Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function.

The ability of cells to migrate and form metastases is one of the fatal hallmarks of cancer that can be conquered only with better understanding of the molecules and regulatory mechanisms involved. The oncogenic PIM kinases have been shown to support cancer cell survival and motility, but the PIM-regulated pathways stimulating cell migration and invasion are less well characterized than those affecting cell survival. Here we have identified the glycogen synthase kinase 3β (GSK3B) and the forkhead box P3 (FOXP3) transcription factor as direct PIM targets, whose tumour-suppressive effects in prostate cancer cells are inhibited by PIM-induced phosphorylation, resulting in increased cell migration. Targeting GSK3B is also essential for the observed PIM-enhanced expression of the prostaglandin-endoperoxide synthase 2 (PTGS2), which is an important regulator of both cell migration and adhesion. Accordingly, selective inhibition of PIM activity not only reduces cell migration, but also affects integrin-mediated cell adhesion. Taken together, these data provide novel mechanistic insights on how and why patients with metastatic prostate cancer may benefit from therapies targeting PIM kinases, and how such approaches may also be applicable to inflammatory conditions.

We have analyzed the structure and function of the integrin α(1)I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α(1)I C139S/E317A was a higher avidity collagen binder than α(1)I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α(1)I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α(2)I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α(2)I structure, has not changed its position in the activated α(1)I variant. During the integrin activation, Glu(335) on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β(1) subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu(335). This indicates that the stabilization of helix 7 into its downward position is not required if the α(1) MIDAS is already open. To conclude, the activated α(1)I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg(287)-Glu(317) ion pair has just broken during the integrin activation.

Collagen binding integrins are an important family of cell surface receptors that mediate bidirectionally signals between the interior of the cell and the extracellular matrix. The protein-protein interactions between cells and collagen are necessary for many physiological functions, but also promote diseases. For example, the interaction of α2β1 integrin and collagen has been shown to have an important role in thrombus formation and cancer spread. The fact that the discovery of small molecules that can block such protein-protein interactions is highly challenging has significantly hindered the discovery of pharmaceutical agents to treat these diseases. Here, we present a rationally designed novel fluorescent molecule that can be synthesized in just a few minutes from commercially available starting materials. This molecule blocks the protein-protein interaction between α2β1 integrin and collagen, and due to its fluorescent properties, it can be employed in wide variety of biological applications.

Adhesion of epithelium to the extracellular matrix is crucial for the maintenance of systemic and oral health. In the oral cavity, teeth or artificial dental implants penetrate the soft tissue of the gingiva. In this interface, gingival soft tissue needs to be well attached via the epithelial seal to the tooth or implant surface to maintain health. After injury or wounding, epithelial tissue rapidly migrates to form the initial epithelial cover to restore the barrier against infection. These events are crucially dependent on deposition of extracellular matrix and proper activation and function of integrin receptors in the epithelial cells. Recent experimental evidence suggests that epithelial integrins also participate in the regulation of periodontal inflammation. In this review, we will discuss the structure and function of epithelial integrins and their extracellular ligands and elaborate on their potential role in disease and repair processes in the oral cavity.

SummaryInteraction of blood platelets with vascular collagen is an initiating event in haemostasis and thrombus formation. Based on molecular modelling of human integrin α2I domain and cell-based screening assays we have developed sulfonamide derivatives, a mechanistically novel class of molecules. These molecules show antiplatelet efficacy by selectively inhibiting α2β1 integrin-mediated collagen binding. One sulfonamide derivative, named BTT-3016, showed inhibitory capacity in several assessments of human platelet interaction with collagen. It inhibited about 90% of the aggregation of gel-filtered magnesium-supplemented platelets and 70% of aggregation in PPACK-anticoagulated platelet-rich plasma when stimulated with collagen but not with ADP. The antiplatelet activity of BTT-3016 was dependent on α2β1 integrin, since in collagen binding test BTT-3016 had no effect on the platelets derived from α2 integrin null mice. When tested in an in vivo model in mice, BTT-3016 clearly reduced thrombus formation on the vessel wall after vascular injury. Furthermore, BTT-3016 prolonged tail-bleeding time in a manner comparable to aspirin. We show that new α2β1 inhibitors exert collagen-specific antiplatelet activity and regulate thrombus growth in vivo without compromising primary haemostasis more than aspirin. We suggest that the α2β1 inhibiting strategy could be further developed for the prevention and treatment of arterial thrombosis.

The emergence of multicellular animals could only take place once evolution had produced molecular mechanisms for cell adhesion and communication. Today, all metazoans express integrin-type adhesion receptors and receptors for growth factors. Integrins recognize extracellular matrix proteins and respective receptors on other cells and, following ligand binding, can activate the same cellular signaling pathways that are regulated by growth factor receptors. Recent reports have indicated that the two receptor systems also collaborate in many other ways. Here, we review the present information concerning the role of integrins as assisting growth factor receptors and the interplay between the receptors in cell signaling and in the orchestration of receptor recycling.

Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336-related conformational changes in alpha2beta1. Furthermore, the mutation E336A did neither prevent EV1 induced and alpha2beta1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on alpha2beta1 clustering, but do not require the conformational regulation of the receptor.

Extracellular matrix (ECM) is composed of large collagen fibrils. Glycoproteins, such as fibronectin, can bind to collagen or form their own networks. Collagen fibrils are also decorated by proteoglycans, proteins that have large glycosaminoglycan sidechains. In addition, extracellular space often contains hyaluronan, a large glycosaminoglycan molecule that has no core protein. Basement membranes represent a specialized form of extracellular matrix. Basement membranes are built by laminin and type IV collagen networks. In multicellular animals cells are anchored to ECM and basement membranes. Cell locomotion during development and after tissue injury is also based on cellular interactions with different matrix molecules. Specific cell surface receptors mediate these interactions. The largest family of receptors, which mediates cell adhesion to fibronectin, laminins and collagens is called the integrins. Several other cellular receptors have also evolved to bind to various matrix components. Here, we review the basic facts about these receptors and shortly describe their role in human diseases, including cancer and inflammation.

Several different receptor molecules act in concert to regulate cell adhesion. Among these are cell-surface proteoglycans and integrins, which collaborate extensively in mediating binding of cells to extracellular matrix molecules fibronectin and vitronectin. However, very little is known about possible functional synergism between proteoglycans and integrins during adhesion of cells to collagen, although collagen is the most abundant protein in the human body. Here we show that cell-surface heparan sulphate proteoglycans (HSPGs) support integrin alpha2beta1-mediated adhesion to collagen. Cells made devoid of HSPGs either by genetic means or by enzymatic digestions were unable to adhere to collagen via alpha2beta1 integrin. HSPG-deficient cells also displayed impaired spreading and actin organization on collagen. Among different HSPG molecules syndecan-1 was found to play an important role in supporting alpha2beta1 integrin-mediated adhesion. Using overexpression and knock-down experiments we demonstrated that syndecan-1, but not syndecan-2 or -4, enhanced binding of alpha2beta1 to collagen. Moreover, syndecan-1 co-localized with alpha2beta1 integrin and contributed to proper organization of cortical actin. Finally, crosstalk between syndecan-1 and alpha2beta1 integrin was found to enhance the transcription of matrix metalloproteinase-1 in response to collagen binding. Our findings thus suggest that a previously unknown link between integrin alpha2beta1 and syndecan-1 is important in regulating cell adhesion to collagen and in triggering integrin downstream signalling.

Collagen receptor integrins alpha 1 beta 1 and alpha 2 beta 1 can selectively recognize different collagen subtypes. Here we show that their alpha I domains can discriminate between laminin isoforms as well: alpha 1I and alpha 2I recognized laminin-111, -211 and -511, whereas their binding to laminin-411 was negligible. Residue Arg-218 in alpha1 was found to be instrumental in high-avidity binding. The gain-of-function mutation E318W makes the alpha 2I domain to adopt the "open" high-affinity conformation, while the wild-type alpha 2I domain favors the "closed" low-affinity conformation. The E318W mutation markedly increased alpha 2I domain binding to the laminins (-111, -211 and -511), leading us to propose that the activation state of the alpha 2 beta 1 integrin defines its role as a laminin receptor. However, neither wild-type nor alpha 2IE318W domain could bind to laminin-411. alpha 2IE318W also bound tighter to all collagens than alpha 2I wild-type, but it showed reduced ability to discriminate between collagens I, IV and IX. The corresponding mutation, E317A, in the alpha 1I domain transformed the domain into a high-avidity binder of collagens I and IV. Thus, our results indicate that conformational activation of integrin alpha 1I and alpha 2I domains leads to high-avidity binding to otherwise disfavored collagen subtypes.

The collagen-binding integrins alpha1beta1 and alpha2beta1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that alpha1beta1 negatively regulates integrin alpha2beta1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins alpha1beta1 and alpha2beta1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin alpha2beta1, but not alpha1beta1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin alpha2 subunit with that of alpha1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin alpha2 cytoplasmic tail with that of alpha1, decreases cell migration and cord formation, but increases proliferation. When integrin alpha1 and alpha2 subunits are co-expressed in UB cells, the alpha1 subunit negatively regulates integrin alpha2beta1-dependent cord formation, adhesion and migration and this inhibition requires expression of both alpha1 and alpha2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the alpha2 integrin subunit, as well as the alpha1 integrin subunit, regulate integrin alpha2beta1 cell function.

Integrin alpha2beta1 is a potential target molecule in drug development. We have established "design" criteria for molecules that bind to the "closed" conformation of alpha2I domain via Mg(2+) in MIDAS (metal ion dependent adhesion site) while simultaneously forming interactions with neighboring amino acid residues. Specific tetracyclic Streptomyces products belonging to the group of aromatic polyketides fulfill our criteria and inhibit alpha2beta1 integrin. All previously described inhibitors of alphaI domain integrins act in an allosteric manner.

Integrins αLβ2, αMβ2 and αXβ2 are expressed on leukocytes. Their primary ligands are counter transmembrane receptors or plasma proteins, such as intercellular cell adhesion molecule-1 (ICAM-1) or components of complement system (iC3b, iC4b), respectively. Function blocking antibodies for these integrins may also reduce cell adhesion to collagens. To make the first systematical comparison of human αLβ2, αMβ2 and αXβ2 as collagen receptors, we produced the corresponding integrin αI domains both in wild-type and activated form and measured their binding to collagens I-VI. In the "closed" (wild-type) conformation, the αLI and αMI domains bound with low avidity to their primary ligands, and the interaction with collagens was also very weak. Gain-of-function mutations αL I306G, αL K287C/K294C and αM I316G are considered to mimic "open", activated αI domains. The binding of these activated αI domains to the primary ligands was clearly stronger and they also recognized collagens with moderate avidity (Kd

The plasma protein histidine-rich glycoprotein (HRG) affects the morphology and function of both endothelial cells (ECs) and monocytes/macrophages in cancer. Here, we examined the mechanism of action of HRG's effect on ECs. HRG suppressed adhesion, spreading and migration of ECs specifically on collagen I (COL I) whereas ECs seeded on other extracellular matrix proteins were insensitive to HRG. HRG did not bind specifically to COL I or to the α-integrin binding site on collagen, GFOGER. Furthermore, HRG's inhibition of EC adhesion was not dependent upon heparan sulfate (HS) moieties as heparitinase-treated ECs remained sensitive to HRG. C2C12 cells expressing α2 integrin, the major collagen-binding α-integrin subunit in ECs, showed increased binding of HRG compared with wild type C2C12 cells lacking the α2 subunit. Recombinant α2 I-domain protein bound HRG and to a higher extent when in active conformation. However, the α2 I-domain bound weakly to HRG compared with COL I and the purified α2β1 ectodomain complex failed to retain HRG. We conclude that HRG binds to α2 integrin through low-affinity interactions in a HS-independent manner, thereby blocking EC-adhesion to COL I.

Ross River (RR) virus is an alphavirus endemic to Australia and New Guinea and is the aetiological agent of epidemic polyarthritis or RR virus disease. Here we provide evidence that RR virus uses the collagen-binding alpha1beta1 integrin as a cellular receptor. Infection could be inhibited by collagen IV and antibodies specific for the beta1 and alpha1 integrin proteins, and fibroblasts from alpha1-integrin-/- mice were less efficiently infected than wild-type fibroblasts. Soluble alpha1beta1 integrin bound immobilized RR virus, and peptides representing the alpha1beta1 integrin binding-site on collagen IV inhibited virus binding to cells. We speculate that two highly conserved regions within the cell-receptor binding domain of E2 mimic collagen and provide access to cellular collagen-binding receptors.

Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.

The expression of integrin alphaV subunit on 4 melanoma-derived cell lines (A2058, SK-mel-5, WM-115 and WM-266-4) was analyzed. WM-115 cells, which originate from a primary tumor, were negative, whereas all 3 metastasis-derived lines had high levels of alphaV. To study alphaV integrins in the survival of melanoma cells, we developed a novel strategy that is exempt from extracellular inhibitors of ligand binding, which can activate integrin signaling and have integrin-independent effects on apoptosis. A recombinant adenovirus was used to transfer cDNA coding for a single-chain intracellular anti-alphaV integrin antibody into the melanoma cells. Anti-alphaV integrin adenovirus effectively inhibited the cell surface expression of alphaV integrins. In cell culture experiments, the depletion of alphaV integrins detached cells from extracellular matrix and induced apoptosis. Moreover, it prevented WM-266-4 cells from forming tumors in severe combined immunodeficiency mice but it could not prevent the growth of tumors that were formed by alphaV-negative WM-115 cells. Our results indicate that in primary melanomas there are cells that survive without alphaV integrins, whereas during the progression of disease cells can develop a dependency on these receptors. Furthermore, the data oppose the possibility that in melanoma cells apoptosis could occur due to direct activation of caspases by ligand-free alphaV integrins on the cell surface.

The four collagen receptor integrins, alpha1beta1, alpha2beta1, alpha10beta1 and alpha11beta1, form a structurally and functionally distinct subgroup when compared to other members of the integrin family. In this review, we discuss the structures of these receptors and their differences in collagen binding and signalling function.

Exogenous heat shock proteins may modify cell behavior of infected epithelium. The effect of heat shock protein 60 (hsp60) of Actinobacillus actinomycetemcomitans and Escherichia coli, and human recombinant hsp60 on migration of HaCaT skin keratinocytes was studied using the Boyden chamber assay. Hsp60 from different species increased cell migration by two- to fivefold and this effect was inhibited by ERK inhibitor PD 98059, p38 inhibitor SB 203580, and a function-blocking epidermal growth factor receptor (EGFR) antibody. Hsp60 reduced the expression of alpha6-integrin mRNA and its protein levels on the cell surface but had no effect on the expression of beta4, beta1, alpha1, alpha5 or alphav integrin subunits. Hsp60 also significantly inhibited cell adhesion to laminin-5, a ligand of alpha6beta4 integrin. These results suggest that exogenous hsp60 released from bacteria or inflammatory cells may promote epithelial cell migration through activation of EGFR and MAP kinases, and inhibition of alpha6beta4 integrin expression.

Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.

The expression pattern of integrin-type cell adhesion receptors is often changed during malignant transformation. In the present work, we studied the prognostic significance of beta1 and alphav integrin chains for survival of patients with metastatic melanoma. The expression levels of beta1 integrin were also compared with those of Bcl-2, an anti-apoptotic protein, the presence of which is associated with treatment response and survival in melanoma. The expression of beta1 and alphav integrins in 68 melanoma metastases obtained from 55 patients treated with combined chemoimmunotherapy was studied by immunohistochemistry using anti-beta1 and anti-alphav antibodies. The patients were divided into two groups (using a cut-off point of >/= 81%) for beta1 integrin expression levels and into three categories (negative/low, median, high) for alphav integrin expression levels. All tumours were positive for beta1 integrin, and the tumours (n = 6) which had the highest alphav score were also strongly positive for beta1 (94%; P = 0.0055). Patients (n = 43) with 80% or less beta1 integrin-positive tumour cells in their samples had a median disease-free survival (DFS) of 17.0 months, and patients (n = 12) with 81% or more beta1 integrin-positive tumour cells had a DFS of only 5.7 months (P = 0.0001). Patients (n = 32) with low alphav integrin expression levels had shorter DFS (median 12.3 months; P = 0.0146) than patients (n = 20) with median expression levels (median 16.7 months; P = 0.0146). However, three patients who had a very strong alphav expression in their tumours had a median DFS of only 1.8 months (P = 0.0146). Median level expression of beta1 integrin was associated with the presence of Bcl-2 in tumour cells (P = 0.0033). Our results suggest that beta1 and alphav integrin chains are independently expressed in metastatic melanoma and may have an effect on the metastatic potential of melanoma cells.

Integrin alphavbeta6 is an epithelial cell-specific receptor that is not normally expressed by resting epithelium but its expression is induced during wound healing. The function of alphavbeta6-integrin in wound repair is not clear. In the present study, we showed that beta6-integrin expression was strongly up-regulated in the epidermis in human chronic wounds but not in different forms of skin fibrosis. To test whether increased beta6-integrin expression plays a role in abnormal wound healing we developed four homozygous transgenic mouse lines that constitutively expressed human beta6-integrin in the epithelium. The mice developed normally and did not show any histological abnormalities in the skin. The rate of experimental skin wound closure was unaltered and the wounds healed without significant scar formation. However, during breeding program 16.1 to 27.0% of transgenic mice developed spontaneous, progressing fibrotic chronic ulcers. None of the wild-type animals developed these lesions. The chronic lesions had areas with severe fibrosis and numerous activated macrophages and fibroblasts expressing transforming growth factor (TGF)-beta. The level of TGF-beta1 was significantly increased in the lesions as compared with normal skin. The findings suggest that increased alphavbeta6-integrin in keratinocytes plays an active part in abnormal wound healing possibly through a mechanism involving increased activation of TGF-beta.

Integrin alpha7beta1 is a laminin receptor, both subunits of which have alternatively spliced, developmentally regulated variants. In skeletal muscle beta1 has two major splice variants of the intracellular domain (beta1A and beta1D). alpha7X1 and alpha7X2 represent variants of the alpha7 ectodomain, whereas alpha7A and alpha7B are variants of the intracellular domain. Previously we showed that during early regeneration after transection injury of muscle alpha7 integrin mediates dynamic adhesion of myofibers along their lateral aspects to the extracellular matrix. Stable attachment of myofibers to the extracellular matrix occurs during the third week after injury, when new myotendinous junctions develop at the ends of the regenerating myofibers. Now we have analyzed the relative expression of beta1A/beta1D and alpha7A/alpha7B and alpha7X1/alpha7X2 isoforms during regeneration for 2 to 56 days after transection of rat soleus muscle using reverse transcriptase-polymerase chain reaction and immunohistochemistry. During early regeneration beta1A was the predominant isoform in both the muscle and scar tissue. Expression of muscle-specific beta1D was detected in regenerating myofibers from day 4 onwards, ie, when myogenic mitotic activity began to decrease, and it became more abundant with the progression of regeneration. alpha7B isoform predominated on day 2. Thereafter, the relative expression of alpha7A transcripts increased until day 7 with the concomitant appearance of alpha7A immunoreactivity on regenerating myofibers. Finally, alpha7B again became the predominant variant in highly regenerated myofibers. Similarly as in the controls, alpha7X1 and alpha7X2 isoforms were both expressed throughout the regeneration with a peak in alpha7X1 expression on day 4 coinciding with the dynamic adhesion stage. The results suggest that during regeneration of skeletal muscle the splicing of beta1 and alpha7 integrin subunits is regulated according to functional requirements. alpha7A and alpha7X1 appear to have a specific role during the dynamic phase of adhesion, whereas alpha7B, alpha7X2, and beta1D predominate during stable adhesion.

The integrin beta1 subunit can form a heterodimer with 12 different alpha subunits. According to the present model, the expression level of any alphabeta complex is regulated by the availability of the specific alpha subunit, whereas beta1 subunit is constantly present in a large excess. The expression of several heterodimers containing the alphaV subunit seems to be regulated by an identical mechanism. The fact that many cells express alphaVbeta1 heterodimer, and that this fibronectin/vitronectin receptor may be selectively regulated, compromises the present model of the regulation of beta1 and alphaV integrins. We have tried to solve this problem by assuming that distinct alphabeta heterodimers are formed with different tendency. To test the hypothesis, we analyzed WM-266-4 melanoma cells transfected with a cDNA construct coding for an intracellular single-chain anti-alphaV integrin antibody. We could see 70-80% reduction in the cell surface expression of alphaV subunit. However, the only one of the alphaV integrins reduced on the cell surface was alphaVbeta1. This suggests that the cell surface expression level of alphaVbeta1 is dependent on the number of alphaV subunits available after the formation of other alphaV-containing heterodimers. Thus, there seems to be a hierarchy in the complex formation between alphaV and its different beta-partners. These observations explain how alphaVbeta1 can be specifically regulated without concomitant changes in the expression of other alphaV or beta1 integrins.

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor α2β1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, α2β1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.

The four collagen-binding αI domain integrins form their own subgroup among cell adhesion receptors. The signaling functions of α1β1 and α2β1 integrins have been analyzed in many experimental models, whereas less studies are available about the more recently found α10β1 and α11β1 heterodimers. Interestingly, collagen binding by α1β1 and α2β1 often generates opposite cellular responses. For example α1β1 has often been reported to promote cell proliferation and to suppress collagen synthesis, whereas α2β1 can in many model systems inhibit growth and promote collagen synthesis. There are obviously cell type dependent factors modifying the signaling. Additionally the structure and the organization of collagenous matrix play a critic role. Many recent studies have also stressed the importance of the crosstalk between the integrins and other cell surface receptors.

Collagen XXII, a FACIT (fibril-associated collagen with interrupted triple helices), is expressed at the myotendinous junction and the articular surface of joint cartilage. Cellular receptors like collagen-binding integrins are known to bind collagens with distinct binding motifs following the sequence GXOGER. In the present study, we demonstrate the sequences GLQGER and GFKGER as novel binding motifs between collagen XXII and collagen-binding integrins, especially α2β1 integrin. Solid-phase assays and surface plasmon resonance spectroscopy revealed a direct interaction between α2β1 integrin and the motif GFKGER. In addition, immunohistochemical analysis demonstrated partial co-localization of collagen XXII, α2β1 integrin and α11β1 integrin at the myotendinous junction. Furthermore, computational modelling of the motifs GLQGER and GFKGER showed perfect fitting of the sequences into the binding pocket of collagen-binding integrins. Taken together, we demonstrated that collagen XXII interacts with collagen-binding integrins via the new motifs GLQGER and GFKGER.

Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collagen binding preferences. The integrin alpha(2)I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin alpha(1)I had the opposite binding pattern. The integrin alpha(10)I domain was similar to the alpha(1)I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the alpha(1)I and alpha(2)I domains, we modeled the structure of the alpha(10)I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the alphaI domains, and their binding to types I, IV, and VI collagen was tested. In the alpha(2)I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the alpha(1)I and alpha(10)I domains is oppositely charged (Arg-218). The mutation D219R in the alpha(2)I domain changed the ligand binding pattern to resemble that of the alpha(1)I and alpha(10)I domains and, vice versa, the R218D mutation in the alpha(1)I and alpha(10)I domains created an alpha(2)I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.

Heat shock proteins (hsp) have important roles in the regulation and protection of both prokaryotic and eukaryotic cells, especially during environmental stress. Hsps are also important bacterial virulence factors. We investigated whether bacterial hsp60 can alter epithelial cell mitogen-activated protein kinase (MAPK) signaling and cell proliferation. Human skin keratinocytes (HaCaT cell line) were cultured in the presence of hsp60 purified from Actinobacillus actinomycetemcomitans, an important oral pathogen. Protein kinases in the ERK1/2 and p38 MAPK signaling pathways were probed with kinase-specific and phosphorylation-site-specific antibodies on Western blots. In quiescent cultures, hsp60 increased ERK1/2 phosphorylation in a sustained manner and p38 phosphorylation transiently. Hsp60 also increased epithelial cell proliferation by about 30%. Inhibition of the ERK1/2 pathway by PD 98059 (a MEK1 inhibitor) reversed partially ERK1/2 phosphorylation and totally cell proliferation indicating that the ERK1/2 MAPK pathway is involved in the hsp60-induced cell growth. This was supported by findings that hsp60 stimulated phosphorylation of RSK1/2 and cyclic AMP response element-binding protein and increased expression of transcription factors c-Jun and c-Fos. Recombinant human hsp60 did not alter ERK1/2 or p38 phosphorylation and had no effect on epithelial cell proliferation. Inhibition of p38 MAPK pathway by SB 203580 increased both ERK1/2 phosphorylation and cell proliferation demonstrating that the inhibitor can either directly or indirectly activate the ERK1/2 MAPK pathway. The results show that exogenous bacterial hsp60 is able to activate ERK1/2 phosphorylation and thereby cause increased epithelial proliferation. In case of mucosal infection this effect may either lead to increased wound repair or participate in the pathological mechanism of some bacterial diseases that involve increased epithelial proliferation.

Integrin alpha1beta1, one of the cellular collagen receptors, can participate in the regulation of collagen accumulation by acting as a negative feedback regulator. The molecular mechanism behind this phenomenon has been unknown. We have plated cells inside three-dimensional collagen and analyzed a set of chemical inhibitors for various signal transduction pathways. Only two wide-spectrum serine/threonine kinase inhibitors, H-7 and iso-H-7 could prevent the down-regulation of alpha1(I) collagen mRNA levels in cells exposed to three-dimensional collagen. In monolayer iso-H-7 slightly down-regulated collagen gene expression, indicating that inside collagen it affected integrin signaling rather than having a direct stimulatory effect on collagen mRNA levels. The effect of iso-H-7 was not dependent on its ability to inhibit protein kinases A, C, or G. H-7 and iso-H-7 could also inhibit collagen gel contraction, but this mechanism was independent of collagen gene regulation. Three-dimensional collagen could also up-regulate the mRNA levels of several matrix metalloproteinases (MMPs) but H-7 and iso-H-7 had no effect on the regulation of MMP genes. Our data indicate that three-dimensional collagenous matrix regulates distinct cellular signaling pathways and that collagen gene regulation is independent of the other effects of the matrix.

The expression of integrin-type cell adhesion receptors is frequently changed in malignant transformation. Despite their important role in cancer cell behaviour, the value of integrins as prognostic markers is mostly unknown. We have examined the expression of beta1 integrins in 38 metastatic melanomas obtained from 27 patients treated with combined chemoimmunotherapy. On the basis of beta1 integrin expression, the melanoma samples were divided into two groups: beta1-negative tumours ( or = 10% positive cells). Patients with beta1-positive tumours (n = 15) had significantly longer disease-free survival (median 38 versus 7 months, P < 0.0001) and overall survival (median 70 versus 23 months, P = 0.0001) evaluated after the diagnosis of primary disease compared with patients with beta1-negative metastases (n = 11). Moreover, the survival of the patients with beta1-positive tumours after the initiation of chemoimmunotherapy was significantly prolonged (median 18 versus 9 months, P = 0.017). The independent nature of beta1 integrin expression as a significant prognostic factor for survival after therapy was confirmed using Cox's multivariate analysis (P = 0.014). Our results indicate that the expression of beta1 integrins might have some major tumour growth regulatory role and can be used as a predictor for prognosis in patients with metastatic melanoma.

Two integrin-type collagen receptors, α1β1 and α2β1, are structurally very similar. However, cells can concomitantly express the both receptors and they might have independent functions. Here, Chinese hamster ovary (CHO) cells, which lack endogenous collagen receptors, were transfected with either α1 or α2 integrin cDNA. Cells were allowed to adhere to various collagen types and their integrin function was tested by observing the progression of cell spreading. The cells expressing α1β1 integrin could spread on collagen types I, III, IV, and V but not on type II, while α2β1 integrin could mediate cell spreading on collagen types I-V. Type XIII is a transmembrane collagen and its interaction with the integrins has not been previously studied. CHO-α1β1 cells could spread on human recombinant type XIII collagen, unlike CHO-α2β1 cells. Integrins α1β1 and α2β1recognize collagens with the specific αI domains. The α1I and α2I domains were produced as recombinant proteins, labeled with europium and used in a sensitive solid-phase binding assay based on time-resolved fluorescence. α1I domain, unlike the α2I domain, could attach to type XIII collagen. The results indicate, that α1β1 and α2β1have different ligand binding specificity. Distinct recognition of different collagen subtypes by the αI domains can partially explain the differences seen in cell spreading. However, despite the fact that CHO-α1β1 cells could not spread on type II collagen α1I domain could bind to this collagen type. Thus, the cell spreading on collagens may also be regulated by factors other than the integrins.