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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.

Background Tumor microenvironment or stroma has the potency to regulate the behavior of malignant cells. Fibroblast-like cells are abundant in tumor stroma and they are also responsible for the synthesis of many extracellular matrix components. Fibroblast-cancer cell interplay can modify the functions of both cell types. Methods We applied mass spectrometry and proteomics to unveil the matrisome in 3D spheroids formed by DU145 prostate cancer cells, PC3 prostate cancer cells, or prostate-derived fibroblasts. Similarly, DU145/fibroblast and PC3/fibroblast coculture spheroids were also analyzed. Western blot analysis and immunofluorescence were used to confirm the presence of specific proteins in spheroids. Cancer dissemination was studied by utilizing "out of spheroids" migration and invasion assays. Results In the spheroid model cancer cell-fibroblast interplay caused remarkable changes in the extracellular matrix and accelerated the invasion of DU145 cells. Fibroblasts produced structural matrix proteins, growth factors, and matrix metalloproteinases. In cancer cell/fibroblast cocultures basement membrane components, including laminins (α3, α5, β2, and β3), heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII accumulated in a prominent manner when compared with spheroids that contained fibroblasts or cancer cells only. Furthermore, collagen XVIII was intensively processed to different endostatin-containing isoforms by cancer cell-derived cathepsin L. Conclusions Fibroblasts can promote carcinoma cell dissemination by several different mechanisms. Extracellular matrix and basement membrane proteins provide attachment sites for cell locomotion promoting adhesion receptors. Growth factors and metalloproteinases are known to accelerate cell invasion. In addition, cancer cell-fibroblast interplay generates biologically active fragments of basement membrane proteins, such as endostatin.

Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.

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.

INTRODUCTION AND OBJECTIVES:Extracellular vesicles (EV) have raised interest as a potential source of biomarker discovery. We developed techniques for high-purity isolation of tumor tissue EV (tEV)...

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.

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

Gene copy number alterations, tumor cell stemness, and development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stemness phenotypes involving Wnt-beta-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous genetic gains in KRAS, MYC, and FAK (KMF) genes, in a new aggressive murine model of ovarian cancer. Noncanonical signaling via FAK 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 neoadjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance, triggering tumor cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 genes elevated by a FAK activity-dependent, beta-catenin, and Myc signaling axis including pluripotency and DNA repair genes. Identified target increases in HGSOC tumors may reflect oncogenic FAK signaling. Citation Format: Carlos J. Díaz Osterman, Duygu Ozmadenci, Elizabeth G. Kleinschmidt, Kristin N. Taylor, Allison M. Barrie, Shulin Jiang, Lisa M. Bean, Florian J. Sulzmaier, Jian Li, Xiao Lei Chen, Guo Fu, Marjaana Ojalill, Pekka Rappu, Jyrki Heino, Adam A. Mark, Guorong Xu, Kathleen M. Fisch, David T. Weaver, Jonathan A. Pachter, Balázs Győrffy, Michael T. McHale, Denise C. Connolly, Alfredo Molinolo, Dwayne G. Stupack, David D. Schlaepfer. FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A61.

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.

Conformational activation of integrins is generally required for ligand binding and cellular signalling. However, we have previously reported that the nonactivated conformation of α2β1 integrin can also bind to large ligands, such as human echovirus 1. In this study, we show that the interaction between the nonactivated integrin and a ligand resulted in the activation of focal adhesion kinase (FAK) in a protein kinase C dependent manner. A loss-of-function mutation, α2E336A, in the α2-integrin did not prevent the activation of FAK, nor did EDTA-mediated inactivation of the integrin. Full FAK activation was observed, since phosphorylation was not only confirmed in residue Y397, but also in residues Y576/7. Furthermore, initiation of downstream signaling by paxillin phosphorylation in residue Y118 was evident, even though this activation was transient by nature, probably due to the lack of talin involvement in FAK activation and the absence of vinculin in the adhesion complexes formed by the nonactivated integrins. Altogether these results indicate that the nonactivated integrins can induce cellular signaling, but the outcome of the signaling differs from conventional integrin signaling.

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.

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.

Introduction Pim kinases are constitutively active serine/threonine kinases that are upregulated by the JAK/STAT pathway and are often overexpressed in advanced human haematological and solid cancers. There they have been observed to enhance cancer cell growth, survival and energy metabolism. Using both cell-based and animal models, we have shown that PIM kinases also efficiently promote cancer cell migration, invasion and metastasis formation. In addition, we have identified several substrates to mediate the PIM-dependent effects. Material and methods Motility of PC-3 prostate cancer cells was analysed by cell-based assays, such as wound healing assays with single cell tracking method, Boyden chamber assays and adhesion assays. The effects of PIM kinases on tumour growth were studied using subcutaneous and orthotopic mouse models for human prostate cancer. The interactions of PIM kinases and their substrates were investigated by in vitro kinase assays as well as microscopic techniques such as proximity ligation assay and fluorescence lifetime imaging. To reveal PIM-dependent functions, we used PIM-selective chemical inhibitors, transient or stable PIM upregulation as well as PIM silencing by RNA interference or CRISPR/Cas9-based gene editing. Results and discussions PIM upregulation was shown to promote prostate cancer cell motility both in cell-based wound healing assays and in the orthotopically inoculated xenografts. By contrast, inhibition of PIM kinase activity by the pyrrolocarbazole compound DHPCC-9 decreased cell migration, invasion and adhesion as well as tumour growth, angiogenesis, lymphangiogenesis and formation of metastases. Furthermore, the PIM-dependent effects were shown to be mediated by the ability of PIM kinases to phosphorylate several key substrates, such as CXCR4, FOXP3, GSK3B, NFATC1 and NOTCH1. More recently, we have observed PIM kinases to influence also the actin cytoskeleton and have identified actin-regulating proteins as novel PIM substrates. Conclusion PIM kinases are promising targets for anti-cancer drug development, not only for their roles as pro-survival factors but also as pro-migratory factors. By inhibiting PIM kinases, it may be possible to simultaneously regulate many different PIM substrates that are essential for metastatic tumour growth.

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.

Wound healing is regulated by a complex, spatiotemporally controlled interplay between resident and circulating cells, extracellular ­matrix (ECM), chemical, biochemical, electrochemical, and mechanical signals from the ECM and soluble mediators. Wound healing starts with coagulation and haemostasis that stops the bleeding. Platelets in the clot are activated and release growth factors and cytokines that stimulate resident cells to start re-epithelialization, angiogenesis, and connective tissue repair. In addition, platelet-derived factors, activated complement, and bacterial products trigger an inflammatory reaction with neutrophil and macrophage influx to clear the ­bacteria. How effectively the inflammation is resolved will play a crucial role in determining the healing outcome. Fast re-­epithelialization that establishes the barrier function and neutrophil-macrophage interactions are important for this process. Initial influx of macrophages contains mainly cells that produce proinflammatory cytokines while the reparative macrophages that support granulation tissue formation, angiogenesis, and remodeling dominate the later stages of wound healing. Macrophages also stimulate the differentiation of resident cells, possibly fibroblasts and/or pericytes, to myofibroblasts, which produce matrix that, after remodeling, turns to either scar in skin or normal mucosal tissue in palatal oral mucosa. Current understanding about wound healing is largely based on data obtained from skin that does not always replicate oral mucosal wound healing. In general, however, the wound healing stages and key principles are shared among different tissues. Therefore, in this chapter we will discuss the general principles of wound healing and include information that is distinct to oral mucosal wound healing when relevant.

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 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 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.

In shearing injury both the myofibres and connective tissue framework are breached and the muscle tendon continuity is disrupted. During regeneration the firm myofibre to extracellular matrix (ECM) adhesion must be re-established. We have analysed the expression of selected molecules implementing this adhesion in regenerating myofibres 2-56 days after transection of rat soleus muscle using quantitative immunohistochemistry and Northern blotting. Beta1 integrin mRNA level and alpha7 integrin and vinculin immunoreactivities were transiently increased in both the intact and regenerating parts of the transected myofibres by day 5-7 with normalization by day 10-14. After day 14, alpha7 integrin and vinculin accumulated at the tips of the regenerating myofibres, indicating formation of new mini-myotendinous junctions (mMTJ). Immunoreactivities for dystrophin and associated proteins as well as merosin appeared in regenerating myotubes by day 3-4 reaching control levels by day 56. Our results suggest that integrin and dystrophin associated molecules are complementary in myofibre-ECM adhesion. During regeneration, ruptured myofibres temporarily reinforce their integrin mediated lateral adhesion until mMTJs are formed. Thereby the load on the newly formed scar and the risk of rerupture are reduced. Dystrophin associated molecules appear later and replace integrin on the lateral aspects, while both complexes are abundant at the mMTJs. These molecular events correspond to our previous results on tensile strength.

Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCaT cells used alphavbeta6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFbeta1-treated cells, the untreated cells also needed alpha5beta1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFbeta1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on alphavbeta6 integrin, while alphavbeta1 and alpha5beta1 integrins played a lesser role both in untreated and TGFbeta1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by beta1 integrins, and alphavbeta6 integrin showed a minor role. The migration process appeared to involve a number of beta1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.

In normal adult human skin, expression of epidermal integrins is confined to keratinocytes in the basal layer. However, suprabasal expression of alpha 2, alpha 3 and beta 1 integrin subunits is noted in hyperproliferative epidermis in wound repair and psoriasis. In this study, we examined the effect of topical all-trans-retinoic acid (RA), known to induce epidermal hyperplasia, on expression of integrins in human epidermis. Immunostaining of vehicle-treated skin revealed expression of alpha 2, alpha 3 and beta 1, as well as alpha 6 and beta 4 integrin subunits entirely on basal keratinocytes. Topical application of RA (0.1%) for 2 weeks resulted in marked suprabasal expression of alpha 2, alpha 3 and beta 1 integrin subunits, whereas alpha 6 and beta 4 staining remained on basal keratinocytes. Staining for putative ligands of alpha 2 beta 1 and alpha 3 beta 1 integrins, i.e. type IV collagen, laminin-5 and fibronectin, was not detected in the epidermal layer in RA- or vehicle-treated skin. Treatment of HaCaT keratinocytes in culture with RA (1 mumol/L) enhanced alpha 2 and beta 1 mRNA abundance. Furthermore, RA slightly up-regulated the expression of alpha 2, alpha 3 and beta 1 integrin subunits on primary epidermal keratinocytes and HaCaT cells in culture with no effect on cell proliferation. These results provide evidence that RA-elicited epidermal hyperplasia is associated with aberrant suprabasal expression of alpha 2 beta 1 and alpha 3 beta 1 integrins, and that this also involves direct stimulation of keratinocyte integrin expression by RA.

Induction of immediate-early genes c-jun, junB, and c-fos was demonstrated during echovirus 1 infection in a human osteogenic sarcoma (HOS) cell line. Tenfold induction was seen at 10 h postinfection, corresponding approximately to the end of the first replication cycle of the virus. Echovirus 1 uses VLA-2 integrin as its cellular receptor, and ligand binding by integrin is known to trigger signal transduction pathways ultimately activating immediate-early genes. In the present study, however, VLA-2 binding alone was not sufficient to induce their expression; viral replication was needed. This conclusion was based on the observations that no stimulation of the immediate-early genes occurred in the MG-63 cell line where the virus attached only to VLA-2 but was not able to replicate and that induction of these genes was observed when the HOS cells were infected with echovirus type 7, known to use a different cellular receptor. Induction was not seen in the presence of the antiviral compound WIN 54954, which evidently inhibits the uncoating but not receptor binding of echovirus 1, suggesting that viral replication triggers the activation of the immediate-early genes. The induction of these genes may have a role in viral replication and in the pathogenesis of infection.

Regeneration of periodontal structures is a complex challenge for periodontal wound healing. As by Wikesjo et al. in this issue, wound space provision, primary stability, and healing by primary intention serve as critical factors for attempts to regenerate the periodontium. Barrier membranes for space maintenance can be cumbersome to use in a clinical setting and have proven to provide only partial success in regenerative therapies. Not surprisingly, investigators and clinicians have started to search for biological molecules and cell technologies that could help to either speed up or modify the healing process that depends on migration and differentiation of several different cell populations. In this chapter, we will briefly review the technologies that are currently in use or in clinical trials. Readers are welcome to review in-depth publications for further details on this complex and evolving area of research.

Significance: Regulation of cell adhesions during tissue repair is fundamentally important for cell migration, proliferation, and protein production. All cells interact with extracellular matrix proteins with cell surface integrin receptors that convey signals from the environment into the nucleus, regulating gene expression and cell behavior. Integrins also interact with a variety of other proteins, such as growth factors, their receptors, and proteolytic enzymes. Re-epithelialization and granulation tissue formation are crucially dependent on the temporospatial function of multiple integrins. This review explains how integrins function in wound repair. Recent Advances: Certain integrins can activate latent transforming growth factor beta-1 (TGF-β1) that modulates wound inflammation and granulation tissue formation. Dysregulation of TGF-β1 function is associated with scarring and fibrotic disorders. Therefore, these integrins represent targets for therapeutic intervention in fibrosis. Critical Issues: In...

Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with ameloblast plasma membrane. Although it is known that integrins mediate cell-matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. Thus, we hypothesized that αvβ6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both β6 integrin mRNA and protein. The maxillary incisors of Itgb6−/− mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6−/− mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6−/− enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6−/− mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, which did not use αvβ6 integrin as an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin αvβ6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition/turnover and subsequent enamel biomineralization.

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins α1β1 and α2β1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only α1β1 integrin, MG-63 cells, which express only α2β1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, α1(I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with α2 integrin cDNA produced cell clones overexpressing α2β1 integrin. Transfection of MG-63 cells with α2 integrin cDNA in an antisense orientation reduced the expression level of α2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking α2β1 integrin were forced to express it, and this prevented the down-regulation in the levels of α1(I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor α2β1 integrin. The down-regulation of collagen α1(I) is mediated by another receptor. Integrin α2β1 may compete with it and thus be a positive regulator of collagen synthesis.

Cancer-testis (CT) antigens are predominantly expressed in testis or placenta, but absent in most adult tissues. During malignant transformation CT genes are often activated. CT antigen 16 (CT16, PAGE5) is frequently expressed in advanced melanoma but its biological function has been unknown. To examine the role of CT16 in cell survival we knocked it down in A2058 melanoma cells using specific siRNAs and exposed the cells to cancer drug cisplatin known to induce apoptosis. As a result, cell survival was markedly decreased. To study the effects of CT16 on cell survival in more detail, the cellular gene expression profiles were investigated after CT16 silencing in CT16 positive A2058 melanoma cells, as well as after CT16 overexpression in CT16 negative WM-266-4 melanoma cells. Among the 11 genes both upregulated by CT16 silencing and downregulated by CT16 overexpression or vice versa, 4 genes were potentially apoptotic or antiapoptotic genes. CT16 was recognized as a positive regulator of antiapoptotic metallothionein 2A and interleukin 8 genes, whereas it inhibited the expression of apoptosis inducing dickkopf 1 (DKK1) gene. In addition CT16 enhanced the expression of fatty acid binding protein 7, a known promoter of melanoma progression. The effect of CT16 on DKK1 expression was p53 independent. Furthermore, CT16 did not regulate apoptotic genes via DNA methylation. In twenty melanoma metastasis tissue samples average DKK1 mRNA level was shown to be significantly (p

Cellular receptors for collagens belong to the family of β(1) integrins. In the epidermis, integrin α(2)β(1) is the only collagen-binding integrin present. Its expression is restricted to basal keratinocytes with uniform distribution on the cell surface of those cells. Although α(2)β(1) receptors localized at the basal surface interact with basement membrane proteins collagen IV and laminin 111 and 332, no interaction partners have been reported for these integrin molecules at the lateral and apical membranes of basal keratinocytes. Solid phase binding and surface plasmon resonance spectroscopy demonstrate that collagen XXIII, a member of the transmembrane collagens, directly interacts with integrin α(2)β(1) in an ion- and conformation-dependent manner. The two proteins co-localize on the surface of basal keratinocytes. Furthermore, collagen XXIII is sufficient to induce adhesion and spreading of keratinocytes, a process that is significantly reduced in the absence of functional integrin α(2)β(1).

Cancer-testis antigens (CTAs) are expressed mainly in various cancer tissues and in testis or placenta. Because of their restricted expression pattern, the CTAs can be potentially used for vaccine development and diagnostic applications. CTA CT16 has been found to be expressed in lung and renal cancers as well as in melanomas. Detection of CT16 protein directly from patient serum could facilitate monitoring of tumor growth and response to therapy in CT16-positive patients. A highly sensitive time-resolved fluorescence-based immunoassay measuring CTA CT16 in serum was developed. Generally, CTAs have not been measured directly from body fluids. CT16 level was detectable in 14 of 23 (61%) patients with metastatic melanoma, whereas none of the nine healthy volunteers collected by us had measurable CT16 level. For an unknown reason, 1 of 20 commercial control serum samples gave a positive result. The Wilcoxon-Mann-Whitney exact test showed statistically significant difference when patients with metastatic melanoma were compared to our control group (p = 0.006) or to the commercial set (p < 0.001). Four melanoma patients had exceptionally high serum CT16 level. CT16 did not correlate either with S100B, a recognized marker of progressing melanoma, or with unspecific serum marker lactate dehydrogenase. Elevation of CT16 titers preceded or followed the clinical diagnosis of disease progression in four patients with metastatic melanoma. As a conclusion, our results show that CT16 protein can be measured directly from patient serum, and the developed assay has a potential for clinical use.

The effects of recombinant human Interleukin-1 alpha (IL-1 alpha), Interleukin-1 beta (IL-1 beta), and Tumor Necrosis Factor-alpha (TNF-alpha) on collagen biosynthesis were studied in vitro using dermal fibroblast cultures. Both forms of IL-1 and TNF-alpha induced a dose-dependent inhibition of both types I and III collagen synthesis, as measured by radioimmunoassay, gel electrophoresis, or collagenase-sensitive material. This effect was accompanied by a significant release of prostaglandin E2 into the culture medium. However, indomethacin, a potent inhibitor of prostaglandin synthesis, could not prevent the inhibitory effect of the three cytokines on collagen synthesis. Measurement of type I and type III procollagen mRNA levels in IL-1 treated cells revealed that both IL-1 alpha and IL-1 beta were potent enhancers of procollagen gene expression at pretranslational level. On the other hand, TNF-alpha was found to reduce the steady-state levels of type I and III procollagen mRNA in a dose-dependent manner. Quantitation of IL-1 beta and TNF-alpha transcripts following TNF-alpha treatment of fibroblasts indicated that this cytokine can induce IL-1 beta gene expression in these cells. By contrast, TNF-alpha mRNA remained at a constant level after TNF-alpha exposure. These data suggest that IL-1 and TNF-alpha, two cytokines that share several biologic activities, modulate collagen deposition in dermal fibroblasts by mechanisms that are clearly different: TNF-alpha appears to act at a transcriptional level to inhibit collagen synthesis, whereas IL-1 inhibitory action involves important translational regulation, still unknown, that counterbalances its stimulatory effect on procollagen mRNA levels. Moreover, our data suggest the existence of local fibroblastic cytokine production that may be involved in the modulation of extracellular matrix deposition.

Collagens are large, triple-helical proteins that form fibrils and network-like structures in the extracellular matrix. The collagens may have participated in the evolution of the metazoans from their very earliest origins. Cell adhesion receptors, such as the integrins, are at least as old as the collagens. Still, the early metazoan cells might not have been able to anchor directly to collagen fibrils, since the integrin-type collagen receptors have only been identified in vertebrates. Instead, the early metazoans may have used integrin-type receptors in the recognition of collagen-binding glycoproteins. It is possible that specialized, high-avidity collagen-receptor integrins have become instrumental for the evolution of bone, cartilage, circulatory and immune systems in the chordates. In vertebrates, specific collagen-binding receptor tyrosine kinases send signals into cells after adhesion to collagen. These receptors are members of the discoidin domain receptor (DDR) group. The evolutionary history of DDRs is poorly known at this time. DDR orthologs have been found in many invertebrates, but their ability to function as collagen receptors has not yet been tested. The two main categories of collagens, fibrillar and non-fibrillar, already exist in the most primitive metazoans, such as the sponges. Interestingly, both integrin and DDR families seem to have members that favor either one or the other of these two groups of collagens.