Osteosarcoma Cells Secrete CXCL14 That Activates Integrin α11ß1 on Fibroblasts to Form a Lung Metastatic Niche.

Cooperation between primary malignant cells and stromal cells can mediate the establishment of lung metastatic niches. Here, we characterized the landscape of cell populations in the tumor microenvironment in treatment-naïve osteosarcoma using single-cell RNA sequencing and identified a stem cell-like cluster with tumor cell-initiating properties and prometastatic traits. CXCL14 was specifically enriched in the stem cell-like cluster and was also significantly upregulated in lung metastases compared with primary tumors. CXCL14 induced stromal reprogramming and evoked a malignant phenotype in fibroblasts to form a supportive lung metastatic niche. Binding of CXCL14 to heterodimeric integrin α11ß1 on fibroblasts activated actomyosin contractility and matrix remodeling properties. CXCL14-stimulated fibroblasts produced TGFß and increased osteosarcoma invasion and migration. mAbs targeting the CXCL14-integrin α11ß1 axis inhibited fibroblast TGFß production, enhanced CD8+ T cell-mediated antitumor immunity, and suppressed osteosarcoma lung metastasis. Taken together, these findings identify cross-talk between osteosarcoma cells and fibroblasts that promotes metastasis and demonstrate that targeting the CXCL14-integrin α11ß1 axis is a potential strategy to inhibit osteosarcoma lung metastasis. SIGNIFICANCE: Cooperation between stem-like osteosarcoma cells and fibroblasts mediated by a CXCL14-integrin α11ß1 axis creates a tumor-supportive lung metastatic niche and represents a therapeutic target to suppress osteosarcoma metastasis.

Novel roles for cooperating collagen receptor families in fibrotic niches.

Recent data indicate that integrin and non-integrin collagen receptors cooperate in the fibrosis-specific microenvironment (i.e., the fibrotic niche). In certain tumor types, DDR1 can regulate the interaction with collagen III to regulate dormancy and metastasis, whereas in other tumor types, DDR1 can be shed and used to reorganize collagen. DDR1 expressed on tumor cells, together with DDR2 and α11ß1 integrin expressed on cancer-associated fibroblasts, can increase tumor tissue stiffness. Integrin α1ß1 and α2ß1 are present on immune cells where they together with the immunosuppressive collagen receptor LAIR-1 can mediate binding to intratumor collagens. In summary, collagen-binding integrins together with DDRs, can create fibrillar collagen niches that act as traps to hinder immune cell trafficking into the tumor cell mass. Binding of collagens via LAIR-1 on immune cells in turn results in CD8+T-cell exhaustion. Continued studies of these complex interactions are needed for successful new stroma-based therapeutic interventions. In the current review, we will summarize recent data on collagen receptors with a special focus on their potential role in tumor fibrosis and highlight their collaborative roles in tumor fibrotic niches.

PACSIN2 regulates platelet integrin ß1 hemostatic function.

BACKGROUND: Upon vessel injury, platelets adhere to exposed matrix constituents via specific membrane receptors, including the von Willebrand factor receptor glycoprotein (GP)Ib-IX-V complex and integrins ß1 and ß3. In platelets, the Fes/CIP4-homology Bin-Amphiphysin-Rvs protein PACSIN2 associates with the cytoskeletal and scaffolding protein filamin A (FlnA), linking GPIbα and integrins to the cytoskeleton. OBJECTIVES: Here we investigated the role of PACSIN2 in platelet function. METHODS: Platelet parameters were evaluated in mice lacking PACSIN2 and platelet integrin ß1. RESULTS: Pacsin2-/- mice displayed mild thrombocytopenia, prolonged bleeding time, and delayed thrombus formation in a ferric chloride-mediated carotid artery injury model, which was normalized by injection of control platelets. Pacsin2-/- platelets formed unstable thrombi that embolized abruptly in a laser-induced cremaster muscle injury model. Pacsin2-/- platelets had hyperactive integrin ß1, as evidenced by increased spreading onto surfaces coated with the collagen receptor α2ß1-specific peptide GFOGER and increased binding of the antibody 9EG7 directed against active integrin ß1. By contrast, Pacsin2-/- platelets had normal integrin αIIbß3 function and expressed P-selectin normally following stimulation through the collagen receptor GPVI or with thrombin. Deletion of platelet integrin ß1 in Pacsin2-/- mice normalized platelet count, hemostasis, and thrombus formation. A PACSIN2 peptide mimicking the FlnA-binding site mediated the pull-down of a FlnA rod 2 construct by integrin ß7, a model for integrin ß-subunits. CONCLUSIONS: Pacsin2-/- mice displayed severe thrombus formation defects due to hyperactive platelet integrin ß1. The data suggest that PACSIN2 binding to FlnA negatively regulates platelet integrin ß1 hemostatic function.

An update on novel therapies for treating patients with arterial thrombosis.

INTRODUCTION: Antithrombotic therapy field is undergoing rapid and significant changes during the past decade. In addition to new therapeutic strategies with existing targets, investigators are exploring the potential use of new targets to address unmet needs to treat patients with arterial diseases. AREAS COVERED: We aim to provide an update on and a comprehensive review of the antithrombic agents that are being explored in patients with arterial diseases. We discuss latest developments with respect to upstream antiplatelet agents, and collagen and thrombin pathway inhibitors. We searched PubMed databases for English language articles using keywords: antiplatelet agents, thrombin pathway inhibitors, collagen receptors, arterial disease. EXPERT OPINION: Despite implementation of potent P2Y12 inhibitors, there are numerous unmet needs in the treatment of arterial diseases including ceiling effect of currently available antiplatelet agents along with and an elevated risk of bleeding. The latter observations encouraged investigators to explore new targets that can attenuate the generation of platelet-fibrin clot formation and subsequent ischemic event occurrences with minimal effect on bleeding. These targets include collagen receptors on platelets and thrombin generation including FXa, FXIa, and FXIIa. In addition, investigators are studying novel antiplatelet agents/strategies to facilitate upstream therapy in high-risk patients.

Control of craniofacial development by the collagen receptor, discoidin domain receptor 2.

Development of the craniofacial skeleton requires interactions between progenitor cells and the collagen-rich extracellular matrix (ECM). The mediators of these interactions are not well-defined. Mutations in the discoidin domain receptor 2 gene (DDR2), which encodes a non-integrin collagen receptor, are associated with human craniofacial abnormalities, such as midface hypoplasia and open fontanels. However, the exact role of this gene in craniofacial morphogenesis is not known. As will be shown, Ddr2-deficient mice exhibit defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis and delayed ossification at growth plate synchondroses. These defects were associated with abnormal collagen fibril organization, chondrocyte proliferation and polarization. As established by localization and lineage-tracing studies, Ddr2 is expressed in progenitor cell-enriched craniofacial regions including sutures and synchondrosis resting zone cartilage, overlapping with GLI1 + cells, and contributing to chondrogenic and osteogenic lineages during skull growth. Tissue-specific knockouts further established the requirement for Ddr2 in GLI +skeletal progenitors and chondrocytes. These studies establish a cellular basis for regulation of craniofacial morphogenesis by this understudied collagen receptor and suggest that DDR2 is necessary for proper collagen organization, chondrocyte proliferation, and orientation.

We each have unique facial features that are key to our identities. These features are inherited, but the mechanisms are poorly understood. People with the genetic disease spondylo-meta-epiphyseal dysplasia, or SMED, have characteristic facial and skull abnormalities including a flattened face and shortened skull. SMED is associated with mutations that inactivate the gene encoding a protein called discoidin domain receptor 2 (DDR2), which is a receptor for collagen. Collagen is the major structural protein in the human body, supporting the structure of cells and tissues. It also controls cell behaviors including growth, migration and differentiation, and it helps form tissues such as cartilage or bone. At least some of the effects of collagen on cells depend on its interaction with DDR2. Since the facial and skull abnormalities in mice with mutations that stop DDR2 from working correctly resemble those of SMED patients, these mice can be used to understand the cellular basis for this disease, as well as the role of DDR2 in the embryonic development of the face and skull. Therefore, Mohamed et al. set out to understand how loss of DDR2 causes the characteristic facial and skull defects associated with SMED. Mohamed et al. used mice that had been genetically modified so that DDR2 could be inactivated in skeletal progenitor cells, cartilage cells and bone cells (osteoblasts). Examining these mice, they found that the shortened skulls and flat face characteristic of mice lacking DDR2 are due to bones at the skull base failing to elongate correctly due to defects in the growth centers that depend on cartilage. Mohamed et al. also discovered that the cells that normally produce DDR2 are the progenitors of cartilage and bone-forming cells, which partly explains why lacking this protein leads to issues in growth of these tissues. In addition to shedding light on the causes of SMED, Mohamed et al.'s results also provide general insights into the mechanisms controlling the formation of facial and skull bones that depend on interactions between cells and collagen. This information may help explain how other abnormalities in the face and skull emerge, and provide a basis for how the shape of the skull has changed during human evolution. In the future, it may be possible to manipulate the activity of DDR2 to correct skull defects.

Platelets modulate cardiac remodeling via the collagen receptor GPVI after acute myocardial infarction.

Introduction: Platelets play an important role in cardiovascular diseases. After acute myocardial infarction, platelets display enhanced activation and migrate into the infarct zone. Furthermore, platelets trigger acute inflammation and cardiac remodeling leading to alterations in scar formation and cardiac function as observed in thrombocytopenic mice. GPVI is the major collagen receptor in platelets and important for platelet activation and thrombus formation and stability. Antibody induced deletion of GPVI at the platelet surface or treatment of mice with recombinant GPVI-Fc results in reduced inflammation and decreased infarct size in a mouse model of AMI. However, the role of GPVI has not been fully clarified to date. Methods/Results: In this study, we found that GPVI is not involved in the inflammatory response in experimental AMI using GPVI deficient mice that were analyzed in a closed-chest model. However, reduced platelet activation in response to GPVI and PAR4 receptor stimulation resulted in reduced pro-coagulant activity leading to improved cardiac remodeling. In detail, GPVI deficiency in mice led to reduced TGF-ß plasma levels and decreased expression of genes involved in cardiac remodeling such as Col1a1, Col3a1, periostin and Cthrc1 7 days post AMI. Consequently, collagen quality of the scar shifted to more tight and less fine collagen leading to improved scar formation and cardiac function in GPVI deficient mice at 21d post AMI. Conclusion: Taken together, this study identifies GPVI as a major regulator of platelet-induced cardiac remodeling and supports the potential relevance of GPVI as therapeutic target to reduce ischemia reperfusion injury and to improve cardiac healing.

Collagen Type XI Inhibits Lung Cancer-Associated Fibroblast Functions and Restrains the Integrin Binding Site Availability on Collagen Type I Matrix.

The tumor microenvironment, including cancer-associated fibroblast (CAF), plays an active role in non-small cell lung cancer (NSCLC) development and progression. We previously reported that collagen type XI and integrin α11, a collagen receptor, were upregulated in NSCLC; the latter promotes tumor growth and metastasis. We here explored the role of collagen type XI in NSCLC stroma. We showed that the presence of collagen type XI in collagen type I matrices inhibits CAF-mediated collagen remodeling and cell migration. This resulted in the inhibition of CAF-dependent lung-tumor cell invasion. Among the collagen receptors expressed on CAF, we determined that DDR2 and integrin α2ß1, but not integrin α11ß1, mediated the high-affinity binding to collagen type XI. We further demonstrated that collagen type XI restrained the integrin binding site availability on collagen type I matrices, thus limiting cell interaction with collagen type I. As a consequence, CAFs failed to activate FAK, p38 and Akt one hour after they interacted with collagen type I/XI. We concluded that collagen type XI may have a competitive negative feedback role on the binding of collagen type I to its receptors.

Anti-GPVI Fab reveals distinct roles for GPVI signaling in the first platelet layer and subsequent layers during microfluidic clotting on collagen with or without tissue factor.

The collagen receptor glycoprotein VI (GPVI) drives strong platelet activation, however its role at later stages of clotting remains less clear. Controlled timing of addition of anti-human GPVI Fab (clone E12) with microfluidic venous whole blood flow over collagen (± lipidated tissue factor, TF) produced distinct effects on platelets, fibrin, P-selectin exposure, and phosphatidylserine (PS) exposure. On collagen alone, Fab present initially potently reduced platelet deposition on collagen, while Fab added 90 s after initial platelet deposition, stopped subsequent platelet accumulation (despite the absence of fibrin). With thrombin generation via TF, Fab added at either t = 0 or 90 s had no effect on platelet deposition. However, Fab added initially, but not at 90-s, blocked fibrin formation. Gly-Pro-Arg-Pro ablated fibrin formation without effect on platelet accumulation (regardless of Fab added at t = 0 or 90 s), indicating thrombin signaling can suffice over GPVI signaling. Still, Fab moderately reduced P-selectin exposure with thrombin present and fibrin absent. On collagen/TF, Fab present initially ablated PS exposure, but had no effect when added 30 to 90-s later. The thrombin generated via PS exposure had an important role in driving platelet deposition in the presence of Fab, since inhibition of PS via annexin V binding in the presence of Fab significantly inhibited platelet deposition. We conclude GPVI signaling in the first platelet layer on collagen dictates thrombin and fibrin production, but the role of GPVI at subsequent times after formation of the first monolayer is obscured by thrombin-induced signaling.

Integrin α11ß1 and syndecan-4 dual receptor ablation attenuate cardiac hypertrophy in the pressure overloaded heart.

Pathological myocardial hypertrophy in response to an increase in left ventricular (LV) afterload may ultimately lead to heart failure. Cell surface receptors bridge the interface between the cell and the extracellular matrix (ECM) in cardiac myocytes and cardiac fibroblasts and have been suggested to be important mediators of pathological myocardial hypertrophy. We identify for the first time that integrin α11 (α11) is preferentially upregulated among integrin ß1 heterodimer-forming α-subunits in response to increased afterload induced by aortic banding (AB) in wild-type (WT) mice. Mice were anesthetized in a chamber with 4% isoflurane and 95% oxygen before being intubated and ventilated with 2.5% isoflurane and 97% oxygen. For pre- and postoperative analgesia, animals were administered 0.02-mL buprenorphine (0.3 mg/mL) subcutaneously. Surprisingly, mice lacking α11 develop myocardial hypertrophy following AB comparable to WT. In the mice lacking α11, we further show a compensatory increase in the expression of another mechanoreceptor, syndecan-4, following AB compared with WT AB mice, indicating that syndecan-4 compensated for lack of α11. Intriguingly, mice lacking mechanoreceptors α11 and syndecan-4 show ablated myocardial hypertrophy following AB compared with WT mice. Expression of the main cardiac collagen isoforms col1a2 and col3a1 was significantly reduced in AB mice lacking mechanoreceptors α11 and syndecan-4 compared with WT AB.NEW & NOTEWORTHY Despite their putative importance in stress sensing, the specific integrin α-subunit(s) involved in cardiac hypertrophy has not been identified. Here, we show that α11 and syndecan-4 are critical and interdependent mediators of the hypertrophic response to increased LV afterload. We demonstrate in cells lacking both receptors an interdependent reduction in cell attachment to the major cardiac extracellular matrix components, suggesting that their interplay represents an important mechanism for stress sensing in cardiac cells.

Osteoporosis and its Association with Vitamin D Receptor, Oestrogen α Receptor, Parathyroid Receptor and Collagen Type I alpha Receptor Gene Polymorphisms with Bone Mineral Density: A Pilot Study from South Indian Postmenopausal Women of Tamil Nadu.

The involvement of many putative genetic factors makes osteoporosis a complex disease. With increasing longevity of the Indian population, it's now being realized that, as within the West, osteoporotic fractures are also a significant explanation for morbidity and mortality in postmenopausal women. Studies have suggested that the genetic component liable for bone mass could be linked to single nucleotide polymorphisms. Therefore, this study is aimed to research the role of seven gene polymorphisms previously associated with bone phenotype in a cohort of postmenopausal South Indian women from Tamil Nadu. The subjects for the study (n = 300) included 100 osteoporotic women (age 59.3 ± 9.26), 100 osteopenic women (age 55.6 ± 8.17) and 100 non-osteoporotic women as controls (age 55.4 ± 8.85).Genetic polymorphisms were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Case-control genetic association analysis of BsmI of the VDR and BstBI of the PTH gene showed a significant allelic association with low bone mineral density amongst the osteoporotic postmenopausal women. The association of BMD with the VDR gene polymorphisms revealed that the average BMD in the BsmI polymorphism with the recessive genotype GG in osteoporotic women was significantly reduced compared with the average BMD in osteoporotic women with AA and AG genotypes. In the BstBI polymorphism, the BMD in the osteoporotic subjects were significantly lower in the AA group than in the GA and GG groups. These results provide evidence for an independent association between BMD and rs1544410 in VDR and rs6254 in PTH and may contribute in being a possible genetic marker for predicting the disease susceptibility in the population tested.

Decreased levels of endocytic collagen receptor Endo180 in dermal fibroblasts lead to decreased production of type I collagen and increased expression of matrix metalloproteinase-1.

BACKGROUND: Endo180 is involved in collagen remodeling by incorporating extracellular degraded collagen. Ultraviolet irradiation of dermal fibroblasts reduces Endo180 expression, which affects collagen fiber remodeling. However, it is unclear whether the decrease in Endo180 is directly related to the decrease in type I collagen fibers during photoaging. We aimed to clarify the relationship between Endo180 reduction and the decrease in type I collagen fibers observed in photoaged dermis. METHODS: Endo180 was reduced in normal human dermal fibroblasts using RNAi. Endo180 knockdown cells were inoculated into collagen gels. The influence of Endo180 knockdown was evaluated by measuring mRNA expression of collagen fiber remodeling-related factors and collagen gel contraction. The collagen state and oxidative stress in the collagen gels were also measured. RESULTS: Endo180 knockdown cells, which were confirmed by gelatin uptake inhibition, showed upregulation of matrix metalloproteinase-1 and downregulation of type I collagen mRNA expression when cultured in collagen gels. The contractility of the collagen gel was reduced by Endo180 knockdown. The collagen state in the extracellular matrix of the collagen gels containing Endo180 knockdown fibroblasts showed increased amounts of 3/4 fragmented collagen and denatured collagen and decreased type I collagen synthesis. In addition, an increase in intracellular oxidative stress was observed. CONCLUSIONS: This study confirmed that the decrease in Endo180 caused a failure in collagen fiber formation and a decrease in collagen production, reproducing the photoaging dermal structural changes. This suggests that the decrease in Endo180 may be involved in wrinkle formation, which is a characteristic of photoaged skin.

Profiling oxylipins released from human platelets activated through the GPVI collagen receptor.

In addition to haemostasis, platelets are involved in pathological processes, often driven by material released upon activation. Interaction between collagen and glycoprotein VI (GPVI) is a primary platelet stimulus that liberates arachidonic acid and linoleic acid from membrane phospholipids. These are oxidised by cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) to eicosanoids and other oxylipins with various biological properties. Using liquid chromatography-tandem mass spectrometry we found that GPVI-stimulated platelets released significant levels of ten oxylipins; the well documented TxA2 and 12-HETE, PGD2 and PGE2, as well as 8-, 9-, 11-, and 15-HETE, 9- and 13-HODE.1 Levels of oxylipins released from washed platelets mirrored those from platelets stimulated in the presence of plasma, indicating generation from intracellular, rather than exogenous AA/LA. Inhibition of COX-1 with aspirin, as expected, completely abolished production of TxA2 and PGD/E2, but also significantly inhibited the release of 11-HETE (89 ± 3%) and 9-HODE (74 ± 6%), and reduced 15-HETE and 13-HODE by ∼33 %. Inhibition of 12-LOX by either esculetin or ML355 inhibited the release of all oxylipins apart from 15-HETE. These findings suggest routes to modify the production of bioactive molecules released by activated platelets.

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8ß1 and α11ß1, Induced Specifically on Activated Stellate Cells.

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvß1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFß activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, "disease specificity" has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbß3 on platelets, α4ß1, α4ß7 and αLß2 on leukocytes. Herein, "disease specific" integrins would serve as attractive targets. α8ß1 and α11ß1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather "pathology specific" nature of these new integrins. The monoclonal antibodies against α8ß1 and α11ß1 in preclinical examinations may illuminate the road to the first medical agents.

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target.

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients' asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.

Colchicine inhibits ROS generation in response to glycoprotein VI stimulation.

Colchicine inhibits coronary and cerebrovascular events in patients with coronary artery disease (CAD), and although known to have anti-inflammatory properties, its mechanisms of action are incompletely understood. In this study, we investigated the effects of colchicine on platelet activation with a particular focus on its effects on activation via the collagen glycoprotein (GP)VI receptor, P2Y12 receptor, and procoagulant platelet formation. Therapeutic concentrations of colchicine in vitro (equivalent to plasma levels) significantly decreased platelet aggregation in whole blood and in platelet rich plasma in response to collagen (multiplate aggregometry) and reduced reactive oxygen species (ROS) generation (H2DCF-DA, flow cytometry) in response to GPVI stimulation with collagen related peptide-XL (CRP-XL, GPVI specific agonist). Other platelet activation pathways including P-selectin expression, GPIIb/IIIa conformational change and procoagulant platelet formation (GSAO+/CD62P+) (flow cytometry) were inhibited with higher concentrations of colchicine known to inhibit microtubule depolymerization. Pathway specific mechanisms of action of colchicine on platelets, including modulation of the GPVI receptor pathway at low concentrations, may contribute to its protective role in CAD.

Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen.

OBJECTIVE: The platelet collagen receptor glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced via the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI-dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions. METHODS AND RESULTS: ELISA using biotinyl-fibrinogen immobilized on streptavidin-coated wells indicated that GPVI dimers do not bind intact fibrinogen. Clots were formed by adding thrombin to a mixture of near-plasma level of fibrinogen and recombinant GPVI ectodomain: GPVI dimer (GPVI-Fc2 or Revacept) or monomer (GPVI-His: single chain of Revacept GPVI domain, with His tag). Clot-bound proteins were analyzed by SDS-PAGE/immunoblotting. GPVI-dimer bound to noncrosslinked fibrin clots with classical one-site binding kinetics, with µM-level KD , and to crosslinked clots with higher affinity. Anti-GPVI-dimer (mFab-F) inhibited the binding. However, GPVI-His binding to either type of clot was nonsaturable and nearly linear, indicating very low affinity or nonspecific binding. In clots formed in the presence of platelets, clot-bound platelet-derived proteins were integrin αIIbß3, present at high levels, and GPVI. CONCLUSIONS: We conclude that dimeric GPVI is the receptor for fibrin, exhibiting a similar KD to those obtained for its binding to fibrinogen D-fragment and D-dimer, suggesting that fibrin(ogen)'s GPVI-binding site becomes exposed after fibrin formation or cleavage to fragment D. Analysis of platelets bound to fibrin clots indicates that platelet GPVI binds to fibrin fibers comprising the clot.

The expression of two collagen receptor subfamilies, integrins and discoidin domains during osteogenic and chondrogenic differentiation of human mesenehymal stem cells.

BACKGROUND: Collagen receptors are characterized by binding to and being activated by collagens. We know little about the molecular mechanism by which the integrins and discoidin domains (DDRs) recognize collagen. OBJECTIVE: The aim of this study was to investigate the expression of two main collagen receptor subfamilies, integrins and DDRs, during osteogenic and chondrogenic differentiation of human mesenehymal stem cells (hMSCs). METHODS: Using qRT-PCR, Western blots and FACS, the levels of DDR1, DDR2, integrin subunits ß1, α1, α2, α10 and α11 receptors on hMSCs, were assessed upon activation by collagen type I, as well as during osteogenic and chondrogenic differentiation. RESULTS: The expression of DDR2 and integrin α11ß1 was altered compared with other receptors when the cells were cultured under undifferentiated conditions. During osteogenic and chondrogenetic differentiation, DDR2 and α11 were up-regulated during early stages (6 day) of osteogenesis and chondrogenesis, respectively. The expression and activation of DDR2 was concomitant with another receptor integrin subunit ß1 during osteogenetic differentiation. CONCLUSIONS: The results suggested that DDR2 was more specific for osteogenesis than chondrogenesis, while integrin α11ß1 was more specific in chondrogenesis. DDR2 and α11 may play a role in the regulation of osteogenesis and chondrogenesis based on the differential expression of these receptors during lineage-dependent changes.

Integrin α11ß1 is a receptor for collagen XIII.

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 α11ß1 is a collagen receptor known to recognize the GFOGER (O=hydroxyproline) sequence in collagens. Interestingly, collagen XIII and integrin α11ß1 both have a role in the regulation of bone homeostasis. To study whether α11ß1 is a receptor for collagen XIII, we utilized C2C12 cells transfected to express α11ß1 as their only collagen receptor. The interaction between collagen XIII and integrin α11ß1 was also confirmed by surface plasmon resonance and pull-down assays. We discovered that integrin α11ß1 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 α11ß1-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 α11ß1 as a new collagen XIII receptor and demonstrates that this ligand-receptor pair has a role in the maintenance of bone homeostasis.