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.

Integrin α11ß1 regulates cancer stromal stiffness and promotes tumorigenicity and metastasis in non-small cell lung cancer.

Integrin α11ß1 is a stromal cell-specific receptor for fibrillar collagens and is overexpressed in carcinoma-associated fibroblasts (CAFs). We have investigated its direct role in cancer progression by generating severe combined immune deficient (SCID) mice deficient in integrin α11 (α11) expression. The growth of A549 lung adenocarcinoma cells and two patient-derived non-small cell lung carcinoma (NSCLC) xenografts in these α11 knockout (α11(-/-)) mice was significantly impeded, as compared with wild-type (α11(+/+)) SCID mice. Orthotopic implantation of a spontaneously metastatic NCI-H460SM cell line into the lungs of α11(-/-) and α11(+/+) mice showed significant reduction in the metastatic potential of these cells in the α11(-/-) mice. We identified that collagen cross-linking is associated with stromal α11 expression, and the loss of tumor stromal α11 expression was correlated with decreased collagen reorganization and stiffness. This study shows the role of integrin α11ß1, a receptor for fibrillar collagen in differentiation of fibroblasts into CAFs. Furthermore, our data support an important role for α11 signaling pathway in CAFs, promoting tumor growth and metastatic potential of NSCLC cells and being closely associated with collagen cross-linking and the organization and stiffness of fibrillar collagen matrices.

Reduced granulation tissue and wound strength in the absence of α11ß1 integrin.

Previous wound healing studies have failed to define a role for either α1ß1 or α2ß1 integrin in fibroblast-mediated wound contraction, suggesting the involvement of another collagen receptor in this process. Our previous work demonstrated that the integrin subunit α11 is highly induced during wound healing both at the mRNA and protein level, prompting us to investigate and dissect the role of the integrin α11ß1 during this process. Therefore, we used mice with a global ablation of either α2 or α11 or both integrin subunits and investigated the repair of excisional wounds. Analyses of wounds demonstrated that α11ß1 deficiency results in reduced granulation tissue formation and impaired wound contraction, independently of the presence of α2ß1. Our combined in vivo and in vitro data further demonstrate that dermal fibroblasts lacking α11ß1 are unable to efficiently convert to myofibroblasts, resulting in scar tissue with compromised tensile strength. Moreover, we suggest that the reduced stability of the scar is a consequence of poor collagen remodeling in α11(-/-) wounds associated with defective transforming growth factor-ß-dependent JNK signaling.

Integrin α11ß1: a major collagen receptor on fibroblastic cells.

Integrin α11 is the last addition to the vertebrate integrin family. In this chapter we will summarize some basic facts about this integrin and update with information that has been gained in the last decade. Integrin α11ß1 is a major collagen receptor on a subset of fibroblasts. Extensive characterization of the expression pattern in developing mouse embryos has demonstrated expression restricted to subsets of fibroblasts and a transient expression in odontoblasts, but comprehensive characterization of corresponding expression in adult tissues is still lacking. Mice lacking integrin α11 are dwarfed, primarily due to defective incisor eruption defect, which can be traced back to need for α11 on periodontal ligament fibroblasts during incisor eruption. Separate studies have suggested reduced levels of IGF-1 in mice lacking α11. Analysis of lung cancer has identified α11ß1 as a functional important collagen receptor on carcinoma associated fibroblasts (CAFs) and a number of disease models are awaiting analysis to see the importance of this collagen receptor in pathological models.

von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbß3.

von Willebrand disease type 2B (vWD-type 2B) is characterized by gain-of-function mutations in von Willebrand factor (vWF) that enhance its binding to the glycoprotein Ib-IX-V complex on platelets. Patients with vWD-type 2B have a bleeding tendency that is linked to loss of vWF multimers and/or thrombocytopenia. In this study, we uncovered evidence that platelet dysfunction is a third possible mechanism for bleeding tendency. We found that platelet aggregation, secretion, and spreading were diminished due to inhibition of integrin αIIbß3 in platelets from mice expressing a vWD-type 2B-associated vWF (vWF/p.V1316M), platelets from a patient with the same mutation, and control platelets pretreated with recombinant vWF/p.V1316M. Impaired platelet function coincided with reduced thrombus growth. Further, αIIbß3 activation and activation of the small GTPase Rap1 were impaired by vWF/p.V1316M following exposure to platelet agonists (thrombin, ADP, or convulxin). Conversely, thrombin- or ADP-induced Ca2+ store release, which is required for αIIbß3 activation, was normal, indicating that vWF/p.V1316M acts downstream of Ca2+ release and upstream of Rap1. We found normal Syk phosphorylation and PLCγ2 activation following collagen receptor signaling, further implying that vWF/p.V1316M acts directly on or downstream of Ca2+ release. These data indicate that the vWD-type 2B mutation p.V1316M is associated with severe thrombocytopathy, which likely contributes to the bleeding tendency in vWD-type 2B.

α2ß1 integrin regulates Th17 cell activity and its neutralization decreases the severity of collagen-induced arthritis.

Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently shown that α2ß1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of α2ß1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that α2ß1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of α2ß1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF-κB ligand levels and osteoclast numbers, and reduction of bone loss. We further show that α2ß1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor-related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that α2ß1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.

Role of integrins in the periodontal ligament: organizers and facilitators.

The periodontal ligament is the tissue that connects teeth to bone. The periodontal ligament is a fascinating tissue from a cell biologist's point of view, and because of its special properties and stem-cell content it has also come into the limelight in emerging fields of regenerative medicine. An increased range of genetically modified mouse models offer new tools for studying molecular mechanisms of tooth development. However, owing to species-specific organization of the tooth apparatus, the use of genetic animal models to study the role of the periodontal ligament in normal human tooth physiology and tooth pathology is challenging.

A tale of two collagen receptors, integrin ß1 and discoidin domain receptor 1, in epithelial cell differentiation.

As increase in collagen deposition is no longer taken as simply a consequence but, rather, an inducer of disease progression; therefore, the understanding of collagen signal transduction is fundamentally important. Cells contain at least two types of collagen receptors: integrins and discoidin domain receptors (DDRs). The integrin heterodimers α(1)ß(1), α(2)ß(1), α(10)ß(1), and α(11)ß(1) are recognized as the non-tyrosine kinase collagen receptors. DDR1 and 2, the tyrosine kinase receptors of collagen, are specifically expressed in epithelium and mesenchyme, respectively. While integrin ß(1) and DDR1 are both required for cell adhesion on collagen, their roles in epithelial cell differentiation during development and disease progression seem to counteract each other, with integrin ß(1) favoring epithelium mesenchyme transition (EMT) and DDR1 inducing epithelial cell differentiation. The in vitro evidence shows that the integrin ß(1) and DDR1 exert opposing actions in regulation of membrane stability of E-cadherin, which itself is a critical regulator of epithelial cell differentiation. Here, we review the functional roles of integrin ß(1) and DDR1 in regulation of epithelial cell differentiation during development and disease progression, and explore the underlining mechanisms regarding to the regulation of membrane stability of E-cadherin.

An acquired defect associated with abnormal signaling of the platelet collagen receptor glycoprotein VI.

INTRODUCTION: Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. AIM: Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. METHODS: Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. RESULTS: Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an ∼40-kDa Syk fragment. CONCLUSIONS: These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.

Dense fibrillar collagen matrices to analyse extracellular matrix receptor function.

AIM: The goal of this study was to understand whether dense fibrillar collagen matrices, with a hierarchical structure resembling native collagen matrices, could be useful to study collagen receptor function, in a more physiological context. The receptor analysed here was integrin α11ß1, already shown to be involved in cell attachment and migration on collagen-coated plastic, and also in contraction of loose fibrillar collagen hydrogels. MATERIALS AND METHODS: Collagen matrices prepared here corresponded to dense fibrillar hydrogels concentrated at 5mg/ml. The behaviour of α11ß1 deficient fibroblasts seeded on these concentrated matrices was assessed in terms of adhesion, morphology and migration, then compared to that observed on classical hydrogels at 1mg/ml, corresponding to loose collagen matrices. RESULTS: Short-term attachment assays showed disturbed interactions between α11ß1 deficient cells and collagen matrices in a concentration-dependent manner. Long-term assays revealed reduced cell spreading of alpha 11(-/-) cells on the dense collagen matrices, associated with a disturbed cytoskeleton network. Moreover, anoikis was observed when alpha 11(-/-) cells were seeded on 5mg/ml matrices, and not on looser 1mg/ml matrices. In scratch wound in vitro assays, carried out with cells on 5mg/ml fibrillar collagen matrices, alpha 11(-/-) cells migrated much better than their wild-type counterparts. In contrast, no significant difference was observed between wild and knock-out cells seeded on plastic. CONCLUSIONS: The present study demonstrates the validity of in vivo-like dense fibrillar collagen matrices to evaluate cell receptor functions more significantly than with 2D cell cultures or loose hydrogels.

[Congenital thrombocytopathies]. / Angeborene Thrombozytenfunktionsstörungen.

Inherited thrombocytopathies are much less frequent in comparison to acquired platelet function disorders. However, congenital disorders can lead to severe bleeding tendency and are often not diagnosed. They are induced by different platelet defects based on disorders of platelet adhesion, receptors, secretion and signal transduction. In some cases they are associated with thrombocytopenia, giant platelets and various comorbidities. This article gives an overview regarding diverse defects, their diagnosis and treatment options.

Extracellular matrix-induced transforming growth factor-beta receptor signaling dynamics.

Matrix remodeling, degradation, inflammation and invasion liberate peptide fragments that can subsequently interact with cells in an attachment-independent manner. Such 'soluble' matrix components, including collagens, fibronectin and laminin, induced Smad activation (termed crosstalk signaling), which follows a similar chronological sequence and R-Smad specificity as induced by transforming growth factor (TGF)-beta1. Smad4 nuclear translocation occurred in response to collagen binding, indicating downstream signal propagation. TGF-beta scavenging antibody affected only TGF-beta1, but not crosstalk-induced responses. TGF-beta type II receptor mutation (DR26Delta25), which is deficient in TGF-beta type I receptor recruitment to the ligand, induced a heterotetramer signaling complex, and propagated Smad2 activation only through collagen induction and not TGF-beta signaling. Consequentially, TGF-beta ligand participation is not required for crosstalk signaling. This signaling requires a functional integrin beta1 receptor as showed by RNA interference. Co-immunoprecipitation (co-IP) and fluorescent microscopy indicate the involvement of focal adhesion kinase (FAK) and Src activity in collagen-induced signal propagation, and suggest a membrane signaling complex formation that includes both TGF-beta receptors and integrins. The related gene expressional responses are distinct from that evoked by TGF-beta1, supporting its separate function. This signaling mechanism expands and partially explains TGF-beta receptor dynamics and consequential signaling diversity-related gene expressional plasticity.

A role for alpha11beta1 integrin in the human periodontal ligament.

We previously demonstrated a role for alpha11beta1 integrin in periodontal ligament (PDL)-driven tooth eruption in the mouse. To explore a possible role for alpha11beta1 in the human periodontium, we have characterized the expression and function of alpha11 in human PDL tissue, in human PDL fibroblasts (hPDLF), and in human gingival fibroblasts (hGF). alpha11 expression was detected in PDL tissue, in hPDLF, and in hGF cells. Platelet-derived growth factor-BB and insulin-like growth factor II stimulated contraction of collagen lattices by both types of fibroblasts. alpha2 integrin blocking antibodies and the use of alpha11 siRNA demonstrated a role for both alpha2beta1 and alpha11beta1 in collagen lattice remodeling. Analysis of the proximal ITGA11 promoter from persons with chronic periodontal disease failed to reveal any polymorphism. Analysis of our data shows that alpha11beta1 is a major collagen receptor on cultured human PDL cells and implies that it is also functionally important in the PDL in vivo.

The inhibitory collagen receptor LAIR-1 (CD305).

The immune system protects the body from invaders such as viruses and bacteria. Immune cells must be activated in the correct context to function properly. It is critical that the receptors, costimulatory molecules, and cytokines that orchestrate this activation are carefully regulated to prevent uncontrolled inflammation and autoimmunity. Inhibitory receptors play an important role in regulation of immune cell function, usually upon interaction with ligands present on other cells. In contrast, the function of the inhibitory leukocyte-associated Ig-like receptor (LAIR)-1 can be regulated by extracellular matrix collagens. LAIR-1 is expressed on most cells of the immune system, and its function has been studied on multiple cell types. This review summarizes current literature about LAIR-1, a receptor that potentially is able to regulate multiple steps of an immune response.

Platelet collagen receptors, signaling and antagonism: emerging approaches for the prevention of intravascular thrombosis.

Collagen, one of the major proteins of sub-endothelial vasculature get exposed following endothelium denudement, is a potent stimulator of platelet adhesion and aggregation. Adhesion of platelets following endothelial injury is the primary event usually associated with uncontrolled platelet activation culminating into intravascular thrombosis, thus needs to be intervened to prevent the pathology related to various peripheral, myocardial and cerebral ischemic episodes. Recent advances in the understanding of collagen mediated platelet adhesion and aggregation have led to the identification of two prominent receptors, glycoprotein Ia/IIa (GPIa/IIa or integrin alpha(2)beta(1)) and glycoprotein VI (GPVI) and associated intracellular signaling, which are undoubtedly the new emerging targets for the development of more effective antithrombotic drugs. The optimism for collagen antagonism is based on results obtained so far by the use of monoclonal and polyclonal antibodies, peptide inhibitors, knockouts models and collagen-mimetics in various in vitro test systems and animal models. These findings have revealed that collagen receptor inhibition is an attractive and secure strategy for the new drug development to prevent intravascular thrombosis.

Physiology and pathology of collagen receptors.

Just before the transition from pre-genomic to the post-genomic era, the two latest members of the mammalian integrin family were identified. These integrins, which were named alpha10beta1 and alpha11beta1, are both collagen receptors and are related. Rather than being twins, they can be regarded as close cousins. They both belong to the subfamily of integrins that contain an I-domain in the alpha subunit. This domain is also the part that endows these integrins with the capacity to bind the GFOGER sequence in collagens. In the current review, we summarize and update the current knowledge about the in vitro and in vivo functions of these integrins.

Adhesion mechanisms in platelet function.

Platelet adhesion is an essential function in response to vascular injury and is generally viewed as the first step during which single platelets bind through specific membrane receptors to cellular and extracellular matrix constituents of the vessel wall and tissues. This response initiates thrombus formation that arrests hemorrhage and permits wound healing. Pathological conditions that cause vascular alterations and blood flow disturbances may turn this beneficial process into a disease mechanism that results in arterial occlusion, most frequently in atherosclerotic vessels of the heart and brain. Besides their relevant role in hemostasis and thrombosis, platelet adhesive properties are central to a variety of pathophysiological processes that extend from inflammation to immune-mediated host defense and pathogenic mechanisms as well as cancer metastasis. All of these activities depend on the ability of platelets to circulate in blood as sentinels of vascular integrity, adhere where alterations are detected, and signal the abnormality to other platelets and blood cells. In this respect, therefore, platelet adhesion to vascular wall structures, to one another (aggregation), or to other blood cells, represent different aspects of the same fundamental biological process. Detailed studies by many investigators over the past several years have been aimed to dissect the complexity of these functions, and the results obtained now permit an attempt to integrate all the available information into a picture that highlights the balanced diversity and synergy of distinct platelet adhesive interactions.

Alpha11 beta1 integrin-dependent regulation of periodontal ligament function in the erupting mouse incisor.

The fibroblast integrin alpha11beta1 is a key receptor for fibrillar collagens. To study the potential function of alpha11 in vivo, we generated a null allele of the alpha11 gene. Integrin alpha11(-/-) mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors. Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary defect in the incisor ligament leads to halted tooth eruption. alpha11beta1-defective embryonic fibroblasts displayed severe defects in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen I, (ii) reduced ability to retract collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed disturbed MMP13 and MMP14 synthesis in alpha11(-/-) cells. We show that alpha11beta1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that alpha11beta1 integrin is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement. Our data show a unique role for alpha11beta1 integrin during tooth eruption.

Novel platelet and vascular roles for immunoreceptor signaling.

The immunoreceptor signaling pathway has classically been defined by its role in mediating intracellular signals downstream of immune receptors on circulating cells, but recent studies have revealed new and unexpected roles for this pathway in vascular biology. In platelets the immunoreceptor signaling pathway is coupled to 2 structurally distinct platelet collagen receptors, glycoprotein VI and integrin alpha2beta1, and is required for the activation of platelets after exposure to vessel wall collagen during plaque rupture. During vascular development immunoreceptor signaling is required for proper formation of the lymphatic system, a role that has revealed the contribution of hematopoietic endothelial progenitors to that process. In conjunction with the identification of new biological roles in vascular cell types, new molecular mechanisms of activating this signaling pathway have been discovered, including activation by integrins and immunoreceptor tyrosine activation motifs (ITAMs) on receptors that do not function as part of the immune response. Here we discuss some of these recent findings and their implications for vascular biology and the treatment of human vascular diseases.