[8] Analysis of collagen receptors

Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the α₂β₁ integrin (α₂β₁) and the glycoprotein VI (GPVI)/FcRγ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (α2-CRP) containing the α₂β₁-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to α2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was α₂β₁-dependent and GPVI/FcRγ-independent as revealed in experiments with α₂β₁- or FcRγ-deficient mouse platelets. We further show that suboptimal activation of other platelet G_q-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to α2-CRP. The enhanced α₂β₁-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of α_IIbβ₃ integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet G_q-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α₂β₁ binding to collagens containing GFOGER sites.

Data from several investigators suggest that the α2β1 integrin, a receptor for collagens, laminins, decorin, E-cadherin, matrix metalloproteinase-1, endorepellin, and several viruses, is required for innate immunity and regulation of autoimmune/allergic disorders. We demonstrated that the innate immune response to Listeria monocytogenes required α2β1 integrin expression by peritoneal mast cells (PMCs). Ligation of the α2β1 integrin by C1q contained in immune complexes comprised of Listeria and antibody was required for PMC activation in vitro and in vivo. However, ligation of the α2β1 integrin alone was insufficient to activate cytokine secretion, suggesting that one or more additional signals emanating from a coreceptor were required for PMC activation. Here, we demonstrate that C1q, but neither other complement proteins nor FcRγ, is required for early innate immune response to Listeria. The binding of Listeria's Internalin B (InlB) to hepatocyte growth factor receptor (HGF-R)/c-met provides the costimulatory function required for PMC activation. Either HGF or Listeria InlB bound to c-met and either C1q or type I collagen bound to α2β1 integrin stimulates PMC activation. These findings suggest that crosstalk between c-met and the α2β1 integrin may contribute to mast-cell activation in autoimmune and inflammatory disorders.

The α2β1 integrin, a collagen/laminin receptor, is expressed at high level in the basal cell layer of the epidermis. To define the role of the α2β1 integrin in wound healing, wound repair was extensively evaluated in wild-type and α2-null mice in vivo. In addition, the impact of α2β1 integrin-deficiency on the function of primary murine keratinocytes in vitro was analyzed. Our in vivo findings demonstrate that genetic deletion of the α2β1 integrin does not significantly alter the rate of re-epithelialization, collagen deposition, or tensile strength during wound closure in mice. In marked contrast to the observed similarities in wound healing, deletion of the α2β1 integrin resulted in a dramatic increase in neoangiogenesis in the wound microenvironment. In contrast to in vivo studies, primary keratinocytes from α2-null mice adhered poorly and displayed impaired migration on type I collagen in vitro. We demonstrate that α2β1 integrin-ligation negatively regulates expression of genes including matrix metalloproteinases both in vivo and in vitro. Furthermore, the changes in gene expression could potentially account for relatively normal wound healing in the α2-deficient mouse and our recent observation that suggests an antiangiogenic role for the α2β1 integrin in vivo.

Mast cells play a critical role in innate immunity, allergy, and autoimmune diseases. The receptor/ligand interactions that mediate mast cell activation are poorly defined. The α₂β₁ integrin, a receptor for collagens, laminins, decorin, E-cadherin, matrix metalloproteinase-1 (MMP-1), endorepellin, and several viruses, has been implicated in normal developmental, inflammatory, and oncogenic processes. We recently reported that α₂ integrin subunit–deficient mice exhibited markedly diminished neutrophil and IL-6 responses during Listeria monocytogenes–and zymosan-induced peritonitis. Peritoneal mast cells require α₂β₁ integrin expression for activation in response to pathogens, yet the ligand and molecular mechanisms by which the α₂β₁ integrin induces activation and cytokine secretion remain unknown. We now report that the α₂β₁ integrin is a novel receptor for multiple collectins and the C1q complement protein. We demonstrate that the α₂β₁ integrin provides a costimulatory function required for mast cell activation and cytokine secretion. This finding suggests that the α₂β₁ integrin is not only important for innate immunity but may serve as a critical target for the regulation of autoimmune/allergic disorders.

Tumor–stromal interactions induce expression of matrix metalloproteinases and serine proteases and, as shown recently, the cysteine protease cathepsin B. We speculate that such interactions upregulate the transcription factor Ets1, resulting in increased cathepsin B expression. This would be consistent with the observed concomitant upregulation of matrix metalloproteinases and serine proteases as well as with the ability of extracellular matrices and their binding partners to alter cathepsin B expression and secretion. Using a confocal assay to analyze the contribution of tumor–stromal interactions to proteolysis, we have been able to confirm enhanced degradation of extracellular matrices by all three classes of proteases.

Interactions of integrin cellular adhesion molecules with matrix proteins play important roles in complex bidirectional signaling pathways. To investigate these interactions, a novel flow-cytometry-based cellular adhesion assay has been developed. Based on the concept of microcarrier cell culture, derivatized polystyrene microspheres (9.6 μm) are used as a substrate for the immobilization of type I collagen to which cells then adhere. Using cytometric detection, the extent of cellular adhesion can be precisely determined by comparison of adhered and nonadhered populations based on the side scatter properties of the microspheres. In combination with immunostaining, the novel format of this assay enables the correlation of adhesive function to other cellular characteristics such as surface expression. In this work, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was used to stimulate increased adhesion in Chinese hamster ovary cells stably transfected with the collagen receptor integrin α2β1. Multiple clones of varying expression distributions were analyzed, and correlations of adherent populations versus receptor distributions show a threefold increase in functional cellular adhesion to collagen upon treatment with TPA. Probability binning analysis of duplexed data revealed subtle changes in adhesion versus receptor distribution mediated by TPA which otherwise would not have been detectable.