Requirement for the collagen receptor Endo180 in collagen gel contraction mediated by corneal fibroblasts

Highlights

• Endo180 is required for collagen gel contraction mediated by corneal fibroblasts.

• Endo180 is up-regulated in migrating keratocytes during corneal wound healing.

• Interaction of Endo180 with collagen influences corneal fibroblast morphology.

Abstract

The interaction of keratocytes with extracellular matrix components plays an important role in the maintenance of corneal transparency and shape as well as in the healing of corneal wounds. In particular, the interaction of these cells with collagen and cell-mediated collagen contraction contribute to wound closure. Endo180 is a receptor for collagen that mediates its cellular internalization. We have now examined the role of Endo180 in collagen contraction mediated by corneal fibroblasts (activated keratocytes). Antibodies to Endo180 inhibited the contractile activity of mouse corneal fibroblasts embedded in a three-dimensional collagen gel and cultured in the presence of serum, with this effect being both concentration and time dependent and essentially complete at an antibody concentration of 0.2 μg/ml. Whereas corneal fibroblasts cultured in a collagen gel manifested a flattened morphology with prominent stress fibers under control conditions, they showed a spindlelike shape with few stress fibers in the presence of antibodies to Endo180. Antibodies to Endo180 had no effect on the expression of α–smooth muscle actin or the extent of collagen degradation in collagen gel cultures of corneal fibroblasts. Immunohistofluorescence analysis did not detect the expression of Endo180 in the unwounded mouse cornea. However, Endo180 expression was detected in keratocytes migrating into the wound area at 3 days after a corneal incisional injury. Together, our results suggest that Endo180 is required for the contraction of collagen matrix mediated by corneal fibroblasts and that its expression in these cells may contribute to the healing of corneal stromal wounds.

Introduction

The cornea plays an important role in vision as a refractory component of the eye (Nishida et al., 2017). The transparency and dome-shaped structure of the cornea are both important for this role and are dependent on the arrangement of corneal cells—including epithelial cells, stromal keratocytes, and endothelial cells—and the extracellular matrix (ECM) as well as on interactions between the cells and ECM components. In particular, the molecular structure and alignment of collagen fibers (mostly composed of type I collagen) and the interactions of these fibers with keratocytes and other ECM proteins such as proteoglycans are key to the maintenance of both corneal transparency and shape (Chen et al., 2015; Hassell and Birk, 2010). In contrast to the skin, in which wound healing is associated with the formation of fibrotic scars, the healing of corneal wounds must take place without fibrotic changes and scarring if the transparency and shape of the tissue are to be maintained.

The interaction between cells including activated keratocytes (corneal fibroblasts) and collagen has been studied in a three-dimensional (3D) culture system in which the cells are embedded in a collagen matrix (Bell et al., 1979; Grinnell, 2003; Nishida et al., 1988). Interaction of collagen molecules in the matrix with the cells results in gel contraction mediated by the cells. This contraction is influenced by multiple factors including cytokines (Assouline et al., 1992), cell differentiation (Kurosaka et al., 1998), cell adhesion molecules (Schiro et al., 1991), and the secretion of proteolytic enzymes (Davis et al., 2001; Margulis et al., 2009; Phillips and Bonassar, 2005; Pins et al., 2000; Scott et al., 1998).

Endo180, also known as urokinase-type plasminogen activator (uPA) receptor–associated protein (uPARAP), is related to the macrophage mannose receptor (East and Isacke, 2002), is expressed predominantly on mesenchymal cells (Isacke et al., 1990; Sheikh et al., 2000), and plays a pivotal role in collagen internalization by cells (Behrendt et al., 2000; Engelholm et al., 2009). It binds to collagen and enhances the adhesion of cells to this ECM protein (Engelholm et al., 2003; Thomas et al., 2005). Furthermore, Endo180 promotes the migration of fibroblasts on collagen (Engelholm et al., 2003), and it interacts with the complex formed by pro-uPA and the uPA receptor (Engelholm et al., 2001).

The mechanism of collagen gel contraction mediated by fibroblasts in 3D culture involves repetitive engagement and disengagement of the cells with the collagen matrix (Bell et al., 1979). The principal cellular receptors for collagen are thought to be integrins (Santoro and Zutter, 1995; Zeltz and Gullberg, 2016), with integrin α2β1 being thought to be largely responsible for the reorganization and contraction of collagen mediated by cells (Klein et al., 1991; Schiro et al., 1991; Tian et al., 2002). Given that Endo180 also functions as a receptor for collagen, however, we have now examined the possible role of Endo180 in collagen contraction mediated by mouse corneal fibroblasts in 3D culture.