Equarin is involved in cell adhesion by means of heparan sulfate proteoglycan during lens development

Authors

Errata

This article is corrected by:

  1. Errata: Equarin Is Involved in Cell Adhesion by Means of Heparan Sulfate Proteoglycan During Lens Development Volume 242, Issue 3, 301, Article first published online: 8 February 2013

Abstract

Background: Adhesion molecules are known to be instructive for both development and differentiation. During lens differentiation, epithelial cells undergo vertical elongation, with the anterior and posterior tips of the elongating fiber cells sliding along the epithelium and capsule, respectively. These cellular processes are highly coordinated through cell adhesive interactions, actin cytoskeletal reorganization and contractile force generation. Alterations in extracellular matrix composition that interfere with these interactions can lead to defects that alter tissue morphogenesis and the state of differentiation. We have demonstrated that Equarin, which is a secreted molecule expressed in the equator region of the lens, plays an important role in chick lens fiber differentiation through fibroblast growth factor signaling. Results: Here, we explored the function of Equarin in chick lens cell adhesion. Equarin protein was expressed in the extracellular region of lens differentiating cells. We found that Equarin promoted lens cell adhesion through heparan sulfate proteoglycan. By biochemical analysis, we found that Equarin directly binds syndecan-3, which displayed a similar expression pattern to Equarin. Overexpression of Equarin resulted in altered actin localization. Conclusions: Equarin is involved in cell adhesion during fiber differentiation and development. Developmental Dynamics 242:23–29, 2013. © 2012 Wiley Periodicals, Inc.

INTRODUCTION

Cell adhesion is responsible for assembling cells together and is known to be instructive for both development and differentiation. During lens differentiation, epithelial cells undergo vertical elongation, with the anterior and posterior tips of the elongating fiber cells sliding along the epithelium and capsule, respectively, as these cells migrate inward. These cellular processes are highly coordinated through cell adhesive interactions, actin cytoskeletal reorganization and contractile force generation (Piatigorsky, 1981; Taylor et al., 1996; Bassnett et al., 1999; Kuszak et al., 2004; Zelenka, 2004). The ability of a cell to recognize and interact with specific extracellular matrix (ECM) components is a fundamental requirement for cell migration and differentiation. Cells systematically create and dissolve cell–cell and cell–matrix adhesions, form connections between these adhesions and the cytoskeleton and generate contractile force. Because errors in cell adhesion may lead to aberrant lens shape or misplacement of the lens sutures, the precise regulation of each step is essential for the optical quality of the lens (Kuszak et al., 1994). Alterations in extracellular matrix (ECM) composition that interfere with these interactions can lead to defects that alter tissue morphogenesis and the state of differentiation (Juliano and Haskill, 1993).

Examination of the expression patterns of extracellular matrix components, integrins, and adhesion proteins in the lens suggests that many of the proteins involved in fibroblast migration will have similar roles in the lens (Holly et al., 2000; Etienne-Manneville and Hall, 2002). The lens is surrounded by an elaborate basement membrane composed of collagen IV, laminin, fibronectin (Cammarata et al., 1986), and a variety of proteoglycans, which are necessary for proper adhesion and migration. Integrins capable of binding these ECM proteins are expressed along fiber cell membranes (Menko and Philp, 1995; Menko et al., 1998; Menko and Walker, 2004). Focal adhesion proteins, such as FAK, MLCK, caldesmon, and paxillin, are arranged at the basal tips of elongating fiber cells in an unusual, two-dimensional array referred to as the basement membrane complex (Bassnett et al., 1999). In addition, many signaling proteins known to regulate aspects of fibroblast migration, such as Rho family proteins, are also expressed in the lens. Thus, the expected cellular adhesion machinery and the signaling pathways that coordinate and regulate its operation are present in the lens.

Although important insights have emerged regarding the identity of genes controlling lens induction and differentiation, the signaling mechanism(s) regulating fiber cell adhesive interactions are presently far from clearly understood. Previously, we identified Equarin as a novel lens differentiation inducer through fibroblast growth factor (FGF) signaling (Song et al., 2012). Mouse CCDC80 (coiled-coil domain-containing protein 80; also known as DRO1 and URB), which is highly homologous and structurally similar to chick Equarin, is involved in cell adhesion and migration (Manabe et al., 2008). Additionally, immunocytochemical studies show that Equarin protein is present in the extracellular region of transient Equarin expressing cells (Mu et al., 2003). Taken together, these data led us to hypothesize the involvement of Equarin in cell–cell or cell–matrix adhesion.

In this study, we demonstrate that Equarin protein is present in the extracellular region of differentiating lens cells. Equarin can mediate lens cell adhesion through heparan sulfate proteoglycan, and overexpression of Equarin altered actin localization. Taken together, these results suggest that Equarin is involved in lens adhesion during chick lens differentiation.

RESULTS

Localization of Equarin Protein in Chick Lens Cells

The processes of lens fiber cell differentiation are highly dependent on the adhesion proteins that are expressed. Several ECM components, including laminin, collagen IV, Fibronectin and proteoglycans, are localized in the lens (Cammarata et al., 1986). Many ECM molecules are secreted and immobilized outside the cells. As shown in Mu et al., 2003, COS-7 cells transfected with Equarin cDNAs exhibit intense Equarin immunoreactivity around the cells. We further confirmed the localization of Equarin protein in lens cells. In primary embryonic chick lens dissociated cell-derived monolayer (DCDML) cultures, lens cells form a cuboidal packed epithelium when they begin to differentiate (Menko et al., 1984). At this stage, Equarin protein was localized in the extracellular region of the lens cells and cell–cell borders (Fig. 1) as secreted Equarin protein was detected, without fixation, in the peripheral region of the cells (Song et al., 2012). This expression pattern of differentiating fiber cells raised the question of whether Equarin might play a role in cell–cell or cell–matrix interactions of lens fiber cells.

Figure 1.

Localization of Equarin protein in chick lens cells. Equarin protein was visualized in DCDMLs that had been cultured for 3 days, by using an anti-Equarin antibody without fixation. Note that Equarin protein was localized in the extracellular region of the lens cells. Scale bar = 50 μm.

Equarin Promotes Adhesion of the Lens Cells

To investigate the role of Equarin in cell adhesion, we used an in vitro system. A standard cell attachment assay was used in which cells are allowed to attach for 1 hr in wells coated with respective substrates. As expected, we found that lens cells attached to Fibronectin in a dose-dependent manner (Fig. 2B). Of interest, similar to Fibronectin, Equarin protein also served as an adhesion substrate for lens cells (Fig. 2A). We showed that the attachment of lens cells to Equarin protein was concentration dependent and that the strong attachment was obtained at concentrations of 100 to 200 nM (Fig. 2B). To confirm this adhesion ability of Equarin, we tested a large series of tumor cell lines. We found that although the ability to attach to Equarin or Fibronectin differs by cell type, HT1080, RD, C2C12, T98G, and A549 cells adhered well to both Equarin and Fibronectin (Supp. Fig. S1A–E, which is available online).

Figure 2.

Equarin mediates lens cell adhesion. Nunc-Immuno 96-well plates were coated with different concentration of Equarin protein. The dissociated lens epithelial cell suspension (1 × 105/ml) was allowed to each well. The absorbance was measured at 595 nm. A: Lens cells attachment to Equarin at different concentrations: 0, 3, 10, 30, 100, and 300 nM. B: Dose-dependent cell attachment of lens cells to Equarin or Fibronectin. Blue: Equarin; Red: Fibronectin. The ordinate represents the O.D. values at 595 nm, and the abscissa represents the concentration of the substrate in nM as indicated. Scale bar = 50 μm.

Equarin Mediates Lens Cell Adhesion by Means of Cell Surface Heparan Sulfate Proteoglycan

Next, we examined which receptor(s) and molecular mechanism(s) are involved in Equarin-induced cell adhesion. Members of the integrin family of cell-surface proteins are primary participants in cell adhesion to extracellular matrix molecules and other cells (Hynes et al., 1992; Plow et al., 2000). We found that the RGD peptide, an inhibitor of integrins (α5, 8, IIb, V)-ligand interactions, did not inhibit the attachment to Equarin, whereas RGD efficiently perturbed the attachment of lens cells to Fibronectin (Fig. 3). Another candidate cell receptor family is the sulfated cell membrane proteoglycan family (Bernfield et al., 1992). The cell attachment to Equarin was almost completely inhibited by heparin when heparin was preincubated with Equarin-coated wells for 30 min (Fig. 3). Because the heparin incubation occurred before the addition of the cells, heparin did not bind to the cell surface; rather, heparin bound to Equarin and thereby blocked cell adhesion to Equarin. No significant heparin effect was observed regarding cell attachment to Fibronectin (Fig. 3). These results suggest that the heparan sulfate chain is critically involved in Equarin-mediated cell attachment. Other tumor cell lines, such as HT1080 cells, were tested, and the same inhibitory effect by heparin on Equarin-mediated adhesion was also observed (Fig. S1F).

Figure 3.

Inhibitory activity of human IgG (10 μg/ml), RGD peptide (1 mM) or heparin (100 μg/ml) on the attachment of lens cells to Equarin (50 nM) or Fibronectin (10 nM). Note that the RGD peptide inhibited the attachment to Fibronectin, and heparin completely perturbed the attachment to Equarin. Blue: Equarin; Red: Fibronectin. The ordinates represent the percentage adhesion of the control. The data in B and C represent the mean ± SEM of triplicate wells.

The data shown above indicate that Equarin may interact with lens cell surface heparan sulfate proteoglycans. This concept is further suggested by the binding of Equarin to heparin Sepharose (Song et al., 2012). Previously, we demonstrated that Equarin binds heparin Sepharose CL-6B beads, and this binding is eliminated by ionic strength, as was observed for known heparan sulfate-binding proteins. Taken together, lens cell surface heparan sulfate proteoglycan may be the potential receptor that is responsible for Equarin-mediated adhesion activity.

Equarin Interacts With Syndecan-3

Several receptors that contain heparan sulfate chains are expressed on the cell surface, including the syndecan family of cell surface proteoglycans, the phosphatidylinositol-linked glypican, and the part-time proteoglycans betaglycan and CD44E (Carey, 1997; Bernfield et al., 1999). Previous studies demonstrate that syndecan receptors are involved in the regulation of FGF signaling and cell–matrix adhesion (Carey, 1997; Iba et al., 2000; Rapraeger, 2001). Their heparan sulfate chains bind to a large number of molecules, including ECM components (e.g., Fibronectin and collagen) and heparin-binding growth factors (e.g., FGFs). In the chick lens, only syndecan-3 displays expression by the lens fiber cells after the head ectoderm differentiates into the lens placode (Gould et al., 1995). By co-precipitation assay, we demonstrated that Equarin binds to syndecan-3 but not syndecan-2. Therefore, these data suggest that syndecan-3 may provide the heparan sulfate chain for Equarin to mediate cell adhesion (Fig. 4).

Figure 4.

Equarin directly binds to syndecan-3 but not syndecan-2. Equarin-Myc-His- and syndecan-3-Flag- or syndecan-2-Flag-containing cell lysates were incubated together, followed by incubation with ProBand Resin. The input amount of each protein was detected using immunoblotting with an anti-tag antibody. After immunoprecipitation, bound syndecan was detected by immunoblotting with an anti-Flag antibody. A: Lane 1 indicates that in the absence of Equarin, syndecan-3-Flag could not be detected. Conversely, Lane 2 indicates that syndecan-3-Flag protein was visualized only in the presence of Equarin, suggesting that syndecan-3 is co-precipitated by Equarin. B: Lane 2 indicates that syndecan-2-Flag protein is not co-precipitated by Equarin.

Effect of FGF on Lens Cell Adhesion

Previously, we have reported that Equarin directly binds to FGF, thereby upregulates FGF signaling pathway during chick lens differentiation (Song et al., 2012). By analysis of cultured explants, FGF has been identified three distinct cellular responses: proliferation, migration, and fiber differentiation, which were induced sequentially as the FGF concentration was increased (McAvoy and Chamberlain, 1989). FGF2 at a concentration of 1 ng/ml (55 pM) only minimally stimulated δ-crystallin expression despite its ability to efficiently enhance cell proliferation. However, FGF2 significantly induces epithelial-to-fiber differentiation at a concentration of 20 ng/ml (1 nM). Here, we further explored the involvement of FGF on lens cell adhesion. We found that dissociated lens cells did not attach to different concentration of FGF2, whereas they attached well to Equarin protein (Fig. 5). Combination coating of Equarin and FGF2 showed similar attachment to Equarin alone (Fig. 5). Therefore, FGF2 does not serves as an adhesion substrate for lens cell, suggesting that FGF is not involved in Equarin mediated adhesion activity.

Figure 5.

Fibroblast growth factor (FGF) does not function as an adhesion substrate for lens cells. Lens cell attachment on phosphate buffered saline (PBS), Equarin or FGF2 at different concentration were tested. The ordinate represents the OD values at 595 nm, and the abscissa represents the concentration of the substrate in nM as indicated. Note while Equarin promotes lens cell attachment at 50 nM or 100 nM, almost no cell attached to FGF protein. The well coated with Equarin and FGF together showed same OD values with Equain alone, indicating Equarin-mediated adhesion was not affected in the presence of FGF. N.S., No statistical significance.

Overexpression of Equarin Alters the Cytoskeletal Organization of Chick Lens

The formation of intimate, extensive adhesive contacts between cells or between cells and matrix results from a cooperation between adhesive systems and the actin cytoskeleton. At the point of contact between cells and the extracellular matrix, transmembrane integrin adhesion receptors assemble to constitute focal adhesions, which provide a structural link between the cytoskeleton and the extracellular matrix. The dynamic assembly of actin filaments that underlies the generation of force and drives the interactions of cells with the extracellular matrix is critical for the control of cell adhesion and migration. Actin is one of the major cytoskeletal proteins in the lens (Ireland et al., 1983). To investigate the in vivo function of Equarin on adherens junction integrity, we examined actin cytoskeleton localization in differentiating fiber cells in Equarin-electroporated chick embryos. The pCAGGS-Equarin-Myc-His vector was electroporated into one side of the head ectoderm of Hamburger-Hamilton stage (HH) 10 chick embryos together with pCAGGS-GFP. Embryos were cultured to HH20 for analysis. In the nonelectroporated lenses, phalloidin staining for F-actin revealed strong staining at apical margins of the epithelial-fiber surface (arrowhead), and staining was also detectable along the lateral membranes and basolateral margins of fiber cells (open arrowhead) where epithelial and fiber cells abut the lens capsule (Fig. 6). Equarin-electroporated lenses displayed altered F-actin staining. We found increased staining for F-actin in the apical margin (arrowhead) and basolateral surfaces of the fibers (open arrowhead). In fibers, the cytoplasm and basal regions of the fibers also displayed weak actin localization (Fig. 6). Our data indicate that the increased actin localization in the lens is likely due to overexpression of Equarin, which promotes the adhesion activity.

Figure 6.

Altered actin localization in lens cells after overexpression of Equarin. HH 10 embryos were electroporated with pCAGGS-Equarin-Myc-His vector together with pCAGGS-GFP vector and were incubated until HH18. Serial sections were made. GFP signal represents the electroporation area. Myc-stained area stained indicated Equarin protein distribution. F-actin was visualized by phalloidin staining. In the contralateral lens of electroporated embryos, F-actin was observed at apical margins of the epithelial-fiber surface (arrowhead), and basolateral margins of fiber cells (open arrowhead), where epithelial and fiber cells abut the lens capsule. In Equarin-electroporated embryos, enhanced F-actin staining was observed in the apical margin (arrowhead) and basolateral surfaces of the fibers (open arrowhead). In fibers, the cytoplasm and basal regions of the fibers also showed weak actin staining. Scale bar = 100 μm.

DISCUSSION

Cell adhesion is crucial for the assembly of individual cells into the three-dimensional tissues of animals. Adhesion-dependent signaling is important in developing tissues, because highly localized signals in the ECM are needed to control cell growth and differentiation. There is abundant evidence that adhesion molecules participate in a large variety of signal transduction events important for regulating cell adhesion and cell motility, cell growth, and differentiation (Juliano and Haskill, 1993; Ruoslahti and Reed, 1994). After the lens epithelial cells migrate to the equator and initiate differentiation, the differentiating fiber cells elongate along the apical and basal surfaces, respectively. Adherens complexes form during differentiating fiber cell elongation. Cells systematically create and dissolve cell–cell and cell–matrix adhesions. How these processes are regulated depends on the particular array of matrix components and adhesion proteins that are expressed through their signaling pathways (Roskelley et al., 1995; Lauffenburger and Horwitz, 1996). Recently, mouse CCDC80, which is a homolog of Equarin, was reported to be involved in the assembly of the extracellular matrix and the mediation of cell adhesion (Manabe et al., 2008). Additionally, Equarin protein is present on the extracellular region of cells. To test the ability of Equarin to mediate cell adhesion, we used an in vitro attachment assay. We found that Equarin can serve as a cell adhesion protein in a dose-dependent manner. This result suggests that Equarin is involved in cell adhesion during chick lens differentiation.

We further explored the molecular mechanisms of Equarin-mediated cell adhesion. We found that lens cells attach to Equarin by cell surface heparan sulfate proteoglycans. This conclusion is based on the demonstration that the attachment of lens epithelium cells to Equarin was completely inhibited by heparin but not perturbed by the RGD peptide. Previous biochemical analysis shows that Equarin binds to heparan sulfate proteoglycan (Song et al., 2012). Therefore, Equarin mediates and triggers cell adhesion through heparan sulfate proteoglycan (Fig. 7). Because heparin incubation occurred before the addition of the cells, heparin bound to Equarin and thereby blocked cell adhesion to Equarin. Therefore, Equarin may interact with lens cell surface heparan sulfate proteoglycans. Among all the cell surface heparan sulfate proteoglycans, we demonstrated that syndecan-3 is likely the receptor that provides the heparan sulfate chains for Equarin-mediated adhesion. This concept is based on the following observations: (1) only syndecan-3 persists to be expressed in the lens fiber cells; and (2) Equarin directly binds to syndecan-3. Functional analysis to block syndecan-3 expression is possible to further support this hypothesis. Moreover, integrins and heparan sulfate proteoglycans are the primary ECM adhesion receptors that coordinate signaling events and determine the signaling outcomes (Iba et al., 2000; Kim et al., 2011). Regarding the specific mechanism that is involved, we need to confirm whether integrin is responsible for the transduction of the Equarin-mediated cell adhesion response.

Figure 7.

Model describing the role of Equarin in chick lens cell differentiation.

The functional units of cell adhesion are typically multiprotein complexes made up of three general classes of proteins: the cell adhesion molecules/adhesion receptors, the extracellular matrix proteins, and the cytoplasmic proteins. We further demonstrated the altered cytoskeletal protein localization after overexpression of Equarin. It is also essential to examine the in vivo requirement of Equarin in lens cell adhesion by loss-of-function analysis.

An important issue to address is why there is need for signaling by cell adhesion molecules, which are especially suited to mediate physical interactions between cells and matrix. An important reason is adhesion molecules to localize the signal to a specific region of the cell surface or ECM. Furthermore, signals generated locally by adhesion junctions can interact with classic signal transduction pathways to help control cell growth and differentiation. It's useful to distinguish two types of signaling events mediated by adhesion molecules: signals that control local cytoplasmic processes and signals that influence cell growth and differentiation. Synergy between cell adhesion-mediated and growth-factor-triggered signals may be even more significant mechanisms for regulating cell growth and differentiation. A particular example is the synergy between adhesion to Fibronectin and platelet-derived growth factor (PDGF) in stimulating signaling pathways in fibroblasts (McNamee et al., 1993; Schwartz et al., 1995). In nonadherent cells, PDGF cannot trigger downstream events in the phospholipase C (PLC) pathway. Adhesion to Fibronectin stimulates the activity of PLC substrate. Thus, the growth factor receptor regulates the upstream signaling components, while cell attachment controls the cellular sensitivity to growth factor. This coupling between physical adhesion and developmental signaling provides a mechanism to tightly integrate physical aspects of tissue morphogenesis with cell growth and differentiation, a coordination that is essential to achieve the intricate patterns of cells in tissues.

During lens development FGFs are known to play key roles in influencing cell behavior and cell fates, including proliferation, migration, and fiber differentiation. In this study, we clarified that FGF does not work as attachment substrate for lens cells. Because Equarin protein is found to be localized around the cell surface, we speculate that Equarin is involved in two aspects of chick lens cell differentiation: (1) Equarin associates cell surface heparin sulfate proteoglycans to mediate cell adhesion activity; and (2) Equarin binds to FGF and upregulates FGF signaling to promote lens fiber differentiation (Fig. 7). Further investigation will be necessary to identify the functional domains of Equarin that mediate the interactions of Equarin with FGF and heparan sulfate proteoglycans.

EXPERIMENTAL PROCEDURES

Embryos

Fertilized White Leghorn chicken embryos obtained from a local supplier were incubated at 38°C in a humidified incubator. The embryos were staged as previously described by Hamburger and Hamilton (1951).

Equarin Protein Purification

Equarin protein purification was performed as previously described (Song et al., 2012).

Immunohistochemistry

Dissected embryos were fixed overnight in 4% paraformaldehyde (PFA) and embedded in Optimal Cutting Temperature compound (Sakura, Torrance, CA) before being snap-frozen. Immunostaining was performed using specific primary antibodies, followed by incubation with fluorescent secondary antibodies and Hoechst 33342 staining to label the nuclei. The following primary antibodies were used for immunostaining: anti-Equarin monoclonal antibody (Song et al., 2012); anti-Myc monoclonal antibody (9E10; the Developmental Studies Hybridoma Bank [DSHB], University of Iowa, Iowa City, IA); and anti-Flag-M2 monoclonal antibody (Sigma-Aldrich, St Louis, MO).

Attachment Assay

Purified Equarin protein and Fibronectin that was derived from bovine plasma (Sigma) were used as substrates. The negative control consisted of 1% bovine serum albumin (BSA) in DMEM. Nunc-Immuno 96-well plates with a MaxiSorp surface (Nunc) were coated with different substrates overnight at 4°C, rinsed with phosphate buffered saline (PBS) and incubated with 1% BSA in DMEM medium for 1 hr at 37°C. Subsequently, 100 μl of the dissociated lens epithelial cell suspension (1 × 105/ml) was added to each well, and this was incubated at 37°C in a 5% CO2 incubator. After allowing the cells to attach for 1 hr, the wells were rinsed twice with DMEM, fixed for 20 min in 4% PFA, rinsed in PBS, and stained with 0.05% Toluidine Blue. Finally, the cells were solubilized in 1% sodium dodecyl sulfate (SDS) buffer, and the absorbance was measured with a UV-Visible Spectrophotometer 3000 (Pharmacia Biotech, Kenilworth, NJ) at 595 nm. Each assay point was derived from three separate wells. For the inhibition assay, the cells or wells were preincubated for 30 min with human IgG (10 μg/ml), RGD peptide (1 mM, Peptide Institute, Inc., Minoh-shi, Osaka, Japan) or heparin (100 μg/ml, Sigma).

Co-precipitation

COS-7 cells were transfected with Myc-His-tagged Equarin and either Flag-tagged syndecan-2 or Flag-tagged syndecan-3. The conditioned media from Equarin transfected COS-7 cells and cell lysates from Flag-tagged syndecan-2 or syndecan-3 were incubated together overnight at 4°C, and this was followed by incubation with ProBand Resin (Invitrogen, Carlsbad, CA) on a rotator for 2 hr. Next, the beads were washed in IP buffer (0.1% BSA, 150 mM NaCl, 20 mM Tris-HCl [pH 7.5], 1.5 mM CaCl2, 1.5 mM MgCl2, 0.1% Triton X-100 and 0.1% CHAPS), and the protein-bound beads were analyzed using SDS-polyacrylamide gel electrophoresis. The Myc-tagged and Flag-tagged proteins were detected using 9E10 (DSHB) and M2 (Sigma) antibodies, respectively.

Acknowledgements

We thank Yohei Shinmyo and Rie Kawano for critical comments; and all members of our laboratories for their valuable help. K.O. was funded by Grant-in-Aid for Scientific Research on Innovative Areas.

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