β1 and β3 Integrins Cooperate to Induce Syndecan-4-Containing Cross-linked Actin Networks in Human Trabecular Meshwork Cells

The actin cytoskeleton plays a fundamental role in regulating a variety of cell processes including morphogenesis, motility, secretion, endocytosis, and cytokinesis.1 Actin-based processes may also play an important role in regulating aqueous humor outflow through the trabecular meshwork (TM).2–4 Agents that disrupt the cytoskeleton, such as cytochalasin, H-7, BDM, latrunculins, ethacrynic acid, vinblastine, and Y-27632,2,4–6 can increase aqueous humor outflow facility. Recent studies suggest that alterations in the organization of the actin cytoskeleton may also play a role in outflow facility and may be involved in the pathogenesis of steroid-induced glaucoma7–9 and possibly other forms of POAG as well.7–9 Treatment with glucocorticoids such as dexamethasone (DEX) can increase intraocular pressure (IOP) both in vivo and in cultured anterior segments.10–14 Examination of the actin cytoskeleton in DEX-treated anterior segment cultures14 and TM cell cultures8,9,15 show that the actin cytoskeleton undergoes reorganization into highly cross-linked actin networks (CLANs) or actin geodesic domes. Similar structures have been observed in glaucomatous TM cells in the absence of DEX treatment,16 suggesting that CLANs may also be the result of pressure-induced changes in the actin network. It has been suggested by others that DEX-induced CLAN formation in TM cells decreases the overall contractility of the tissue by making the cells more rigid and unable to change shape and “relax” under pressure.14 Alternatively, it could affect other actin-mediated biological processes of the TM needed for normal outflow facility, such as gene expression.17 CLANs are composed of a series of interconnected F-actin bundles that radiate outward from central vertices and resemble a geodesic dome. Besides actin, CLANs are known to be composed of α-actinin, tropomyosin, and filamin.18–20 CLANs are located just beneath the apical cell surface and may be physically attached to the plasma membrane.18,20–22 They are often found within lamellipodia, but they can also be anywhere throughout the cell.9,19,22 CLANs can be found in both spreading19 and nonspreading cells22,23 and were originally thought to be precursors to actin stress fibers19 or reorganized sarcomeres.24 CLANs have also been proposed to be specialized structures that help maintain cellular tensegrity.25 The function of CLANs, however, remains unclear, and little is known about the structural components and signaling pathways that regulate their formation and disassembly. The formation of other actin-containing structures such as filopodia, lamellipodia, and stress fibers is regulated by signaling pathways involving Rho family GTPases26 and cell surface receptors, such as integrin family members. Integrins are heterodimeric transmembrane glycoproteins composed of an α and β subunit. They bind several extracellular matrix (ECM) proteins via a tripeptide sequence, arg-gly-asp (RGD) in the ECM protein, whereas their cytoplasmic tails interact with a variety of tyrosine kinases, adaptor proteins, and actin-binding proteins. Thus, integrins not only form an important physical link between the extracellular environment and the actin cytoskeleton, but the protein complexes associated with their cytoplasmic tails regulate the organization of actin-containing structures.27,28 Recent work has demonstrated that syndecans act as coreceptors, along with integrins, to regulate the organization of the actin cytoskeleton.29–31 Syndecans are a family of type I membrane proteins, with cytoplasmic and transmembrane domains that are highly conserved. Of the four members in the family, syndecans-3 and -4 are the predominant members that are in the anterior segment.32 Their extracellular domains bear multiple heparan sulfate glycosaminoglycan chains. Syndecans, like integrins, bind ECM proteins and regulate the organization of the actin cytoskeleton via association with downstream signaling molecules. Most of the signaling activity of syndecans can be linked, either directly or indirectly, to Rho family GTPase-mediated signaling pathways and involves interactions between the cytoplasmic domains of syndecans and signaling molecules like phosphatidylinositol 4,5-bisphosphate (PIP2) and protein kinase C (PKC). In this study, we examined the structure of CLANs in HTM cells and the possible role that syndecans and integrins play in their formation. These studies show that α-actinin, syndecan-4, PIP2, and filamin are structural components of CLANs and the formation of these structures is regulated by a novel β1/β3 integrin signaling pathway.