A syndecan-4 binding peptide derived from laminin 5 uses a novel PKCε pathway to induce cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells

Abstract

In this study, we examined the role(s) of syndecan-4 in regulating the formation of an actin geodesic dome structure called a cross-linked actin network (CLAN) in which syndecan-4 has previously been localized. CLANs have been described in several different cell types, but they have been most widely studied in human trabecular meshwork (HTM) cells where they may play a key role in controlling intraocular pressure by regulating aqueous humor outflow from the eye. In this study we show that a loss of cell surface synedcan-4 significantly reduces CLAN formation in HTM cells. Analysis of HTM cultures treated with or without dexamethasone shows that laminin 5 deposition within the extracellular matrix is increased by glucocorticoid treatment and that a laminin 5-derived, syndecan-4-binding peptide (PEP75), induces CLAN formation in TM cells. This PEP75-induced CLAN formation was inhibited by heparin and the broad spectrum PKC inhibitor Ro-31-7549. In contrast, the more specific PKCα inhibitor Go 6976 had no effect, thus excluding PKCα as a downstream effector of syndecan-4 signaling. Analysis of PKC isozyme expression showed that HTM cells also expressed both PKCγ and PKCε. Cells treated with a PKCε agonist formed CLANs while a PKCα/γ agonist had no effect. These data suggest that syndecan-4 is essential for CLAN formation in HTM cells and that a novel PKCε-mediated signaling pathway can regulate formation of this unique actin structure.

Steroid-induced ocular hypertension/glaucoma: Focus on pharmacogenomics and implications for precision medicine

“…These findings are consistent with the morphological changes of basement membrane type extracellular matrix deposition in SIOH leading to SIG discussed above in the Morphology section. Proteins deposited include LAMA1 (laminin-1) (Dickerson et al, 1998), LAMA5 (laminin-5) (Filla et al, 2014) and COL4A1 (collagen type IV A1) (Zhou et al, 1998), as well as other extracellular matrix components including FN1 (fibronectin) (Steely et al, 1992; Zhou et al, 1998), TSP1 (thrombospondin-1) (Flugel-Koch et al, 2004), and ELN (elastin) (Yun et al, 1989). Glucocorticoid treatment further increases the resistance of glycosaminoglycans to enzymatic degradation (Johnson et al, 1990) and reduces hyaluronan synthesis (Engelbrecht-Schnur et al, 1997).…”

“…These cross-linked actin networks contain a nidus of signaling proteins including SDC4 (syndecan-4), PIK3R1 (Phosphatidylinositol 3-kinase regulatory subunit α) and PDLIM1 at the regions where actin filaments intersect, suggesting that they are more than a structural component and may represent a signaling mechanism to sense changes in IOP. CLANs are regulated by interactions with the extracellular matrix through integrins especially the αvβ3 integrin (Filla et al, 2011; Filla et al, 2009; Filla et al, 2006), which is upregulated and activated by dexamethasone (Clark et al, 2013; Faralli et al, 2013) and syndecans (Filla et al, 2014) in cultured trabecular meshwork cells. This group showed that a number of integrin binding matrix proteins upregulated by glucocorticoid treatment of trabecular meshwork cells increase CLAN formation (Filla et al, 2006).…”

Life under pressure: The role of ocular cribriform cells in preventing glaucoma

“…(Clark et al, 1994; Filla et al, 2011), dependent upon the cell surface laminin 5-derived syndecan-4 peptide. (Filla et al, 2014; Read et al, 2007). …”

The Effects of Glucocorticoids on Trabecular Meshwork and its Role in Glaucoma

“…Additional studies have demonstrated that DEX treatment plays a role in increased Cross-Linked Actin Network (CLAN) formation by increasing laminin-5 deposition within the ECM (Filla et al, 2014). Although the exact function of CLANs are unknown, they are found with increased frequency in the TM of glaucoma patients (Hoare et al, 2008).…”

“…Syndecans, which are a four-member family of transmembrane heparan sulfate proteoglycans (HSPG), contain extracellular domains that bind ECM proteins and other extracellular ligands. Novel studies show that laminin 5-derived PEP75, which binds to syndecan-4, activates a novel PKCε-signaling pathway that triggers CLAN formation (Filla et al, 2014). However, it is unknown whether these changes result in reorganization of the ECM that leads to increase outflow resistance or they are a stress response to increased IOP.…”