Regulation of Cross-linked Actin Network (CLAN) Formation in Human Trabecular Meshwork (HTM) Cells by Convergence of Distinct 1 and 3 Integrin Pathways

Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
Investigative ophthalmology & visual science (Impact Factor: 3.4). 08/2009; 50(12):5723-31. DOI: 10.1167/iovs.08-3215
Source: PubMed


To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM.
Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2.
HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it.
Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.

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Available from: Mark S Filla, Sep 29, 2015
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    • "It was not entirely unexpected that a matrix fragment like laminin 5-derived PEP75 could induce CLAN formation. A recent study [31] found that a thrombospondin-1-derived peptide, 4N1K, also induced CLAN formation in HTM cells. This peptide binds to the αvβ3 integrin co-receptor CD47 (Fig. 1A). "
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    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 Gö 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.
    Experimental Cell Research 08/2014; 327(2). DOI:10.1016/j.yexcr.2014.07.035 · 3.25 Impact Factor
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    • "β1-mediated CLAN formation is PI-3 K dependent, whereas β3-mediated CLAN formation is CD47 and Rac1/Trio-dependent and might be regulated by thrombospondin-1. Both integrin pathways are Src dependent [58, 71]. Therefore, integrin-mediated signaling events can modulate the organization of the actin cytoskeleton in TM cells and consequently participate in regulation of cytoskeletal events previously demonstrated to be involved in regulation of outflow facility [42, 51]. "
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    ABSTRACT: Integrins are a family of membrane-spanning proteins that are important receptors for cell adhesion to extracellular matrix proteins. They also provide connections between the extracellular environment and intracellular cytoskeletons and are responsible for activation of many intracellular signaling pathways. In vitro and in vivo data strongly indicate that integrin-mediated signaling events can modulate the organization of the actin cytoskeleton in trabecular meshwork (TM) cells and are associated with astrocyte migration and microglia activation of the optic nerve head in patients with primary open angle glaucoma. Consequently, increase in resistance in the TM outflow pathways and remodeling of the optic nerve head occur, which in turn increases intraocular pressure (IOP), adds additional mechanical stress and strain to optic nerve axons, and accelerates damage of axons initially caused by optic nerve head remodeling. Integrins appear to be ideal candidates for translating physical stress and strain into cellular responses known to occur in glaucomatous optic neuropathy.
    03/2013; 2013(11):202905. DOI:10.1155/2013/202905
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    • "Morphologically, we observed an increased formation of CLANs and intracellular stress fibers after H2O2 stimulation, a frequent finding in glaucomatous TM cells [5], [45], [46], [47], [48], [49], [50]. Here, only preemptive application of ω-3 had a preventive effect on formation of these stress indicators. "
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    ABSTRACT: Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H₂O₂, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H₂O₂ further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H₂O₂ stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H₂O₂ mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H₂O₂ induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease.
    PLoS ONE 02/2012; 7(2):e31340. DOI:10.1371/journal.pone.0031340 · 3.23 Impact Factor
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