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: 3.66). 08/2009; 50(12):5723-31. DOI: 10.1167/iovs.08-3215
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Mark S Filla, Jun 24, 2015
  • Source
    [Show abstract] [Hide abstract]
    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; DOI:10.1016/j.yexcr.2014.07.035 · 3.37 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The trabecular meshwork (TM) is located in the anterior segment of the eye and is responsible for regulating the outflow of aqueous humor. Increased resistance to aqueous outflow causes intraocular pressure to increase, which is the primary risk factor for glaucoma. TM cells reside on a series of fenestrated beams and sheets through which the aqueous humor flows to exit the anterior chamber via Schlemm's canal. The outer trabecular cells are phagocytic and are thought to function as a pre-filter. However, most of the outflow resistance is thought to be from the extracellular matrix (ECM) of the juxtacanalicular region, the deepest portion of the TM, and from the inner wall basement membrane of Schlemm's canal. It is becoming increasingly evident that the extracellular milieu is important in maintaining the integrity of the TM. In glaucoma, not only have ultrastructural changes been observed in the ECM of the TM, and a significant number of mutations in ECM genes been noted, but the stiffness of glaucomatous TM appears to be greater than that of normal tissue. Additionally, TGFβ2 has been found to be elevated in the aqueous humor of glaucoma patients and is assumed to be involved in ECM changes deep with the juxtacanalicular region of the TM. This review summarizes the current literature on trabecular ECM as well as the development and function of the TM. Animal models and organ culture models targeting specific ECM molecules to investigate the mechanisms of glaucoma are described. Finally, the growing number of mutations that have been identified in ECM genes and genes that modulate ECM in humans with glaucoma are documented. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Experimental Eye Research 04/2015; 133. DOI:10.1016/j.exer.2014.07.014 · 3.02 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Purified C. botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors. Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in E. coli and purified. HTM cells were incubated with up to 10µg/ml C3cs or with 5 μg of C3rec and/or viral vector (MOI=25). Cells were then fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hrs, 24 hrs, and 3-4 days following bolus injection of AdV (1.6x10e7 transducing units) and/or 2.5µg C3rec. HTM cells treated for 4hrs with C3cs (all doses) or for 24hrs with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time-point. Cells exposed to C3rec and co-transduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hrs post-exposure, which persisted at 24 hrs. In MOCAS, the AdV vector alone had no effect on outflow facility but enhanced the response to C3rec at 4 hours. Co-administration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors could potentially be utilized to increase the bioavailability of proteins for cells that are difficult to transfect. Copyright © 2015 by Association for Research in Vision and Ophthalmology.
    Investigative ophthalmology & visual science 03/2015; DOI:10.1167/iovs.14-15909 · 3.66 Impact Factor