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Originally published In Press as doi:10.1167/iovs.08-3215 on July 30, 2009
 (Investigative Ophthalmology and Visual Science. 2009;50:5723-5731.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-3215
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Regulation of Cross-linked Actin Network (CLAN) Formation in Human Trabecular Meshwork (HTM) Cells by Convergence of Distinct β1 and β3 Integrin Pathways

Mark S. Filla,1 Marie K. Schwinn,2 Nader Sheibani,1 Paul L. Kaufman,1 and Donna M. Peters1,2

From the Departments of 1Ophthalmology and Visual Sciences and 2Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin.

Corresponding author: Donna M. Peters, University of Wisconsin-Medical School, Department of Pathology, 1300 University Avenue, Madison, WI 53706; dmpeter2{at}wisc.edu.

Purpose. To determine the β1/β3 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.

Methods. Expression of CD47 (an {alpha}vβ3 integrin coreceptor/thrombospondin-1 receptor) and integrins {alpha}vβ3 and β1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a β1 integrin ligand) in the absence or presence of the β3 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.

Results. HTM cells expressed CD47 and integrins {alpha}vβ3 and β1. β3 Integrin or CD47 activation significantly increased CLAN formation over β1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased β3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased β1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it.

Conclusions. Distinct β1 and {alpha}vβ3 integrin signaling pathways converge to enhance CLAN formation. β1-Mediated CLAN formation was PI-3K dependent, whereas β3-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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