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 αvβ3 integrin coreceptor/thrombospondin-1 receptor) and integrins αvβ3 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 αvβ3 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 αvβ3 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.