TGFβ2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways

PURPOSE. Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor beta 2 (TGF beta 2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGF beta 2 signaling pathways in CLAN formation. METHODS. Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGF beta 2, with or without the inhibitors of TGF beta receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGF beta 2 plus inhibitors for 10 days or pretreated with TGF beta 2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 40,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. RESULTS. TGF beta 2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGF beta receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGF beta receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. CONCLUSIONS. TGF beta 2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.