Abstract
PurposeIncreased 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 β2 (TGFβ2) 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 TGFβ2 signaling pathways in CLAN formation.MethodsCultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ 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 TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4′,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test.ResultsTGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ 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 TGFβ 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.ConclusionsTGFβ2-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.
Highlights
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)
nonglaucomatous human TM (NTM) cells were cotreated with Transforming growth factor b2 (TGFb2) plus inhibitors for 10 days or pretreated with TGFb2 for 10 days followed by 1-hour inhibitor treatment
TGFb2 significantly induced CLAN formation (n 1⁄4 6 to 12, P < 0.05), which was completely inhibited by TGFb receptor, Smad[3], and extracellular signal regulated kinase (ERK) inhibitors, as well as completely or partially inhibited by Jun N-terminal kinases (JNK), P38, and Rho-associated protein kinase (ROCK) inhibitors, depending on cell strains
Summary
Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFb2, with or without the inhibitors of TGFb receptor, Smad[3], c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFb2 plus inhibitors for 10 days or pretreated with TGFb2 for 10 days followed by 1-hour inhibitor treatment. Treatments were administered in high glucose DMEM with 1% FBS. High glucose medium was used to improve cell survival, and a low concentration of FBS minimized confounding effects of endogenous growth factors that may be present in the serum. Confluent NTM cells were treated with TGFb2 (5 ng/mL) (RD Systems, Minneapolis, MN, USA) to induce CLAN formation or DMEM with or without dimethyl sulfoxide (DMSO; vehicle control for MAPK inhibitors). To inhibit the Smad pathway, NTM cells were cotreated with TGFb2 and the Smad[3] phosphorylation inhibitor
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