title: TGFβ2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways.
abstract: 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 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.
  
  
  
  Cultured 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.
  
  
  
  TGFβ2 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.
  
  
  
  TGFβ2-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.
authors.affiliation: North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors.forename: Michela
authors.initials: M
authors.lastname: Montecchi-Palmer
authors.affiliation: North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors.forename: Jaclyn Y
authors.initials: JY
authors.lastname: Bermudez
authors.affiliation: North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors.forename: Hannah C
authors.initials: HC
authors.lastname: Webber
authors.affiliation: North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors.forename: Gaurang C
authors.initials: GC
authors.lastname: Patel
authors.affiliation: North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
authors.affiliation: North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States.
authors.forename: Weiming
authors.initials: W
authors.lastname: Mao
mesh_terms: D000199:Actins / Q000378:metabolism*; D000704:Analysis of Variance; D001082:Aqueous Humor / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta; D015398:Signal Transduction / Q000502:physiology; D051900:Smad3 Protein / Q000378:metabolism*; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000037:antagonists & inhibitors / Q000494:pharmacology* / Q000502:physiology
chemical_list: D000199:Actins; D018125:Receptors, Transforming Growth Factor beta; D051900:Smad3 Protein; D053781:Transforming Growth Factor beta2; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II