title: Elasticity-dependent modulation of TGF-β responses in human trabecular meshwork cells.
abstract: OBJECTIVE
  To gain further insight into a possible role of biomechanical cues in glaucoma, the authors assessed the influence of extracellular matrix (ECM) elasticity on TGF-β2-induced changes in trabecular meshwork (TM) cells.
  
  
  METHODS
  Human TM cells derived from donor cornea rings were plated on rigid collagen-coated tissue culture plastic or polyacrylamide gels of different elasticity and treated with vehicle or TGF-β2. Activation of Smad-2/3, ERK, and AKT signaling and expression of α-SMA and PAI-1 proteins were assessed by Western blot analysis. Subcellular localization of α-SMA was determined by confocal immunofluorescence microscopy. Transcription of collagen-I, -IV, and -VI, α-SMA, PAI-1, fibronectin, fibrillin-1, cochlin, and MGP-1 was studied by RT-qPCR. The contribution of non-Smad signaling pathways to TGF-β-induced α-SMA and PAI-1 expression was assessed using the small molecule inhibitors U0126 and LY294002.
  
  
  RESULTS
  TGF-β2-induced activation of Smad-2/3, ERK, and AKT signaling as well as expression of collagen-1, α-SMA, fibulin, and MGP-1 were attenuated with increasing elasticity. In contrast, TGF-β2-induced collagen-6, fibronectin, PAI-1, and cochlin expression were enhanced on elastic substrates. The MEK-inhibitor U0126 blocked TGF-β-induced PAI-1 expression, whereas α-SMA expression was enhanced. PI3K inhibition with LY294002 reduced α-SMA expression.
  
  
  CONCLUSIONS
  ECM elasticity modulates TGF-β-induced signaling and protein expression in human TM cells. Non-Smad signaling contributes to TGF-β-induced alterations. Increasing ECM elasticity in vitro promotes protein expression patterns reminiscent of patterns reported in primary open-angle glaucoma. Therefore, changes in TM elasticity and mechanical load may have a significant role in primary open-angle glaucoma.
authors.affiliation: Department of Ophthalmology, Würzburg University Hospital, Würzburg, Germany.
authors.forename: Hong
authors.initials: H
authors.lastname: Han
authors.forename: Thomas
authors.initials: T
authors.lastname: Wecker
authors.forename: Franz
authors.initials: F
authors.lastname: Grehn
authors.forename: Günther
authors.initials: G
authors.lastname: Schlunck
mesh_terms: D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D004548:Elasticity / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005902:Glaucoma, Open-Angle / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D004791:Enzyme Inhibitors; D017395:Plasminogen Activator Inhibitor 1; C051321:SERPINE1 protein, human; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases