title: Keratoconus in vitro and the key players of the TGF-β pathway.
abstract: OBJECTIVE
  Keratoconus (KC) is a corneal thinning disease of unknown etiology whose pathophysiology is correlated with the presence of a thin corneal stroma and altered extracellular matrix (ECM). Transforming growth factor-β (TGF-β) signaling is a key regulator of ECM secretion and assembly in multiple tissues, including the anterior segment of the eye, and it has been linked to KC. We have previously shown that human keratoconus cells (HKCs) have a myofibroblast phenotype and altered ECM assembly compared to normal human corneal fibroblasts (HCFs). Moreover, TGF-β3 treatment promotes assembly of a more normal stromal ECM and modulates the fibrotic phenotype in HKCs. Herein, we identify alterations in TGF-β signaling that contribute to the observed fibrotic phenotype in HKCs.
  
  
  METHODS
  HCFs and HKCs were stimulated with TGF-β1, TGF-β2, or TGF-β3 isoforms (0.1 ng/mL) in the presence of a stable vitamin C derivative (0.5 mM) for 4 weeks. All samples were examined using RT-PCR and western blotting to quantify changes in the expressions of key TGF-β signaling molecules between HCFs and HKCs.
  
  
  RESULTS
  We found a significant downregulation in the SMAD6 and SMAD7 expressions by HKCs when compared to HCFs (p≤0.05). Moreover, stimulation of HKCs with any of the three TGF-β isoforms did not significantly alter the expressions of SMAD6 or SMAD7. HCFs also showed an upregulation in TGF-βRI, TGF-βRII, and TGF-βRIII following TGF-β3 treatment, whereas HKCs showed a significant two-fold downregulation.
  
  
  CONCLUSIONS
  Overall, our data shows the decreased expressions of the regulatory SMADs SMAD6 and SMAD7 by HKCs contribute to the pathological ECM structure observed in KC, and TGF-β3 may attenuate this mechanism by downregulating the expression of the key profibrotic receptor, TGF-βRII. Our study suggests a significant role of altered regulation of TGF-β signaling in KC progression and that it may enable novel therapeutic developments targeting TGF-β receptor regulation.
authors.affiliation: Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
authors.forename: Shrestha
authors.initials: S
authors.lastname: Priyadarsini
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
authors.forename: Tina B
authors.initials: TB
authors.lastname: McKay
authors.affiliation: Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
authors.forename: Akhee
authors.initials: A
authors.lastname: Sarker-Nag
authors.affiliation: Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK ; Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Karamichos
mesh_terms: D016022:Case-Control Studies; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism; D015536:Down-Regulation; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007640:Keratoconus / Q000235:genetics / Q000378:metabolism*; D058628:Myofibroblasts / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism; D051905:Smad6 Protein / Q000235:genetics / Q000378:metabolism; D051906:Smad7 Protein / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D011509:Proteoglycans; D018125:Receptors, Transforming Growth Factor beta; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; C497802:SMAD6 protein, human; C497806:SMAD7 protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D051905:Smad6 Protein; D051906:Smad7 Protein; D016212:Transforming Growth Factor beta; C071736:betaglycan; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II