title: PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts.
abstract: The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor β3 (TGF-β3) and TGF-β1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-β3's response to wounding.
  
  
  
  A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-β1 or -β3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated.
  
  
  
  In HCF, TGF-β3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-β1, while in HCF-P, both TGF-β1 and -β3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-β3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF.
  
  
  
  These results show that TGF-β3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-β3 treatment. Understanding the role of PDGFRα in TGF-β3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-β1 and TGF-β3 in corneal wound healing.
authors.affiliation: The Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Sriniwas
authors.initials: S
authors.lastname: Sriram
authors.affiliation: The Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Tran
authors.affiliation: The Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Guo
authors.affiliation: The Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Audrey E K
authors.initials: AE
authors.lastname: Hutcheon
authors.affiliation: The Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Hetian
authors.initials: H
authors.lastname: Lei
authors.affiliation: F. Hoffmann-La Roche AG, Basel, Switzerland.
authors.forename: Andrius
authors.initials: A
authors.lastname: Kazlauskas
authors.affiliation: The Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: James D
authors.initials: JD
authors.lastname: Zieske
mesh_terms: D000199:Actins / Q000378:metabolism*; D000704:Analysis of Variance; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology / Q000502:physiology*; D053782:Transforming Growth Factor beta3 / Q000494:pharmacology / Q000502:physiology*; D014945:Wound Healing / Q000502:physiology
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D053773:Transforming Growth Factor beta1; D053782:Transforming Growth Factor beta3; D020796:Receptor, Platelet-Derived Growth Factor alpha