title: Transduction of PTEN proteins using the tat domain modulates TGF-β1-mediated signaling pathways and transdifferentiation in subconjunctival fibroblasts.
abstract: OBJECTIVE
  This study investigated the effects of the tumor suppressor protein PTEN (phosphatase and tensin homolog) on transforming growth factor (TGF)-β1-mediated signaling pathways and the transdifferentiation of human subconjunctival fibroblasts (SCFs) after the transduction of this protein containing a transactivator of transcription (Tat) domain.
  
  
  METHODS
  The Tat-PTEN expression vector was constructed to express the Tat domain of HIV-1 fused to PTEN. After transduction of the fusion protein and TGF-β1 stimulation, the dose-dependent effect of the transduced Tat-PTEN fusion protein on Akt phosphorylation and the stability of the Tat-PTEN fusion protein in SCF cells were evaluated by Western blot analysis. The effect of the Tat-PTEN fusion protein on the TGF-β1-stimulated expression of α-SMA and fibronectin was also evaluated by Western blot analysis and immunocytochemistry.
  
  
  RESULTS
  To increase the efficiency of enzyme activity and to successfully deliver this protein to cells, the authors used a PTEN fusion protein that contained the transduction domain of the Tat protein from HIV-1. By Western blot analysis, the transduced Tat-PTEN fusion protein was found to modulate TGF-β1 signaling in SCF cells and result in the suppression of Akt phosphorylation. Furthermore, the transduction of the Tat-PTEN fusion protein was found to suppress the TGF-β1-stimulated expression of α-SMA and fibronectin by Western blot analysis and immunocytochemical staining, and the effects of the transduced fusion protein could be controlled in a dose-dependent manner.
  
  
  CONCLUSIONS
  The Tat-PTEN fusion proteins were successfully transduced into the SCF cells and induced the suppression of transdifferentiation and fibrosis through the regulation of TGF-β-mediated signaling. The ability of the Tat-PTEN fusion protein to regulate cell survival could potentially be applied to protein therapy to counteract postoperative scarring in glaucoma surgery.
authors.affiliation: Department of Ophthalmology, National Health Insurance Corporation Ilsan Hospital, Gyounggi-do, Korea.
authors.forename: Eun Jee
authors.initials: EJ
authors.lastname: Chung
authors.forename: Hyung Keun
authors.initials: HK
authors.lastname: Lee
authors.forename: Sun-Ah
authors.initials: SA
authors.lastname: Jung
authors.forename: Seung Jae
authors.initials: SJ
authors.lastname: Lee
authors.forename: Hee Youn
authors.initials: HY
authors.lastname: Chee
authors.forename: Yong Ho
authors.initials: YH
authors.lastname: Sohn
authors.forename: Joon H
authors.initials: JH
authors.lastname: Lee
mesh_terms: D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D054338:Cell Transdifferentiation / Q000187:drug effects; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D015696:Gene Products, tat / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D018613:Microscopy, Confocal; D051059:PTEN Phosphohydrolase / Q000235:genetics*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000187:drug effects; D014161:Transduction, Genetic; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D005353:Fibronectins; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D053773:Transforming Growth Factor beta1; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase