title: 2-Methoxyestradiol causes functional repression of transforming growth factor β3 signaling by ameliorating Smad and non-Smad signaling pathways in immortalized uterine fibroid cells.
abstract: OBJECTIVE
  To investigate the effects and the mechanism of action of 2-methoxyestradiol (2ME(2)) on transforming growth factor (TGF) β3-induced profibrotic response in immortalized human uterine fibroid smooth muscle (huLM) cells.
  
  
  METHODS
  Laboratory study.
  
  
  METHODS
  University research laboratory.
  
  
  METHODS
  Not applicable.
  
  
  METHODS
  Not applicable.
  
  
  METHODS
  huLM cells were treated with TGF-β3 (5 ηg/mL) in the presence or absence of specific Smad3 inhibitor SIS3 (1 μmol/L), inhibitor of the PI3K/Akt (LY294002, 10 μmol/L), or 2ME(2) (0.5 μmol/L), and the expression of collagen (Col) type I(αI), Col III(αI), plasminogen activator inhibitor (PAI) 1, connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) were determined by real-time reverse-transcription polymerase chain reaction and immunoblotting. The effect of 2ME(2) on Smad-microtubule binding was evaluated by coimmunoprecipitation.
  
  
  RESULTS
  Our data revealed that TGF-β3-induced fibrogenic response in huLM is mediated by both Smad-dependent and Smad-independent PI3K/Akt/mTOR signaling pathways. 2ME(2) abrogates TGF-β3-induced expression of Col I(αI), Col III(αI), PAI-1, CTGF, and α-SMA. Molecularly, 2ME(2) ameliorates TGF-β3-induced Smad2/3 phosphorylation and nuclear translocation. In addition, 2ME(2) inhibits TGF-β3-induced activation of the PI3K/Akt/mTOR pathway.
  
  
  CONCLUSIONS
  TGF-β3-induced profibrotic response in fibroid cells is mediated by Smad-dependent and Smad-independent PI3K/Akt/mTOR pathways. 2ME(2) inhibits TGF-β3 profibrotic effects in huLM cells by ameliorating both Smad-dependent and Smad-independent signaling pathways.
authors.affiliation: Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030, USA. salama@bcm.tmc.edu
authors.forename: Salama A
authors.initials: SA
authors.lastname: Salama
authors.forename: Concepcion R
authors.initials: CR
authors.lastname: Diaz-Arrastia
authors.forename: Gokhan S
authors.initials: GS
authors.lastname: Kilic
authors.forename: Marwa W
authors.initials: MW
authors.lastname: Kamel
mesh_terms: D000077584:2-Methoxyestradiol; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D005355:Fibrosis / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007889:Leiomyoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism*; D053782:Transforming Growth Factor beta3 / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000502:physiology; D014404:Tubulin / Q000378:metabolism
chemical_list: D051785:Smad Proteins; D053782:Transforming Growth Factor beta3; D014404:Tubulin; D004958:Estradiol; D000077584:2-Methoxyestradiol