title: PITX2 is involved in stress response in cultured human trabecular meshwork cells through regulation of SLC13A3.
abstract: OBJECTIVE
  Mutations of the PITX2 gene cause Axenfeld-Rieger syndrome (ARS) and glaucoma. In this study, the authors investigated genes directly regulated by the PITX2 transcription factor to gain insight into the mechanisms underlying these disorders.
  
  
  METHODS
  RNA from nonpigmented ciliary epithelium cells transfected with hormone-inducible PITX2 and activated by mifepristone was subjected to microarray analyses. Data were analyzed using dCHIP algorithms to detect significant differences in expression. Genes with significantly altered expression in multiple microarray experiments in the presence of activated PITX2 were subjected to in silico and biochemical analyses to validate them as direct regulatory targets. One target gene was further characterized by studying the effect of its knockdown in a cell model of oxidative stress, and its expression in zebrafish embryos was analyzed by in situ hybridization.
  
  
  RESULTS
  Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) was identified as 1 of 47 potential PITX2 target genes in ocular cells. PITX2 directly regulates SLC13A3 expression, as demonstrated by luciferase reporter and chromatin immunoprecipitation assays. Reduction of PITX2 or SLC13A3 levels by small interfering RNA (siRNA)-mediated knockdown augmented the death of transformed human trabecular meshwork cells exposed to hydrogen peroxide. Zebrafish slc13a3 is expressed in anterior ocular regions in a pattern similar to that of pitx2.
  
  
  CONCLUSIONS
  The results indicate that SLC13A3 is a direct downstream target of PITX2 transcriptional regulation and that levels of PITX2 and SLC13A3 modulate responses to oxidative stress in ocular cells.
authors.affiliation: Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: M Hermina
authors.initials: MH
authors.lastname: Strungaru
authors.forename: Tim
authors.initials: T
authors.lastname: Footz
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Fred B
authors.initials: FB
authors.lastname: Berry
authors.forename: Pascal
authors.initials: P
authors.lastname: Belleau
authors.forename: Elena V
authors.initials: EV
authors.lastname: Semina
authors.forename: Vincent
authors.initials: V
authors.lastname: Raymond
authors.forename: Michael A
authors.initials: MA
authors.lastname: Walter
mesh_terms: D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology*; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D017403:In Situ Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics*; D018384:Oxidative Stress / Q000502:physiology*; D010957:Plasmids; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters / Q000235:genetics*; D014129:Trabecular Meshwork / Q000378:metabolism*; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D015027:Zebrafish / Q000196:embryology; D000097577:Homeobox Protein PITX2
chemical_list: D018398:Homeodomain Proteins; D006861:Hydrogen Peroxide; D029363:Organic Anion Transporters, Sodium-Dependent; D034741:RNA, Small Interfering; D027981:Symporters; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2; C568530:SLC13A3 protein, human