ZFIN ID: ZDB-PUB-110901-26
PITX2 is involved in stress response in cultured human trabecular meshwork cells through regulation of SLC13A3
Strungaru, M.H., Footz, T., Liu, Y., Berry, F.B., Belleau, P., Semina, E.V., Raymond, V., and Walter, M.A.
Date: 2011
Source: Investigative ophthalmology & visual science   52(10): 7625-33 (Journal)
Registered Authors: Semina, Elena
Keywords: none
MeSH Terms:
  • Animals
  • Cell Survival
  • Cells, Cultured
  • Gene Expression Regulation/physiology*
  • Homeodomain Proteins/physiology*
  • Humans
  • Hydrogen Peroxide/toxicity
  • In Situ Hybridization
  • Oligonucleotide Array Sequence Analysis
  • Organic Anion Transporters, Sodium-Dependent/genetics*
  • Oxidative Stress/physiology*
  • Plasmids
  • RNA, Small Interfering/pharmacology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Symporters/genetics*
  • Trabecular Meshwork/metabolism*
  • Transcription Factors/physiology*
  • Transcriptional Activation
  • Transfection
  • Zebrafish/embryology
PubMed: 21873665 Full text @ Invest. Ophthalmol. Vis. Sci.

Purpose: Mutations of the PITX2 transcription factor gene cause Axenfeld-Rieger Syndrome (ARS) and glaucoma. In this study we investigated genes directly regulated by PITX2 to gain insight into the mechanisms underlying these disorders.

Methods: RNA from non-pigmented ciliary epithelium cells (NPCEs) transfected with hormone-inducible PITX2 and activated by mifepristone was subjected to microarray analyses using Affymetrix U133A arrays. 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 analyzed by in situ hybridization.

Results: Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) was identified as one of 47 potential PITX2 target genes in ocular cells. PITX2 directly regulates SLC13A3 expression as demonstrated by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Reduction of PITX2 or SLC13A3 levels via small interfering RNA (siRNA)-mediated knockdown augmented the death of transformed human trabecular meshwork (HTM) 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 indicate that levels of PITX2 and SLC13A3 modulate responses to oxidative stress in ocular cells.