Interferon gamma decreases intestinal epithelial aquaporin 3 expression through downregulation of constitutive transcription

Aquaporin (AQP) 3 expression is altered in inflammatory bowel diseases, although the exact mechanisms regulating AQP abundance are unclear. Although interferon gamma (IFN) is centrally involved in intestinal inflammation, the effect of this cytokine on AQP3 expression remains unknown. HT-29 human colonic epithelial cells were treated with IFN to assess AQP3 mRNA expression by real-time RT-PCR and functional protein expression through the uptake of radiolabelled glycerol. Transient knockdown of signal transducer and activator of transcription 1 (STAT1), STAT3, Sp1, and Sp3 were performed to determine the involvement of these transcription factors in the IFN-induced signalling cascade. AQP3 promoter regions involved in the response to IFN were assessed using a luciferase reporter system. Likewise, enteroids derived from human colonic biopsies were also treated with IFN to assess for changes in AQP3 mRNA expression. IFN decreased AQP3 mRNA expression in HT-29 cells in a time- and concentration-dependent manner and reduced functional AQP3 protein expression (decreased H-3-labelled glycerol uptake). IFN also reduced AQP3 expression in enteroids derived from human colonic biopsies. Knockdown of STAT1 partially prevented the IFN-induced downregulation of AQP3 expression, whereas STAT3 and Sp3 knockdowns resulted in increased baseline expression of AQP3 but did not alter IFN-induced downregulation. Constitutive transcription of AQP3 is downregulated by IFN as demonstrated using the luciferase reporter system, with Sp3 bound to the AQP3 promoter as shown by chromatin immunoprecipitation. AQP3 constitutive transcription in intestinal epithelial cells is downregulated by IFN. This response requires STAT1 that is postulated to drive the downregulation of AQP3 expression through increased acetylation or decreased deacetylation the AQP3 promoter, ultimately resulting in decreased constitutive transcription of AQP3.Key messages center dot IFN suppresses the expression of AQP3 in intestinal epithelial cells.center dot Proximal AQP3 promoter elements are sufficient to drive constitutive expression and mediate the IFN-induced downregulation of AQP3 mRNA expression.center dot IFN-induced suppression of AQP3 is dependent upon STAT1 expression, but not STAT3, Sp1, or Sp3.