Interferon-γ suppresses intestinal epithelial aquaporin-1 expression via Janus kinase and STAT3 activation.

Inflammatory bowel diseases are associated with dysregulated electrolyte and water transport and resultant diarrhea. Aquaporins are transmembrane proteins that function as water channels in intestinal epithelial cells. We investigated the effect of the inflammatory cytokine, interferon-γ, which is a major player in inflammatory bowel diseases, on aquaporin-1 expression in a mouse colonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mL interferon-γ and aquaporin-1 mRNA and protein expressions were measured by real-time PCR and western blot, respectively. In other experiments, CMT93 cells were pretreated with inhibitors or were transfected with siRNA to block the effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2, prior to treatment with interferon-γ. Interferon-γ decreased aquaporin-1 expression in mouse intestinal epithelial cells in a manner that did not depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on an alternate Janus kinase (likely JAK1) as well as on STAT3. The pro-inflammatory cytokine, interferon-γ may contribute to diarrhea associated with intestinal inflammation in part through regulation of the epithelial aquaporin-1 water channel via a non-classical JAK/STAT receptor signalling pathway

Inhibition of JAK2 prior to treatment with IFNγ does not significantly prevent the decrease in AQP1 expression in CMT93 cells.

<p>Immunoblots of (A) phosphorylated (phospho) STAT1, total STAT1, AQP1 and actin in the presence or absence of a JAK2 inhibitor (20 μM for 2 hr) +/- IFNγ (10 ng/mL for 24 hr). (B) Densitometry graph of phospho-STAT1 normalized to total (t) STAT1 expression. (C) Densitometry graph of AQP1 normalized to actin expression. *p < 0.05, **p < 0.01, ***p < 0.001 vs. DMSO alone; blots are representative of 3 separate experiments.</p

The STAT1/IRF-1 pathway is not required for the IFNγ-induced reduction in AQP1 expression.

<p>(A) Immunoblots showing STAT1, IRF-1, AQP1 and actin expression in CMT93 cells. Cells were pretreated with STAT1 or scrambled siRNA (80 nM for 24 hr) or with the transfection control medium (Lipofectamine Control), followed by either serum-free medium or IFNγ (10 ng/mL for 24 hr). (B) Densitometry graph of total (t) STAT1 normalized to actin expression. (C) Densitometry graph of AQP1 normalized to actin expression. *p < 0.05, **p < 0.01; blots are representative of 3 separate experiments.</p

A pan-JAK inhibitor prevents the decrease in AQP1 expression following treatment with IFNγ.

<p>(A) Immunoblots showing expression of phosphorylated and total STAT1, phosphorylated and total STAT3, AQP1 and actin in CMT93 cells pretreated with a pan-JAK inhibitor (20 μM for 2 hr) or DMSO vehicle followed by treatment with or without IFNγ (10 ng/mL for 24 hr). (B) Densitometry graph of phospho-STAT1 normalized to total (t) STAT1 expression. (C,D) Densitometry graph of phospho-STAT3α and phospho-STAT3β each normalized to total (t) STAT3 expression. (E) Densitometry graph of AQP1 normalized to actin expression. *p < 0.05, **p < 0.01, ***p < 0.001 vs. DMSO + IFNγ; ^$$$p < 0.001 vs. DMSO alone and pan-JAK Inhibitor alone; ^#p < 0.05 vs. DMSO alone. Blots are representative of 4 separate experiments.</p

Epithelial AQP1 expression is decreased following treatment of CMT93 epithelial cells with IFNγ.

<p>Confocal immunocytochemistry was performed to detect AQP1 in CMT93 cells treated with either vehicle or IFNγ (10 ng/mL for 24 hr). Constitutive AQP1 immunoreactivity was observed apically and laterally in control cells, which co-localized with E-cadherin. An overall decrease in AQP1 expression in the cell monolayers was observed after treatment with IFNγ. Much of the remaining AQP1 appeared to be re-localized from cell membranes to vesicular cytosolic structures. Images are representative of 4 monolayers per group.</p

IRF-2 appears to regulate baseline AQP1 expression and is not involved in the effects of IFNγ on AQP1 expression.

<p>(A) Immunoblots of IRF-2, AQP1 and actin levels in CMT93 cells pretreated with IRF-2 or scrambled siRNA (30 nM for 48 hr) or with transfection medium alone (Lipofectamine Control) in the presence or absence of IFNγ (10 ng/mL for 24 hr) (B) Densitometry graph of total IRF-2 normalized to actin expression. (C) Densitometry graph of AQP1 normalized to actin expression. ^#p < 0.05, ^###p < 0.001 vs. scrambled siRNA + media; **p < 0.01 vs. scrambled siRNA + IFNγ; blots are representative of 3 separate experiments.</p

Knockdown of STAT3 partially restores the IFNγ-induced decrease in AQP1 expression.

<p>(A) Immunoblots of phospho-STAT3, total STAT3, AQP1 and actin levels in CMT93 cells pretreated with STAT3 or scrambled siRNA (80 nM for 24 hr) or with transfection medium alone (Lipofectamine Control) in the presence or absence of IFNγ (10 ng/mL for 24 hr) (B) Densitometry graph of total STAT3 normalized to actin expression. (C) Densitometry graph of AQP1 normalized to actin expression. **p < 0.01 vs. scrambled siRNA + media; ^##p < 0.01 vs. scrambled siRNA + IFNγ blots are representative of 3 separate experiments.</p

Treatment of CMT93 cells with IFNγ leads to a decrease in IRF-2 protein expression.

<p>The same samples used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118713#pone.0118713.g006" target="_blank">Fig. 6</a> were run for these immunoblots. (A) Representative blots of CMT93 cells pretreated with STAT3 siRNA (80 nM, 24 hrs) and then IFNγ (10 ng/mL, 24 hrs) (B) Densitometry graph of total IRF-2 normalized to actin expression; blots are representative of 3 separate experiments.</p

Schematic of proposed mechanism of IFNγ-induced suppression of AQP-1.

<p>While IFNγ activates both STAT1 and STAT3 in a JAK-dependent manner, only activation of STAT3 plays a role in the reduced expression of AQP1. IRF2 may play a role in the constitutive expression of AQP1. IFNγ suppresses IRF2, but this does not appear to play a role in reduced AQP1 expression.</p