RETRACTED: REDOX regulation of IL-13 signaling in intestinal epithelial cells: usage of alternate pathways mediates distinct gene expression patterns

Abstract

In the classic view interleukin-13 (IL-13) binds to a heterodimer protein complex of the IL-13Ralpha1 and IL-4Ralpha chains and signals through a Janus kinase 1 (JAK1)-signal transducer and activator of transcription 6 (STAT6) mechanism. We recently reported that IL-13 also signals through the IL-13Ralpha2 chain initiating all three mitogen activated protein kinase (MAPK) pathways, and the relative expression of IL-13Ralpha1 and IL-13Ralpha2 modulates one another's transduction pathway. Therefore we investigated whether generation of reactive oxygen species (ROS) as second messengers may serve as a common nexus between these two pathways emanating from the individual IL-13 receptor chains in intestinal epithelial cells (IEC). IL-13 stimulates intracellular ROS synthesis within 5min via IL-13Ralpha1-JAK1-STAT6- and IL-13Ralpha2-MEK1/2-ERK1/2-dependent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1 (NOX-1). IL-13-induced ROS generation in turn positively regulates phosphorylation of ERK1/2 and STAT6, yielding a feed forward amplification loop. IL-13 also stimulates the stable, long-term gene expression of two other NADPH oxidases, NOX-4 and DUOX-2, which along with constitutive NOX-1, might facilitate elevated, continuous production of ROS in IL-13-activated IEC. The contribution of each signal transduction pathway initiated by IL-13 engagement to such biological functions as wound healing, inflammation, and apoptosis was mapped for representative, responsive genes. Distinct usage patterns were observed, demonstrating not only that IL-13 signal transduction through STAT6, MAPK, and ROS is regulated in both an antagonistic and cyclic fashion, but also that each pathway plays a specific role in modulating the wound healing and anti-apoptotic capabilities of the intestinal epithelium.

Figure 1. JAK1 and ERK activation are required for IL-13-induced ROS generation

HT-29.19A cells were loaded with 5 μM CM-H₂DCFDA and stimulated with 20 ng/ml IL-13 or PBS, and ROS-dependent fluorescence measured by flow cytometry at the indicated times. MFI values are plotted as mean ± SE. A) IL-13 signaling generates intracellular ROS. HT-29.19A cells were stimulated with 20 ng/ml IL-13 or PBS, and intracellular ROS was measured (n=4, * p < 0.01). B) IL-13-induced ROS generation requires JAK1 and MEK 1/2 activation. HT-29.19A were pretreated with JAK1 inhibitor (25 μM for 6 h), U1026 (10 μM for 2 h), or the DMSO vehicle, and IL-13-induced ROS was measured (n=3, * p < 0.01). C) IL-13 induced ROS generation is independent of the Src family of non-receptor tyrosine kinases and partially dependent on PI-3 kinase. HT-29.19A cells were pre-treated with PP2 inhibitor (10 μM for 2 h), LY294002 (10 μM for 2 h), or the DMSO vehicle, and IL-13-induced ROS was measured (n=3). D) IL-13Rα1 is required for ROS generation. HT-29.19A cells were pre-treated with anti-IL-13Rα1 mAb (60 μg/ml for 1 h), stimulated with IL-13 for the indicated times, and ROS measured (n=3, * p < 0.01). E) IL-13Rα2 is required for IL-13-induced ROS generation. HT-29.19A cells were untreated or transfected with IL-13Rα2 or control siRNA for 72 h, and ROS was measured after IL-13 stimulation (n=3, * p < 0.01). RNA was isolated in a parallel culture, and expression of IL-13Rα2 and GAPDH mRNA determined by RT-PCR.

Figure 2. IL-13 generates ROS through NADPH oxidase, which positively regulates ERK and STAT6 phosphorylation

A) HT-29.19A cells were pre-treated for 2 h with apocynin (500 μM), DPI (10 μM), or DMSO, and ROS measured after IL-13 stimulation is presented as mean MFI ± SE (n=3, * p < 0.01). B) DPI inhibits IL-13 mediated activation of ERK and STAT6. HT-29.19A cells were pre-treated with DPI (10 μM for 2 h) and stimulated with IL-13 for 10 min. Whole cell extracts were fractioned by SDS-PAGE and immunoblotted for phospho-specific and total ERK 1/2 and STAT6. Data is representative of two experiments.

Figure 3. NOX-1 is required for IL-13-induced ROS generation and phosphorylation of ERK 1/2 and STAT6

A) NOX-1 is constitutively expressed in HT-29.19A. Total RNA from HT-29.19A, stimulated with IL-13 for 4 or 24 h, was isolated and probed for all NOX and DUOX family genes, p22^PHOX, or GAPDH expression via RT-PCR. A representative gel pattern from three consecutive experiments is shown. B) Silencing of NOX-1 expression reduces IL-13-induced ROS generation. HT-29.19A cells were untreated or transfected with NOX-1 or control siRNA for 72 h, and ROS was measured after IL-13 stimulation (n=3, * p < 0.01). RNA was isolated in a parallel culture, and expression of NOX-1 and GAPDH mRNA determined by RT-PCR. C) Silencing of NOX-1 reduces IL-13-induced ERK and STAT6 phosphorylation. HT-29.19A cells were left untreated or transfected with NOX-1 or control siRNA. After 72 h cells were stimulated to IL-13 (20 ng/ml) for 10 min. Whole cell extracts were fractioned by SDS-PAGE and immunoblotted for phospho-specific and total anti-ERK and STAT6. Data is representative of two experiments.

Figure 4. IL-13 induces mRNA expression of NOX-4 and DUOX-2 in HT-29.19A and primary cultures of intestinal epithelial cells

RT-PCR and real-time PCR were performed on RNA harvested from HT-29.19A cells after treatment with PBS or 20 ng/ml IL-13 using NOX-4 (Panel A), DUOX-2 (Panel B), or GAPDH (both panels) specific primers (n=3). The lane labeled + is a positive control for NOX-4 or DUOX-2 mRNA in samples in which these genes are not expressed. C) RNA was extracted from freshly isolated IEC of control donors after IL-13 stimulation, and expression of NOX-4, NOX-1, DUOX-2, p22^PHOX, and GAPDH mRNA was measured by RT-PCR (n=3).

Figure 5. Distinct signaling pathways used for alternate gene expression in IL-13-stimulated intestinal epithelial cells

A) IL-13 stimulates TFF3 and Bcl-xl expression in intestinal epithelial cells. HT-29.19A were treated with IL-13 for 4, 24, and 48 h, and TFF3, Bcl-xl, MUC2, COX2, and GAPDH mRNA expression analyzed by RT-PCR (n=2). B) HT-29.19A cells were pre-treated with U1026 (10 μM for 2 h), JAK1 inhibitor (25 μM for 6 h), Apocynin (500 μM for 2h), or DPI (10 μM for 2h). After exposure to IL-13 for 4 or 24 h as indicated, RT-PCR was performed using NOX-4, DUOX-2, TFF3 or Bcl-xl specific primers, and the amplicons separated by gel electrophoresis (n=2). C) Bcl-xl expression is dependent on IL-13Rα1 signaling. HT-29.19A were pre-treated with neutralizing anti-IL-13Rα1 (60 μg/ml for 2 h), stimulated with IL-13 for 4 h, and expression of NOX-4, DUOX-2, TFF3, or Bcl-xl mRNA was measured by RT-PCR. D) NOX-4 and TFF3 expression is initiated by IL-13Rα2. HT-29.19A cells were left untreated or transfected with NOX-1 or control siRNA. After 72 h cells were stimulated to IL-13 (20 ng/ml) for 4 h and expression of NOX-4, DUOX-2, TFF3, or Bcl-xl were measured by RT-PCR. Data in Panels C and D are representative of two experiments.

Figure 6. Role of ROS in IL-13-mediated signaling

Ligation of IL-13 with IL-13Rα1 and IL-13Rα2 leads to activation of STAT6 and MAPK pathways, respectively, which in turn activate NOX-1 to produce intracellular ROS. IL-13-induced ROS then facilitates the phosphorylation of STAT6 and ERK, leading to a feed forward amplification. Selective use of each of these signaling branches distinctly regulates gene expression of critical epithelial cell responses that include apoptosis, cell cycle progression, wound healing, and immune protection.