Kosin Med J.  2022 Jun;37(2):134-139. 10.7180/kmj.22.102.

STAT3 inhibition decreases ATP-induced MUC8 gene expression in human airway epithelial cells

• Kim CH
• Song KS

• KMID: 2532043
• DOI: http://doi.org/10.7180/kmj.22.102

Abstract

Background
Contact between the human pulmonary system and bacteria, viruses, or other pathogens can induce airway diseases. Although pathogen-induced mucus oversecretion and hyperproduction are frequently observed in the human respiratory tract, the molecular mechanisms of pathogen-induced mucus hypersecretion and overproduction remain unclear. The objective of this study was to investigate the physiological signaling mechanism of ATP-induced MUC8 gene expression in human airway epithelial cells.
Methods
Real-time reverse transcription polymerase chain reaction, a cytokine array, and a Ca2+ concentration assay were performed to investigate the ATP/P2Y2-induced MUC8 gene expression levels in human airway epithelial cells.
Results
The ATP/P2Y2 complex robustly secreted interleukin (IL)-6 in a time-dependent manner, whereas siRNA-P2Y2 did not. Moreover, ATP/P2Y2 induced MUC8 gene expression. IL-6 secreted by ATP strongly elevated ATP/P2Y2-induced MUC8 gene expression compared to ATP/P2Y2. Interestingly, a specific STAT3 inhibitor, 5,15-DPP, dramatically inhibited ATP/P2Y2/IL-6-induced STAT3 phosphorylation and resulted in an approximately 5-fold decrease in MUC8 gene expression.
Conclusions
We showed that IL-6-activated STAT6 is essential for ATP/P2Y2-induced MUC8 gene expression as part of inflammatory signaling by cytokines during airway inflammation. Our results provide a new molecular understanding of the signaling mechanism of MUC8 gene expression during airway inflammation.

Keyword

Airway inflammation; ATP; P2Y2; STAT3

Figure

• 

  Fig. 1. ATP induces IL-6 secretion in a P2Y2-dependent manner in NCI-H292 cells. (A) Cells were transfected with either wild-type P2Y2 or a siRNA-P2Y2 construct and were then incubated with ATP for 24 hours before the generation of total cell lysates; MUC8 transcripts were assessed by real-time RT-PCR. (B) A construct expressing wild-type P2Y2 or siRNA-P2Y2 was transiently transfected into NCI-H292 cells. The cells were washed, serum-starved overnight, and treated with ATP for 4 hours for a cytokine assay. The positive control was used as a loading control. (C) After transfection with a construct expressing wild-type P2Y2, NCI-H292 cells were incubated in a time-dependent manner. The supernatants were obtained to perform specific IL-6 ELISA. All figures are representative of three independent experiments. ATP, adenosine triphosphate; IL, interleukin; RT-PCR, reverse transcription polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay. a)p<0.05 compared to the control; b)p<0.05 compared to ATP only; c)p<0.05 compared to P2Y2-transfected cells.

• 

  Fig. 2. Cotreatment with ATP and IL-6 dramatically induces MUC8 gene expression via the Gαq-Ca2+ pathway. (A) Cells were transfected with a wild-type P2Y2 construct and were then incubated with ATP (10 μM) and IL-6 (50 ng/mL) for 24 hours before the generation of total cell lysates; MUC8 transcripts were assessed by real-time RT-PCR. (B) Cells were plated in six-well plates 1 day before transfection with a construct driving the expression of P2Y2. Twenty-four hours after transfection, serum-starved cells were treated with either ATP or ATP/IL-6 for 1 hour. The cells were assayed for Ca2+ concentration according to the kit’s instructions. All figures are representative of three independent experiments. ATP, adenosine triphosphate; IL, interleukin; RT-PCR, reverse transcription polymerase chain reaction. a)p<0.05 compared to the control, b)p<0.05 compared to ATP only; c)p<0.05 compared to P2Y2-transfected cells.

• 

  Fig. 3. STAT3 inhibition strongly decreases ATP/P2Y2-induced MUC8 gene expression. The cells were transfected with a wild-type P2Y2 construct, pre-incubated with 5,15-DPP for 4 hours, and then treated with ATP (10 μM) and IL-6 (50 ng/mL) for 24 hours to generate total cell lysates. Then, MUC8 transcripts were assessed by real-time RT-PCR. The figure is representative of three independent experiments. STAT3, signal transducer and activator of transcription 3; ATP, adenosine triphosphate; IL, interleukin; RT PCR, reverse transcription polymerase chain reaction. a)p<0.05 compared to the control.

Reference

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