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

Affiliations
  • 1Department of Pediatrics, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwon, Korea
  • 2Department of Medical Science, Kosin University College of Medicine, Busan, Korea

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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