Clin Exp Otorhinolaryngol.  2019 May;12(2):181-189. 10.21053/ceo.2018.00493.

Endoplasmic Reticulum Stress Induces MUC5AC and MUC5B Expression in Human Nasal Airway Epithelial Cells

• Kim MH
• Bae CH
• Choi YS
• Na HG
• Song SY
• Kim YD

• KMID: 2447427
• DOI: http://doi.org/10.21053/ceo.2018.00493

Abstract

OBJECTIVES
Endoplasmic reticulum (ER) stress is known to be associated with inflammatory airway diseases, and three major transmembrane receptors: double-stranded RNA-activated protein kinase-like ER kinase, inositol requiring enzyme 1, and activating transcription factor 6 (ATF6) play important roles in ER stress-related proinflammatory signaling. However, the effects of ER stress and these three major signaling pathways on the regulation of the production of airway mucins in human nasal airway epithelial cells have not been elucidated.
METHODS
In primary human nasal epithelial cells, the effect of tunicamycin (an ER stress inducer) and 4-phenylbutyric acid (4-PBA, ER stress inhibitor) on the expression of MUC5AC and MUC5B was investigated by reverse transcriptasepolymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and immunoblot analysis. Small interfering RNA (siRNA) transfection was used to identify the mechanisms involved.
RESULTS
Tunicamycin increased the expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules, including spliced X-box binding protein 1 (XBP-1), transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP), and ATF6. In addition, 4-PBA attenuated the tunicamycin-induced expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules. Furthermore, siRNA knockdowns of XBP-1, CHOP, and ATF6 blocked the tunicamycin-induced mRNA expressions and glycoprotein productions of MUC5AC and MUC5B.
CONCLUSION
.: These results demonstrate that ER stress plays an important role in the regulation of MUC5AC and MUC5B via the activations of XBP-1, CHOP, and ATF6 in human nasal airway epithelial cells.

Keyword

Endoplasmic Reticulum Stress; X-Box Binding Protein 1; Transcription Factor CHOP; Activating Transcription Factor 6; Mucins

MeSH Terms

Activating Transcription Factor 6
Carrier Proteins
CCAAT-Enhancer-Binding Proteins
Endoplasmic Reticulum Stress*
Endoplasmic Reticulum*
Epithelial Cells*
Glycoproteins
Humans*
Immunoenzyme Techniques
Inositol
Mucins
Phosphotransferases
Real-Time Polymerase Chain Reaction
RNA, Messenger
RNA, Small Interfering
Transcription Factor CHOP
Transcription Factors
Transfection
Tunicamycin
Activating Transcription Factor 6
CCAAT-Enhancer-Binding Proteins
Carrier Proteins
Glycoproteins
Inositol
Mucins
Phosphotransferases
RNA, Messenger
RNA, Small Interfering
Transcription Factor CHOP
Transcription Factors
Tunicamycin

Figure

• 

  Fig. 1. Effects of tunicamycin (TM; an endoplasmic reticulum stress inducer) on the expressions of MUC5AC and MUC5B in human nasal epithelial cells. (A, B) Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR showed that TM significantly increased the mRNA expressions of MUC5AC and MUC5B at all concentrations examined. (C) Time-dependent changes, real-time PCR showed that TM induced the expressions of MUC5AC and MUC5B mRNA at all times. However, peak times differed; MUC5AC peaked after 4 hours of exposure to TM, whereas MUC5B peaked after 8 hours of exposure. (D) Enzyme-linked immunosorbent assay (ELISA) showed TM induced the productions of MUC5AC and MUC5B glycoproteins. Effects of 4-phenylbutyric acid (4-PBA; an endoplasmic reticulum stress inhibitor) on the TM-induced expressions of MUC5AC and MUC5B in human nasal epithelial cells. (E-G) RT-PCR and real-time PCR showed that 4-PBA significantly attenuated the TM-induced expressions of MUC5AC and MUC5B mRNA. (H) ELISA also showed that 4-PBA significantly attenuated TM-induced the productions of MUC5AC and MUC5B glycoprotein. The images shown are representative of three separate experiments performed in triplicate. Bars represent the averages±standard deviation of three independent experiments performed in triplicate. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. a)P<0.05 vs. baseline. b)P<0.05 vs. TM (0.5 µg/mL) alone.

• 

  Fig. 2. The roles of the three major endoplasmic reticulum stress-mediated signaling pathways (IRE1/XBP-1, PERK/ATF4/CHOP, and ATF6) on the expressions of MUC5AC and MUC5B in human nasal epithelial cells. (A-D) Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR showed that the mRNA expressions of sXBP-1, CHOP, or ATF6 were significantly increased at all doses of tunicamycin (TM; 0.25, 0.5, 1, 2.5 µg/mL) administered and that these increases were attenuated by 4-phenylbutyric acid (4-PBA). (E-G) Western blot also showed that TM induced XBP-1, CHOP and ATF6 and attenuated by 4-PBA. The images shown are representative of three separate experiments performed in triplicate. Bars represent the averages±standard deviation of three independent experiments performed in triplicate. sXBP-1, spliced X-box binding protein 1; CHOP, CCAAT-enhancer-binding protein homologous protein; ATF6, activating transcription factor 6; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; XBP-1, X-box binding protein 1; IRE1, inositol requiring enzyme 1; PERK, RNA-activated protein kinase-like endoplasmic reticulum kinase; ATF4, activating transcription factor 4. a)P<0.05 vs. baseline. b)P<0.05 vs. TM (0.5 µg/mL) alone.

• 

  Fig. 3. Contributions of the three major pathways to the upregulation of MUC5AC expression by endoplasmic reticulum stress in human nasal epithelial cells. (A-I) Reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and enzyme-linked immunosorbent assay showed that small interfering RNA (siRNA) knockdowns of XBP-1, CHOP, or ATF6 significantly blocked the endoplasmic reticulum stress-induced MUC5AC mRNA expression and glycoprotein production. (A-C) Inhibition by XBP-1 siRNA was greatest. (D-I) Inhibitions by CHOP and ATF6 siRNA were limited, and inhibition by CHOP siRNA was weakest. The images shown are representative of three separate experiments performed in triplicate. Bars represent the averages±standard deviation of three independent experiments performed in triplicate. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TM, tunicamycin; XBP-1, X-box binding protein 1; Con, control; CHOP, CCAAT-enhancer-binding protein homologous protein; ATF6, activating transcription factor 6. a)P<0.05 vs. control siRNA. b)P<0.05 vs. TM (0.5 µg/mL) with control siRNA.

• 

  Fig. 4. Contributions of the three major pathways to the upregulation of MUC5B expression by endoplasmic reticulum stress in human nasal epithelial cells. (A-I) Reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and enzyme-linked immunosorbent assay showed that small interfering RNA (siRNA) knockdowns of XBP-1, CHOP, or ATF6 significantly blocked the endoplasmic reticulum stress-induced MUC5B mRNA expression and glycoprotein production. (D-F) Inhibition by CHOP siRNA was greatest. (A-C, G-I) Inhibitions by XBP-1 and ATF6 siRNA were limited, and inhibition by XBP-1 siRNA was weakest. The images shown are representative of three separate experiments performed in triplicate. Bars represent the averages±standard deviation of three independent experiments performed in triplicate. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TM, tunicamycin; Con, control; XBP-1, X-box binding protein 1; CHOP, CCAAT-enhancer-binding protein homologous protein; ATF6, activating transcription factor 6. a)P<0.05 vs. control siRNA. b)P<0.05 vs. TM (0.5 µg/mL) with control siRNA.

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