Clin Exp Otorhinolaryngol.  2018 Jun;11(2):124-132. 10.21053/ceo.2017.00493.

Clusterin Induces MUC5AC Expression via Activation of NF-κB in Human Airway Epithelial Cells

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

• KMID: 2412670
• DOI: http://doi.org/10.21053/ceo.2017.00493

Abstract

OBJECTIVES
Clusterin (CLU) is known as apolipoprotein J, and has three isoforms with different biological functions. CLU is associated with various diseases such as Alzheimer disease, atherosclerosis, and some malignancies. Recent studies report an association of CLU with inflammation and immune response in inflammatory airway diseases. However, the effect of CLU on mucin secretion of airway epithelial cells has not yet been understood. Therefore, the effect and brief signaling pathway of CLU on MUC5AC (as a major secreted mucin) expression were investigated in human airway epithelial cells.
METHODS
In the tissues of nasal polyp and normal inferior turbinate, the presence of MUC5AC and CLU was investigated using immunohistochemical stain and Western blot analysis. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effect and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway of CLU on MUC5AC expression were investigated using immunohistochemical stain, reverse transcription-polymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and Western blot analysis.
RESULTS
In the nasal polyps, MUC5AC and CLU were abundantly present in the epithelium on immunohistochemical stain, and nuclear CLU (nCLU) was strongly detected on Western blot analysis. In human NCI-H292 airway epithelial cells or the primary cultures of normal nasal epithelial cells, recombinant nCLU increased MUC5AC expression, and significantly activated phosphorylation of NF-κB. And BAY 11-7085 (a specific NF-κB inhibitor) and knockdown of NF-κB by NF-κB siRNA (small interfering RNA) significantly attenuated recombinant nCLU-induced MUC5AC expression.
CONCLUSION
These results suggest that nCLU induces MUC5AC expression via the activation of NF-κB signaling pathway in human airway epithelial cells.

Keyword

Clusterin; Nasal Polyps; MUC5AC; NF-kappa B; Epithelial Cells

MeSH Terms

Alzheimer Disease
Atherosclerosis
B-Lymphocytes
Bays
Blotting, Western
Clusterin*
Epithelial Cells*
Epithelium
Humans*
Immunoenzyme Techniques
Inflammation
Mucins
Nasal Polyps
NF-kappa B
Phosphorylation
Protein Isoforms
Real-Time Polymerase Chain Reaction
RNA, Small Interfering
Turbinates
Clusterin
Mucins
NF-kappa B
Protein Isoforms
RNA, Small Interfering

Figure

• 

  Fig. 1. The presence of MUC5AC and clusterin (CLU) in the normal inferior turbinate and the nasal polyp. (A, B) Results of immunohistochemical stain showed that MUC5AC and CLU were present in the epithelium of the normal inferior turbinate. (C, D) Results of immunohistochemical stain showed that MUC5AC and CLU were abundantly present in the epithelium of the nasal polyp. (E) Results of semi-quantitative histological analysis, immunoreactivity scores of MUC5AC and CLU in the nasal polyp were significantly higher than those in normal interior turbinate. (F) Results of Western blot showed that the secretory CLU (sCLU; alpha chain, 40 kDa) and nuclear CLU (nCLU; 50 kDa) constitutively found in human nasal epithelium. The expression level of the nCLU is higher than that of sCLU. And the level of nCLU of chronic rhinosinusitis with nasal polyps group was higher than that of healthy control. On the other hand, there was no difference in the expression levels of sCLU between the two groups. Immunopositive cells appeared brown (original magnification, ×400). Images are representative of three separate experiments performed in triplicate. Bars indicate the average±standard deviation of three independent experiments performed in triplicate. a)P<0.05 compared with normal inferior turbinate group. b)P<0.05 compared with sCLU from normal inferior turbinate. c)P<0.05 compared with nCLU from normal interior turbinate.

• 

  Fig. 2. The effect of nuclear clusterin (nCLU) on MUC5AC expression in human NCI-H292 airway epithelial cells. (A, B) Results of real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) showed that MUC5AC mRNA expression and protein production were significantly increased at all concentrations of recombinant nCLU (R-nCLU), and peaked at 5 µg/mL of R-nCLU regardless of dose-dependent manner. (C, D) Results of real-time PCR and ELISA showed that R-nCLU significantly increased MUC5AC mRNA expression and protein production regardless of time-dependent manner; MUC5AC mRNA expression and protein production were peaked at 8 hours after exposure to R-nCLU. Images are representative of three separate experiments performed in triplicate. Bars indicate the average±standard deviation of three independent experiments performed in triplicate. a)P<0.05 compared with zero value.

• 

  Fig. 3. The activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) on nuclear clusterin (nCLU)-induced MUC5AC expression in human NCI-H292 airway epithelial cells. (A) Results of Western blot showed that the phosphorylation of NF-κB was significantly increased at 60 minutes and 120 minutes after treatment with recombinant nCLU (R-nCLU). (B, C) Results of reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that BAY 11-7085 significantly attenuated R-nCLU-induced MUC5AC mRNA expression and protein production. (D) Results of RT-PCR showed that the knockdown of NF-κB by NF-κB siRNA (small interfering RNA) significantly blocked R-nCLU-induced MUC5AC mRNA expression. Images are representative of three separate experiments performed in triplicate. Bars indicate the average±standard deviation of three independent experiments performed in triplicate. p-NF-κB, phosphorylated NF-κB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Con, control. a)P<0.05 compared with zero value. b)P<0.05 compared with R-nCLU alone. c)P<0.05 compared with control siRNA. d)P<0.05 compared with R-nCLU and control siRNA.

• 

  Fig. 4. The effect of nuclear clusterin (nCLU) on MUC5AC expression in human nasal epithelial cells. (A, B) Results of reverse transcriptionpolymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that recombinant nCLU (R-nCLU) significantly increased MUC5AC mRNA expression and protein production. (C, D) Results of RT-PCR and ELISA showed that BAY 11-7085 significantly attenuated R-nCLU-induced MUC5AC mRNA expression and protein production. Images are representative of three separate experiments performed in triplicate. Bars indicate the average±standard deviation of three independent experiments performed in triplicate. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. a)P<0.05 compared with zero value. b)P<0.05 compared with recombinant nCLU alone.

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