Limits...
Apigenin Inhibits Tumor Necrosis Factor-α-Induced Production and Gene Expression of Mucin through Regulating Nuclear Factor-Kappa B Signaling Pathway in Airway Epithelial Cells.

Seo HS, Sikder MA, Lee HJ, Ryu J, Lee CJ - Biomol Ther (Seoul) (2014)

Bottom Line: Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-α in NCI-H292 cells.To elucidate the action mechanism of apigenin, effect of apigenin on TNF-α-induced nuclear factor kappa B (NF-κB) signaling pathway was also investigated by western blot analysis.Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Chungnam National University, Daejeon 301-131, Republic of Korea.

ABSTRACT
In the present study, we investigated whether apigenin significantly affects tumor necrosis factor-α (TNF-α)-induced production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with apigenin for 30 min and then stimulated with TNF-α for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-α in NCI-H292 cells. To elucidate the action mechanism of apigenin, effect of apigenin on TNF-α-induced nuclear factor kappa B (NF-κB) signaling pathway was also investigated by western blot analysis. Apigenin inhibited NF-κB activation induced by TNF-α. Inhibition of inhibitory kappa B kinase (IKK) by apigenin led to the suppression of inhibitory kappa B alpha (IκBα) phosphorylation and degradation, p65 nuclear translocation. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1). These results suggest that apigenin can regulate the production and gene expression of mucin through regulating NF-κB signaling pathway in airway epithelial cells.

No MeSH data available.


Related in: MedlinePlus

Effect of apigenin on TNF-α-induced phosphorylation of IκBα, degradation of IκBα and phosphorylation of IKKα/β. NCI-H292 cells were incubated with 20 μM apigenin for 24 h and treated with 50 ng/mL TNF-α for the indicated periods. Cytoplasmic extracts were fractionated and then subjected to western blot analysis using phospho-specific IκBα (Ser 32/36) antibody (A) or antibody against anti-IκBα (B). NCI-H292 cells were incubated with 20 μM apigenin for 24 h and treated with 25 ng/mL TNF-α for the indicated periods. Whole cell lysates (100 μg) were prepared and then subjected to western blot analysis using phospho-specific IKKα/β (Ser 176/180) antibody (C). The results shown are the representative of three independent experiments. Equal protein loading was evaluated by β-actin levels. *significantly different from TNF-α alone at each time point (p<0.05, Student’s t-test) (cont: control, Api: apigenin, TNF: TNF-α).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256032&req=5

f3-bt-22-525: Effect of apigenin on TNF-α-induced phosphorylation of IκBα, degradation of IκBα and phosphorylation of IKKα/β. NCI-H292 cells were incubated with 20 μM apigenin for 24 h and treated with 50 ng/mL TNF-α for the indicated periods. Cytoplasmic extracts were fractionated and then subjected to western blot analysis using phospho-specific IκBα (Ser 32/36) antibody (A) or antibody against anti-IκBα (B). NCI-H292 cells were incubated with 20 μM apigenin for 24 h and treated with 25 ng/mL TNF-α for the indicated periods. Whole cell lysates (100 μg) were prepared and then subjected to western blot analysis using phospho-specific IKKα/β (Ser 176/180) antibody (C). The results shown are the representative of three independent experiments. Equal protein loading was evaluated by β-actin levels. *significantly different from TNF-α alone at each time point (p<0.05, Student’s t-test) (cont: control, Api: apigenin, TNF: TNF-α).

Mentions: Apigenin appears to affect the phosphorylation and degradation of IκBα which is required for NF-κB dimerization and maximal activation of transcription (Fig. 3A, B). NF-κB activation involves the phosphorylation of inhibitory kappa B alpha (IκBα) by inhibitory kappa B kinases (IKKs), resulting in IκBα degradation. As a result, NF-κB subunits are released and translocated to the nucleus. Therefore, we investigated the effect of apigenin on the phosphorylation of IκBα. As shown in Fig. 3A, TNF-α increased the phosphorylation of IκBα to its maximal level at 5 min. However, preincubation of NCIH292 cells with apigenin prior to TNF-α exposure dramatically attenuated the phosphorylation of IκBα. On the other hand, IκBα degradation is required for the activation of NF-κB. Consequently, we determined whether apigenin inhibits TNF-α-induced NF-κB activation by inhibition of IκBα degradation. As shown in Fig. 3B, at 5 min after treatment, TNF-α showed the maximal induction of IκBα degradation. However, preincubation of NCI-H292 cells with apigenin prior to TNF-α exposure suppressed the degradation of IκBα. For most agents that activate NF-κB through a common pathway based on phosphorylation, proteaseome mediated the degradation of IκB. The key regulatory step in this pathway involves the activation of IkB kinase (IKK) complex. Activation of IKK depends on phosphorylation. Therefore, we investigated whether apigenin inhibits the TNF-α-induced activity of IKKα/β. As shown in Fig. 3C, TNF-α activated the IKKα/β, although apigenin suppressed its activation by regulating the serine 176/180 phosphorylation of IKKα/β.


Apigenin Inhibits Tumor Necrosis Factor-α-Induced Production and Gene Expression of Mucin through Regulating Nuclear Factor-Kappa B Signaling Pathway in Airway Epithelial Cells.

Seo HS, Sikder MA, Lee HJ, Ryu J, Lee CJ - Biomol Ther (Seoul) (2014)

Effect of apigenin on TNF-α-induced phosphorylation of IκBα, degradation of IκBα and phosphorylation of IKKα/β. NCI-H292 cells were incubated with 20 μM apigenin for 24 h and treated with 50 ng/mL TNF-α for the indicated periods. Cytoplasmic extracts were fractionated and then subjected to western blot analysis using phospho-specific IκBα (Ser 32/36) antibody (A) or antibody against anti-IκBα (B). NCI-H292 cells were incubated with 20 μM apigenin for 24 h and treated with 25 ng/mL TNF-α for the indicated periods. Whole cell lysates (100 μg) were prepared and then subjected to western blot analysis using phospho-specific IKKα/β (Ser 176/180) antibody (C). The results shown are the representative of three independent experiments. Equal protein loading was evaluated by β-actin levels. *significantly different from TNF-α alone at each time point (p<0.05, Student’s t-test) (cont: control, Api: apigenin, TNF: TNF-α).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256032&req=5

f3-bt-22-525: Effect of apigenin on TNF-α-induced phosphorylation of IκBα, degradation of IκBα and phosphorylation of IKKα/β. NCI-H292 cells were incubated with 20 μM apigenin for 24 h and treated with 50 ng/mL TNF-α for the indicated periods. Cytoplasmic extracts were fractionated and then subjected to western blot analysis using phospho-specific IκBα (Ser 32/36) antibody (A) or antibody against anti-IκBα (B). NCI-H292 cells were incubated with 20 μM apigenin for 24 h and treated with 25 ng/mL TNF-α for the indicated periods. Whole cell lysates (100 μg) were prepared and then subjected to western blot analysis using phospho-specific IKKα/β (Ser 176/180) antibody (C). The results shown are the representative of three independent experiments. Equal protein loading was evaluated by β-actin levels. *significantly different from TNF-α alone at each time point (p<0.05, Student’s t-test) (cont: control, Api: apigenin, TNF: TNF-α).
Mentions: Apigenin appears to affect the phosphorylation and degradation of IκBα which is required for NF-κB dimerization and maximal activation of transcription (Fig. 3A, B). NF-κB activation involves the phosphorylation of inhibitory kappa B alpha (IκBα) by inhibitory kappa B kinases (IKKs), resulting in IκBα degradation. As a result, NF-κB subunits are released and translocated to the nucleus. Therefore, we investigated the effect of apigenin on the phosphorylation of IκBα. As shown in Fig. 3A, TNF-α increased the phosphorylation of IκBα to its maximal level at 5 min. However, preincubation of NCIH292 cells with apigenin prior to TNF-α exposure dramatically attenuated the phosphorylation of IκBα. On the other hand, IκBα degradation is required for the activation of NF-κB. Consequently, we determined whether apigenin inhibits TNF-α-induced NF-κB activation by inhibition of IκBα degradation. As shown in Fig. 3B, at 5 min after treatment, TNF-α showed the maximal induction of IκBα degradation. However, preincubation of NCI-H292 cells with apigenin prior to TNF-α exposure suppressed the degradation of IκBα. For most agents that activate NF-κB through a common pathway based on phosphorylation, proteaseome mediated the degradation of IκB. The key regulatory step in this pathway involves the activation of IkB kinase (IKK) complex. Activation of IKK depends on phosphorylation. Therefore, we investigated whether apigenin inhibits the TNF-α-induced activity of IKKα/β. As shown in Fig. 3C, TNF-α activated the IKKα/β, although apigenin suppressed its activation by regulating the serine 176/180 phosphorylation of IKKα/β.

Bottom Line: Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-α in NCI-H292 cells.To elucidate the action mechanism of apigenin, effect of apigenin on TNF-α-induced nuclear factor kappa B (NF-κB) signaling pathway was also investigated by western blot analysis.Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Chungnam National University, Daejeon 301-131, Republic of Korea.

ABSTRACT
In the present study, we investigated whether apigenin significantly affects tumor necrosis factor-α (TNF-α)-induced production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with apigenin for 30 min and then stimulated with TNF-α for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-α in NCI-H292 cells. To elucidate the action mechanism of apigenin, effect of apigenin on TNF-α-induced nuclear factor kappa B (NF-κB) signaling pathway was also investigated by western blot analysis. Apigenin inhibited NF-κB activation induced by TNF-α. Inhibition of inhibitory kappa B kinase (IKK) by apigenin led to the suppression of inhibitory kappa B alpha (IκBα) phosphorylation and degradation, p65 nuclear translocation. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1). These results suggest that apigenin can regulate the production and gene expression of mucin through regulating NF-κB signaling pathway in airway epithelial cells.

No MeSH data available.


Related in: MedlinePlus