Elsevier

Cytokine

Volume 46, Issue 1, April 2009, Pages 96-99
Cytokine

IL-13 induces translocation of NF-κB in cultured human bronchial smooth muscle cells

https://doi.org/10.1016/j.cyto.2008.12.021Get rights and content

Abstract

Background and purpose: Interleukin-13 (IL-13), a major Th2 cytokine, plays an important role in bronchial asthma, including mucus production, inflammation and airway hyperresponsiveness. Although IL-13 through its binding to IL-4 receptor α (IL-4Rα/IL-13Rα1 uses the canonical signal transducer and activator of transcription 6 (STAT6)-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved in. In the present study, whether IL-13 induces an activation of nuclear factor (NF)-κB, inflammatory transcription factor, was investigated in human bronchial smooth muscle cells (hBSMCs). Methods: Nuclear proteins were extracted from cultured hBSMCs treated with tumor necrosis factor (TNF)-α (10 ng/mL) or IL-13 (100 ng/mL), and assayed for activated NF-κB and STAT6 by Western blotting. Result: Treatments with TNF-α and IL-13 induced a translocation of NF-κB to nuclei in hBSMCs. In addition, coincubation with BMS-345541 (0.3 μM), an inhibitor of NF-κB (IκB) kinase (IKK) inhibitor, markedly inhibited the translocation of NF-κB. Conclusion: Our results suggest for the first time that IL-13 activates NF-κB in hBSMCs.

Introduction

T helper 2 (Th2) cytokines such as interleukin-4 (IL-4), IL-5 and IL-13 are important mediators of bronchial asthma, a complex chronic disease of the airways, which is characterized by airway inflammation, airway remodeling and airway hyperresponsiveness (AHR). In murine models of the asthmatic response, IL-13 has been shown to be both necessary and sufficient for the generation of asthma-like deleterious tissue alterations [1], [2], [3], [4]. The mechanisms of these responses have not been fully defined. However, because IL-13 induces a greater number of genes in airway smooth muscle as compared with other lung cell types, including smooth muscle myosin light chain (MLC), phospholipase A2 and IL-13 receptor α1 (IL-13Rα1), airway smooth muscle cells have the capacity to respond to direct stimulation by IL-13 [5].

IL-13 binds to a polymeric receptor that contains IL-4Rα and IL-13Rα1 subunits. The majority of the studies of this receptor have focused on its ability to induce signal transducer and activator of transcription 6 (STAT6) tyrosine phosphorylation and subsequent signaling. In murine models, IL-13 has been shown to mediate many of its asthma-relevant responses through the activation of the STAT6 pathway in bronchial epithelial cells [6]. However, other signal transduction pathways also contribute to the pathogenesis of asthma-like Th2 responses including airway hyperresponsiveness and inflammation [7], [8]. Extracellular signal-regulated kinase (ERK) 1/2 mitogen activated protein kinase (MAPK) activation is required for optimal IL-13 stimulation of specific chemokines, matrix metalloproteinases (MMPs), and protease inhibitors in the murine lung [9].

The nuclear transcription factor NF-κB, typical of p50 and p65 heterodimer, regulates over 150 genes involved in immune and inflammatory responses. Current evidence suggests an essential role in NF-κB overactivation for increased expression of many inflammatory genes and for airway inflammation in asthma [9], [10]. One well established mechanism of NF-κB suppression is the export of nuclear NF-κB by inhibitor of NF-κB (IκB) a, thereby rapidly repressing NF-κB activation [11]. In general, NF-κB has been known to be activated through phosphorylation and degradation of IκBα by tumor necrosis factor (TNF)-α. However, it is reported that NF-κB is activated by not only TNF-α but also IL-1β, UV-light and stress and has an important role in the immune response including inflammation [12], [13], [14].

We hypothesized that NF-κB plays a critical role in the pathogenesis of IL-13-induced signal transduction. To test this hypothesis, we investigated whether IL-13 induces an activation of NF-κB in human bronchial smooth muscle cells (hBSMCs). As the results, both TNF-α and IL-13 induced activations of NF-κB and STAT6, respectively. Interestingly, IL-13 also induced an activation of NF-κB, which was demonstrated by the NF-κB translocation to nuclei. These phenomena were significantly inhibited by combination with BMS-345541, IκB kinase (IKK) inhibitor. Therefore, our results suggest that both the IL-13 signaling and TNF-α signaling regulate the activation of NF-κB in hBSMCs.

Section snippets

Chemicals

All biochemicals were of analytical grade and were purchased from commercial suppliers: rhTNF-α (Peprotech, Paris, France), rhIL-13 (Peprotech, Paris, France) and BMS-345541 (Sigma, MO, USA).

Cell culture

Human bronchial smooth muscle cells (hBSMCs, Cambrex, MD, USA) were maintained in SmBM (Cambrex) medium supplemented with 5% fetal bovine serum (FBS), 1 mg/mL hFGF-B, 0.5 mg/mL hEGF, 5 mg/mL insulin and gentamycin/amphotericin B. The cells plated at a density of 2.7 × 104 per one well of a 6-well dish in FBS.

Result

To determine whether TNF-α and IL-13 are capable of activating STAT6 and NF-κB in hBSMCs, the levels of STAT6 phosphorylation and nuclear translocation of p65 were assessed by immunoblottings.

As shown in Fig. 1, IL-13 caused distinct phosphorylation of STAT6 in the hBSMCs: the level of phosphorylated STAT6 in the IL-13 (100 ng/mL)-treated cells was significantly increased as compared with that in the vehicle-treated control cells (p < 0.001). On the other hand, TNF-α (10 ng/mL) had no effect on the

Discussion

IL-13 is a Th2 cytokine that has emerged as a critical regulator of inflammatory immune responses, with key roles in asthma and parasite immunity [15], [16]. IL-13 can be detected in the bronchial tissue [17], nasal lavage fluid [18], and induced sputum [17] of asthmatics. Following segmental allergen challenge, bronchoalveolar lavage (BAL) fluid contains IL-13 mRNA [19] and IL-13 protein [20], indicating that the cytokine is generated in the lung in response to respiratory provocation.

References (25)

  • Prescott G. Woodruff

    Gene expression in asthmatic airway smooth muscle

    Proc Am Thorac Soc

    (2008)
  • D.A. Kuperman et al.

    Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma

    Nat Med

    (2002)
  • Cited by (24)

    • Allergic inflammation in lungs and nasal epithelium of rat model is regulated by tissue-specific miRNA expression

      2022, Molecular Immunology
      Citation Excerpt :

      In our experiments, we used human IL-13 to induce allergic inflammation in ALI cell culture, that induces MUC5AC overproduction by STAT6/SPDEF pathway. Additionally, as it was shown by Goto et al. (2009), this cytokine, also increases the activity of NF-kB, presumably by affecting IKK (validated target for has-miR-223–3p), and therefore induces cumulative overexpression of MUC5AC. Interestingly, as we showed in our study, transfection with hsa-miR-223–3p inhibitor resulted in upregulation of MUC5AC expression which suggests the role of this miRNA in IL-13-induced allergic inflammation.

    • A semisynthetic diterpenoid lactone inhibits NF-κB signalling to ameliorate inflammation and airway hyperresponsiveness in a mouse asthma model

      2016, Toxicology and Applied Pharmacology
      Citation Excerpt :

      Our present data show that SRS27 significantly reduced the level of IL-4, IL-5, IL-13, and CCL11 in BAL fluids form OVA-challenged mice. Repression of NF-κB signalling pathway has been shown to block IL-13-induced CCL11 production in cultured human airway smooth muscle cells (Goto et al., 2009). Our findings also revealed a significant inhibition of p65 nuclear translocation and NF-κB DNA binding activity by SRS27 in OVA-challenged lungs in vivo.

    • Reversal of IL-13-induced inflammation and Ca<sup>2+</sup> sensitivity by resolvin and MAG-DHA in association with ASA in human bronchi

      2015, Prostaglandins and Other Lipid Mediators
      Citation Excerpt :

      Once phosphorylated, STAT6 is translocated to the nucleus, where it regulates gene expression [42]. Mutational studies have revealed that STAT6 and NFκB are required for the upregulation of RhoA induced by IL-13 and TNFα in human BSM cells [4,6]. Other reports have shown that inhibition of STAT-6 prevents IL-13-induced Rho activation [29,41].

    • Cytokine effects on cell survival and death of A549 lung carcinoma cells

      2013, Cytokine
      Citation Excerpt :

      Notable reduction of p-JNK was not detected in samples treated with IL-13 after pretreatment with UO126 (ERK1/2 inhibitor) or SB203580 (p38 inhibitor) or SP600125 (JNK inhibitor) or BAY-117082 (NF-κB inhibitor) or LY-294002 (PI3-K inhibitor) (Figs. 9 and 10). Cytokines play important roles in the regulation of the immunity, the inflammatory response and the cell growth and death of normal and cancerous cells [6–41,42–45,48–64]. Therefore, we investigated the effects of IL-13, TNF-α, IL-1β alone and in combination with Fas on cell viability, cycle and death of A549 lung carcinoma cells, as well as major signaling pathways involved in these effects.

    View all citing articles on Scopus
    View full text