IL-13 induces translocation of NF-κB in cultured human bronchial smooth muscle cells
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.
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Cytokine effects on cell survival and death of A549 lung carcinoma cells
2013, CytokineCitation 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.