Abstract

IL-17F is involved in asthma, but its biological function and signaling pathway have not been fully elucidated. IL-11 is clearly expressed in the airway of patients with allergic airway diseases such as asthma and plays an important role in airway remodeling and inflammation. Therefore, we investigated the expression of IL-11 by IL-17F in bronchial epithelial cells. Bronchial epithelial cells were cultured in the presence or absence of IL-17F and/or Th2 cytokines (IL-4 and IL-13) or various kinase inhibitors to analyze the expression of IL-11. Next, activation of mitogen- and stress-activated protein kinase (MSK) 1 by IL-17F was investigated. Moreover, the effect of short interfering RNAs (siRNAs) targeting MSK1 and cAMP response element binding protein (CREB) on IL-17F-induced IL-11 expression was investigated. IL-17F induced IL-11 expression, whereas the costimulation with IL-4 and IL-13 augmented this effect even further. MEK inhibitors PD-98059, U0126, and Raf1 kinase inhibitor I, significantly inhibited IL-11 production, whereas overexpression of a Raf1 dominant-negative mutant inhibited its expression. IL-17F clearly phosphorylated MSK1, whereas PD-98059 inhibited the phosphorylation of IL-17F-induced MSK1. Both MSK1 inhibitors Ro-31-8220 and H89 significantly blocked IL-11 expression. Moreover, transfection of the cells with siRNAs targeting MSK1 inhibited activation of CREB, and the siRNAs targeting MSK1 and CREB blocked expression of IL-11. These data suggest that IL-17F may be involved in airway inflammation and remodeling via the induction of IL-11, and RafI-MEK1/2-ERK1/2-MSK1-CREB is identified as a novel signaling pathway participating in this process. Therefore, the IL-17F/IL-11 axis may be a valuable therapeutic target for asthma.

  • asthma

asthma is a clinical diagnosis based on episodic symptoms and variable airway obstruction. It is also characterized by variable degrees of chronic inflammation and structural alterations in the airways (5, 39). These structural alterations, collectively called airway remodeling, encompass various changes in composition, content, and organization of many cellular and molecular constituents of the airway wall (31). The most important abnormalities are epithelial detachment, goblet cell hyperplasia, subepithelial thickening, hyperplasia and hypertrophy of airway smooth muscle, bronchial gland enlargement, angiogenesis, and alterations in the extracellular matrix components (14). Airway remodeling is caused by, at least partially, a complicated network of cytokines, including IL-11.

IL-11 is derived from many cell types, including bronchial epithelial cells, in response to many stimuli such as cytokines, cysteinyl leukotriene, histamine, and respiratory viruses (9, 10, 11, 27, 42). Several reports have suggested the role of IL-11 in asthma, since its level is significantly elevated in endobronchial biopsy from asthmatics (28). Moreover, its level is correlated with disease severity and a reduced FEV1 (28). Overexpression of IL-11 in the lung was correlated with subepithelial fibrosis and accumulation of myocytes and myofibroblasts (35). These findings suggest that IL-11 is one of the critical cytokines in the pathogenesis of airway inflammation and remodeling. However, the mechanisms by which the expression of IL-11 is regulated have not been fully elucidated.

Discovery of a new cytokine involving airway inflammation and remodeling and identification of its signaling pathway might help to clarify the mechanisms of asthma. We and other groups (13, 21, 32) discovered a member of the IL-17 cytokine family, IL-17F. IL-17F is expressed in activated CD4+ T cells, basophils, and mast cells, three important cell types involved in bronchial asthma (21). Recent reports demonstrated that IL-17F is produced by Th17 cells constituting an independent CD4+ T cell subset (2). Th17 cells express both IL-17A and IL-17F, but not IL-4 and IFNγ, and may play a role in tissue inflammation and host protection against extracellular pathogens (2). Th17 cells provided a new insight into the molecular mechanisms involved in airway diseases (38). Emerging reports have indicated that IL-17A and IL-17F, as the markers of the novel Th17 cells, may play important and unique roles in the exertion of Th17 functions. In the airway of asthmatics, the IL-17F gene is clearly upregulated (21). We have recently found that a coding region variant (H161R) of the IL-17F gene is inversely associated with asthma and encodes an antagonist for the wild-type IL-17F (12, 22). These findings suggest that IL-17F is one of the crucial cytokines regulating the development of allergic airway inflammation. However, the role of IL-17F in asthma has not been elucidated. We have demonstrated that IL-17F is able to induce various cytokines, chemokines, and adhesion molecules in several cell types, such as bronchial epithelial cells, via activation of RafI-MEK1/2-ERK1/2-p90 ribosomal S6 kinase (p90RSK)-cAMP response element binding protein (CREB) pathway (15, 16, 18, 19, 20). However, inhibition of this pathway did not completely abrogate biological activity of IL-17F (16). This suggests the potential involvement of other signaling molecules, such as mitogen- and stress-activated protein kinase (MSK) 1. To gain further understanding of the function of IL-17F and its signaling pathway, expression of IL-11 by IL-17F was investigated. Moreover, the effect of Th2 cytokines on IL-17F-induced IL-11 expression was investigated since Th2 cytokines, IL-4 and IL-13, have been associated with airway inflammation in asthma (24). Herein, we demonstrated for the first time that bronchial epithelial cells express IL-11 in response to IL-17F via the activation of MSK1-CREB pathway.

MATERIALS AND METHODS

Cell culture.

Two different bronchial epithelial cells were used in this study. A bronchial epithelial cell line, BEAS-2B, was cultured in Hanks’ F-12/DMEM (Biofluids, Rockville, MD) with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 ng/ml streptomycin (Life Technologies-BRL, Gaithersburg, MD). Normal human bronchial epithelial cells (NHBEs) were purchased from Lonza (Walkersville, MD) and cultured in bronchial epithelial basal medium according to the manufacturer's instructions. The cells were cultured for no more than three passages before the analysis.

Analysis of IL-11 gene and protein expression.

Total RNA was extracted using RNeasy (Qiagen, Chatsworth, CA) from 1 × 106 cells at 2 h after stimulation with 10 and 100 ng/ml of IL-17F, and cDNAs were synthesized from 500 ng of total RNA and subjected for PCR. The sequences of PCR primers for IL-11: forward, 5′-AGCTCTACAGCTCCCAGGTG-3′, reverse, 5′-TCACAGCCGCCGAGTCTTCAGC-3′; G3PDH: forward, 5′-ACCACAGTCCATGCCATCAC-3′, reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. The amplification reaction was performed for 30 cycles with denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s. The expected size for IL-11 was 343 bp, and for G3PDH it was 450 bp. PCR products were detected by ethidium bromide staining and quantified by video densitometry using Image 1.61 software (NIH Public Software; National Institutes of Health, Bethesda, MD). The level of IL-11 gene expression was quantified by calculating the ratio of densitometric readings of the band intensity for IL-11 and G3PDH from the same cDNA sample. The values are expressed as means ± SD (n = 3 experiments). IL-11 protein levels in the supernatants and cell lysates of IL-17F-stimulated cells were determined with a commercially available ELISA kit (Biosource, Camarillo, CA) according to the manufacturer's instructions and expressed as the amount recovered per 106 cells. The cell lysates were generated as described previously (20). The values are expressed as means ± SD (n = 6 experiments).

Effect of Th2 cytokines on IL-17F-induced IL-11 expression.

BEAS-2B cells were treated with 100 ng/ml of each of the cytokines: IL-17F generated as described previously (21), IL-4, or IL-13 (all purchased from R&D Systems, Minneapolis, MN) or a combination of IL-17F (100 ng/ml) with either IL-4 (100 ng/ml) or IL-13 (100 ng/ml) for 24 h. IL-11 protein levels in the supernatants were determined as described above. The values are expressed as means ± SD (n = 6 experiments).

Effect of inhibitors on the expression of IL-11.

For analysis of involvement of the Raf1-MEK-ERK1/2-MSK1 pathway, the cells were treated in the presence or absence of the following kinase inhibitors at varying doses: MEK1/2 inhibitors PD-98059 and U0126, MSK1 inhibitors H89 and Ro-31-8220, and a vehicle control, DMSO (Me2SO), for 1 h before treatment with 100 ng/ml IL-17F (all purchased from Calbiochem, La Jolla, CA) (4, 6, 7, 36). The final concentration of Me2SO did not exceed 0.1% (vol/vol). On the other hand, MSK1 is activated by other MAP kinase family members such as p38 and JNK (23, 41). Therefore, p38 inhibitor SB-202190 and JNK inhibitor SP-600125 were used (all purchased from Calbiochem) (1, 4). The cell supernatants were harvested at 24 h after stimulation for analyses by ELISA. IL-11 protein levels in the supernatants were determined as described above. The values are expressed as means ± SD (n = 4 experiments). The total number of cells and cell viability at the end of the culture period for each experiment were similar among all culture conditions, as determined by trypan blue exclusion assay, suggesting that the inhibition of IL-17F-induced IL-11 expression did not result from cytotoxicity of those inhibitors.

Detection of MSK1.

For analysis of activation of MSK1, the cells were treated with IL-17F (100 ng/ml) and in some cases with or without treatment with the MEK inhibitor PD-98059 or a vehicle control (Me2SO) for 1 h. Following treatment, the total cellular extracts (1 × 106 cell equivalents/lane) were subjected to 4–20% Tris-glycine gel electrophoresis (NOVEX, San Diego, CA), followed by transfer onto polyvinylidene difluoride membranes (Bio-Rad, Tokyo, Japan) as previously described (20). The antibodies (Ab) used were rabbit anti-MSK1 Ab and anti-phospho-MSK1 Ab (Cell Signaling Technology, Beverly, MA).

Overexpression of Raf1 dominant negative vector.

The plasmid encoding pCMV-RafS621A vector (dominant negative mutant of Raf-1) cloned into pCMV and a control vector were purchased from Clontech (San Diego, CA). The plasmids were prepared by using Qiagen plasmid DNA preparation kit. BEAS-2B cells were cultured on 100-mm plates and were transfected by an Effectene Reagent (Qiagen) according to the manufacturer's instructions. The cells were selected with 500 ng/ml Geneticin (G418, Gibco/BRL). After selection, the cells were seeded into six-well culture plates. The cells were near confluent, and the cell supernatants were then harvested at 24 h after stimulation with 100 ng/ml IL-17F for analyses by ELISA. IL-11 protein levels in the supernatants were determined as described above. The values are expressed as means ± SD (n = 3 experiments).

Knockdown of MSK1 and CREB with siRNA.

Predesigned siRNAs for MSK1 and CREB and control siRNAs were purchased from Ambion and BioLabs, respectively (Tokyo, Japan). siRNA transfection into the cells was performed according to the manufacturer's instructions. The cells and cell supernatants were then harvested at 20 min and 24 h after stimulation with 100 ng/ml IL-17F and subjected to Western blotting and ELISA analyses, respectively. For analysis of the interrelationship between MSK1 and CREB, Western blotting was performed as described above. The antibodies used were anti-CREB antibody and anti-phospho-CREB antibody (Cell Signaling Technology). IL-11 protein levels in the supernatants were determined as described above. The values are expressed as means ± SD (each n = 4 experiments).

Data analysis.

The statistical significance of differences was determined by ANOVA. The values are expressed as means ± SD from independent experiments. Any difference with P values less than 0.05 was considered significant. When ANOVA indicated a significant difference, the Scheffé F-test was used to determine the difference between groups.

RESULTS

To examine whether IL-17F is able to induce IL-11 expression, bronchial epithelial cells were stimulated with varying doses of IL-17F at five different time points, and the levels of IL-11 gene and protein expression were analyzed. While IL-11 gene was not expressed in control culture, IL-17F induced, in a dose-dependent manner, the gene expression of IL-11 at the 2-h time point in BEAS-2B cells (Fig. 1A). In the time course experiments, IL-11 gene expression peaked at the 2-h time point in IL-17F (100 ng/ml)-treated BEAS-2B cells (Fig. 1B) and returned to baseline 24 h after stimulation. IL-11 proteins were weakly detected in untreated BEAS-2B cells, but in the presence of IL-17F, the levels of IL-11 in cell lysates were significantly increased and peaked at the 12-h time point (Fig. 1C), whereas the levels of IL-11 in the supernatants peaked at 24 h and decreased at 48 h after stimulation (Fig. 1D). Similarly, NHBEs also induced IL-11 expression in response to IL-17F and showed the same kinetics as BEAS-2B cells (Fig. 1E).

Fig. 1.

Expression of IL-11 gene and protein in bronchial epithelial cells. A: IL-11 gene expression by IL-17F in BEAS-2Bs. *P < 0.05 vs. control. **P < 0.05 vs. 10 ng/ml IL-17F-stimulated cells (n = 3). B: time course of IL-11 gene expression in BEAS-2B cells. *P < 0.05 vs. the level at 0.5-h time point (n = 3). IL-11 protein levels are shown in lysates (C) and supernatants (D) in BEAS-2B cells. E: IL-11 protein levels in supernatants in normal human bronchial epithelial cells (NHBEs). *P < 0.05 vs. medium control. **P < 0.05 vs. 10 ng/ml IL-17F-stimulated cells (n = 6). Values are expressed as means ± SD.

Next we investigated whether IL-17F modulates the expression of IL-11 in cells stimulated with Th2 cytokines (IL-4 and IL-13). As shown in Fig. 2, the Th2 cytokines (IL-4 and IL-13) did not induce IL-11 expression. However, both IL-4 and IL-13 showed an augmenting effect in combination with IL-17F compared with that seen in cells stimulated with IL-17F alone.

Fig. 2.

Effect of Th2 cytokines (IL-4 and IL-13) on IL-17F-induced IL-11 expression. BEAS-2B cells were stimulated as indicated, and IL-11 protein levels in supernatants were measured by ELISA. Values are expressed as means ± SD (n = 6). *P < 0.05 vs. control. **P < 0.05 vs. IL-17F-stimulated cells.

To investigate IL-17F-mediated signaling events leading to the induction of IL-11, the activation of the Raf1-MEK1/2-ERK1/2 pathway was investigated, since this pathway is a central upstream pathway of IL-17F (15, 16, 18, 19, 20). The results showed that pretreatment of the cells for 1 h with each of the selective MEK inhibitors, PD-98059 (10 μM) and U0126 (5 μM), and Raf1 kinase inhibitor I (1 nM) significantly decreased the levels of IL-17F-induced IL-11 expression, whereas a 1-h pretreatment of the cells with vehicle alone (DMSO) did not affect IL-11 expression. In addition, the protein levels of IL-11 were unchanged in IL-17F-treated cells in the presence of varying doses of a p38 kinase inhibitor, SB-202190, and a JNK inhibitor, SP-600125 (Fig. 3). While induction of IL-11 is only partially inhibited by PD-98059, U0126, or Raf1 kinase inhibitor I, even at relatively high doses (50 μM, 10 μM, and 10 nM, respectively), the combination of 10 μM PD-98059 and 1 nM Raf1 kinase inhibitor I inhibited, to a significantly greater degree, the production of IL-11 (Fig. 3).

Fig. 3.

Effect of various inhibitors on IL-11 expression in BEAS-2B cells. The cells were pretreated with the inhibitors PD-98059 (PD), U0126, Raf1 kinase inhibitor I (Raf I), SB-202190 (SB), SP-600125 (SP), H89, or Ro-31-8220. Values are expressed as means ± SD (n = 4). *P < 0.05 vs. IL-17F-stimulated cells. **P < 0.05 vs. the presence of inhibitors (PD, U0126, or Raf I).

Next, the involvement of Raf1 kinase in IL-17F-induced IL-11 expression was further confirmed by the use of a Raf1 dominant negative mutant with serine to alanine substitution at position 621. The results showed that overexpression of Raf1 dominant negative mutants in BEAS-2B cells significantly inhibited IL-17F-induced IL-11 expression (Fig. 4), whereas the cells transfected with a control vector showed no significant change in the level of IL-11 expression. Therefore, these results suggest that IL-17F expresses IL-11 through Raf1-mediated pathway.

Fig. 4.

Effect of overexpression of Raf1 dominant-negative mutants on IL-11 protein expression in BEAS-2B cells. Values are expressed as means ± SD (n = 3). *P < 0.05 vs. IL-17F-stimulated cells without vector.

To date, the downstream signaling pathway of IL-17F has not been fully understood. In this study, a transient phosphorylation of MSK1 was observed upon stimulation of the cells with IL-17F, reaching the maximum at 20 min after stimulation (Fig. 5A). To establish the interrelationship between ERK1/2 and MSK1, the cells were treated with a MEK inhibitor, PD-98059, before the stimulation with IL-17F, since the current study and a previous report (20) have demonstrated that IL-17F activates only ERK1/2 in bronchial epithelial cells. As shown in Fig. 5B, pretreatment of the cells with PD-98059 markedly inhibited the phosphorylation of MSK1, indicating that ERK1/2 is a critical upstream kinase responsible for activation of MSK1.

Fig. 5.

A: kinetic activation of MSK1 by IL-17F in BEAS-2B cells. Western blotting was performed with antibodies against MSK1 and phosphorylated (p)-MSK1. B: effect of PD-98059 on IL-17F-induced phosphorylation of MSK1. Western blotting analysis was performed with antibodies against total (t)-MSK1 and p-MSK1. The results shown are representative of 3 separate experiments.

Next, to determine whether MSK1 affects IL-17F-induced IL-11 expression, effects of MSK1 inhibitors were investigated. Pretreatments with MSK1 inhibitors Ro-31-8220 and H89 significantly blocked IL-17F-induced IL-11 expression (Fig. 6), suggesting that MSK1 regulates this expression.

Fig. 6.

Effect of MSK1 inhibitors H89 and Ro-31-8220 on IL-17F-induced IL-11 in BEAS-2B cells. The levels of IL-11 protein production were measured by ELISA. Values are expressed as means ± SD (n = 4). *P < 0.05 vs. IL-17F-stimulated cells in the absence of the inhibitor.

Finally, to further confirm whether MSK1 plays a role in IL-17F-induced IL-11 expression, total MSK1 expression was first diminished in the cells by transfecting with siRNA targeting MSK1 (Fig. 7A). Next, the interrelationship between MSK1 and CREB was investigated by using siRNAs targeting MSK1, since CREB is located downstream of MSK1 (40). Knockdown of MSK1 in cells abrogated the IL-17F-induced activation of CREB (Fig. 7B). This suggests that CREB is located downstream of MSK1. As shown in Fig. 7C, the involvement of MSK1 and CREB for induction of IL-11 expression by IL-17F was then analyzed. Induction of IL-11 expression by IL-17F was significantly inhibited in cells transfected with siRNA targeting MSK1, whereas no significant difference was seen in wild-type cells and cells transfected with a control siRNA.

Fig. 7.

Regulation of IL-17F-induced IL-11 by MSK1-CREB in BEAS-2B cells. The validation of its blocking by siRNA for MSK1 was performed by Western blotting. The results shown are representative of 3 separate experiments. A: effect of siRNA for MSK1 on IL-17F-induced phosphorylation of CREB. Western blotting analysis was performed with antibodies against t-CREB and p-CREB. The results shown are representative of 4 separate experiments. B: the levels of IL-11 protein production were measured by ELISA. C: values are expressed as means ± SD (n = 4). *P < 0.05 vs. non-transfected cells.

DISCUSSION

In this report, we demonstrated that IL-17F induces expression of IL-11 in bronchial epithelial cells through the activation of the Raf1-MEK-ERK1/2-MSK1-CREB signaling pathway, providing a novel functional linkage between two cytokines that are involved in airway inflammation and remodeling. Bronchial epithelial cells have been shown to be a major cell source of IL-11 (10). Bronchial epithelial cells did not induce IL-11 in response to IL-4 and IL-13. However, IL-4 and IL-13 augmented IL-17F-induced IL-11 expression. These findings suggest that IL-17F is a novel inducer of IL-11 and induces airway remodeling and inflammation either alone or in combination with Th2 cytokines.

IL-11 plays a key role in airway remodeling and inflammation in asthma. A significantly increased expression of IL-11 mRNA and protein within the airways of subjects with severe asthma is seen when compared with those with mild asthma (28). The number of cells expressing IL-11 mRNA was inversely correlated to the FEV1 (28). IL-11 induces subepithelial fibrosis, accumulation of fibroblasts, myofibroblasts and myocytes, and deposition of type I and III collagen (35). Furthermore, a more recent report demonstrated that IL-11, signaling via IL-11Rα, plays an important role in aeroallergen-induced Th2 inflammation and mucus metaplasia (26). These findings suggest, therefore, that IL-11 contributes both to development of airway fibrosis and subsequent decline in lung function. The IL-17F-IL-11 axis may be especially important in the pathophysiological events of airway remodeling and inflammation in asthma. IL-11 is derived from various cell types, including bronchial epithelial cells, fibroblasts, eosinophils, mast cells, and airway smooth muscle cells, which play an important role in the pathogenesis of asthma (33, 43). In addition, IL-11 is induced by many asthma-related stimuli such as IL-1, TGF-β, histamine, eosinophil major basic protein, and prostaglandin E2 (43). Moreover, IL-11 is also induced by rhinovirus, respiratory syncytial virus, influenza virus A, and parainfluenza virus, which are important causes of asthma exacerbation (8, 10, 30). Here, we provided evidence that IL-17F is a novel inducer of IL-11 and is involved in pathogenesis of asthma via the induction of IL-11. Uncovering interactions between IL-17F and IL-11 may be valuable in understanding and treating asthma.

IL-17F is derived from activated CD4+ T cells, basophils, and mast cells, which are important regulatory cell types for allergic airway inflammation (21). Upregulated expression of IL-17F is seen in the bronchoalveolar lavage cells from asthmatics following segmental allergen challenge (21). Similarly, IL-17F is also clearly upregulated in the mouse model of asthma (34). Moreover, IL-17F has been shown to be able to induce pulmonary neutrophilia and produces an additive effect on antigen-induced allergic inflammatory responses (29). These findings suggest a role of IL-17F in the enhancement of allergic airway inflammation. Recently, we have demonstrated that IL-17F is a candidate gene for asthma susceptibility (12, 22). Moreover, IL-17F is able to induce several cytokines and chemokines in bronchial epithelial cells, vein endothelial cells, and fibroblasts (13, 15, 16, 18, 19, 20, 32). A recent report demonstrated that IL-17F also acts on eosinophils, one of the most important inflammatory cells in allergic airway inflammation and remodeling, to induce several cytokines and chemokines such as IL-1β, IL-6, IL-8, GROα, and MIP-1β (3). These findings suggest that these cell types play crucial roles in asthma in response to IL-17F. The potential involvement of IL-17F in airway inflammation is, therefore, likely mediated, in part, through the induction of IL-11. The release of IL-11 from epithelial cells by IL-17F may contribute both to the development of airway remodeling and to inflammation in asthma. The IL-17F-IL-11 axis provides new insight into the molecular mechanisms involved in airway remodeling and inflammation.

So far, asthma has been believed to be an example of Th2-mediated airway inflammation. Recent reports demonstrated that a new CD4+ T cell subset, Th17 cells, are involved in the pathogenesis of asthma (38). Current and previous data show that Th2 cytokines are able to augment the function of IL-17F (16). These findings suggest that both Th2 and Th17 cytokines orchestrate many features of asthma. The combination of Th2 cytokines and IL-17F may thus perpetuate allergic inflammation and cause airway remodeling via enhancement of IL-11 expression.

Unlike IL-17A, little information has been available regarding the signaling pathway of IL-17F. Similar to IL17A, IL-17F utilizes a heterodimer of IL17RA and IL17RC as its receptor (37). In the upstream signaling pathway, we have previously demonstrated that IL-17F activates the Raf1-MEK1/2-ERK1/2 pathway (15, 16, 18, 19, 20). Similarly, IL-17F induced IL-11 expression through this pathway. This pathway has been shown to be a central one for IL-17F-induced cytokine and chemokine expression in bronchial epithelial cells. Therefore, ERK1/2 may serve as a crucial signaling molecule for IL-17 family members, since IL-17A also activates ERK1/2 in bronchial epithelial cells (17). These findings suggest that regulation of the Raf1-MEK1/2-ERK1/2 pathway may constitute a useful therapeutic target for IL-17 family cytokine-associated diseases. Moreover, we have demonstrated that p90RSK-CREB pathway is an important downstream signaling pathway for IL-17F (16). However, inhibition of p90RSK did not completely abrogate biological activity of IL-17F, suggesting an additional signaling pathway to CREB (23). Here, we identified that MSK1-CREB is a novel signaling pathway of IL-17F. MSK1 is located downstream of the Raf1-MEK1/2-ERK1/2 cascade, since a MEK inhibitor, PD-98059, blocked the phosphorylation of MSK. Similarly, CREB is the downstream signaling molecule of MSK1, since the siRNA targeting MSK1 inhibited the activation of CREB. Moreover the activation of MSK1 is essential for IL-11 expression by IL-17F, since MSK1 inhibitors Ro-31-8220 and H89 and siRNA targeting MSK1 blocked its expression. These data suggest the candidacy of the MSK1-CREB pathway as a potential pharmacological target in IL-17F-induced airway inflammation. On the other hand, it is reported that ERK1/2 and p38MAPK are able to activate MSK1 in skeletal myoblasts in response to growth factors and cellular stress (23). However, IL-17F did not phosphorylate p38MAPK (20), and a p38MAPK inhibitor, SB-202190, did not elicit a significant response in the present study. These data suggest that MSK1 is regulated by ERK1/2 in the case of IL-17F in bronchial epithelial cells.

In conclusion, this study has revealed that IL-17F induces an airway remodeling-related cytokine, IL-11, in bronchial epithelial cells, and induces IL-11 via the activation of the Raf1-MEK1/2-ERK1/2-MSK1-CREB pathway. The respective role of IL-17F and IL-11 in allergic airway diseases has been suggested, and our study provides evidence for a functional linkage between these two cytokines, further strengthening their role in the regulation of airway inflammation. IL-17F may contribute to amplification and persistence of allergic airway inflammatory processes. Activation of the IL-17F-IL-11 axis in asthma may have important therapeutic implications and contribute to the development of airway remodeling and inflammation.

GRANTS

This work was supported by Grant-in-Aid for Young Scientists B 20790697 and The Uehara Memorial Foundation. S. K. Huang was supported, in part, by National Institute of Allergy and Infectious Diseases Grants AI-052468 and AI-073610.

Acknowledgments

We thank Hiroko Takeuchi, Hideaki Watanabe, and Miho Kawaguchi for excellent technical assistance.

Footnotes

  • * M. Kawaguchi and J. Fujita contributed equally to this work.

REFERENCES

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