ERK1/2 mitogen-activated protein kinase selectively mediates IL-13-induced lung inflammation and remodeling in vivo

Abstract

IL-13 dysregulation plays a critical role in the pathogenesis of a variety of inflammatory and remodeling diseases. In these settings, STAT6 is believed to be the canonical signaling molecule mediating the tissue effects of IL-13. Signaling cascades involving MAPKs have been linked to inflammation and remodeling. We hypothesized that MAPKs play critical roles in effector responses induced by IL-13 in the lung. We found that Tg IL-13 expression in the lung led to potent activation of ERK1/2 but not JNK1/2 or p38. ERK1/2 activation also occurred in mice with null mutations of STAT6. Systemic administration of the MAPK/ERK kinase 1 (MEK1) inhibitor PD98059 or use of Tg mice in which a dominant-negative MEK1 construct was expressed inhibited IL-13-induced inflammation and alveolar remodeling. There were associated decreases in IL-13-induced chemokines (MIP-1alpha/CCL-3, MIP-1beta/CCL-4, MIP-2/CXCL-1, RANTES/CCL-5), MMP-2, -9, -12, and -14, and cathepsin B and increased levels of alpha1-antitrypsin. IL-13-induced tissue and molecular responses were noted that were equally and differentially dependent on ERK1/2 and STAT6 signaling. Thus, ERK1/2 is activated by IL-13 in the lung in a STAT6-independent manner where it contributes to IL-13-induced inflammation and remodeling and is required for optimal IL-13 stimulation of specific chemokines and proteases as well as the inhibition of specific antiproteases. ERK1/2 regulators may be useful in the treatment of IL-13-induced diseases and disorders.

Figure 1

Effect of IL-13 on MAPK activation. Western blots for phosphorylated (p) STAT6, ERK1/2, JNK1/2, and p38 were performed on lung lysates from CC10–rtTA–IL-13 mice (IL-13⁺) fed dox water for 0 to 3 days. Antibodies against the relevant total protein were used to control for loading. Data are representative of n = 4 for each group.

Figure 2

IL-13–induced ERK1/2 activation occurs in the absence of STAT6. Western blots for phosphorylated ERK1/2 and STAT6 were performed on lung lysates from (A) constitutive CC10–IL-13⁺ or (B) CC10–rtTA–IL-13⁺ Tg mice. In each, we compare Tg^– (–) and Tg⁺ (+) IL-13 mice with wild-type (+/+) and homozygous null (–/–) STAT6 loci. β-tubulin was used as loading control. Data are representative of n = 4 for each group.

Figure 3

ERK1/2 inhibition in IL-13 Tg mice. Western blots for phosphorylated ERK1/2 and STAT6 were performed on lung lysates from IL-13^– and CC10–rtTA–IL-13 Tg⁺ (+) mice. (A) Mice treated with PD or its vehicle control. (B) Mice with and without the CC10–dnMEK1 Tg construct. Data are representative of n = 4 for each group. (C) Levels of IL-13 protein in BAL fluids from IL-13^– and IL-13⁺ mice with and without PD or dnMEK1. Data are expressed as mean ± SEM. n = 4–5 for each group. *P < 0.05 compared with IL-13⁺.

Figure 4

Role of ERK1/2 MAPK in IL-13–induced inflammation. BAL fluids and lungs were obtained from IL-13^– and CC10–rtTA–IL-13 Tg⁺ (IL-13⁺) mice. (A) BAL-fluid cellularity of mice treated with PD or vehicle control (veh). (B) BAL-fluid cellularity of IL-13⁺ mice with and without dnMEK1. (C) MBP immunostaining and morphometric eosinophil quantification were used in comparing lungs from IL-13^– and IL-13⁺ mice after PD administration or with and without dnMEK1. Data are expressed as mean ± SEM and are representative of n = 4–16 for each group. *P < 0.05 compared with IL-13⁺.

Figure 5

Role of STAT6 in IL-13–induced inflammation. BAL fluids and lung tissues were obtained from IL-13^– and CC10–rtTA–IL-13 Tg⁺ (IL-13⁺) mice with wild-type or null STAT6 loci. (A) BAL fluids were obtained for total cell counts and differentials. (B) MBP immunostaining and morphometric lung tissue eosinophil quantification were undertaken. Data are expressed as mean ± SEM and are representative of n = 4–5 for each group. *P < 0.05 compared with IL-13⁺/STAT6^+/+ mice.

Figure 6

Role of ERK1/2 MAPK in IL-13–induced chemokine mRNA and protein expression. (A and B) Whole-lung RNA was used for RT-PCR to detect mRNA for IL-13–regulated chemokines in lungs from IL-13^– and IL-13⁺ mice. Comparisons are made of mice administered PD and vehicle control (A) and mice with and without dnMEK1 (B). β-actin mRNA expression was used as a loading control. (C and D) Levels of the noted chemokines were assessed by ELISA in BAL fluids from IL-13^– and IL-13⁺ mice. Comparisons are made of mice administered PD or vehicle control (C) and mice with and without dnMEK1 (D). Data are expressed as mean ± SEM and represent n = 5 for each group. *P < 0.05 compared with IL-13⁺.

Figure 7

Role of STAT6 in IL-13–induced chemokine mRNA and protein expression. IL-13^– and CC10–rtTA–IL-13 Tg⁺ (IL-13⁺) mice with wild-type or null STAT6 loci were generated. (A) Whole-lung RNA was processed for RT-PCR to detect mRNA for IL-13–regulated chemokines. β-actin mRNA expression was used as a loading control. (B) Levels of the noted chemokines in BAL fluids were evaluated by ELISA. Data are expressed as mean ± SEM and represent n = 4–5 for each group. *P < 0.05 compared with IL-13⁺/STAT6^+/+ mice.

Figure 8

Role of ERK1/2 MAPK in IL-13–induced tissue remodeling responses. Parameters of remodeling were assessed in IL-13^– and CC10–rtTA–IL-13 Tg⁺ (IL-13⁺) mice. (A–C) Comparison of (A) alveolar histology (H&E), (B) lung volume, and (C) alveolar chord length in mice treated with PD or vehicle control. Original magnification, ×10. (D–F) Comparison of (D) alveolar histology (H&E), (E) lung volume, and (F) alveolar chord length in mice with and without dnMEK1. Original magnification, ×10. (G) PAS with diastase staining and (H) levels of mucin and Gob-5 mRNA in these animals. β-actin mRNA expression was used as a loading control. Original magnification, ×40. Arrow highlights PAS with diastase staining of epithelial cells in IL-13⁺/STAT6^–/– animals. Values in graphs illustrate the mean ± SEM and represent n = 4–6 for each group. *P < 0.05 compared with IL-13⁺.

Figure 9

Role of STAT6 in IL-13–induced tissue remodeling responses. IL-13^– and CC10–rtTA–IL-13 Tg⁺ (IL-13⁺) mice with wild-type or null STAT6 loci were generated. Lungs were processed for (A) alveolar histology (H&E), (B) lung volume, (C) alveolar chord length, (D) PAS with diastase staining, (E) mucin gene expression, (F) BAL-fluid Muc-5ac content. Arrow highlights residual PAS⁺ epithelial cells in IL-13⁺/STAT6^–/– lungs. Original magnification, ×10 (A); ×40 (D). For E, β-actin mRNA expression was used as loading control. Graphical values illustrate the mean ± SEM and represent n = 4–5 for each group. *P < 0.05 compared with IL-13⁺/STAT6^+/+ mice.

Figure 10

Role of ERK1/2 MAPK in IL-13 regulation of proteases and antiproteases. (A–D) Whole-lung RNA was used for RT-PCR (A and B) and real-time RT-PCR (C and D) to detect mRNA for proteases and anti-proteases in IL-13 (–) and IL-13 (+) mice. Comparisons are made of mice treated with PD or vehicle control (A and C) and mice with and without dnMEK1 (B and C). β-actin mRNA expression was used as a loading control. Data are expressed as mean ± SEM and represent n = 4–5 for each group. *P < 0.05 compared with IL-13⁺.

Figure 11

Role of STAT6 in IL-13 regulation of proteases and antiproteases. IL-13 (–) and CC10–rtTA–IL-13 Tg (+) mice with wild-type or null STAT6 loci were generated. Protease and antiprotease mRNA levels were assessed using RT-PCR (A) and real-time RT-PCR (B). β-actin mRNA expression was used as a loading control. Data are expressed as mean ± SEM and represent n = 4–5 for each group. *P < 0.05 compared with IL-13⁺/STAT6^+/+ mice.