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Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement.

Alrashdan YA, Alkhouri H, Chen E, Lalor DJ, Poniris M, Henness S, Brightling CE, Burgess JK, Armour CL, Ammit AJ, Hughes JM - Am. J. Physiol. Lung Cell Mol. Physiol. (2012)

Bottom Line: However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation.We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation.However, in both, JNK activation did not regulate early events leading to NF-κB activation.

View Article: PubMed Central - PubMed

Affiliation: Respiratory Research Group, Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia.

ABSTRACT
CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.

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JNK activation and the effects of its inhibitor SP600125 in ASM cells. Confluent serum-deprived asthmatic and nonasthmatic ASM cells were stimulated with cytomix for up to 1 h. Levels of phosphorylated P-46 and P-54 JNK in the cell lysates were detected using immunoblotting and quantified using densitometry. Differences in phosphorylated P-46 and P-54 levels (expressed as fold change over 0 h) between A (n = 6) and (n = 7) cells (A). Representative immunoblots and densitometry of changes with time in cytomix-induced P-46 and P-54 JNK phosphorylation (expressed as percentage of maximum phosphorylation) and the effects of SP600125 on it for A (n = 4, B) and NA (n = 6, C) ASM cells. The cells were treated with SP600125 or vehicle for 45 min before and during the cytomix stimulation. White lines indicate where immunoblot images have been spliced. Bars, mean ± SE; vehicle 0.1% vol/vol DMSO.
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Figure 5: JNK activation and the effects of its inhibitor SP600125 in ASM cells. Confluent serum-deprived asthmatic and nonasthmatic ASM cells were stimulated with cytomix for up to 1 h. Levels of phosphorylated P-46 and P-54 JNK in the cell lysates were detected using immunoblotting and quantified using densitometry. Differences in phosphorylated P-46 and P-54 levels (expressed as fold change over 0 h) between A (n = 6) and (n = 7) cells (A). Representative immunoblots and densitometry of changes with time in cytomix-induced P-46 and P-54 JNK phosphorylation (expressed as percentage of maximum phosphorylation) and the effects of SP600125 on it for A (n = 4, B) and NA (n = 6, C) ASM cells. The cells were treated with SP600125 or vehicle for 45 min before and during the cytomix stimulation. White lines indicate where immunoblot images have been spliced. Bars, mean ± SE; vehicle 0.1% vol/vol DMSO.

Mentions: There was a significant difference between asthmatic and nonasthmatic cells in JNK phosphorylation (Fig. 5A). Both JNK isoforms P-54 and P-46 were rapidly phosphorylated, reaching a peak over 10–30 min following cytomix stimulation (Fig. 5, A–C). However, P46 and P54 phosphorylation were lower in asthmatic compared with nonasthmatic cells (Fig. 5A). The JNK inhibitor SP600125 reduced the early P-54 phosphorylation more than P-46 phosphorylation in the asthmatic cell lysates (Fig. 5B). In nonasthmatic cells SP600125 reduced P-54 and P-46 phosphorylation in a similar way (Fig. 5C).


Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement.

Alrashdan YA, Alkhouri H, Chen E, Lalor DJ, Poniris M, Henness S, Brightling CE, Burgess JK, Armour CL, Ammit AJ, Hughes JM - Am. J. Physiol. Lung Cell Mol. Physiol. (2012)

JNK activation and the effects of its inhibitor SP600125 in ASM cells. Confluent serum-deprived asthmatic and nonasthmatic ASM cells were stimulated with cytomix for up to 1 h. Levels of phosphorylated P-46 and P-54 JNK in the cell lysates were detected using immunoblotting and quantified using densitometry. Differences in phosphorylated P-46 and P-54 levels (expressed as fold change over 0 h) between A (n = 6) and (n = 7) cells (A). Representative immunoblots and densitometry of changes with time in cytomix-induced P-46 and P-54 JNK phosphorylation (expressed as percentage of maximum phosphorylation) and the effects of SP600125 on it for A (n = 4, B) and NA (n = 6, C) ASM cells. The cells were treated with SP600125 or vehicle for 45 min before and during the cytomix stimulation. White lines indicate where immunoblot images have been spliced. Bars, mean ± SE; vehicle 0.1% vol/vol DMSO.
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Figure 5: JNK activation and the effects of its inhibitor SP600125 in ASM cells. Confluent serum-deprived asthmatic and nonasthmatic ASM cells were stimulated with cytomix for up to 1 h. Levels of phosphorylated P-46 and P-54 JNK in the cell lysates were detected using immunoblotting and quantified using densitometry. Differences in phosphorylated P-46 and P-54 levels (expressed as fold change over 0 h) between A (n = 6) and (n = 7) cells (A). Representative immunoblots and densitometry of changes with time in cytomix-induced P-46 and P-54 JNK phosphorylation (expressed as percentage of maximum phosphorylation) and the effects of SP600125 on it for A (n = 4, B) and NA (n = 6, C) ASM cells. The cells were treated with SP600125 or vehicle for 45 min before and during the cytomix stimulation. White lines indicate where immunoblot images have been spliced. Bars, mean ± SE; vehicle 0.1% vol/vol DMSO.
Mentions: There was a significant difference between asthmatic and nonasthmatic cells in JNK phosphorylation (Fig. 5A). Both JNK isoforms P-54 and P-46 were rapidly phosphorylated, reaching a peak over 10–30 min following cytomix stimulation (Fig. 5, A–C). However, P46 and P54 phosphorylation were lower in asthmatic compared with nonasthmatic cells (Fig. 5A). The JNK inhibitor SP600125 reduced the early P-54 phosphorylation more than P-46 phosphorylation in the asthmatic cell lysates (Fig. 5B). In nonasthmatic cells SP600125 reduced P-54 and P-46 phosphorylation in a similar way (Fig. 5C).

Bottom Line: However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation.We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation.However, in both, JNK activation did not regulate early events leading to NF-κB activation.

View Article: PubMed Central - PubMed

Affiliation: Respiratory Research Group, Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia.

ABSTRACT
CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.

Show MeSH
Related in: MedlinePlus