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Inhibition of PDE4B suppresses inflammation by increasing expression of the deubiquitinase CYLD.

Komatsu K, Lee JY, Miyata M, Hyang Lim J, Jono H, Koga T, Xu H, Yan C, Kai H, Li JD - Nat Commun (2013)

Bottom Line: Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defence response.Importantly, ototopical post-inoculation administration of a PDE4 inhibitor suppresses inflammation in this animal model, thus demonstrating the therapeutic potential of targeting PDE4.These studies provide insights into how inflammation is tightly regulated via the inhibition of its negative regulator and may also lead to the development of new anti-inflammatory therapeutics that upregulate CYLD expression.

View Article: PubMed Central - PubMed

Affiliation: Center for Inflammation, Immunity & Infection and Department of Biology, Georgia State University, 100 Piedmont Avenue, Atlanta, Georgia 30303, USA.

ABSTRACT
The deubiquitinase CYLD acts as a key negative regulator to tightly control overactive inflammation. Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defence response. Here, we show that inhibition of phosphodiesterase 4B (PDE4B) markedly enhances upregulation of CYLD expression in response to bacteria, thereby suggesting that PDE4B acts as a negative regulator for CYLD. Interestingly, in Cyld-deficient mice, inhibition of PDE4B no longer suppresses inflammation. Moreover, PDE4B negatively regulates CYLD via specific activation of JNK2 but not JNK1. Importantly, ototopical post-inoculation administration of a PDE4 inhibitor suppresses inflammation in this animal model, thus demonstrating the therapeutic potential of targeting PDE4. These studies provide insights into how inflammation is tightly regulated via the inhibition of its negative regulator and may also lead to the development of new anti-inflammatory therapeutics that upregulate CYLD expression.

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PDE4B negatively regulates CYLD expression via specific activation of JNK2 but not JNK1.CYLD and IL-1β mRNA expression was measured in HMEEC (a), mouse middle ear (b) pretreated with SP600125 (5 μM for 1 h in vitro, 5 mg kg−1 for 2 h in vivo) and stimulated with NTHi. (c) CYLD, IL-1β mRNA expression in cells was measured after NTHi stimulation pretreated with Rolipram (10 μM) and SP600125 (5 μM). (d) Cells were pretreated with Rolipram, followed by NTHi stimulation, and cell lysates were analysed by immunoblotting with the indicated antibodies. (e) Cells were transfected with siCON or siPDE4B, followed by NTHi stimulation, and cell lysates were analysed by immunoblotting with the indicated antibodies. (f) Cells transfected with siCON, siJNK1 or siJNK2 were pretreated with Rolipram (10 μM), and CYLD mRNA expression was measured post NTHi treatment. (g) IL-1β mRNA expression was measured in MEF cells from Cyld+/+, Cyld−/− mice after NTHi stimulation pretreated with SP600125 (5 μM). (h) TNF-α mRNA expression was measured in middle ear tissues from Cyld+/+, Cyld−/− mice preinoculated with SP600125 (5 mg kg−1) and inoculated with NTHi. (i) Haematoxylin and eosin staining of middle ear tissues from Cyld+/+, Cyld−/− mice preinoculated with SP600125 and inoculated with NTHi (magnification × 400). Scale bars, 20 μm. (j) Thickness of middle ear mucosa in Cyld+/+, Cyld−/− mice preinoculated with SP600125 and inoculated with NTHi was measured from 15 middle ear tissue sections per experimental group. Data in a–c, f–h and j are mean±s.d. (n=3 in a–c, f–h and 15 in j). *P<0.05. Statistical analysis was performed using Student’s t-test. Data are representative of three or more independent experiments. CON, control; NS, nonsignificant.
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f5: PDE4B negatively regulates CYLD expression via specific activation of JNK2 but not JNK1.CYLD and IL-1β mRNA expression was measured in HMEEC (a), mouse middle ear (b) pretreated with SP600125 (5 μM for 1 h in vitro, 5 mg kg−1 for 2 h in vivo) and stimulated with NTHi. (c) CYLD, IL-1β mRNA expression in cells was measured after NTHi stimulation pretreated with Rolipram (10 μM) and SP600125 (5 μM). (d) Cells were pretreated with Rolipram, followed by NTHi stimulation, and cell lysates were analysed by immunoblotting with the indicated antibodies. (e) Cells were transfected with siCON or siPDE4B, followed by NTHi stimulation, and cell lysates were analysed by immunoblotting with the indicated antibodies. (f) Cells transfected with siCON, siJNK1 or siJNK2 were pretreated with Rolipram (10 μM), and CYLD mRNA expression was measured post NTHi treatment. (g) IL-1β mRNA expression was measured in MEF cells from Cyld+/+, Cyld−/− mice after NTHi stimulation pretreated with SP600125 (5 μM). (h) TNF-α mRNA expression was measured in middle ear tissues from Cyld+/+, Cyld−/− mice preinoculated with SP600125 (5 mg kg−1) and inoculated with NTHi. (i) Haematoxylin and eosin staining of middle ear tissues from Cyld+/+, Cyld−/− mice preinoculated with SP600125 and inoculated with NTHi (magnification × 400). Scale bars, 20 μm. (j) Thickness of middle ear mucosa in Cyld+/+, Cyld−/− mice preinoculated with SP600125 and inoculated with NTHi was measured from 15 middle ear tissue sections per experimental group. Data in a–c, f–h and j are mean±s.d. (n=3 in a–c, f–h and 15 in j). *P<0.05. Statistical analysis was performed using Student’s t-test. Data are representative of three or more independent experiments. CON, control; NS, nonsignificant.

Mentions: We next sought to determine how PDE4B mediates NTHi-induced inflammation via negatively regulating CYLD expression. As the mitogen-activated protein kinase (MAPK) JNK has an important role in mediating bacteria-induced host response333435, we initially determined the involvement of JNK in regulating CYLD using specific JNK inhibitor, SP600125. As shown in Fig. 5a, inhibition of JNK using SP600125 markedly enhanced NTHi-induced upregulation of CYLD and suppressed inflammation in HMEEC cells in vitro and in the middle ear of mouse in vivo. Interestingly, PDE4 inhibition using Rolipram no longer enhanced NTHi-induced upregulation of CYLD and suppressed inflammation in HMEEC cells that had been already pretreated with SP600125 (Fig. 5c). These results suggest that PDE4B negatively regulates CYLD expression and mediates inflammation via the JNK pathway.


Inhibition of PDE4B suppresses inflammation by increasing expression of the deubiquitinase CYLD.

Komatsu K, Lee JY, Miyata M, Hyang Lim J, Jono H, Koga T, Xu H, Yan C, Kai H, Li JD - Nat Commun (2013)

PDE4B negatively regulates CYLD expression via specific activation of JNK2 but not JNK1.CYLD and IL-1β mRNA expression was measured in HMEEC (a), mouse middle ear (b) pretreated with SP600125 (5 μM for 1 h in vitro, 5 mg kg−1 for 2 h in vivo) and stimulated with NTHi. (c) CYLD, IL-1β mRNA expression in cells was measured after NTHi stimulation pretreated with Rolipram (10 μM) and SP600125 (5 μM). (d) Cells were pretreated with Rolipram, followed by NTHi stimulation, and cell lysates were analysed by immunoblotting with the indicated antibodies. (e) Cells were transfected with siCON or siPDE4B, followed by NTHi stimulation, and cell lysates were analysed by immunoblotting with the indicated antibodies. (f) Cells transfected with siCON, siJNK1 or siJNK2 were pretreated with Rolipram (10 μM), and CYLD mRNA expression was measured post NTHi treatment. (g) IL-1β mRNA expression was measured in MEF cells from Cyld+/+, Cyld−/− mice after NTHi stimulation pretreated with SP600125 (5 μM). (h) TNF-α mRNA expression was measured in middle ear tissues from Cyld+/+, Cyld−/− mice preinoculated with SP600125 (5 mg kg−1) and inoculated with NTHi. (i) Haematoxylin and eosin staining of middle ear tissues from Cyld+/+, Cyld−/− mice preinoculated with SP600125 and inoculated with NTHi (magnification × 400). Scale bars, 20 μm. (j) Thickness of middle ear mucosa in Cyld+/+, Cyld−/− mice preinoculated with SP600125 and inoculated with NTHi was measured from 15 middle ear tissue sections per experimental group. Data in a–c, f–h and j are mean±s.d. (n=3 in a–c, f–h and 15 in j). *P<0.05. Statistical analysis was performed using Student’s t-test. Data are representative of three or more independent experiments. CON, control; NS, nonsignificant.
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Related In: Results  -  Collection

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Show All Figures
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f5: PDE4B negatively regulates CYLD expression via specific activation of JNK2 but not JNK1.CYLD and IL-1β mRNA expression was measured in HMEEC (a), mouse middle ear (b) pretreated with SP600125 (5 μM for 1 h in vitro, 5 mg kg−1 for 2 h in vivo) and stimulated with NTHi. (c) CYLD, IL-1β mRNA expression in cells was measured after NTHi stimulation pretreated with Rolipram (10 μM) and SP600125 (5 μM). (d) Cells were pretreated with Rolipram, followed by NTHi stimulation, and cell lysates were analysed by immunoblotting with the indicated antibodies. (e) Cells were transfected with siCON or siPDE4B, followed by NTHi stimulation, and cell lysates were analysed by immunoblotting with the indicated antibodies. (f) Cells transfected with siCON, siJNK1 or siJNK2 were pretreated with Rolipram (10 μM), and CYLD mRNA expression was measured post NTHi treatment. (g) IL-1β mRNA expression was measured in MEF cells from Cyld+/+, Cyld−/− mice after NTHi stimulation pretreated with SP600125 (5 μM). (h) TNF-α mRNA expression was measured in middle ear tissues from Cyld+/+, Cyld−/− mice preinoculated with SP600125 (5 mg kg−1) and inoculated with NTHi. (i) Haematoxylin and eosin staining of middle ear tissues from Cyld+/+, Cyld−/− mice preinoculated with SP600125 and inoculated with NTHi (magnification × 400). Scale bars, 20 μm. (j) Thickness of middle ear mucosa in Cyld+/+, Cyld−/− mice preinoculated with SP600125 and inoculated with NTHi was measured from 15 middle ear tissue sections per experimental group. Data in a–c, f–h and j are mean±s.d. (n=3 in a–c, f–h and 15 in j). *P<0.05. Statistical analysis was performed using Student’s t-test. Data are representative of three or more independent experiments. CON, control; NS, nonsignificant.
Mentions: We next sought to determine how PDE4B mediates NTHi-induced inflammation via negatively regulating CYLD expression. As the mitogen-activated protein kinase (MAPK) JNK has an important role in mediating bacteria-induced host response333435, we initially determined the involvement of JNK in regulating CYLD using specific JNK inhibitor, SP600125. As shown in Fig. 5a, inhibition of JNK using SP600125 markedly enhanced NTHi-induced upregulation of CYLD and suppressed inflammation in HMEEC cells in vitro and in the middle ear of mouse in vivo. Interestingly, PDE4 inhibition using Rolipram no longer enhanced NTHi-induced upregulation of CYLD and suppressed inflammation in HMEEC cells that had been already pretreated with SP600125 (Fig. 5c). These results suggest that PDE4B negatively regulates CYLD expression and mediates inflammation via the JNK pathway.

Bottom Line: Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defence response.Importantly, ototopical post-inoculation administration of a PDE4 inhibitor suppresses inflammation in this animal model, thus demonstrating the therapeutic potential of targeting PDE4.These studies provide insights into how inflammation is tightly regulated via the inhibition of its negative regulator and may also lead to the development of new anti-inflammatory therapeutics that upregulate CYLD expression.

View Article: PubMed Central - PubMed

Affiliation: Center for Inflammation, Immunity & Infection and Department of Biology, Georgia State University, 100 Piedmont Avenue, Atlanta, Georgia 30303, USA.

ABSTRACT
The deubiquitinase CYLD acts as a key negative regulator to tightly control overactive inflammation. Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defence response. Here, we show that inhibition of phosphodiesterase 4B (PDE4B) markedly enhances upregulation of CYLD expression in response to bacteria, thereby suggesting that PDE4B acts as a negative regulator for CYLD. Interestingly, in Cyld-deficient mice, inhibition of PDE4B no longer suppresses inflammation. Moreover, PDE4B negatively regulates CYLD via specific activation of JNK2 but not JNK1. Importantly, ototopical post-inoculation administration of a PDE4 inhibitor suppresses inflammation in this animal model, thus demonstrating the therapeutic potential of targeting PDE4. These studies provide insights into how inflammation is tightly regulated via the inhibition of its negative regulator and may also lead to the development of new anti-inflammatory therapeutics that upregulate CYLD expression.

Show MeSH
Related in: MedlinePlus