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AMPK Activation by A-769662 Controls IL-6 Expression in Inflammatory Arthritis.

Guma M, Wang Y, Viollet B, Liu-Bryan R - PLoS ONE (2015)

Bottom Line: Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects.The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Allergy and Immunology, UC San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT

Objective: AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase critically involved in the regulation of cellular energy homeostasis. It is a central regulator of both lipid and glucose metabolism. Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects. In this study, we assessed whether targeted activation of AMPK inhibits inflammatory arthritis in vivo.

Methods: We tested the effect of A-769662, a specific AMPK agonist (60mg/kg/bid) in mouse models of antigen-induced arthritis (AIA) and passive K/BxN serum-induced arthritis. The passive K/BxN serum-induced arthritis model was also applied to AMPKα1-deficient mice. Joints were harvested and subjected to histological analysis. IL-6 expression was measured in both joint tissues and sera by ELISA. The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.

Results: AMPK activation by A-769662 reduced inflammatory infiltration and joint damage in both mouse models. IL-6 expression in serum and arthritic joints was significantly decreased in A-769662-treated mice. AMPKα1 deficient mice mildly elicited an increase of clinical arthritis. IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

Conclusions: AMPK activation by specific AMPK agonist A-769662 suppressed inflammatory arthritis in mice as well as IL-6 expression in serum and arthritic joints. These data suggest that targeted activation of AMPK has a potential to be an effective therapeutic strategy for IL-6 dependent inflammatory arthritis.

No MeSH data available.


Related in: MedlinePlus

A-769662 activated AMPKα and regulated NF-κB and MAPK signaling in BMDMs.BMDMs were cultured in the presence of different concentrations of A-769662 for 2 hours (A), or BMDMs were pre-treated with A-769662 for 2 hours before stimulated with LPS (1 μg/ml) (B). Lysates of BMDMs were prepared and analyzed for the expression of the indicated proteins by Western blot (WB). Semi-quantitative densitometry analysis of Western blots (arbitrary densitometry units) for phosphorylation of indicated proteins normalized to total protein is shown. Results are representative of three independent experiments.
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pone.0140452.g001: A-769662 activated AMPKα and regulated NF-κB and MAPK signaling in BMDMs.BMDMs were cultured in the presence of different concentrations of A-769662 for 2 hours (A), or BMDMs were pre-treated with A-769662 for 2 hours before stimulated with LPS (1 μg/ml) (B). Lysates of BMDMs were prepared and analyzed for the expression of the indicated proteins by Western blot (WB). Semi-quantitative densitometry analysis of Western blots (arbitrary densitometry units) for phosphorylation of indicated proteins normalized to total protein is shown. Results are representative of three independent experiments.

Mentions: As macrophages are considered the principal innate immune effector cells of rheumatoid arthritis, we determined the effect in vitro of the specific AMPK pharmacological activator A-769662 on BMDMs. We first evaluated activation of AMPKα in cultured primary BMDMs stimulated with A-769662. As shown in Fig 1A, phosphorylation of AMPKα Thr172 was enhanced by A-769662 at all doses tested. A-769662 (100 μM) was able to inhibit LPS-induced phosphorylation p65 NF-κB, phosphorylation of Erk1/2 MAPK, and phosphorylation of p38 MAPK (Fig 1B and S1 Fig) in BMDM stimulated with LPS. It also inhibited IL-6 secretion of BMDMs stimulated with Pam3Cys4 and LPS (Fig 2A), NO release (Fig 2B) and iNOS and IL-6 mRNA expression (Fig 2C and 2D) after LPS stimulation. However other cytokines such as TNF and chemokines such as CXCL1 were not affected by A-769662 treatment (Fig 2E and 2F). These data suggest that activation of AMPK attenuated inflammatory responses to TLR2 or TLR4 agonists in macrophages in vitro.


AMPK Activation by A-769662 Controls IL-6 Expression in Inflammatory Arthritis.

Guma M, Wang Y, Viollet B, Liu-Bryan R - PLoS ONE (2015)

A-769662 activated AMPKα and regulated NF-κB and MAPK signaling in BMDMs.BMDMs were cultured in the presence of different concentrations of A-769662 for 2 hours (A), or BMDMs were pre-treated with A-769662 for 2 hours before stimulated with LPS (1 μg/ml) (B). Lysates of BMDMs were prepared and analyzed for the expression of the indicated proteins by Western blot (WB). Semi-quantitative densitometry analysis of Western blots (arbitrary densitometry units) for phosphorylation of indicated proteins normalized to total protein is shown. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608670&req=5

pone.0140452.g001: A-769662 activated AMPKα and regulated NF-κB and MAPK signaling in BMDMs.BMDMs were cultured in the presence of different concentrations of A-769662 for 2 hours (A), or BMDMs were pre-treated with A-769662 for 2 hours before stimulated with LPS (1 μg/ml) (B). Lysates of BMDMs were prepared and analyzed for the expression of the indicated proteins by Western blot (WB). Semi-quantitative densitometry analysis of Western blots (arbitrary densitometry units) for phosphorylation of indicated proteins normalized to total protein is shown. Results are representative of three independent experiments.
Mentions: As macrophages are considered the principal innate immune effector cells of rheumatoid arthritis, we determined the effect in vitro of the specific AMPK pharmacological activator A-769662 on BMDMs. We first evaluated activation of AMPKα in cultured primary BMDMs stimulated with A-769662. As shown in Fig 1A, phosphorylation of AMPKα Thr172 was enhanced by A-769662 at all doses tested. A-769662 (100 μM) was able to inhibit LPS-induced phosphorylation p65 NF-κB, phosphorylation of Erk1/2 MAPK, and phosphorylation of p38 MAPK (Fig 1B and S1 Fig) in BMDM stimulated with LPS. It also inhibited IL-6 secretion of BMDMs stimulated with Pam3Cys4 and LPS (Fig 2A), NO release (Fig 2B) and iNOS and IL-6 mRNA expression (Fig 2C and 2D) after LPS stimulation. However other cytokines such as TNF and chemokines such as CXCL1 were not affected by A-769662 treatment (Fig 2E and 2F). These data suggest that activation of AMPK attenuated inflammatory responses to TLR2 or TLR4 agonists in macrophages in vitro.

Bottom Line: Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects.The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Allergy and Immunology, UC San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT

Objective: AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase critically involved in the regulation of cellular energy homeostasis. It is a central regulator of both lipid and glucose metabolism. Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects. In this study, we assessed whether targeted activation of AMPK inhibits inflammatory arthritis in vivo.

Methods: We tested the effect of A-769662, a specific AMPK agonist (60mg/kg/bid) in mouse models of antigen-induced arthritis (AIA) and passive K/BxN serum-induced arthritis. The passive K/BxN serum-induced arthritis model was also applied to AMPKα1-deficient mice. Joints were harvested and subjected to histological analysis. IL-6 expression was measured in both joint tissues and sera by ELISA. The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.

Results: AMPK activation by A-769662 reduced inflammatory infiltration and joint damage in both mouse models. IL-6 expression in serum and arthritic joints was significantly decreased in A-769662-treated mice. AMPKα1 deficient mice mildly elicited an increase of clinical arthritis. IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

Conclusions: AMPK activation by specific AMPK agonist A-769662 suppressed inflammatory arthritis in mice as well as IL-6 expression in serum and arthritic joints. These data suggest that targeted activation of AMPK has a potential to be an effective therapeutic strategy for IL-6 dependent inflammatory arthritis.

No MeSH data available.


Related in: MedlinePlus