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AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs.

El Hadri K, Denoyelle C, Ravaux L, Viollet B, Foretz M, Friguet B, Rouis M, Raymondjean M - PLoS ONE (2015)

Bottom Line: Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far.Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex.Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universités, Université Pierre et Marie Curie, Biological Adaptation and Ageing (B2A) CNRS UMR8256/INSERM ERL-U1064, F-75005 Paris, France.

ABSTRACT
Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

No MeSH data available.


Related in: MedlinePlus

AICAR and phenformin treatments induce the phosphorylation of AMPK and ACC in isolated rat VSMCs in primary culture.After 1 hour pretreatment with AICAR (2mM) or phenformin (1mM), the cells were incubated or not with IL-1β (10ng/ml) for 30 minutes. Total proteins (20μg/lane) were separated by SDS PAGE and western blotted with specific antibodies (part A) against phospho-AMPK, total AMPK (65 KDa), and (part B) phospho-ACC, total ACC. β–actin was used as loading control. Representative blots of 3 independent experiments are shown. Data of the quantification are expressed as mean +/- SEM. *, p < 0,05; **, p < 0,01, AICAR or Phenformin treated vs control cells.
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pone.0132498.g001: AICAR and phenformin treatments induce the phosphorylation of AMPK and ACC in isolated rat VSMCs in primary culture.After 1 hour pretreatment with AICAR (2mM) or phenformin (1mM), the cells were incubated or not with IL-1β (10ng/ml) for 30 minutes. Total proteins (20μg/lane) were separated by SDS PAGE and western blotted with specific antibodies (part A) against phospho-AMPK, total AMPK (65 KDa), and (part B) phospho-ACC, total ACC. β–actin was used as loading control. Representative blots of 3 independent experiments are shown. Data of the quantification are expressed as mean +/- SEM. *, p < 0,05; **, p < 0,01, AICAR or Phenformin treated vs control cells.

Mentions: Experiments were conducted with primary cultures of VSMCs isolated from rat aorta. These cells undergo phenotypic changes in response to proinflammatory conditions. Indeed, in response to proinflammatory cytokines, these cells express several biomarkers of inflammation such as VCAM-1, MCP1, extracellular metalloproteinases and acute phase enzymes such as secreted sPLA2 and COX-2 [7,37–39]. Moreover, we have previously observed that prostaglandins E2 combined with IL-1β progressively synergizes the secretion of sPLA2 and causes a complete disorganization of the cytoskeletal framework [9]. To explore the effect of AMPK activation on IL-1β-induced sPLA2IIA gene expression and activity, cultured VSMCs were pretreated or not with the AMPK activators AICAR or phenformin prior IL-1β treatment. We chose to use phenformin which is more lipophilic than metformin and more efficiently internalized in cell culture in absence of cationic transporters. We performed immunoblotting to show whether treatment with AICAR or phenformin led to the activation of AMPK, characterized by the phosphorylation of Thr172 within the AMPKα subunit and of Ser79 in acetyl-coA carboxylase (ACC), a well-established target of AMPK [26]. As shown in Fig 1, treatment of VSMCs with 2mM AICAR or 1mM phenformin led to a significant increase in Thr172 AMPK and Ser79 ACC phosphorylation. In the presence of IL-1β, AICAR and phenformin induced phosphorylation of both Thr172 AMPK and Ser79 ACC. These results confirm that AICAR and phenformin stimulate AMPK signaling pathway in primary cultured rat VSMCs, consistent with previous studies showing AMPK activation by the biguanide metformin in BAECs or HUVECs [40].


AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs.

El Hadri K, Denoyelle C, Ravaux L, Viollet B, Foretz M, Friguet B, Rouis M, Raymondjean M - PLoS ONE (2015)

AICAR and phenformin treatments induce the phosphorylation of AMPK and ACC in isolated rat VSMCs in primary culture.After 1 hour pretreatment with AICAR (2mM) or phenformin (1mM), the cells were incubated or not with IL-1β (10ng/ml) for 30 minutes. Total proteins (20μg/lane) were separated by SDS PAGE and western blotted with specific antibodies (part A) against phospho-AMPK, total AMPK (65 KDa), and (part B) phospho-ACC, total ACC. β–actin was used as loading control. Representative blots of 3 independent experiments are shown. Data of the quantification are expressed as mean +/- SEM. *, p < 0,05; **, p < 0,01, AICAR or Phenformin treated vs control cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4498592&req=5

pone.0132498.g001: AICAR and phenformin treatments induce the phosphorylation of AMPK and ACC in isolated rat VSMCs in primary culture.After 1 hour pretreatment with AICAR (2mM) or phenformin (1mM), the cells were incubated or not with IL-1β (10ng/ml) for 30 minutes. Total proteins (20μg/lane) were separated by SDS PAGE and western blotted with specific antibodies (part A) against phospho-AMPK, total AMPK (65 KDa), and (part B) phospho-ACC, total ACC. β–actin was used as loading control. Representative blots of 3 independent experiments are shown. Data of the quantification are expressed as mean +/- SEM. *, p < 0,05; **, p < 0,01, AICAR or Phenformin treated vs control cells.
Mentions: Experiments were conducted with primary cultures of VSMCs isolated from rat aorta. These cells undergo phenotypic changes in response to proinflammatory conditions. Indeed, in response to proinflammatory cytokines, these cells express several biomarkers of inflammation such as VCAM-1, MCP1, extracellular metalloproteinases and acute phase enzymes such as secreted sPLA2 and COX-2 [7,37–39]. Moreover, we have previously observed that prostaglandins E2 combined with IL-1β progressively synergizes the secretion of sPLA2 and causes a complete disorganization of the cytoskeletal framework [9]. To explore the effect of AMPK activation on IL-1β-induced sPLA2IIA gene expression and activity, cultured VSMCs were pretreated or not with the AMPK activators AICAR or phenformin prior IL-1β treatment. We chose to use phenformin which is more lipophilic than metformin and more efficiently internalized in cell culture in absence of cationic transporters. We performed immunoblotting to show whether treatment with AICAR or phenformin led to the activation of AMPK, characterized by the phosphorylation of Thr172 within the AMPKα subunit and of Ser79 in acetyl-coA carboxylase (ACC), a well-established target of AMPK [26]. As shown in Fig 1, treatment of VSMCs with 2mM AICAR or 1mM phenformin led to a significant increase in Thr172 AMPK and Ser79 ACC phosphorylation. In the presence of IL-1β, AICAR and phenformin induced phosphorylation of both Thr172 AMPK and Ser79 ACC. These results confirm that AICAR and phenformin stimulate AMPK signaling pathway in primary cultured rat VSMCs, consistent with previous studies showing AMPK activation by the biguanide metformin in BAECs or HUVECs [40].

Bottom Line: Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far.Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex.Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universités, Université Pierre et Marie Curie, Biological Adaptation and Ageing (B2A) CNRS UMR8256/INSERM ERL-U1064, F-75005 Paris, France.

ABSTRACT
Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

No MeSH data available.


Related in: MedlinePlus