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Phosphatidylinositol 3-kinase interacts with the glucocorticoid receptor upon TLR2 activation.

Arancibia S, Benítez D, Núñez LE, Jewell CM, Langjahr P, Candia E, Zapata-Torres G, Cidlowski JA, González MJ, Hermoso MA - J. Cell. Mol. Med. (2011)

Bottom Line: Mutations of two tyrosine residues in the GR, 598 and 663, to phenylalanine significantly reduced interaction with PI3K and the GC effects on TLR2-induced TNF-α expression.However, these mutations did not alter GR transcriptional activity nor affect cellular localization of the expressed mutant GR in COS-1 cells.Therefore, the PI3K-GR interaction may contribute to the effects of GC on the TLR2 pro-inflammatory signalling cascade, thus defining a novel signalling mechanism with a profound impact on innate immune responses.

View Article: PubMed Central - PubMed

Affiliation: Immunology Disciplinary Program, Biomedical Sciences Institute, School of Medicine, University of Chile, Santiago, Chile.

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Related in: MedlinePlus

PI3K regulates the production of TNF-α and signalling events induced by Pam3-Cys-Ser-Lys4 and dexamethasone. (A) A549 cells transiently trans-fected with a p85 dominant negative mutant (p85-DN) as indicated, were exposed to Pam3-Cys-Ser-Lys4 (0.1 to 20 μg/ml) in the presence or absence of 100 nM dexamethasone for 4 hrs. Brefeldin-A was added 30 min. after exposure to the stimuli to stop cytokine secretion. Intracellular TNF-α production was determined by flow cytometry and normalized to untreated cells (n= 3; *P < 0.05, **P < 0.001 significant over the control). (B) A549 cells were co-transfected with the NF-κB reporter plasmid 3XMHC-Luc, pGL3-hRL and p85-DN mutant constructs indicated. Cells were stimulated for 18 hrs with different concentrations of Pam3-Cys-Ser-Lys4 (0.1–20 μg/ml) in the presence or absence of 100 nM dexamethasone and analysed for NF κ B activation through luciferase activity. (n= 3, *P < 0.05, **P < 0.01 significant over the vehicle treated control). (C) A549 cells were co-transfected with the reporter plasmids AP-1-Luc, pGL3-hRL and p85-DN mutant as indicated, together with vectors expressing c-fos and c-jun. Cells were left either untreated or stimulated for 18 hrs with different concentrations of Pam3-Cys-Ser-Lys4 (0.1–20 μg/ml) in the presence or absence of 100 nM dexamethasone. AP-1 transcriptional activity was assessed through luciferase activity. (n= 3, *P < 0.05, **P < 0.01 significant over the vehicle treated control.)
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fig02: PI3K regulates the production of TNF-α and signalling events induced by Pam3-Cys-Ser-Lys4 and dexamethasone. (A) A549 cells transiently trans-fected with a p85 dominant negative mutant (p85-DN) as indicated, were exposed to Pam3-Cys-Ser-Lys4 (0.1 to 20 μg/ml) in the presence or absence of 100 nM dexamethasone for 4 hrs. Brefeldin-A was added 30 min. after exposure to the stimuli to stop cytokine secretion. Intracellular TNF-α production was determined by flow cytometry and normalized to untreated cells (n= 3; *P < 0.05, **P < 0.001 significant over the control). (B) A549 cells were co-transfected with the NF-κB reporter plasmid 3XMHC-Luc, pGL3-hRL and p85-DN mutant constructs indicated. Cells were stimulated for 18 hrs with different concentrations of Pam3-Cys-Ser-Lys4 (0.1–20 μg/ml) in the presence or absence of 100 nM dexamethasone and analysed for NF κ B activation through luciferase activity. (n= 3, *P < 0.05, **P < 0.01 significant over the vehicle treated control). (C) A549 cells were co-transfected with the reporter plasmids AP-1-Luc, pGL3-hRL and p85-DN mutant as indicated, together with vectors expressing c-fos and c-jun. Cells were left either untreated or stimulated for 18 hrs with different concentrations of Pam3-Cys-Ser-Lys4 (0.1–20 μg/ml) in the presence or absence of 100 nM dexamethasone. AP-1 transcriptional activity was assessed through luciferase activity. (n= 3, *P < 0.05, **P < 0.01 significant over the vehicle treated control.)

Mentions: To determine the role of the PI3K pathway on TLR2-mediated pro-inflammatory cytokine production, we analysed the effect of PI3K on the expression of TNF-α upon TLR2 activation in the presence or absence of dexamethasone. Expression of TNF-α was studied in A549 cells expressing a dominant negative construct of the PI3K subunit p85 (Δ478–511) which lacks the p110-binding domain (p85-DN) stimulated with Pam3-Cys-Ser-Lys4 alone or in combination with dexamethasone. PI3K intervention with the p85-DN induced a significant increase in TNF-α intracellular expression that was greatly enhanced with 20 μg/ml of Pam3-Cys-Ser-Lys4. Dexamethasone addition did not increase TNF-α when PI3K was abrogated (Fig. 2A).


Phosphatidylinositol 3-kinase interacts with the glucocorticoid receptor upon TLR2 activation.

Arancibia S, Benítez D, Núñez LE, Jewell CM, Langjahr P, Candia E, Zapata-Torres G, Cidlowski JA, González MJ, Hermoso MA - J. Cell. Mol. Med. (2011)

PI3K regulates the production of TNF-α and signalling events induced by Pam3-Cys-Ser-Lys4 and dexamethasone. (A) A549 cells transiently trans-fected with a p85 dominant negative mutant (p85-DN) as indicated, were exposed to Pam3-Cys-Ser-Lys4 (0.1 to 20 μg/ml) in the presence or absence of 100 nM dexamethasone for 4 hrs. Brefeldin-A was added 30 min. after exposure to the stimuli to stop cytokine secretion. Intracellular TNF-α production was determined by flow cytometry and normalized to untreated cells (n= 3; *P < 0.05, **P < 0.001 significant over the control). (B) A549 cells were co-transfected with the NF-κB reporter plasmid 3XMHC-Luc, pGL3-hRL and p85-DN mutant constructs indicated. Cells were stimulated for 18 hrs with different concentrations of Pam3-Cys-Ser-Lys4 (0.1–20 μg/ml) in the presence or absence of 100 nM dexamethasone and analysed for NF κ B activation through luciferase activity. (n= 3, *P < 0.05, **P < 0.01 significant over the vehicle treated control). (C) A549 cells were co-transfected with the reporter plasmids AP-1-Luc, pGL3-hRL and p85-DN mutant as indicated, together with vectors expressing c-fos and c-jun. Cells were left either untreated or stimulated for 18 hrs with different concentrations of Pam3-Cys-Ser-Lys4 (0.1–20 μg/ml) in the presence or absence of 100 nM dexamethasone. AP-1 transcriptional activity was assessed through luciferase activity. (n= 3, *P < 0.05, **P < 0.01 significant over the vehicle treated control.)
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fig02: PI3K regulates the production of TNF-α and signalling events induced by Pam3-Cys-Ser-Lys4 and dexamethasone. (A) A549 cells transiently trans-fected with a p85 dominant negative mutant (p85-DN) as indicated, were exposed to Pam3-Cys-Ser-Lys4 (0.1 to 20 μg/ml) in the presence or absence of 100 nM dexamethasone for 4 hrs. Brefeldin-A was added 30 min. after exposure to the stimuli to stop cytokine secretion. Intracellular TNF-α production was determined by flow cytometry and normalized to untreated cells (n= 3; *P < 0.05, **P < 0.001 significant over the control). (B) A549 cells were co-transfected with the NF-κB reporter plasmid 3XMHC-Luc, pGL3-hRL and p85-DN mutant constructs indicated. Cells were stimulated for 18 hrs with different concentrations of Pam3-Cys-Ser-Lys4 (0.1–20 μg/ml) in the presence or absence of 100 nM dexamethasone and analysed for NF κ B activation through luciferase activity. (n= 3, *P < 0.05, **P < 0.01 significant over the vehicle treated control). (C) A549 cells were co-transfected with the reporter plasmids AP-1-Luc, pGL3-hRL and p85-DN mutant as indicated, together with vectors expressing c-fos and c-jun. Cells were left either untreated or stimulated for 18 hrs with different concentrations of Pam3-Cys-Ser-Lys4 (0.1–20 μg/ml) in the presence or absence of 100 nM dexamethasone. AP-1 transcriptional activity was assessed through luciferase activity. (n= 3, *P < 0.05, **P < 0.01 significant over the vehicle treated control.)
Mentions: To determine the role of the PI3K pathway on TLR2-mediated pro-inflammatory cytokine production, we analysed the effect of PI3K on the expression of TNF-α upon TLR2 activation in the presence or absence of dexamethasone. Expression of TNF-α was studied in A549 cells expressing a dominant negative construct of the PI3K subunit p85 (Δ478–511) which lacks the p110-binding domain (p85-DN) stimulated with Pam3-Cys-Ser-Lys4 alone or in combination with dexamethasone. PI3K intervention with the p85-DN induced a significant increase in TNF-α intracellular expression that was greatly enhanced with 20 μg/ml of Pam3-Cys-Ser-Lys4. Dexamethasone addition did not increase TNF-α when PI3K was abrogated (Fig. 2A).

Bottom Line: Mutations of two tyrosine residues in the GR, 598 and 663, to phenylalanine significantly reduced interaction with PI3K and the GC effects on TLR2-induced TNF-α expression.However, these mutations did not alter GR transcriptional activity nor affect cellular localization of the expressed mutant GR in COS-1 cells.Therefore, the PI3K-GR interaction may contribute to the effects of GC on the TLR2 pro-inflammatory signalling cascade, thus defining a novel signalling mechanism with a profound impact on innate immune responses.

View Article: PubMed Central - PubMed

Affiliation: Immunology Disciplinary Program, Biomedical Sciences Institute, School of Medicine, University of Chile, Santiago, Chile.

Show MeSH
Related in: MedlinePlus