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Annexin A1 released from apoptotic cells acts through formyl peptide receptors to dampen inflammatory monocyte activation via JAK/STAT/SOCS signalling.

Pupjalis D, Goetsch J, Kottas DJ, Gerke V, Rescher U - EMBO Mol Med (2011)

Bottom Line: Supernatants from apoptotic neutrophils or the annexin A1 peptidomimetic Ac2-26 significantly reduced IL-6 signalling and the release of TNF-α from endotoxin-challenged monocytes.Ac2-26 activated STAT3 in a JAK-dependent manner, resulting in upregulated SOCS3 levels, and depletion of SOCS3 reversed the Ac2-26-mediated inhibition of IL-6 signalling.This identifies annexin A1 as part of the anti-inflammatory pattern of apoptotic cells and links the activation of FPRs to established signalling pathways triggering anti-inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Biology of Inflammation, and Interdisciplinary Clinical Research Centre, Institute of Medical Biochemistry, University of Muenster, Muenster, Germany.

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Apoptotic cell supernatants induce DNA binding of phospho-STAT3 and an upregulation of SOCS3 expressionNuclear appearance of tyrosine-phosphorylated STAT3 following treatment with apoptotic cell supernatants. Monocytes were either left unstimulated (uns) or stimulated with apopt. SN for the indicated periods of time and cytosolic (20 µg) as well as nuclear protein fractions (5 µg) were probed by immunoblotting with a pY-STAT3 antibody. The cytosolic versus nuclear fractionation was confirmed by analysing the distribution of the cytosolic marker tubulin. A representative result of four independent experiments is shown.EMSA analysis of STAT3 DNA binding activity in the nuclear fractions of either control cells (lane 1) or cells treated with apopt. SN (lane 3). The specificity of STAT3 binding in the complexes was verified by competition with a 100-fold molar excess of unlabelled specific dsDNA probe (lanes 2 and 4). Arrowheads indicate the position of the STAT3/DNA complex. The control lane shows the DNA probe without the addition of protein. A representative result of four independent experiments is shown.Supernatants from apoptotic neutrophils induce an increase in SOCS3 protein levels. Monocytes were stimulated for the indicated periods of time with apopt. SN and SOCS3 protein levels were analysed by Western blotting. Membranes were reprobed with a STAT3 antibody to ensure equal loading. A representative blot of three independent experiments is shown.Apoptotic cell supernatants induce an upregulation of SOCS3 mRNA. Total RNA was extracted from monocytes incubated with apopt. SN with or without prior treatment with JAK Inhibitor 1 and subjected to qPCR. Levels of SOCS3 mRNA were calculated as change over control (unstimulated cells). Data are expressed as means ± SEM of at least three independent experiments. The significance of the differences was evaluated by two-sided t-tests and respective p-values are given.
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fig02: Apoptotic cell supernatants induce DNA binding of phospho-STAT3 and an upregulation of SOCS3 expressionNuclear appearance of tyrosine-phosphorylated STAT3 following treatment with apoptotic cell supernatants. Monocytes were either left unstimulated (uns) or stimulated with apopt. SN for the indicated periods of time and cytosolic (20 µg) as well as nuclear protein fractions (5 µg) were probed by immunoblotting with a pY-STAT3 antibody. The cytosolic versus nuclear fractionation was confirmed by analysing the distribution of the cytosolic marker tubulin. A representative result of four independent experiments is shown.EMSA analysis of STAT3 DNA binding activity in the nuclear fractions of either control cells (lane 1) or cells treated with apopt. SN (lane 3). The specificity of STAT3 binding in the complexes was verified by competition with a 100-fold molar excess of unlabelled specific dsDNA probe (lanes 2 and 4). Arrowheads indicate the position of the STAT3/DNA complex. The control lane shows the DNA probe without the addition of protein. A representative result of four independent experiments is shown.Supernatants from apoptotic neutrophils induce an increase in SOCS3 protein levels. Monocytes were stimulated for the indicated periods of time with apopt. SN and SOCS3 protein levels were analysed by Western blotting. Membranes were reprobed with a STAT3 antibody to ensure equal loading. A representative blot of three independent experiments is shown.Apoptotic cell supernatants induce an upregulation of SOCS3 mRNA. Total RNA was extracted from monocytes incubated with apopt. SN with or without prior treatment with JAK Inhibitor 1 and subjected to qPCR. Levels of SOCS3 mRNA were calculated as change over control (unstimulated cells). Data are expressed as means ± SEM of at least three independent experiments. The significance of the differences was evaluated by two-sided t-tests and respective p-values are given.

Mentions: Activated STAT3 molecules form a homodimer which translocates into the nucleus, where it recognizes specific DNA elements and activates transcription. To assess STAT3 activation, nuclear translocation and DNA binding, we treated monocytes with apoptotic cell culture supernatant, prepared cytosolic and nuclear extracts and probed for tyrosine-phosphorylated STAT3 by Western blotting. As shown in Fig 2A, increased amounts of tyrosine-phosphorylated STAT3 were detected in the nuclear, but not in the cytosolic fraction upon 60–90 min addition of apoptotic cell culture supernatant. Furthermore, electrophoretic mobility shift assay (EMSA) analysis revealed that nuclear extracts from monocytes treated for 60 min with apoptotic cell culture supernatant contained more STAT3 bound to its cognate DNA probe than extracts from untreated control cells (Fig 2B, lanes 1 and 3). The observed STAT3 binding was highly specific, since binding to the DNA was completely inhibited in the presence of a 100-fold molar excess of unlabelled probe (Fig 2B, lanes 2 and 4).


Annexin A1 released from apoptotic cells acts through formyl peptide receptors to dampen inflammatory monocyte activation via JAK/STAT/SOCS signalling.

Pupjalis D, Goetsch J, Kottas DJ, Gerke V, Rescher U - EMBO Mol Med (2011)

Apoptotic cell supernatants induce DNA binding of phospho-STAT3 and an upregulation of SOCS3 expressionNuclear appearance of tyrosine-phosphorylated STAT3 following treatment with apoptotic cell supernatants. Monocytes were either left unstimulated (uns) or stimulated with apopt. SN for the indicated periods of time and cytosolic (20 µg) as well as nuclear protein fractions (5 µg) were probed by immunoblotting with a pY-STAT3 antibody. The cytosolic versus nuclear fractionation was confirmed by analysing the distribution of the cytosolic marker tubulin. A representative result of four independent experiments is shown.EMSA analysis of STAT3 DNA binding activity in the nuclear fractions of either control cells (lane 1) or cells treated with apopt. SN (lane 3). The specificity of STAT3 binding in the complexes was verified by competition with a 100-fold molar excess of unlabelled specific dsDNA probe (lanes 2 and 4). Arrowheads indicate the position of the STAT3/DNA complex. The control lane shows the DNA probe without the addition of protein. A representative result of four independent experiments is shown.Supernatants from apoptotic neutrophils induce an increase in SOCS3 protein levels. Monocytes were stimulated for the indicated periods of time with apopt. SN and SOCS3 protein levels were analysed by Western blotting. Membranes were reprobed with a STAT3 antibody to ensure equal loading. A representative blot of three independent experiments is shown.Apoptotic cell supernatants induce an upregulation of SOCS3 mRNA. Total RNA was extracted from monocytes incubated with apopt. SN with or without prior treatment with JAK Inhibitor 1 and subjected to qPCR. Levels of SOCS3 mRNA were calculated as change over control (unstimulated cells). Data are expressed as means ± SEM of at least three independent experiments. The significance of the differences was evaluated by two-sided t-tests and respective p-values are given.
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Related In: Results  -  Collection

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fig02: Apoptotic cell supernatants induce DNA binding of phospho-STAT3 and an upregulation of SOCS3 expressionNuclear appearance of tyrosine-phosphorylated STAT3 following treatment with apoptotic cell supernatants. Monocytes were either left unstimulated (uns) or stimulated with apopt. SN for the indicated periods of time and cytosolic (20 µg) as well as nuclear protein fractions (5 µg) were probed by immunoblotting with a pY-STAT3 antibody. The cytosolic versus nuclear fractionation was confirmed by analysing the distribution of the cytosolic marker tubulin. A representative result of four independent experiments is shown.EMSA analysis of STAT3 DNA binding activity in the nuclear fractions of either control cells (lane 1) or cells treated with apopt. SN (lane 3). The specificity of STAT3 binding in the complexes was verified by competition with a 100-fold molar excess of unlabelled specific dsDNA probe (lanes 2 and 4). Arrowheads indicate the position of the STAT3/DNA complex. The control lane shows the DNA probe without the addition of protein. A representative result of four independent experiments is shown.Supernatants from apoptotic neutrophils induce an increase in SOCS3 protein levels. Monocytes were stimulated for the indicated periods of time with apopt. SN and SOCS3 protein levels were analysed by Western blotting. Membranes were reprobed with a STAT3 antibody to ensure equal loading. A representative blot of three independent experiments is shown.Apoptotic cell supernatants induce an upregulation of SOCS3 mRNA. Total RNA was extracted from monocytes incubated with apopt. SN with or without prior treatment with JAK Inhibitor 1 and subjected to qPCR. Levels of SOCS3 mRNA were calculated as change over control (unstimulated cells). Data are expressed as means ± SEM of at least three independent experiments. The significance of the differences was evaluated by two-sided t-tests and respective p-values are given.
Mentions: Activated STAT3 molecules form a homodimer which translocates into the nucleus, where it recognizes specific DNA elements and activates transcription. To assess STAT3 activation, nuclear translocation and DNA binding, we treated monocytes with apoptotic cell culture supernatant, prepared cytosolic and nuclear extracts and probed for tyrosine-phosphorylated STAT3 by Western blotting. As shown in Fig 2A, increased amounts of tyrosine-phosphorylated STAT3 were detected in the nuclear, but not in the cytosolic fraction upon 60–90 min addition of apoptotic cell culture supernatant. Furthermore, electrophoretic mobility shift assay (EMSA) analysis revealed that nuclear extracts from monocytes treated for 60 min with apoptotic cell culture supernatant contained more STAT3 bound to its cognate DNA probe than extracts from untreated control cells (Fig 2B, lanes 1 and 3). The observed STAT3 binding was highly specific, since binding to the DNA was completely inhibited in the presence of a 100-fold molar excess of unlabelled probe (Fig 2B, lanes 2 and 4).

Bottom Line: Supernatants from apoptotic neutrophils or the annexin A1 peptidomimetic Ac2-26 significantly reduced IL-6 signalling and the release of TNF-α from endotoxin-challenged monocytes.Ac2-26 activated STAT3 in a JAK-dependent manner, resulting in upregulated SOCS3 levels, and depletion of SOCS3 reversed the Ac2-26-mediated inhibition of IL-6 signalling.This identifies annexin A1 as part of the anti-inflammatory pattern of apoptotic cells and links the activation of FPRs to established signalling pathways triggering anti-inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Biology of Inflammation, and Interdisciplinary Clinical Research Centre, Institute of Medical Biochemistry, University of Muenster, Muenster, Germany.

Show MeSH
Related in: MedlinePlus