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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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The Ac2-26-mediated STAT3 activation is JAK-dependent and induces nuclear translocation and DNA binding of phosphorylated STAT3 as well as increased SOCS3 expressionAc2-26 triggers nuclear appearance of phosphorylated STAT3. Cytosolic and nuclear fractions of monocytes stimulated with Ac2-26 for the indicated periods of time were immunoblotted to detect the subcellular distribution of pY-STAT3. The cytosolic versus nuclear fractionation was verified by analysing the distribution of the cytosolic marker tubulin, which was absent from the nuclear fractions. A representative result of three independent experiments is shown.Ac2-26 induces DNA binding of STAT3. EMSA analysis of STAT3 DNA binding activity in the nuclear fractions of either control (lane 1) or Ac2-26-treated monocytes (lane 3). The specificity of STAT3 binding in the complexes was ensured by competition with a 100-fold molar excess of unlabelled dsDNA probe (lanes 2 and 4). Arrowheads indicate the position of the STAT3/DNA complex. A representative result of three independent experiments is shown.The Ac2-26-mediated STAT3 activation is JAK-dependent. Monocytes were pre-treated with JAK Inhibitor 1 and then stimulated with Ac2-26 for the indicated periods of time. Levels of pY-STAT3 and total STAT3 in the cellular lysates were detected by immunoblotting. A representative blot of three independent experiments is shown.Ac2-26 induces an increase in SOCS3 mRNA expression. Total RNA was extracted from monocytes stimulated with Ac2-26 for 60 or 120 min and subjected to SOCS3-specific qPCR. Levels of SOCS3 mRNA present following Ac2-26 stimulation were calculated as change over control (unstimulated cells).SOCS3 protein levels are increased following Ac2-26 treatment. Monocytes were stimulated for the indicated periods of time with Ac2-26 and SOCS3 protein present in the cellular extracts was identified by Western blotting. Membranes were reprobed with a tubulin antibody to ensure equal loading. A representative blot of three independent experiments is shown.The Ac2-26-induced upregulation of SOCS3 requires JAK activity. Monocytes were stimulated with Ac2-26 for 60 min in the absence or presence of JAK Inhibitor 1 and levels of SOCS3 mRNA were determined by qPCR. Data in (D) and (F) 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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fig06: The Ac2-26-mediated STAT3 activation is JAK-dependent and induces nuclear translocation and DNA binding of phosphorylated STAT3 as well as increased SOCS3 expressionAc2-26 triggers nuclear appearance of phosphorylated STAT3. Cytosolic and nuclear fractions of monocytes stimulated with Ac2-26 for the indicated periods of time were immunoblotted to detect the subcellular distribution of pY-STAT3. The cytosolic versus nuclear fractionation was verified by analysing the distribution of the cytosolic marker tubulin, which was absent from the nuclear fractions. A representative result of three independent experiments is shown.Ac2-26 induces DNA binding of STAT3. EMSA analysis of STAT3 DNA binding activity in the nuclear fractions of either control (lane 1) or Ac2-26-treated monocytes (lane 3). The specificity of STAT3 binding in the complexes was ensured by competition with a 100-fold molar excess of unlabelled dsDNA probe (lanes 2 and 4). Arrowheads indicate the position of the STAT3/DNA complex. A representative result of three independent experiments is shown.The Ac2-26-mediated STAT3 activation is JAK-dependent. Monocytes were pre-treated with JAK Inhibitor 1 and then stimulated with Ac2-26 for the indicated periods of time. Levels of pY-STAT3 and total STAT3 in the cellular lysates were detected by immunoblotting. A representative blot of three independent experiments is shown.Ac2-26 induces an increase in SOCS3 mRNA expression. Total RNA was extracted from monocytes stimulated with Ac2-26 for 60 or 120 min and subjected to SOCS3-specific qPCR. Levels of SOCS3 mRNA present following Ac2-26 stimulation were calculated as change over control (unstimulated cells).SOCS3 protein levels are increased following Ac2-26 treatment. Monocytes were stimulated for the indicated periods of time with Ac2-26 and SOCS3 protein present in the cellular extracts was identified by Western blotting. Membranes were reprobed with a tubulin antibody to ensure equal loading. A representative blot of three independent experiments is shown.The Ac2-26-induced upregulation of SOCS3 requires JAK activity. Monocytes were stimulated with Ac2-26 for 60 min in the absence or presence of JAK Inhibitor 1 and levels of SOCS3 mRNA were determined by qPCR. Data in (D) and (F) 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: To elucidate the effect of Ac2-26-induced STAT3 activation on the nuclear translocation of activated STAT3, we prepared cytoplasmic and nuclear extracts from annexin-treated monocytes and subjected them to immunoblotting with the phospho-STAT3-specific antibody. As shown in Fig 6A, pY-STAT3 was found in the nuclear fraction in response to Ac2-26. We next investigated whether Ac2-26-induced tyrosine phosphorylation of STAT3 correlated with STAT3 DNA binding activity. Therefore, monocytes were stimulated with Ac2-26 for 60 min, and the DNA binding activity of STAT3 was determined by EMSA. Whereas unstimulated cells showed relatively few STAT3/DNA complexes (Fig 6B, lane 1), STAT3 DNA-binding was readily observed in Ac2-26-treated monocytes (Fig 6B, lane 3). Collectively, these data indicate that Ac2-26 induces STAT3 phosphorylation, nuclear translocation and DNA binding via agonistic activation of the FPRs.


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)

The Ac2-26-mediated STAT3 activation is JAK-dependent and induces nuclear translocation and DNA binding of phosphorylated STAT3 as well as increased SOCS3 expressionAc2-26 triggers nuclear appearance of phosphorylated STAT3. Cytosolic and nuclear fractions of monocytes stimulated with Ac2-26 for the indicated periods of time were immunoblotted to detect the subcellular distribution of pY-STAT3. The cytosolic versus nuclear fractionation was verified by analysing the distribution of the cytosolic marker tubulin, which was absent from the nuclear fractions. A representative result of three independent experiments is shown.Ac2-26 induces DNA binding of STAT3. EMSA analysis of STAT3 DNA binding activity in the nuclear fractions of either control (lane 1) or Ac2-26-treated monocytes (lane 3). The specificity of STAT3 binding in the complexes was ensured by competition with a 100-fold molar excess of unlabelled dsDNA probe (lanes 2 and 4). Arrowheads indicate the position of the STAT3/DNA complex. A representative result of three independent experiments is shown.The Ac2-26-mediated STAT3 activation is JAK-dependent. Monocytes were pre-treated with JAK Inhibitor 1 and then stimulated with Ac2-26 for the indicated periods of time. Levels of pY-STAT3 and total STAT3 in the cellular lysates were detected by immunoblotting. A representative blot of three independent experiments is shown.Ac2-26 induces an increase in SOCS3 mRNA expression. Total RNA was extracted from monocytes stimulated with Ac2-26 for 60 or 120 min and subjected to SOCS3-specific qPCR. Levels of SOCS3 mRNA present following Ac2-26 stimulation were calculated as change over control (unstimulated cells).SOCS3 protein levels are increased following Ac2-26 treatment. Monocytes were stimulated for the indicated periods of time with Ac2-26 and SOCS3 protein present in the cellular extracts was identified by Western blotting. Membranes were reprobed with a tubulin antibody to ensure equal loading. A representative blot of three independent experiments is shown.The Ac2-26-induced upregulation of SOCS3 requires JAK activity. Monocytes were stimulated with Ac2-26 for 60 min in the absence or presence of JAK Inhibitor 1 and levels of SOCS3 mRNA were determined by qPCR. Data in (D) and (F) 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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fig06: The Ac2-26-mediated STAT3 activation is JAK-dependent and induces nuclear translocation and DNA binding of phosphorylated STAT3 as well as increased SOCS3 expressionAc2-26 triggers nuclear appearance of phosphorylated STAT3. Cytosolic and nuclear fractions of monocytes stimulated with Ac2-26 for the indicated periods of time were immunoblotted to detect the subcellular distribution of pY-STAT3. The cytosolic versus nuclear fractionation was verified by analysing the distribution of the cytosolic marker tubulin, which was absent from the nuclear fractions. A representative result of three independent experiments is shown.Ac2-26 induces DNA binding of STAT3. EMSA analysis of STAT3 DNA binding activity in the nuclear fractions of either control (lane 1) or Ac2-26-treated monocytes (lane 3). The specificity of STAT3 binding in the complexes was ensured by competition with a 100-fold molar excess of unlabelled dsDNA probe (lanes 2 and 4). Arrowheads indicate the position of the STAT3/DNA complex. A representative result of three independent experiments is shown.The Ac2-26-mediated STAT3 activation is JAK-dependent. Monocytes were pre-treated with JAK Inhibitor 1 and then stimulated with Ac2-26 for the indicated periods of time. Levels of pY-STAT3 and total STAT3 in the cellular lysates were detected by immunoblotting. A representative blot of three independent experiments is shown.Ac2-26 induces an increase in SOCS3 mRNA expression. Total RNA was extracted from monocytes stimulated with Ac2-26 for 60 or 120 min and subjected to SOCS3-specific qPCR. Levels of SOCS3 mRNA present following Ac2-26 stimulation were calculated as change over control (unstimulated cells).SOCS3 protein levels are increased following Ac2-26 treatment. Monocytes were stimulated for the indicated periods of time with Ac2-26 and SOCS3 protein present in the cellular extracts was identified by Western blotting. Membranes were reprobed with a tubulin antibody to ensure equal loading. A representative blot of three independent experiments is shown.The Ac2-26-induced upregulation of SOCS3 requires JAK activity. Monocytes were stimulated with Ac2-26 for 60 min in the absence or presence of JAK Inhibitor 1 and levels of SOCS3 mRNA were determined by qPCR. Data in (D) and (F) 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: To elucidate the effect of Ac2-26-induced STAT3 activation on the nuclear translocation of activated STAT3, we prepared cytoplasmic and nuclear extracts from annexin-treated monocytes and subjected them to immunoblotting with the phospho-STAT3-specific antibody. As shown in Fig 6A, pY-STAT3 was found in the nuclear fraction in response to Ac2-26. We next investigated whether Ac2-26-induced tyrosine phosphorylation of STAT3 correlated with STAT3 DNA binding activity. Therefore, monocytes were stimulated with Ac2-26 for 60 min, and the DNA binding activity of STAT3 was determined by EMSA. Whereas unstimulated cells showed relatively few STAT3/DNA complexes (Fig 6B, lane 1), STAT3 DNA-binding was readily observed in Ac2-26-treated monocytes (Fig 6B, lane 3). Collectively, these data indicate that Ac2-26 induces STAT3 phosphorylation, nuclear translocation and DNA binding via agonistic activation of the FPRs.

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