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Apoptosis signal-regulating kinase 1 in peptidoglycan-induced COX-2 expression in macrophages

Ming-Jen Hsu1, Chia-Kai Chang, Mei-Chieh Chen

  • 1Graduate Institute of Medical Sciences, Department of Pharmacology, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.

Journal of Leukocyte Biology
|March 5, 2010
PubMed

Insights

Peptidoglycan (PGN) activates the PP2A-ASK1-JNK-AP-1-C/EBPbeta pathway, leading to COX-2 expression in macrophages. This pathway involves Toll-like receptor 2 (TLR2) and is crucial for inflammatory responses.

Area of Science:

  • Immunology
  • Cell Biology
  • Molecular Biology

Background:

  • Toll-like receptor 2 (TLR2) agonists, such as peptidoglycan (PGN), play a significant role in innate immune responses.
  • Cyclooxygenase-2 (COX-2) is a key enzyme in inflammatory pathways, and its expression is often upregulated by PGN.
  • The upstream signaling cascades regulating PGN-induced COX-2 expression, particularly involving ASK1 and C/EBPbeta, require further elucidation.

Purpose of the Study:

  • To investigate the role of Apoptosis Signal-regulating Kinase 1 (ASK1) in peptidoglycan (PGN)-induced C/EBPbeta activation and COX-2 expression.
  • To elucidate the signaling pathway mediating PGN-induced COX-2 expression in RAW 264.7 macrophages.
  • To determine the involvement of protein phosphatase 2A (PP2A) and the JNK-AP-1 cascade in this process.

Main Methods:

  • Utilized dominant-negative (DN) constructs of ASK1, JNK1, and JNK2, along with specific inhibitors (SP600125, curcumin).
  • Assessed ASK1 phosphorylation status (Ser967), dissociation from 14-3-3 complex, and activation.
  • Investigated the role of PP2A using okadaic acid and analyzed JNK-AP-1 and C/EBPbeta activation via expression and DNA-binding assays. Employed COX-2 promoter luciferase assays and utilized TLR2 and c-Jun inhibitors.

Main Results:

  • PGN induced time-dependent ASK1 dephosphorylation at Ser967, dissociation from 14-3-3, and activation, which was dependent on PP2A activity.
  • The ASK1-JNK-AP-1 signaling cascade downstream of ASK1 was activated by PGN, leading to increased C/EBPbeta expression and DNA-binding activity.
  • PGN-induced COX-2 expression and promoter activity were significantly attenuated by blocking ASK1, JNK, AP-1, TLR2, or c-Jun, and this pathway was also observed in human peripheral mononuclear cells.

Conclusions:

  • PGN activates the TLR2-mediated PP2A-ASK1-JNK-AP-1-C/EBPbeta signaling cascade in macrophages.
  • This cascade is essential for the subsequent induction of COX-2 expression.
  • The findings provide a detailed molecular mechanism for PGN-induced inflammation via TLR2 signaling.

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