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Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages
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miRNA-Induced Downregulation of IPMK in Macrophages Mediates Lipopolysaccharide-Triggered TLR4 Signaling

Haein Lee1, Eunha Kim1, Seyun Kim1,2,3

  • 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea.

Biomolecules
|February 25, 2023
PubMed

Insights

Lipopolysaccharide (LPS) acutely decreases inositol polyphosphate multikinase (IPMK) in macrophages via miR-181c, suppressing Toll-like receptor 4 (TLR4) signaling and inflammation. Restoring IPMK levels prevents this suppression, highlighting IPMK

Area of Science:

  • Immunology
  • Molecular Biology
  • Cell Biology

Background:

  • Inositol polyphosphate multikinase (IPMK) is crucial for inositol phosphate production.
  • IPMK in macrophages is vital for Toll-like receptor 4 (TLR4) pathway activation and inflammation.
  • IPMK interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6) in TLR4 signaling.

Purpose of the Study:

  • To investigate the dynamic changes in IPMK levels during lipopolysaccharide (LPS)-stimulated macrophages.
  • To elucidate the functional significance of IPMK regulation in macrophage activation.
  • To identify the molecular mechanisms controlling IPMK expression in response to LPS.

Main Methods:

  • Quantitative analysis of IPMK mRNA and protein levels in LPS-stimulated mouse and human macrophages.
  • MicroRNA (miR-181c) binding site analysis in the 3' untranslated region (UTR) of IPMK mRNA.
  • Luciferase reporter assays and gene editing (CRISPR/Cas9) in RAW 264.7 macrophage cell lines.
  • Assessment of TLR4 signaling, TRAF6 ubiquitination, and pro-inflammatory cytokine expression.

Main Results:

  • LPS stimulation led to acute downregulation of both IPMK mRNA and protein in macrophages.
  • A conserved miR-181c binding site in the IPMK 3'UTR was identified, mediating LPS-induced suppression.
  • Genetic deletion of the miR-181c binding site in the IPMK 3'UTR prevented LPS-triggered IPMK reduction.
  • Macrophages with a modified IPMK 3'UTR exhibited impaired TLR4 signaling, reduced TRAF6 activation, and decreased pro-inflammatory cytokine production.

Conclusions:

  • LPS-mediated suppression of IPMK, regulated by miR-181c, is a critical mechanism controlling TLR4 signaling and inflammation in macrophages.
  • IPMK plays a key role in the full activation of the TLR4 pathway by modulating TRAF6 activation.
  • Targeting the IPMK-miR-181c axis offers potential therapeutic strategies for inflammatory diseases.
Keywords:
IPMKTRAF6inflammationmacrophagetoll-like receptor

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