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Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
09:04

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells

Published on: May 22, 2014

Interleukin-3 stimulation of mcl-1 gene transcription involves activation of the PU.1 transcription factor through a

Ju-Ming Wang1, Ming-Zong Lai, Hsin-Fang Yang-Yen

  • 1Institute of Molecular Biology, Academia Sinica, 128 Yen-Jiou Yuan Road, Section 2, Nankang, Taipei 11529, Taiwan, Republic of China.

Molecular and Cellular Biology
|March 4, 2003
PubMed

Insights

Interleukin-3 (IL-3) activates the antiapoptotic MCL-1 gene via the SIE motif by phosphorylating the transcription factor PU.1. This phosphorylation, mediated by p38 MAPK, is crucial for MCL-1 gene transcription.

Area of Science:

  • Cellular and Molecular Biology
  • Gene Regulation
  • Signal Transduction

Background:

  • The antiapoptotic gene MCL-1 is activated by Interleukin-3 (IL-3) in Ba/F3 pro-B cells.
  • IL-3 activation occurs through two promoter elements: CRE-2 and SIE motifs.
  • The CRE-2 pathway involves CREB and is IL-3/PI3K/Akt-dependent, but the SIE pathway remains unclear.

Purpose of the Study:

  • To identify the transcription factor(s) binding to the SIE motif.
  • To elucidate the cytokine activation pathway regulating SIE-mediated transcription.
  • To determine the role of PU.1 phosphorylation in IL-3-induced MCL-1 expression.

Main Methods:

  • Chromatin immunoprecipitation (ChIP) assays to confirm in vivo binding of PU.1 to the mcl-1 promoter SIE motif.
  • Reporter assays to assess transactivation activity of PU.1.
  • Site-directed mutagenesis (S142A) to investigate the role of serine 142 phosphorylation.
  • Western blotting and kinase assays to analyze PU.1 phosphorylation and p38 MAPK pathway activation.

Main Results:

  • PU.1 was identified as a component of the SIE-binding complex.
  • IL-3 stimulation increased PU.1 transactivation activity without altering its binding activity.
  • IL-3 induced PU.1 phosphorylation via a p38 MAPK-dependent pathway.
  • Mutation of serine 142 (S142A) impaired PU.1 phosphorylation and IL-3-induced transactivation of the mcl-1 reporter.

Conclusions:

  • PU.1 binds to the mcl-1 promoter SIE motif in vivo.
  • IL-3-induced transcription of mcl-1 via the SIE motif is mediated by p38 MAPK-dependent phosphorylation of PU.1 at serine 142.

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