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Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis
Published on: June 3, 2016
Expression of adipose differentiation-related protein (ADRP) is conjointly regulated by PU.1 and AP-1 in macrophages
Ping Wei1, Susumu Taniguchi, Yoshiyuki Sakai
1Division of Clinical Immunology, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan.
Abstract:
ADRP is associated with intracellular lipid droplets. We demonstrate the regulatory mechanism for ADRP expression in RAW264.7 macrophages. The ADRP mRNA expression was stimulated by PMA, and synergistically enhanced in association with its protein level in the presence of lipids. A proteasome inhibitor protected the protein from degradation under the lipid-free conditions. One of the possible sites of the PMA action was proved to be an Ets/AP-1 element in the promoter, since mutations of this site reduced the PMA-induced promoter activity, and ligation of this element led to a significant increase in the PMA-responsiveness of homologous or heterologous promoters. Mutations of this site diminished the synergistic effect on the promoter activity induced by PMA and oleic acid, suggesting a possible interaction between this site and the downstream PPARdelta site. EMSA revealed that PU.1 and AP-1 conjointly bound to this site. The juxtaposition of the two sequences was requisite for full activity, since spacer sequences between them decreased the PMA-induced activity. PI3 kinase inhibitor was found to reduce the PMA-induced mRNA expression and promoter activity in parallel with PU.1/AP-1 complex formation on EMSA. From these results, we concluded that the Ets/AP-1 site is an important cis-acting element that regulates the ADRP gene expression in macrophages.
Insights
This study reveals how adipose differentiation-related protein (ADRP) expression is regulated in macrophages. PMA stimulation and lipids synergistically enhance ADRP levels, involving an Ets/AP-1 element and PU.1/AP-1 binding.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Adipose differentiation-related protein (ADRP) is linked to intracellular lipid droplets.
- Understanding ADRP gene regulation is crucial for lipid metabolism research.
Purpose of the Study:
- To elucidate the regulatory mechanism of ADRP expression in RAW264.7 macrophages.
- To identify key cis-acting elements and transcription factors involved in ADRP gene regulation.
Main Methods:
- Stimulation of macrophages with PMA and lipids.
- Analysis of ADRP mRNA and protein levels.
- Site-directed mutagenesis of the ADRP promoter.
- Electrophoretic Mobility Shift Assay (EMSA) to study protein-DNA interactions.
- Inhibition studies using proteasome and PI3 kinase inhibitors.
Main Results:
- Phorbol 12-myristate 13-acetate (PMA) stimulates ADRP mRNA expression, synergistically enhanced by lipids.
- An Ets/AP-1 element in the promoter is critical for PMA-induced ADRP expression and lipid synergy.
- PU.1 and AP-1 transcription factors bind to this Ets/AP-1 site, with proximity being essential for activity.
- PI3 kinase pathway inhibition reduces PMA-induced ADRP expression and transcription factor binding.
Conclusions:
- The Ets/AP-1 element is a key cis-acting regulator of ADRP gene expression in macrophages.
- Macrophage lipid droplet accumulation involves complex transcriptional regulation influenced by PMA and lipid availability.
- The PI3 kinase pathway plays a role in modulating ADRP expression via PU.1/AP-1 complex formation.
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