gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

MicroRNA-dependent regulation of macrophage function

Meeting Contribution

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  • S. Sendelbach - Institute of Nutrition, Friedrich Schiller University Jena, Germany
  • M. B. Maeß - Institute of Nutrition, Friedrich Schiller University Jena, Germany
  • corresponding author S. Lorkowski - Institute of Nutrition, Friedrich Schiller University Jena, Germany

Deutsche Gesellschaft für Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf17

DOI: 10.3205/10dgaf17, URN: urn:nbn:de:0183-10dgaf177

Published: March 23, 2011

© 2011 Sendelbach et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

The lipid metabolite 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) modulates inflammatory response of macrophages via distinct pathways. To unravel new modes of action of 15dPGJ2, we analyzed its effect on expression of miRNAs and their precursor molecules. We identified a miRNA precursor whose expression is induced by 15dPGJ2 independently from PPARγ, but expression of the encoded miRNA was not influenced. Our data suggest a new pathway by which 15dPGJ2 modulates macrophage function. Further studies are required to unravel the relevance of our findings.


Introduction

Macrophages play a crucial role in atherogenesis [1]. In addition to their contribution as foam cells to the formation of necrotic lipid cores, macrophages secrete several pro- and anti-inflammatory factors, interact with neighboring cells, contribute to immune response through diverse activation programs [2], and influence stability of the atherosclerotic plaque by producing proteases [3].

15-deoxy-Δ12,14-prostaglandin J2, a lipid metabolite abundantly produced in macrophages, is known to regulate immune response. In addition to its anti-inflammatory effects, which are basically mediated by PPARγ (peroxisome proliferator-activated receptor γ), 15dPGJ2 influences cellular processes also via PPARγ-independent pathways [4]. The molecular mechanisms by which 15dPGJ2 regulates macrophage function are yet not well understood. We were therefore interested in understanding how 15dPGJ2 regulates expression of microRNAs.

MicroRNAs are evolutionary conserved, small non-coding RNA molecules cut out of a precursor RNA molecule of about 1,000 nucleotides in length via several processing steps in the nucleus and cytoplasm. As part of the RNA-induced silencing complex (RISC) they control gene expression by interfering with mRNA translation (Figure 1 [Fig. 1]). Up to date not much is known about their implications in inflammation and immunity; however, supposedly they expedite the initiation and progression of inflammatory processes [5], and therefore atherosclerosis, as well.

Hence, the major aim of our study was to get insights into the signaling pathways and regulatory mechanisms involved in 15dPGJ2-mediated regulation of expression of selected miRNAs and of their precursors.


Materials and methods

Cell culture: The THP-1 monocytic cell line was purchased from ATCC. Cells were maintained as recommended by the supplier. Cells were differentiated into macrophages using PMA (phorbol-13-myristate-12-acetate) for 96 h and were stimulated with different concentrations of 15dPGJ2, p38/MAPK (mitogen-acticated protein kinase) inhibitor SB203580, PPARγ ligand rosigliatzone, and phosphatase inhibitor calyculin A in different combinations.

RNA isolation and reverse transcription: RNeasy Mini Kit (Qiagen) and RevertAidTM First Strand cDNA Synthesis Kit (Fermentas) were used as previously described [6].

Real-time RT-PCR: Expression analyses by means of quantitative real-time RT-PCR were performed with the QuantiTect SYBR Green PCR Kit (Qiagen) using a Roche LightCycler 480 as described [6].


Results

Our experiments revealed that 15dPGJ2 time- (data not shown) and dose-dependently increased expression of a miRNA-encoding precursor RNA in THP-1 macrophages (Figure 2A [Fig. 2]).

To unravel the signaling pathways, by which expression of the miRNA precursor is regulated, we incubated matured THP-1 cells with p38/MAPK inhibitor SB203580, but no effect was detected (Figure 2B [Fig. 2]). Similar results were obtained by using the synthetic PPARγ agonist rosiglitazone (Figure 2C [Fig. 2]). We also investigated cells treated with calyculin A, an inhibitor of serine/threonine phosphatases 1 and 2A. As shown in Figure 2D [Fig. 2], inhibition of phosphatase activity increased expression of the miRNA precursor.

Similar experiments were performed for analyzing expression of the corresponding miRNA in THP-1 macrophages. Interestingly, in none of the conditions investigated, expression of the miRNA followed the expression of its precursor (data not shown). Although expression of the precursor was upregulated up to about 20-fold, expression of the miRNA remained unchanged (data not shown).


Discussion

Preliminary studies showed that 15dPGJ2 induced dose- and time-dependently expression of a miRNA precursor in mature THP-1 macrophages. Using PPARγ agonist rosiglitazone and p38/MAPK inhibitor SB203580 we ruled out involvement of these regulatory pathways. By contrast, expression was induced by calyculin A, an inhibitor of serine/threonine phosphatases 1 and 2A. Thus, phosphorylation plays an important role in regulating expression of this miRNA precursor. The reason for the lack of correlation between regulation of expression of the miRNA precursor and its encoded miRNA is not clear but similar observations have been made for other miRNAs, such as miR-155 and its precursor BIC non-coding RNA [7], [8]. Further studies are therefore required to unravel the underlying regulatory pathways and the contribution of the investigated miRNA precursor and its encoded miRNA to macrophage function.


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