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. 2010 Apr 16;285(16):11846-53.
doi: 10.1074/jbc.M109.066399. Epub 2010 Feb 17.

MicroRNA-27b contributes to lipopolysaccharide-mediated peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA destabilization

Carla Jennewein  1 , Andreas von Knethen, Tobias Schmid, Bernhard Brüne
Affiliations

MicroRNA-27b contributes to lipopolysaccharide-mediated peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA destabilization

Carla Jennewein et al. J Biol Chem. .

Abstract

Peroxisome proliferator-activated receptor gamma (PPARgamma) gained considerable interest as a therapeutic _target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARgamma expression. Considering that regulation of PPARgamma expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARgamma1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3'-untranslated regions (UTRs), we analyzed the impact of the PPARgamma-3'-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARgamma-3'-UTR, suggesting that PPARgamma1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3'-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARgamma1 mRNA decay, whereas miR-27b overexpression decreased PPARgamma1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARgamma1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARgamma1 mRNA. Understanding molecular mechanisms decreasing PPARgamma might help to better appreciate inflammatory diseases.

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Figures

FIGURE 1.
FIGURE 1.
LPS down-regulates PPARγ in primary macrophages. A, primary monocytes were isolated and cultured for 1 h up to 7 days and then stimulated with 1 μg/ml LPS for 3 h. RNA levels of the PPARγ transcript variants 1 and 3 were determined by qPCR. B, primary macrophages, differentiated for 7 days and C, differentiated THP-1 macrophages were stimulated with 1 μg/ml LPS and total PPARγ1 mRNA was determined by qPCR. D, PPARγ protein was determined by Western analysis after treating primary macrophages with 1 μg/ml LPS up to 16 h. E, PPRE reporter activity was measured in differentiated THP-1 macrophages after pretreatment with 1 μg/ml LPS for 6 h, followed by 5 μm rosiglitazone for 4 h. Data present mean values ± S.E. n ≥ 4. Statistics were analyzed with the unpaired Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 2.
FIGURE 2.
Destabilization of PPARγ1 mRNA. A, PPARγ promoter 1 and 3 activities were determined by reporter assay in THP-1 macrophages, transfected with the promoter constructs and stimulated with 1 μg/ml LPS for 3 and 6 h. B, primary human macrophages were exposed to 100 μm DRB (filled squares) or 1 μg/ml LPS plus DRB (open circles) up to 3 h and total PPARγ1 mRNA (including transcripts 1 and 3) was determined by qPCR. C, differentiated THP-1 cells were transfected with the pGL3-control or pGL3-PPARγ-3′-UTR vector, and reporter activity was analyzed in response to 1 μg/ml LPS. Luciferase activity was normalized to protein and the ratio of pGL3-PPARγ-3′-UTR activity/pGL3-control is displayed. Data present mean values ± S.E., n ≥ 4. Statistics were analyzed with the unpaired Student's t test.*, p < 0.05; **, p < 0.01.
FIGURE 3.
FIGURE 3.
Deletion and mutation of the miR-27b binding site reverses PPARγ1 mRNA decay. A, sequence of the AU-rich PPARγ-3′-UTR. The miR-27 binding site is underlined, whereas ARE 1 sites (AUUUA) are shaded, and ARE 4 (12-mer A/U with maximum one mismatch) sites are marked with boxes. B, alignment of the PPARγ-3′-UTR with the miR-27b sequence and the sequences of the construct pGL3-PPARγ-3′UTR-ΔmiR-27 and pGL3-PPARγ-3′UTR-C83A/U84G. Mutated nucleotides are underlined. C, differentiated THP-1 cells were transfected with pGL3-control, pGL3-PPARγ-3′UTR, pGL3-PPARγ-3′UTR-ΔmiR-27, or pGL3-PPARγ-3′UTR-C83A/U84G and luciferase expression was measured after stimulation with 1 μg/ml LPS for 3 h. Basal activity was set to 1, ratios of 3′-UTR constructs/pGL3-control are displayed. Data present mean values ± S.E., n ≥ 3. Statistics were analyzed with the unpaired Student's t test. *, p < 0.05; **, p < 0.01.
FIGURE 4.
FIGURE 4.
NFκB-dependent miR-27b expression. A, miR-27b expression was measured in primary human macrophages in response to LPS (1 μg/ml, 2 h) by qPCR. To investigate a role of NFκB, cells were prestimulated for 1 h with 10 μm Bay11–7082. Basal expression was set to 1. B, primary human macrophages were pretreated with Bay as in A followed by 3 h of LPS exposure. PPARγ1 mRNA was determined by qPCR. C, temporal pattern of PPARγ1 mRNA and miR-27b expression was measured by qPCR after stimulation with 1 μg/ml LPS. Data represent mean values ± S.E., n ≥ 4. Statistics were analyzed with the paired Student's t test. *, p < 0.05.
FIGURE 5.
FIGURE 5.
Inhibition of miR-27b reverses PPARγ1 mRNA destabilization. A, primary human macrophages were transfected with different concentrations (50, 100, 150 pmol) of anti-miR-27b or a negative control. After transfection, cells were stimulated with 1 μg/ml LPS for 3 h, and PPARγ1 mRNA level was determined by qPCR. B, primary human macrophages were transfected with 150 pmol of anti-miR-27b or negative control and PPARγ1 mRNA half-life was determined by stimulating cells with 100 μm DRB and 1 μg/ml LPS for 1 to 3 h. Data represent mean values ± S.E., n ≥ 4. Statistics were analyzed with the paired Student's t test. **, p < 0.01; ***, p < 0.001.
FIGURE 6.
FIGURE 6.
Overexpression of miR-27b reduces PPARγ1 mRNA expression. A, primary human macrophages were transfected with different concentrations of miR-27b mimic (0.1, 1, or 10 pmol) or siControl, respectively. B, primary human macrophages were transfected with 10 pmol of miR-27b mimic or siControl and stimulated the next day with 1 μg/ml LPS for 3 h. C, primary human macrophages were transfected with 10 pmol of miR-27b mimic or siControl and treated for 1–3 h with 100 μm DRB 2 h after transfection. PPARγ1 mRNA content was determined by qPCR. Data represent mean values ± S.E., n ≥ 4. Statistics were analyzed with the paired Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 7.
FIGURE 7.
Modulating miR-27b expression affects TNFα and IL-6 expression. Primary human macrophages were transfected with (A, C) 10 pmol miR-27b mimic or (B, D) 150 pmol anti-miR-27b or negative controls (siC or neg.), respectively and prestimulated with 1 μm rosiglitazone (Rosi, (A, C)) for 1 h followed by treatment with 1 μg/ml LPS for 3 h. TNFα and IL-6 mRNA expression was determined by qPCR. Data represent mean values ± S.E., n ≥ 5. Statistics were analyzed with the paired Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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