Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jan;20(1):40-7.
doi: 10.1038/oby.2011.259. Epub 2011 Aug 4.

The mRNA-binding protein Zfp36 is upregulated by β-adrenergic stimulation and represses IL-6 production in 3T3-L1 adipocytes

Pavna K Brahma  1 , Huanchun Zhang, Betsy S Murray, Feng-jue Shu, Neil Sidell, Emre Seli, Caleb B Kallen
Affiliations

The mRNA-binding protein Zfp36 is upregulated by β-adrenergic stimulation and represses IL-6 production in 3T3-L1 adipocytes

Pavna K Brahma et al. Obesity (Silver Spring). 2012 Jan.

Abstract

Obesity produces a chronic inflammatory state that contributes to the development of diabetes and atherosclerosis. In obese humans, fat depot adipocytes and macrophages produce inflammatory cytokines and other factors which exert unfavorable local and systemic immune responses. The expression of many cytokines is modulated at the post-transcriptional level by mRNA-binding proteins which recognize AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTR) of these transcripts. One such protein, zinc finger protein 36 (Zfp36), is known to destabilize target mRNAs leading to decreased cytokine expression. Few regulators of Zfp36 expression in adipocytes have been described and mRNA targets of Zfp36 in adipocytes are largely unknown. We found that macrophage-derived inflammatory stimuli enhanced endogenous Zfp36 expression in 3T3-L1 adipocytes. Furthermore, the β-adrenergic receptor agonist isoproterenol (Iso) and the glucocorticoid dexamethasone (Dex) each enhanced Zfp36 expression in adipocytes, the former most likely via a cyclic adenosine monophosphate (cAMP)-dependent pathway. By contrast, Zfp36 expression in murine macrophages (RAW 264.7) was not enhanced by exposure to Dex but was stimulated by retinoic acid (RA). Zfp36 inhibited basal and lipopolysaccharide (LPS)-stimulated interleukin-6 (IL-6) expression in adipocytes. These data reveal important and cell type-specific modulators of Zfp36 expression in adipocytes and macrophages and identify Zfp36 as a potent repressor of adipocyte-derived IL-6. Furthermore, this work identifies new factors that stimulate adipocyte Zfp36 expression that are neither classically inflammatory nor mitogenic. Upregulating an mRNA-binding protein for therapeutic purposes may provide a novel mechanistic approach with which to treat diverse inflammatory disorders including common conditions associated with obesity.

PubMed Disclaimer

Conflict of interest statement

DISCLOSURE

The authors declared no conflict of interest.

Figures

Figure 1
Figure 1
Lipopolysaccharide (LPS) and macrophage-conditioned medium (CM) enhance zinc finger protein 36 (Zfp36) expression in 3T3-L1 adipocytes. Western blot analysis of adipocyte Zfp36. (a) 3T3-L1 adipocytes were serum starved overnight and treated with LPS (10 ng/ml) for the indicated time in serum-free culture medium. Zfp36 expression was enhanced with short (6 h) exposure to LPS but returned to baseline after 24 h exposure to LPS. Serum starved 3T3-L1 adipocytes were also cultured for 6 h with CM from THP-1 macrophage cells grown in serum-free medium. THP-1 cells were pretreated with vehicle or LPS (10 ng/ml) for 24 h before harvesting of medium. THP-1 CM enhanced Zfp36 expression in 3T3-L1 adipocytes and the effect was more pronounced in LPS-stimulated THP-1 cells. (b) Indirect coculture of 3T3-L1 adipocytes with RAW 264.7 macrophages similarly enhances Zfp36 expression in the adipocytes. 3T3-L1 adipocytes were serum starved overnight and then cultured with RAW 264.7 macrophages grown on cell-impermeable inserts (macrophages in the upper chamber, adipocytes in the lower chamber). Coculture was performed in the presence of LPS (10 ng/ml) for 6 h. In addition, adipocytes were treated for 6 h with RAW 264.7 CM that was pretreated with vehicle or LPS for 24 h before harvesting of medium. Quantitative image analysis indicating Zfp36 expression relative to control cells, normalized to actin, is shown. Data in a and b are representative of three independent experiments.
Figure 2
Figure 2
Indirect coculture of macrophages with adipocytes promotes adipocyte mRNA expression of inflammatory factors. Quantitative reverse transcriptase (RT)-PCR was performed using adipocyte mRNA. Adipocyte mRNA was isolated with and without prior indirect coculture with THP-1 macrophages, with and without costimulation using lipopolysaccharide (LPS) (10 ng/ml). mRNA expression of 3T3-L1-expressed IL-6, Mcp-1, and Rantes was enhanced with macrophage coculture and further enhanced by costimulation with LPS. Values represent the means of at least three independent experiments with s.e.m. *P < 0.05.
Figure 3
Figure 3
Dexamethsone, tumor necrosis factor-α (TNFα), and forskolin stimulate zinc finger protein 36 (Zfp36) expression in 3T3-L1 adipocytes. Western blot analysis of Zfp36 expression in 3T3-L1 adipocytes grown in serum-free medium with the following 6 h treatments: vehicle control, TNFα (20 ng/ml), retinoic acid (RA, 1.0 μmol/l), dexamethasone (Dex, 1 μmol/l), fetal calf serum (FCS, 10%), and forskolin (10 μmol/l). Quantitative image analysis indicating Zfp36 expression relative to control cells, normalized to actin, is shown. Data are representative of three independent experiments.
Figure 4
Figure 4
Sulindac and atorvastatin do not enhance zinc finger protein 36 (Zfp36) expression in 3T3-L1 adipocytes. Western blot analysis of Zfp36 expression in 3T3-L1 adipocytes grown in serum-free medium with the following 6 h treatments: vehicle control, dexamethasone (Dex, 1 μmol/l), forskolin (10 μmol/l), the nonsteroidal anti-inflammatory drug (NSAID) sulindac (100 μmol/l), and the lipid-lowering statin drug atorvastatin (5 μmol/l). Quantitative image analysis indicating Zfp36 expression relative to control cells, normalized to actin, is shown. Data are representative of three independent experiments.
Figure 5
Figure 5
The β-adrenergic receptor agonist isoproterenol stimulates zinc finger protein 36 (Zfp36) expression in 3T3-L1 adipocytes. Western blot analysis of Zfp36 expression in 3T3-L1 adipocytes grown in serum-free medium with the following 6 h treatments: vehicle control, β-adrenergic agonist isoproterenol (Iso, 10 μmol/l), β3-adrenergic antagonist SR59230A (5 μmol/l), or Iso plus SR59230A (same concentrations). Quantitative image analysis indicating Zfp36 expression relative to control cells, normalized to actin, is shown. Data are representative of three independent experiments.
Figure 6
Figure 6
Forskolin and retinoic acid (RA), but not dexamethasone, rapidly enhance zinc finger protein 36 (Zfp36) expression in RAW 264.7 macrophages. (a) Western blot analysis of Zfp36 expression in RAW 264.7 cells grown in serum-free medium with the following 6 h treatments: vehicle control, lipopolysaccharide (LPS) (10 ng/ml), dexamethasone (Dex, 1 μmol/l), forskolin (Forsk, 10 μmol/l), and retinoic acid (RA, 1.0 μmol/l). Actin loading control is shown. Data are representative of three independent experiments. (b) Quantitative image analysis of western blots showing Zfp36 expression in RAW 264.7 cells after the same treatments described above. Band intensities were measured and normalized to respective Actin band intensities for three independent experiments. Data are shown relative to control cells. *P< 0.05 compared with control cells.
Figure 7
Figure 7
Zfp36 represses interleukin (IL)-6 expression in basal and LPS-stimulated 3T3-L1 adipoctyes. (a) Lentivirus-mediated zinc finger protein 36 (Zfp36) overexpression and Zfp36-knockdown in 3T3-L1 adipocytes. Western blot analysis of Zfp36 expression in 3T3-L1 adipocytes. Cells were transduced with empty vector-containing lentiviral particles (vector) or human Zfp36-expressing lentiviral particles (Zfp36) or anti-Zfp36-shRNA-expressing lentiviral particles (Zfp36-KD), with and without lipopolysaccharide (LPS) (10 ng/ml) stimulation for 6 h. Actin loading control is also shown and data are representative of three independent experiments. (b) IL-6 was measured in the culture medium of 3T3-L1 adipocytes engineered to over- or under-express Zfp36. Cells were transduced with empty vector-containing lentiviral particles (vector) or human Zfp36-expressing lentiviral particles (Zfp36) or anti-Zfp36-shRNA-expressing lentiviral particles (Zfp36-KD) (see Figure 7a). Cells were grown in serum-free medium for 24 h and then treated with vehicle or LPS (10 ng/ml) for 24 h. Culture medium was collected and IL-6 protein quantitated using enzyme-linked immunosorbent assay (ELISA). IL-6 data were normalized to percent lipid in each well. IL-6 levels are expressed as fold change relative to vector control. Shown are means with s.e.m. of at least three independent experiments performed in triplicate. *P < 0.05 compared with vector-transduced cells. **P < 0.05 compared with LPS-treated vector-transduced cells. shRNA, short hairpin RNA.
Figure 8
Figure 8
Zinc finger protein 36 (Zfp36) reduces adipokine mRNA expression in 3T3-L1 adipocytes. Cells were transduced with empty vector-containing lentiviral particles (vector) or human Zfp36-expressing lentiviral particles as described in Figure 7. Quantitative reverse transcriptase (RT)-PCR was performed and expression was normalized to vector-treated cells (dashed line). mRNA expression of IL-6, Mcp-1, and IL-1β was reduced in Zfp36-expressing adipocytes (black bars). Values represent the means of three independent experiments with s.e.m. *P < 0.05 relative to vector control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ferrante AW., Jr Obesity-induced inflammation: a metabolic dialogue in the language of inflammation. J Intern Med. 2007;262:408–414. - PubMed
    1. Weisberg SP, McCann D, Desai M, et al. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003;112:1796–1808. - PMC - PubMed
    1. Sartipy P, Loskutoff DJ. Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci USA. 2003;100:7265–7270. - PMC - PubMed
    1. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-α: direct role in obesity-linked insulin resistance. Science. 1993;259:87–91. - PubMed
    1. Odegaard JI, Ricardo-Gonzalez RR, Goforth MH, et al. Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance. Nature. 2007;447:1116–1120. - PMC - PubMed

Publication types

MeSH terms