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miR-155 regulates differentiation of brown and beige adipocytes via a bistable circuit.

Chen Y, Siegel F, Kipschull S, Haas B, Fröhlich H, Meister G, Pfeifer A - Nat Commun (2013)

Bottom Line: Brown adipocytes are a primary site of energy expenditure and reside not only in classical brown adipose tissue but can also be found in white adipose tissue.In contrast, transgenic overexpression of microRNA 155 in mice causes a reduction of brown adipose tissue mass and impairment of brown adipose tissue function.These data demonstrate that the bistable loop involving microRNA 155 and CCAAT/enhancer-binding protein β regulates brown lineage commitment, thereby, controlling the development of brown and beige fat cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.

ABSTRACT
Brown adipocytes are a primary site of energy expenditure and reside not only in classical brown adipose tissue but can also be found in white adipose tissue. Here we show that microRNA 155 is enriched in brown adipose tissue and is highly expressed in proliferating brown preadipocytes but declines after induction of differentiation. Interestingly, microRNA 155 and its target, the adipogenic transcription factor CCAAT/enhancer-binding protein β, form a bistable feedback loop integrating hormonal signals that regulate proliferation or differentiation. Inhibition of microRNA 155 enhances brown adipocyte differentiation and induces a brown adipocyte-like phenotype ('browning') in white adipocytes. Consequently, microRNA 155-deficient mice exhibit increased brown adipose tissue function and 'browning' of white fat tissue. In contrast, transgenic overexpression of microRNA 155 in mice causes a reduction of brown adipose tissue mass and impairment of brown adipose tissue function. These data demonstrate that the bistable loop involving microRNA 155 and CCAAT/enhancer-binding protein β regulates brown lineage commitment, thereby, controlling the development of brown and beige fat cells.

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miR-155 and C/EBPβ constitute a bistable feedback loop.(a) Scheme of the 2 kb murine BIC/miR-155 promoter with the putative C/EBP-binding sites (siteA, siteB/C/D and siteE) (purple); primer binding sites, orange; C/EBP binding sites, red. (b) Representative western blot of C/EBPβ expression during in vitro brown fat differentiation. Protein samples were collected at indicated time points. Tubulin served as loading control. (c) Densitometric analysis of C/EBPβ protein levels normalized to tubulin. Data from day −2 were set as one; all data are represented as means±s.e.m. (*P<0.05; **P<0.01; ***P<0.001; one-way analysis of variance (ANOVA); n=3). (d) qRT–PCR analysis of miR-155 expression (normalized to sno202) in cells transduced with LVC/EBPβ or control virus (LVcontrol). Untreated controls (mock) were set as one; data are represented as means±s.e.m. (***P<0.001; one-way ANOVA; n=3). (e) Luciferase assay to analyse regulation of the BIC/miR-155 promoter by C/EBPβ. Cells were infected with LVcontrol or LVC/EBPβ 48 h before BIC/miR-155 promoter luciferase construct transfection. Uninfected controls (mock), transfected with the same reporter construct were set as one; data are represented as means±s.e.m. (*P<0.05; one-way ANOVA; n=4). (f) Brown preadipocytes were transduced with LVcontrol or LVC/EBPβ 48 h prior the Chromatin immunoprecipitation assay. Precipitation was performed with an anti-C/EBPβ antibody or IgG and PolII control antibodies. C/EBPβ-bound BIC/miR-155 promoter fragments were amplified using qRT–PCR primers that span putative binding sites A, B/C/D or E, respectively. Results are normalized to input values. Relative values are represented as means±s.e.m. (*P<0.05; Student’s t-test; n.s., not significant; n=3). (g,h) TG content and cell number of brown adipocytes transduced with different doses of (g) LVmiR155 (0, 62.5, 250 and 500 ng RTase per well in six-well plates) or (h) LVC/EBPβ (0, 50, 200 and 400 ng RTase per well in six-well plates). TG content was normalized to total protein concentration. LVmiRctrl-transduced cells were set as one. All data are represented as means±s.e.m. (#P<0.05; one-way ANOVA; n=3 in TG assay, *P<0.05; one-way ANOVA; n=3 in proliferation assay).
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f3: miR-155 and C/EBPβ constitute a bistable feedback loop.(a) Scheme of the 2 kb murine BIC/miR-155 promoter with the putative C/EBP-binding sites (siteA, siteB/C/D and siteE) (purple); primer binding sites, orange; C/EBP binding sites, red. (b) Representative western blot of C/EBPβ expression during in vitro brown fat differentiation. Protein samples were collected at indicated time points. Tubulin served as loading control. (c) Densitometric analysis of C/EBPβ protein levels normalized to tubulin. Data from day −2 were set as one; all data are represented as means±s.e.m. (*P<0.05; **P<0.01; ***P<0.001; one-way analysis of variance (ANOVA); n=3). (d) qRT–PCR analysis of miR-155 expression (normalized to sno202) in cells transduced with LVC/EBPβ or control virus (LVcontrol). Untreated controls (mock) were set as one; data are represented as means±s.e.m. (***P<0.001; one-way ANOVA; n=3). (e) Luciferase assay to analyse regulation of the BIC/miR-155 promoter by C/EBPβ. Cells were infected with LVcontrol or LVC/EBPβ 48 h before BIC/miR-155 promoter luciferase construct transfection. Uninfected controls (mock), transfected with the same reporter construct were set as one; data are represented as means±s.e.m. (*P<0.05; one-way ANOVA; n=4). (f) Brown preadipocytes were transduced with LVcontrol or LVC/EBPβ 48 h prior the Chromatin immunoprecipitation assay. Precipitation was performed with an anti-C/EBPβ antibody or IgG and PolII control antibodies. C/EBPβ-bound BIC/miR-155 promoter fragments were amplified using qRT–PCR primers that span putative binding sites A, B/C/D or E, respectively. Results are normalized to input values. Relative values are represented as means±s.e.m. (*P<0.05; Student’s t-test; n.s., not significant; n=3). (g,h) TG content and cell number of brown adipocytes transduced with different doses of (g) LVmiR155 (0, 62.5, 250 and 500 ng RTase per well in six-well plates) or (h) LVC/EBPβ (0, 50, 200 and 400 ng RTase per well in six-well plates). TG content was normalized to total protein concentration. LVmiRctrl-transduced cells were set as one. All data are represented as means±s.e.m. (#P<0.05; one-way ANOVA; n=3 in TG assay, *P<0.05; one-way ANOVA; n=3 in proliferation assay).

Mentions: To identify mechanisms that—apart from TGFβ1—might regulate miR-155, we examined the miR-155 promoter sequence for transcription-factor-binding sites. miR-155 is processed from a primary transcript, the B-cell integration cluster (BIC)3435. Analysis of the BIC/miR-155 promoter sequence revealed putative C/EBP-binding motifs (Fig. 3a) indicating that miR-155 might be regulated by its own target through a feedback mechanism. C/EBPβ is tightly regulated during fat cell differentiation: expression is almost undetectable in preadipocytes and is strongly induced by the adipogenic cocktail applied at day 0 (Fig. 3b), the time point when miR-155 expression is beginning to decrease. Overexpression of C/EBPβ in brown preadipocytes caused a dramatic downregulation of miR-155 levels (Fig. 3d). Next, we used BIC/miR-155 promoter reporter constructs, to analyse transcriptional regulation of miR-155 by C/EBPβ in more detail. Overexpression of C/EBPβ led to a pronounced inhibition of BIC/miR-155 promoter activity in HIB-1B brown preadipocytes (Fig. 3e). In contrast, siRNA-mediated knockdown of C/EBPβ (siC/EBPβ) caused a significant increase in miR-155 expression in BAT-derived preadipocytes (Supplementary Fig. S4a,b).


miR-155 regulates differentiation of brown and beige adipocytes via a bistable circuit.

Chen Y, Siegel F, Kipschull S, Haas B, Fröhlich H, Meister G, Pfeifer A - Nat Commun (2013)

miR-155 and C/EBPβ constitute a bistable feedback loop.(a) Scheme of the 2 kb murine BIC/miR-155 promoter with the putative C/EBP-binding sites (siteA, siteB/C/D and siteE) (purple); primer binding sites, orange; C/EBP binding sites, red. (b) Representative western blot of C/EBPβ expression during in vitro brown fat differentiation. Protein samples were collected at indicated time points. Tubulin served as loading control. (c) Densitometric analysis of C/EBPβ protein levels normalized to tubulin. Data from day −2 were set as one; all data are represented as means±s.e.m. (*P<0.05; **P<0.01; ***P<0.001; one-way analysis of variance (ANOVA); n=3). (d) qRT–PCR analysis of miR-155 expression (normalized to sno202) in cells transduced with LVC/EBPβ or control virus (LVcontrol). Untreated controls (mock) were set as one; data are represented as means±s.e.m. (***P<0.001; one-way ANOVA; n=3). (e) Luciferase assay to analyse regulation of the BIC/miR-155 promoter by C/EBPβ. Cells were infected with LVcontrol or LVC/EBPβ 48 h before BIC/miR-155 promoter luciferase construct transfection. Uninfected controls (mock), transfected with the same reporter construct were set as one; data are represented as means±s.e.m. (*P<0.05; one-way ANOVA; n=4). (f) Brown preadipocytes were transduced with LVcontrol or LVC/EBPβ 48 h prior the Chromatin immunoprecipitation assay. Precipitation was performed with an anti-C/EBPβ antibody or IgG and PolII control antibodies. C/EBPβ-bound BIC/miR-155 promoter fragments were amplified using qRT–PCR primers that span putative binding sites A, B/C/D or E, respectively. Results are normalized to input values. Relative values are represented as means±s.e.m. (*P<0.05; Student’s t-test; n.s., not significant; n=3). (g,h) TG content and cell number of brown adipocytes transduced with different doses of (g) LVmiR155 (0, 62.5, 250 and 500 ng RTase per well in six-well plates) or (h) LVC/EBPβ (0, 50, 200 and 400 ng RTase per well in six-well plates). TG content was normalized to total protein concentration. LVmiRctrl-transduced cells were set as one. All data are represented as means±s.e.m. (#P<0.05; one-way ANOVA; n=3 in TG assay, *P<0.05; one-way ANOVA; n=3 in proliferation assay).
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f3: miR-155 and C/EBPβ constitute a bistable feedback loop.(a) Scheme of the 2 kb murine BIC/miR-155 promoter with the putative C/EBP-binding sites (siteA, siteB/C/D and siteE) (purple); primer binding sites, orange; C/EBP binding sites, red. (b) Representative western blot of C/EBPβ expression during in vitro brown fat differentiation. Protein samples were collected at indicated time points. Tubulin served as loading control. (c) Densitometric analysis of C/EBPβ protein levels normalized to tubulin. Data from day −2 were set as one; all data are represented as means±s.e.m. (*P<0.05; **P<0.01; ***P<0.001; one-way analysis of variance (ANOVA); n=3). (d) qRT–PCR analysis of miR-155 expression (normalized to sno202) in cells transduced with LVC/EBPβ or control virus (LVcontrol). Untreated controls (mock) were set as one; data are represented as means±s.e.m. (***P<0.001; one-way ANOVA; n=3). (e) Luciferase assay to analyse regulation of the BIC/miR-155 promoter by C/EBPβ. Cells were infected with LVcontrol or LVC/EBPβ 48 h before BIC/miR-155 promoter luciferase construct transfection. Uninfected controls (mock), transfected with the same reporter construct were set as one; data are represented as means±s.e.m. (*P<0.05; one-way ANOVA; n=4). (f) Brown preadipocytes were transduced with LVcontrol or LVC/EBPβ 48 h prior the Chromatin immunoprecipitation assay. Precipitation was performed with an anti-C/EBPβ antibody or IgG and PolII control antibodies. C/EBPβ-bound BIC/miR-155 promoter fragments were amplified using qRT–PCR primers that span putative binding sites A, B/C/D or E, respectively. Results are normalized to input values. Relative values are represented as means±s.e.m. (*P<0.05; Student’s t-test; n.s., not significant; n=3). (g,h) TG content and cell number of brown adipocytes transduced with different doses of (g) LVmiR155 (0, 62.5, 250 and 500 ng RTase per well in six-well plates) or (h) LVC/EBPβ (0, 50, 200 and 400 ng RTase per well in six-well plates). TG content was normalized to total protein concentration. LVmiRctrl-transduced cells were set as one. All data are represented as means±s.e.m. (#P<0.05; one-way ANOVA; n=3 in TG assay, *P<0.05; one-way ANOVA; n=3 in proliferation assay).
Mentions: To identify mechanisms that—apart from TGFβ1—might regulate miR-155, we examined the miR-155 promoter sequence for transcription-factor-binding sites. miR-155 is processed from a primary transcript, the B-cell integration cluster (BIC)3435. Analysis of the BIC/miR-155 promoter sequence revealed putative C/EBP-binding motifs (Fig. 3a) indicating that miR-155 might be regulated by its own target through a feedback mechanism. C/EBPβ is tightly regulated during fat cell differentiation: expression is almost undetectable in preadipocytes and is strongly induced by the adipogenic cocktail applied at day 0 (Fig. 3b), the time point when miR-155 expression is beginning to decrease. Overexpression of C/EBPβ in brown preadipocytes caused a dramatic downregulation of miR-155 levels (Fig. 3d). Next, we used BIC/miR-155 promoter reporter constructs, to analyse transcriptional regulation of miR-155 by C/EBPβ in more detail. Overexpression of C/EBPβ led to a pronounced inhibition of BIC/miR-155 promoter activity in HIB-1B brown preadipocytes (Fig. 3e). In contrast, siRNA-mediated knockdown of C/EBPβ (siC/EBPβ) caused a significant increase in miR-155 expression in BAT-derived preadipocytes (Supplementary Fig. S4a,b).

Bottom Line: Brown adipocytes are a primary site of energy expenditure and reside not only in classical brown adipose tissue but can also be found in white adipose tissue.In contrast, transgenic overexpression of microRNA 155 in mice causes a reduction of brown adipose tissue mass and impairment of brown adipose tissue function.These data demonstrate that the bistable loop involving microRNA 155 and CCAAT/enhancer-binding protein β regulates brown lineage commitment, thereby, controlling the development of brown and beige fat cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.

ABSTRACT
Brown adipocytes are a primary site of energy expenditure and reside not only in classical brown adipose tissue but can also be found in white adipose tissue. Here we show that microRNA 155 is enriched in brown adipose tissue and is highly expressed in proliferating brown preadipocytes but declines after induction of differentiation. Interestingly, microRNA 155 and its target, the adipogenic transcription factor CCAAT/enhancer-binding protein β, form a bistable feedback loop integrating hormonal signals that regulate proliferation or differentiation. Inhibition of microRNA 155 enhances brown adipocyte differentiation and induces a brown adipocyte-like phenotype ('browning') in white adipocytes. Consequently, microRNA 155-deficient mice exhibit increased brown adipose tissue function and 'browning' of white fat tissue. In contrast, transgenic overexpression of microRNA 155 in mice causes a reduction of brown adipose tissue mass and impairment of brown adipose tissue function. These data demonstrate that the bistable loop involving microRNA 155 and CCAAT/enhancer-binding protein β regulates brown lineage commitment, thereby, controlling the development of brown and beige fat cells.

Show MeSH
Related in: MedlinePlus