Wnt3a disrupts GR-TEAD4-PPARγ2 positive circuits and cytoskeletal rearrangement in a β-catenin-dependent manner during early adipogenesis

Abstract

Adipogenesis is a process which induces or represses many genes in a way to drive irreversible changes of cell phenotypes; lipid accumulation, round cell-shape, secreting many adipokines. As a master transcription factor (TF), PPARγ2 induces several target genes to orchestrate these adipogenic changes. Thus induction of Pparg2 gene is tightly regulated by many adipogenic and also anti-adipogenic factors. Four hours after the treatment of adipogenic hormones, more than fifteen TFs including glucocorticoid receptor (GR), C/EBPβ and AP-1 cooperatively bind the promoter of Pparg2 gene covering 400 bps, termed "hotspot". In this study, we show that TEA domain family transcription factor (TEAD)4 reinforces occupancy of both GR and C/EBPβ on the hotspot of Pparg2 during early adipogenesis. Our findings that TEAD4 requires GR for its expression and for the ability to bind its own promoter and the hotspot region of Pparg2 gene indicate that GR is a common component of two positive circuits, which regulates the expression of both Tead4 and Pparg2. Wnt3a disrupts these mutually related positive circuits by limiting the nuclear location of GR in a β-catenin dependent manner. The antagonistic effects of β-catenin extend to cytoskeletal remodeling during the early phase of adipogenesis. GR is necessary for the rearrangements of both cytoskeleton and chromatin of Pparg2, whereas Wnt3a inhibits both processes in a β-catenin-dependent manner. Our results suggest that hotspot formation during early adipogenesis is related to cytoskeletal remodeling, which is regulated by the antagonistic action of GR and β-catenin, and that Wnt3a reinforces β-catenin function.

Fig. 1. Effects of Wnt3a on early induction of Pparg2.

a Post-confluent 3T3-L1 preadipocytes were induced to undergo adipogenesis by treatment with adipogenic hormones, IBMX (M), dexamethasone (D), and insulin (I) in the presence or absence of recombinant mouse Wnt3a (W3a). The treated cells were harvested at the indicated time points after induction. b Western blot analyses of 3T3-L1 cells using the indicated antibodies. 14-3-3γ was used as the loading control. Arrows indicate two PPARγ proteins (γ1, 54 kDa; γ2, 57 kDa). c, d Optical densities (510 nm) of Oil Red-O stained lipid in the 3T3-L1 cells at 6 days after the hormone treatment. The images of the Oil Red-O stained 3T3-L1 cells are shown in the Supplementary Fig. S1. e qRT-PCR analyses of Pparg2, Cebpa, and Axin2 mRNA levels, which were normalized to 18S rRNA levels as described previously. f Western blot analyses of 3T3-L1 cells using the indicated antibodies. g Relative mRNA levels of Pparg2 to 18S rRNA levels. h Western blot analyses of 3T3-L1 cells using the indicated antibodies. Arrows indicate two active forms of C/EBPβ (36 and 38 kDa respectively). i, j Western analyses of nuclear extracts (NE) of 3T3-L1 cells treated with either MDI or Dex (D; 2 μM) in the presence or absence of W3a. Lamin C was used as the loading control for the nuclear proteins. The relative band intensities of GR, C/EBPβ (36 kDa, the lower band with a black arrowhead), and lamin C were determined using the ImageJ software from four independent western analyses (details in Supplementary Fig. S2A). k, l ChIP-qPCR analyses of GR or C/EBPβ occupancy on Pparg2 (–0.3 kb or +2.6 kb from TSS) in 3T3-L1 cells. qPCR data show mean ± S.E. All data were repeated at least three independent same or similar experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by Students’ t-test; ns, not significant

Fig. 2. Effects of Wnt3a on cooperative binding of C/EBPβ and GR on Pparg2.

a–c NIH-3T3 cells were infected with retrovirus encoding FLAG-tagged C/EBPβ (C/β-NIH cells) or empty vector as a control (EV-NIH cells). a Western blot analyses using anti-FLAG and 14-3-3γ antibodies. b, c Post-confluent C/β-NIH or EV-NIH cells were treated with the indicated adipogenic hormones, Wnt3a conditioned media (W3a-CM; 75%), or RU486 (10 μΜ) for 24 h. b qRT-PCR analyses of Pparg2, Axin2, and Ccnd1 mRNA levels, which were normalized to 18S rRNA levels. c ChIP-qPCR analyses of FLAG (C/EBPβ) or GR occupancy on the –0.3 kb region from TSS of Pparg2. d, e Reporter analyses using C/EBP-Luc or GRE-Luc. The transfected 3T3-L1 cells were treated with Dex (D, 2 μM), W3a-CM (50 or 100%), recombinant mouse Wnt3a (W3a; 20 ng/ml), or RU486 (10 μM) for 24 h. f, g ChIP-qPCR analyses of STAT5, KLF4, c-Jun, p300, or CBP occupancy on Pparg2 (–0.3 or +2.6 kb from TSS) in 3T3-L1 cells. h FAIRE-qPCR analyses on the –0.3 kb region of Pparg2 in 3T3-L1 cells (details in Materials and methods). i ChIP-qPCR analyses of histone H3 on the –0.3 kb region from TSS of Pparg2 in 3T3-L1 cells. qPCR data show mean ± S.E. All data were repeated at least three independent same or similar experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by Students’ t-test

Fig. 3. GR-induced TEAD4.

a qRT-PCR analyses of Tead4 mRNA of 3T3-L1 cells treated with adipogenic hormones as indicated. b Western blot analyses of 3T3-L1 cells using anti-TEAD4 and 14-3-3γ antibodies. c 3T3-L1 preadipocytes were infected with lentiviruses encoding shRNAs against mouse GR (shGR-L1 cells) or control shRNA (shCtrl-L1 cells). Western blot analyses showing GR protein levels in the shCtrl-L1 cells or the shGR-L1 cells. d, e The shCtrl-L1 cells or the shGR-L1 cells were treated with Dex (D, 2 μΜ) for the indicated time points. d Relative mRNA levels of Tead4 to 18S rRNA levels. e Western analyses showing TEAD4 protein levels. f, g The 3T3-L1 cells were treated with MDI or Dex in the presence or absence of W3a or RU486 for the indicated time points. f Relative mRNA levels of Tead4 to 18S rRNA levels. g Western analyses showing TEAD4 protein levels. h Indication of a putative GRE site (closed box) and TBE sites (open boxes) on Tead4 (–5 to +1 kb) at chromosome 6. Black bars indicate ChIP amplicon regions (–0.9 and +0.3 kb from TSS of Tead4 gene). i, j ChIP-qPCR analyses of GR or TEAD4 occupancy on Tead4 (–0.9 or +0.3 kb from TSS) in 3T3-L1, shCtrl-L1, or shTead4-L1 cells. Western analyses showing TEAD4 protein levels of shCtrl-L1 cells or shTead4-L1 cells (right panel). qPCR data show mean ± SE. All data were repeated at least three independent same or similar experiments. *p < 0.05, ** p < 0.01, and *** p < 0.001 by Students’ t-test; ns, not significant

Fig. 4. TEAD4 as a novel hotspot TF for Pparg2 induction.

a ChIP-qPCR analyses of TEAD4 occupancy on the –0.3 kb region from TSS of Pparg2 in 3T3-L1 cells. b–f The shCtrl-L1 cells or the shTead4-L1 cells were induced to undergo adipogenesis by treating with adipogenic hormones for the indicated time points as described in Fig. 1a. b Images and optical densities (510 nm) of Oil Red-O stained lipid. Scale bars, 200 μm. c Relative mRNA levels of Pparg2 to 18S rRNA levels. d, e Western blot analyses using the indicated antibodies. f ChIP-qPCR analyses of GR, C/EBPβ, or TEAD4 occupancy on the –0.3 kb region from TSS of Pparg2. g–j 3T3-L1 cells were treated with MDI for the indicated time points. g Western blot analyses of 3T3-L1 cells using the indicated antibodies. h Relative mRNA levels of Ctgf to 18S rRNA levels. i, j ChIP-qPCR analyses of TEAD4, TAZ, or YAP occupancy on the –0.3 kb region from TSS of Pparg2 and the –0.1 kb region from TSS of Ctgf. k Western blot analyses using the indicated antibodies in the shCtrl-L1 cells and shTaz-L1 cells. l Schematic diagram showing GR-TEAD4-PPARγ2 positive circuits during early adipogenesis. Wnt3a disrupted two mutually related positive circuits by limiting the nuclear localization of GR. qPCR data show mean ± SE. All data were repeated at least three independent same or similar experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by Students’ t-test

Fig. 5. Effects of GR and C/EBPβ overexpression.

a–g 3T3-L1 preadipocytes were infected with lentiviruses encoding HA-tagged GR (GR-L1), Myc-tagged TEAD4 (Tead4-L1) or empty vector as a control (EV-L1 cells). These cells were treated with MDI in the presence or absence of W3a for the indicated time points. a Western analyses of TEAD4 and HA-tagged GR protein. b Relative levels of Tead4 mRNA to 18S rRNA. c Western analyses of PPARγ and Myc-tagged TEAD4. d Relative levels of Pparg2 mRNA to 18S rRNA. e Western analyses of nuclear extracts (NE) of the EV-L1 cells or the Tead4-L1 cells treated with MDI in the presence or absence of W3a for 24 h using the indicated antibodies. Lamin C was used as the loading control for the nuclear proteins. f Western blot analyses using the indicated antibodies. g Relative levels of Pparg2 mRNA to 18S rRNA. h–k 3T3-L1 preadipocytes were infected with lentiviruses encoding FLAG-tagged C/EBPβ (Cβ-L1) or empty vector as a control (EV-L1 cells). Cβ-L1 cells and EV-L1 cells were further infected with retrovirus encoding HA-tagged GR (GR/Cβ-L1) or empty vector (EV/EV-L1 cells), respectively. These cells were treated with MDI in the presence or absence of W3a for the indicated time points. h Images and optical densities (510 nm) of Oil Red-O stained lipid. Scale bars, 200 μm. i Western analyses using the indicated antibodies. j ChIP-qPCR analyses of GR or HA-tagged GR occupancy on the –0.3 kb region from TSS of Pparg2. p = 0.012 for GR and p = 0.085 for HA (GR). k Western analyses of nuclear extracts (NE) of the EV/EV-L1 cells or GR/Cβ-L1 cells treated with MDI in the presence or absence of W3a. H3 was used as the loading control for nuclear proteins. The relative band intensities of GR and H3 were determined using the ImageJ software from two independent western blot analyses. qPCR data show mean ± SE. All data were repeated at least three independent same or similar experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by Students’ t-test; ns, not significant

Fig. 6. Effects of β-catenin knockdown.

a–k 3T3-L1 preadipocytes were infected with a lentivirus encoding shRNA against mouse β-catenin (shβ-cat-L1 cells) or control shRNA (shCtrl-L1 cells). a Western blot analyses showing β-catenin level. b qRT-PCR analyses showing relative mRNA levels of β-catenin, Ccnd1, and Axin2 to 18S rRNA levels. c–k The shCtrl-L1 or shβ-cat-L1 cells were induced to undergo adipogenesis by treating with adipogenic hormones for the indicated time points in the presence or absence of W3a (5 ng/ml). c Images and optical densities (510 nm) of Oil Red-O stained lipid. Scale bars, 200 μm. d, f Relative mRNA levels of Pparg2 and Tead4 to 18S rRNA levels. e, g Western blot analyses using the indicated antibodies. h Western blot analyses of nuclear extracts (NE) of the shCtrl-L1 or shβ-cat-L1 cells using the indicated antibodies. Lamin C was used as the loading control for nuclear proteins. i, k ChIP-qPCR analyses of C/EBPβ, GR, or H3 occupancy on the –0.3 kb region from TSS of Pparg2. j FAIRE-qPCR analyses on the –0.3 kb region from TSS of Pparg2. qPCR data show mean ± SE. All data were repeated at least three independent same or similar experiments. *p < 0.05, **p < 0.01, and ***p < 0.001, † p = 0.065 by Students’ t-test; ns, not significant

Fig. 7. Cytoskeletal rearrangement during early adipogenesis.

3T3-L1 cells were treated with MDI for the indicated time points in the presence or absence of W3a (5 ng/ml) as described in Fig. 1a. a Confocal microscopic images of cellular filamentous actin (F-actin) and β-catenin (upper panel). F-actin structures in individual cells were categorized into three groups. S (stress fiber), where F-actin stress fibers were observed in both nuclei and cytoplasm; T (transition status), where F-actin stress fibers were observed in the cytoplasm but not in the nucleus; C (cortical structure), where F-actin stress fibers were observed neither in the nucleus nor in the cytoplasm, but F-actin was observed near the cellular membrane. Cells (13–48) in each treatment were observed and categorized into three groups. The graph indicates the percentage of cells in each category (lower panel). b Western blot analyses of 3T3-L1 cells using the indicated antibodies. c Confocal microscopic images of cellular F-actin and β-catenin (upper panel). The graph indicates the percentage of cells in each category (S, T, and C described in Fig. 7a) (lower panel). d–j 3T3-L1 preadipocytes were infected with retrovirus encoding HA-tagged S37A-β-catenin (S37A-β-L1 cells) or empty vector (EV-L1 cells) as a control. d Western blot analyses of the EV-L1 cells or the S37A-β-L1 cells using anti-HA and 14-3-3γ antibodies. e Confocal microscopic images of the EV-L1 cells or the S37A-β-L1 cells immunostained with either anti-β-catenin antibody or anti-HA antibody. The cells were treated with W3a (5 ng/ml) for 24 h. The nuclei were stained with Hoechst 33258 (blue). f qRT-PCR analyses showing relative mRNA levels of Axin2 and Ccnd1 to 18S rRNA. g–j The EV-L1 cells or the S37A-β-L1 cells were treated with MDI for the indicated time points in the presence or absence of W3a (5 ng/ml). g Relative mRNA levels of Pparg2 and Axin2 to 18S rRNA. h Western blot analyses using the indicated antibodies. i ChIP-qPCR analyses of C/EBPβ or GR occupancy on the –0.3 kb region from TSS of Pparg2. j Confocal microscopic images of the cells immunostained with fluorescent phalloidin conjugates (green) and with Hoechst 33258 (blue) (left panel). Graph indicating the percentage of cells in each category (S, T, and C described in Fig. 7a) (right panel). qPCR data show mean ± SE. All data were repeated at least three independent same or similar experiments

Fig. 8. Effects of GR on cytoskeletal rearrangement.

The shCtrl-L1 or shGR-L1 cells were induced to undergo adipogenesis by treating with adipogenic hormones for the indicated time points in the presence or absence of W3a (5 ng/ml). a Confocal microscopic images of F-actin stress fibers (left panel). The graph indicates the percentage of cells in each category (S, T, and C described in Fig. 7a) (right panel). b qRT-PCR analyses of Pparg2 mRNA levels to 18S rRNA levels. c ChIP-qPCR analyses of C/EBPβ or GR occupancy on the –0.3 kb region from TSS of Pparg2. d Confocal microscopic images of cellular F-actin stress fibers (left panel) in the EV/EV-L1 or GR/Cβ-L1 cells. The graph indicates the percentage of cells in each category (S, T, and C described in Fig. 7a) (right panel). e–g C3H10T1/2 cells were treated with Dex (2 μM) or MDI for the indicated time points in the presence or absence of W3a (5 ng/ml). e qRT-PCR analyses of Tead4 and Pparg2 mRNA levels to 18S rRNA levels. f Western blot analyses showing TEAD4 and PPARγ protein levels. g Confocal microscopic images of cellular F-actin stress fibers (left panel) in C3H10T1/2 cells. The graph indicates the percentage of cells in each category (S, T, and C described in Fig. 7a) (right panel). h Schematic diagram showing the inhibitory effects of Wnt3a/β-catenin on positive circuits of GR-TEAD4-PPARγ2 and cytoskeletal remodeling during early adipogenesis (details in Results). qPCR data show mean ± SE. All data were repeated at least three independent same or similar experiments. ***p < 0.001 by Students' t-tests