Fig. 4: ESRRA represses SPP1 transcriptional expression via interrupting with E2/ESR1 signaling in adipocytes.
From: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow
a Plasma SPP1 levels of Esrrafl/fl and EsrraAKO mice at 8 weeks post-OVX or sham operation. n = 7 mice per group. b Representative images and analysis of SPP1 and leptin co-staining of gWAT from OVX mice studied in (a) (scale bar: 100 μm). n = 7 mice per group. c mRNA expression of Spp1, Leptin, Adipoq, Pparg, Cebpa and Fabp4 of gWAT from OVX mice. n = 7 mice per group. d Protein levels of SPP1, leptin and ESRRA of gWAT from Esrrafl/fl and EsrraAKO mice at 4 and 8 weeks post-OVX or sham operation. e Schematic diagram displays the potential binding sites of ESR1 within the Spp1 promoter, including S1, S2 and S3. Fragments for ChIP assay shown as region 1 (R1) and region 2 (R2). f Luciferase reporter activities of the Spp1 promoter in adipogenesis induced 3T3-L1 cells transfected with Esrra or Esr1 expressing plasmids in the presence of E2 or not. n = 3 biologically independent experiments. The consensus sequence binding motifs for ESR1 response element (ERE) and ESRRA response element (ERRE) are presented. g Luciferase reporter activities of the Spp1 promoter regulated by E2/ESR1 in the presence of wild-type (WT) or DNA-binding domain-deleted ESRRA construct (ESRRA-ΔDBD). n = 4 biologically independent experiments. h ChIP assay with ESR1 antibody in BMSCs from Esrrafl/fl and EsrraAKO mice after adipogenic induction for 4 days along with or without E2. n = 3 biologically independent experiments. i Luciferase reporter activities of the R2 deleted-Spp1 promoter (ΔR2-luc) as compared to Spp1 promoter (WT-luc). n = 4 biologically independent experiments. j Enrichment of ESRRA in R2 of Spp1 promoter in adipogenesis induced 3T3-L1 cells with the indicated treatments. n = 3 biologically independent experiments. Spp1 mRNA in murine BMAds (k), matured 3T3-L1 adipocytes (l) or human BMSCs-derived BMAds (m) infected with adenovirus expressing ESRRA or GFP with E2 treatment for 2 days. n = 4, 6, 4 biologically independent experiments, respectively. n Diagram illustrating the mechanism of ESRRA-regulated repression of Spp1 transcriptional expression via interfering with E2/ESR1 signaling in adipocytes (schematic created with BioRender.com. Agreement number: BH26KF823M). Data are shown as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical analysis is performed using unpaired two-tailed Student’s t test (c), one-way ANOVA followed by Bonferroni’s post hoc tests (k, l, m). Source data are provided as a Source Data file.
