Fig. 3: 11β-HSD1-mediated GC signaling overactivation restrains the early growth response 2 (Egr2)-governed osteogenic activity and glucose uptake in osteoblast.
From: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity
a, b Osteogenic activity of MC3T3-E1 osteoblastic cells with 11β-HSD1 overexpression (MC3T3-HSD1 cells) and transfected control cells (MC3T3-GFP cells). a Alkaline phosphatase (ALP) and alizarin red staining. b The mRNA expression of RUNX family transcription factor 2 (Runx2) and bone gamma-carboxyglutamate protein (Bglap). c, d Combination analysis of RNA sequencing (RNA-seq) and assays for transposase-accessible chromatin sequencing (ATAC-seq) in MC3T3-GFP cells and MC3T3-HSD1 cells. c Intersection of differential genes from RNA-seq and ATAC-seq. d Biological processes associated with Egr2. e The mRNA and protein expression of Egr2 in MC3T3-GFP cells and MC3T3-HSD1 cells. f The mRNA expression of Egr2 during osteogenic differentiation. g, h The osteogenic activity of MC3T3-HSD1 cells with Egr2 overexpressing. g Alizarin red staining. h Bglap mRNA expression. i The glucose uptake test of MC3T3-GFP cells and MC3T3-HSD1 cells. j, k The mRNA expression of key glucose transporter proteins and components of insulin-dependent glucose uptake pathway in MC3T3-GFP cells and MC3T3-HSD1 cells. j Glucose transporter type 1 (Glut1), 3 (Glut3) and 4 (Glut4). k: Insulin receptor (Ir), Insulin receptor substrate 1 (Irs1), Phosphatidylinositol-3-kinase catalytic subunit regulatory subunit 1 (Pik3r), alpha (Pik3ca) and subunit beta (Pik3cb). l, m The glucose uptake test and mRNA expression of Glut4 and Pik3cb in MC3T3-HSD1 cells with Egr2 overexpressing. l Glucose uptake test. m Glut4 and Pik3cb. n Dual-luciferase reporter analysis and ChIP quantitative polymerase chain reaction (ChIP-qPCR) analysis of Ege2 target interactions for Pik3cb and Glut4 gene promotors. n Pik3cb and Glut4 gene promotors. o ChIP-qPCR analysis. p Pik3cb and Glut4 gene promotors with different mutations. Note: Data were presented as mean value ± SEM for (a, b, e–m, p). n = 3 biologically independent samples for RNA-seq, ATAC-seq, western blot analysis, glucose uptake test and osteogenic staining; n = 6 biologically independent samples for RT-qPCR analysis. Statistical significance was calculated using two-tailed Student’s t-test (e), one-way ANOVA followed by Tukey’s post-hoc test (n, p), and two-way ANOVA followed by a two-stage step-up method by Benjamini, Krieger and Yekutieli (a, b, f–m) to adjust for multiple comparisons. All tests were two-sided.
