Azoramide, a novel regulator, favors adipogenesis against osteogenesis through inhibiting the GLP-1 receptor-PKA-β-catenin pathway

Abstract

The reciprocal fate decision of mesenchymal stem cells (MSCs) to either bone or adipocytes is determined by Wnt-related signaling and the glucagon-like peptide-1 receptor (GLP-1R). Azoramide, an ER stress alleviator, was reported to have an antidiabetic effect. In this study, we investigated the function of azoramide in regulating the lineage determination of MSCs for either adipogenic or osteogenic differentiation. Microcomputed tomography and histological analysis on bone morphogenetic protein (BMP)2-induced parietal periosteum bone formation assays, C3H10T1/2 and mouse bone marrow MSC-derived bone formation and adipogenesis assays, and specific staining for bone tissue and lipid droplets were used to evaluate the role of azoramide on the lineage determination of MSC differentiation. Cells were harvested for Western blot and quantitative real-time polymerase chain reaction (PCR), and immunofluorescence staining was used to explore the potential mechanism of azoramide for regulating MSC differentiation. Based on MSC-derived bone formation assays both in vivo and in vitro, azoramide treatment displayed a cell fate determining ability in favor of adipogenesis over osteogenesis. Further mechanistic characterizations disclosed that both the GLP-1R agonist peptide exendin-4 (Ex-4) and GLP-1R small interfering (si)RNA abrogated azoramide dual effects. Moreover, cAMP-protein kinase A (PKA)-mediated nuclear β-catenin activity was responsible for the negative function of azoramide on bone formation in favor of adipogenesis. These data provide the first evidence to show that azoramide may serve as an inhibitor against GLP-1R in MSC lineage determination.

Keywords: Adipogenesis; Azoramide; Glucagon-like peptide-1 receptor; MSC; Osteogenesis; Wnt.

Fig. 1

Reduced BMP2-induced bone formation in azoramide-treated mice. A Schematic of a bone morphogenetic protein (BMP)2 calvarial injection model. B Micro-CT 3D reconstruction and cross-sectional image of control (Con) and Azoramide-treated (Azo) calvarium (red arrows) following treatment with BMP2 (n = 6 biologically independent samples in each group). C micro-CT quantification of newly formed bone in response to BMP2. D Representative hematoxylin and eosin staining to count osteoblasts, and toluidine blue staining to visualize adipocytes (yellow arrows) of BMP-induced newly formed bone from control (Con) and Azoramide-treated (Azo) mice. Scale bar = 100 μm. E Histomorphometric quantification of osteoblast numbers (N.Ob) per bone perimeter (B.Pm) and adipocyte numbers. N = 3 biologically independent experiments; *p < 0.05, **p < 0.01, versus control mice. i.p. intraperitoneal

Fig. 2

Inhibited osteogenic differentiation potential of C3H10T1/2 cells with azoramide treatment in vitro. A, B Alkaline phosphatase (ALP) staining and quantification of ALP activity of cellular extracts subjected to different concentrations of azoramide (Azo). Scale bar = 100 μm. C, D Alizarin red S staining for mineral deposition and its quantification with different concentrations of azoramide. Scale bar = 100 μm. Images are representatives of three independent experiments with biological duplicates. E Real-time qPCR of Runt-related transcription factor 2 (Runx2), Sp7, integrin binding sialoprotein (Ibsp), and bone gamma-carboxyglutamate protein (Bglap) mRNA in C3H10T1/2 cells subjected to 15 μM azoramide. f Western blot of Runx2 protein in C3H10T1/2 cells subjected to 15 μM azoramide. n = 3 biologically independent experiments. *p < 0.05, **p < 0.01, azoramide versus vehicle

Fig. 3

Enhanced adipogenic differentiation potential of C3H10T1/2 cells and mouse-derived MSCs with azoramide treatment in vitro. A, B Oil red O staining for lipid production and its quantification with different concentrations of azoramide (Azo). Scale bar = 100 μm. Images are representatives of three independent experiments with biological duplicates. C Real-time qPCR of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid binding protein 4 (Fabp4), and adiponectin (Adipoq) mRNA in C3H10T1/2 cells subjected to 15 μM azoramide. D Western blot of PPARγ and Fabp4 proteins in C3H10T1/2 cells subjected to 15 μM azoramide. Scale bar = 100 μm. *p < 0.05, **p < 0.01, azoramide versus vehicle. n = 3 biologically independent experiments. Bglap bone gamma-carboxyglutamate protein, Ibsp integrin binding sialoprotein, Runx2 Runt-related transcription factor 2

Fig. 4

Ex-4 treatment attenuated azoramide effects on suppressing C3H10T1/2 cell osteoblast differentiation and promoting their differentiation into adipocytes. A Immunofluroscence of GLP-1R and ERp57 in C3H10T1/2 cells after azoramide treatment. B–D Real-time qPCR of Runt-related transcription factor 2 (Runx2), peroxisome proliferator-activated receptor gamma (PPARγ), and fatty acid binding protein 4 (Fabp4) mRNA in C3H10T1/2 cells subjected to different treatments. E–G Western blot of Runx2, PPARγ, and Fabp4 proteins in C3H10T1/2 cells subjected to different treatments. Bars represent the protein quantitative data normalized to β-actin. H, I Alizarin red S for mineral deposition and its quantification. Scale bar = 100 μm. J, K Oil red O staining for lipid production and its quantification. Scale bar = 100 μm. C3H10T1/2 cells were treated with vehicle (Con), 15 μM azoramide alone (Azo), 10 nM exendin-4 alone (Ex-4), and pretreated with azoramide followed by Ex-4 (Azo + Ex-4). Images are representatives of three independent experiments with biological duplicates. *p < 0.05, **p < 0.01, versus Con; #p < 0.05, ##p < 0.01, versus Ex-4

Fig. 5

GLP-1R silencing abolished the azoramide (Azo) regulatory effects of suppressing C3H10T1/2 cell osteoblast differentiation and promoting their differentiation into adipocytes. A–C Alkaline phosphatase (ALP) staining and quantification subject to azoramide with or without glucagonlike peptide-1 receptor (GLP-1R) knock-down. Scale bar = 100 μm. D–F Real-time qPCR of Runt-related transcription factor 2 (Runx2), peroxisome proliferator-activated receptor gamma (PPARγ), and fatty acid binding protein 4 (Fabp4) mRNA in C3H10T1/2 cells subjected to different treatments. G–I Western blot of Runx2, PPARγ, and Fabp4 proteins in C3H10T1/2 cells subjected to different treatments. Bars represent the protein quantitative data normalized to β-actin. n = 3 biologically independent experiments. *p < 0.05, **p < 0.01, versus control (Con); #p < 0.05, ##p < 0.01, versus Azo. si small interfering. Images are representatives of three independent experiments with biological duplicates

Fig. 6

Decreased expression levels of protein kinase A (PKA) with azoramide (Azo) treatment. A, B Western blot of Runt-related transcription factor 2 (Runx2) and PKAc proteins in C3H10T1/2 cells subjected to different treatments. C Alkaline phosphatase (ALP) staining of cells pretreated with azoramide followed by Forskolin treatment. Scale bar = 100 μm. Images are representatives of three independent experiments with biological duplicates

Fig. 7

Azoramide inhibited β-catenin nuclear translocation. A Azoramide (Azo) treatment inhibited β-catenin nuclear localization. C3H10T1/2 cells were pretreated with osteogenic differentiation medium for 12 h followed by azoramide treatment for 6 h and cells were then fixed for immunofluorescence with anti-β-catenin antibody. Scale bar = 20 μm. B Western blot of phosphorylated β-catenin in C3H10T1/2 cells stimulated by 15 μM azoramide at the indicated times. C Western blot of phosphorylated β-catenin in C3H10T1/2 cells subjected to different treatments. D Western blot of Runt-related transcription factor 2 (Runx2) and peroxisome proliferator-activated receptor gamma (PPARγ) proteins in C3H10T1/2 cells treated with azoramide and Wnt agonist 1, a small-molecule agonist of the Wnt/β-catenin signaling pathway. E Alizarin red S staining for mineral deposition of cells subjected to different treatments. F Relative level of TCF7L2 mRNA in C3H10T1/2 cells subjected to different treatments. Cells were cultured in osteogenic differentiation medium. n = 3 biologically independent experiments. Images are representatives of three independent experiments with biological duplicates. *p < 0.05, **p < 0.01, versus control (Con); #p < 0.05, versus Azo. glp-1r glucagon-like peptide-1 receptor, si small interfering