BMP8B Activates Both SMAD2/3 and NF-κB Signals to Inhibit the Differentiation of 3T3-L1 Preadipocytes into Mature Adipocytes

Abstract

Bone morphogenetic protein 8B (BMP8B) has been found to regulate the thermogenesis of brown adipose tissue (BAT) and the browning process of white adipose tissue (WAT). However, there is no available information regarding the role of BMP8B in the process of adipocyte differentiation. Here, we showed that BMP8B down-regulates transcriptional regulators PPARγ and C/EBPα, thereby impeding the differentiation of 3T3-L1 preadipocytes into fully mature adipocytes. BMP8B increased the phosphorylation levels of SMAD2/3, and TP0427736 HCl (SMAD2/3 inhibitor) significantly reduced the ability of BMP8B to inhibit adipocyte differentiation, suggesting that BMP8B repressed adipocyte differentiation through the SMAD2/3 pathway. Moreover, the knockdown of BMP I receptor ALK4 significantly reduced the inhibitory effect of BMP8B on adipogenesis, indicating that BMP8B triggers SMAD2/3 signaling to suppress adipogenesis via ALK4. In addition, BMP8B activated the NF-κB signal, which has been demonstrated to impede PPARγ expression. Collectively, our data demonstrated that BMP8B activates both SMAD2/3 and NF-κB signals to inhibit adipocyte differentiation. We provide previously unidentified insight into BMP8B-mediated adipogenesis.

Keywords: 3T3-L1; BMP8B; NF-κB; SMAD2/3; adipocyte differentiation.

Figure 1

Overexpressing BMP8B inhibits adipocyte differentiation. (A) 3T3-L1 preadipocyte differentiation protocol. (B) The expression pattern of Bmp8b during adipogenesis. (C,D) Effective increased and inhibited Bmp8b expression in 3T3-L1 adipocytes. (E) On Day 8, Oil Red O staining revealed lipid contents in cells (Mock, LV-ZsGreen1, and LV-Bmp8b). (F). Quantification of lipid content in cells after adipogenic differentiation (Day 8). (G–J) qRT-PCR analyses of the expression of adipogenic marker genes (Cebpα, Pparγ, and Fasn) on Day 0 (G), Day 2 (H), Day 4 (I), and Day 8 (J). (K–M) Protein levels of PPARγ (L) and C/EBPα (M) detected through immunoblotting. β-actin was used as a loading control. ImageJ software was used to quantify protein levels. Scale bar = 20 µm. The symbols in the charts represent three biological replicates. The data were presented as mean ± SD and analyzed using one-way ANOVA (ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Knockdown BMP8B promotes adipogenesis. (A) On Day 8, Oil Red O staining revealed lipid contents (Mock, LV-shRNA-scrambled, and LV-shRNA-Bmp8b#1). (B) Quantification of lipid content in cells after adipogenic differentiation (Day 8). (C–F) qRT-PCR analyses of the expression of adipogenic marker genes (Cebpα, Pparγ, and Fasn) on Day 0 (C), Day 2 (D), Day 4 (E), and Day 8 (F). (G–I) On the day after induction, the protein levels of PPARγ (H) and C/EBPα (I) were detected through immunoblotting. Scale bar = 20 µm. The symbols in the charts represent three biological replicates. The data were presented as mean ± SD and analyzed using one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

BMP8B triggers SMAD2/3 signaling to suppress adipogenesis. (A,B) Analysis using immunoblotting and quantification was conducted to assess the protein levels of p-SMAD1/5/8, p-SMAD2/3, p-ERK1/2, p-p38 MAPK, and p-JNK in LV-Bmp8b. (C) A model of BMPs-associated signal transduction. (D) Quantification was performed to determine the luciferase reporter activity driven by BRE, which pCMV-Bmp8b cotransfected with pCMV-Alk2, pCMV-Alk3, pCMV-Bmpr2, pCMV-Acrv2a, respectively. (E) Quantification was performed to determine the luciferase reporter activity driven by CAGA, which pCMV-Bmp8b cotransfected with pCMV-Alk2, pCMV-Alk4, pCMV-Alk5, pCMV-Alk7, pCMV-Tgfβr2, pCMV-Acrv2a, and pCMV-Acrv2b, respectively. (F,G) In the presence of DMH1 or TP0427736 HCL, the cells were induced to differentiate into adipocytes. On Day 8, Oil Red O staining was performed (F). Quantification of lipid content after adipogenic differentiation (G). Scale bar = 20 µm. The symbols in the charts represent three biological replicates. The data were presented as mean ± SD and analyzed using one-way ANOVA (ns not significant, ** p < 0.01, *** p < 0.001).

Figure 4

BMP8B triggers SMAD2/3 signaling to suppress adipose differentiation via ALK4. (A) Schematic representation of wild-type and GS motif mutants. (B,C) Western blots and quantification of p-SMAD1/5/8 in Mock, LV-ZsGreen1, LV-Bmp8b, and LV-Bmp8b +Alk3-ΔGS cells. (D,E) Western blots and quantification of p-SMAD2/3 in Mock, LV-ZsGreen1, LV-Bmp8b, and LV-Bmp8b + Alk4-ΔGS cells. (F,G) Western blots and quantification of p-SMAD2/3 in Mock, LV-ZsGreen1, LV-Bmp8b, and LV-Bmp8b + Alk5-ΔGS cells. (H,I) Knock down of ALK3, ALK4, and ALK5 in LV-Bmp8b. On Day 8, cells were stained with Oil Red O for quantification. Scale bar = 20 µm. The symbols in the charts represent three biological replicates. The data were presented as mean ± SD and analyzed using one-way ANOVA (ns not significant, *** p < 0.001).

Figure 5

BMP8B represses PPARγ transcription via SMAD2/3 signaling, resulting in a block to adipocyte differentiation. (A) After induction of adipogenic differentiation, the KEGG pathway was downregulated in LV-Bmp8b vs LV-ZsGreen1 3T3-L1 cells. (B) The KEGG mapper showed downregulated genes (rectangles highlighted in green) in the PPAR signaling pathway. (C) The PPARγ promoter region is shown in a schematic representation. Three binding sites and sequences of SMAD2/3 TF are predicted. (D) Wild-type and mutation plasmids of predicted SMAD2/3 TF binding sites are shown in a schematic drawing. (E) Quantification the activity of pGL3-Pparγ-promoter and mutation promoter plasmids with pCMV-Bmp8b or vectors in HEK293T. Renilla was used as the internal control. The symbols in the charts represent three biological replicates. The data were presented as mean ± SD and analyzed using one-way ANOVA (ns not significant, *** p < 0.001).

Figure 6

BMP8B triggers NF-κB signaling to suppress adipogenesis. (A) The upregulated KEGG pathway in LV-Bmp8b vs LV-ZsGreen1. (B,C) Western blots and quantification of p-IKKα/β and p-p65 in Mock, LV-ZsGreen1, and LV-Bmp8b. (D,E) Representative photographs of Oil Red O staining were taken to visualize lipids in LV-Bmp8b exposed to JSH-23 with DMSO as a vehicle. The staining intensity was quantified by measuring the optical density at OD₄₉₂. Scale bar = 20 µm. The symbols in the charts represent three biological replicates. Mean ± SD was used to present the data, which were analyzed using one-way ANOVA (** p < 0.01, *** p < 0.001).

Figure 7

Schematic illustration of BMP8B’s role in modulating adipocyte differentiation. BMP8B binds to the ALK4 to activate SMAD2/3 signaling to suppress the expression of PPARγ to inhibit adipogenesis. Additionally, BMP8B can trigger the NF-κB signal to decrease the activation of PPARγ to inhibit adipogenesis.