Sal B targets TAZ to facilitate osteogenesis and reduce adipogenesis through MEK-ERK pathway

Abstract

Salvianolic acid B (Sal B), a major bioactive component of Chinese herb, was identified as a mediator for bone metabolism recently. The aim of this study is to investigate the underlying mechanisms by which Sal B regulates osteogenesis and adipogenesis. We used MC3T3-E1 and 3T3-L1 as the study model to explore the changes of cell differentiation induced by Sal B. The results indicated that Sal B at different concentrations had no obvious toxicity effects on cell proliferation during differentiation. Furthermore, Sal B facilitated osteogenesis but inhibited adipogenesis by increasing the expression of transcriptional co-activator with PDZ-binding motif (TAZ). Accordingly, TAZ knock-down offset the effects of Sal B on cell differentiation into osteoblasts or adipocytes. Notably, the Sal B induced up-expression of TAZ was blocked by U0126 (the MEK-ERK inhibitor), rather than LY294002 (the PI3K-Akt inhibitor). Moreover, Sal B increased the p-ERK/ERK ratio to regulate the TAZ expression as well as the cell differentiation. In summary, this study suggests for the first time that Sal B targets TAZ to facilitate osteogenesis and reduce adipogenesis by activating MEK-ERK signalling pathway, which provides evidence for Sal B to be used as a potential therapeutic agent for the management of bone diseases.

Keywords: MEK-ERK pathway; Salvianolic acid B; TAZ; adipogenesis; osteogenesis.

Figure 1

Sal B at different concentrations had no obvious toxicity effects on cell proliferation during osteogenesis and adipogenesis. (A) Cell viability was measured after 3 days’ introduction of Sal B at different concentrations (0.1 μmol/L, 1 μmol/L and 10 μmol/L Sal B) with osteogenic medium or (B) adipogenic cocktail. (C) Flow cytometry experiments were performed during osteogenic differentiation or (D) adipogenic differentiation. (E) The representative percentages of cells in G1, S and G2 phases of the cell cycles were shown in each individual graph of the MC3T3‐E1 cells or (F) of the 3T3‐L1 cells. Bar graphs showed the means ± SD from at least three independent experiments. (n ≥ 3) *P < 0.05 vs the control group (cells cultured in osteogenic or adipogenic medium without Sal B)

Figure 2

Sal B facilitated osteogenic differentiation and increased the TAZ expression in MC3T3‐E1 cells. (A) The relative expression of the TAZ, RUNX2 and OCN mRNA levels to GAPDH were presented in Day 3 and (B) in Day 7 after the induction of osteogenic medium in the absence or presence of Sal B at different concentrations (0.1 μmol/L, 1 μmol/L and 10 μmol/L Sal B). (C) The alizarin red staining (AR‐S) reflected that the calcium droplets in Day 14 after the treatment. (D) The relative protein levels of TAZ, RUNX2 and OCN to GAPDH were presented in Day 3, in Day 7 and in Day 14 after the treatment. (E‐G) Bar graphs showed the means ± SD of the relative protein levels of TAZ, RUNX2 and OCN to GAPDH from 3 independent experiments. (n = 3) *P < 0.05 vs the control group (cells cultured in osteogenic medium without Sal B)

Figure 3

0.1 μmol/L Sal B inhibited adipogenic differentiation by increasing TAZ expression in 3T3‐L1 cells. (A) The relative expression of the TAZ, C/EBPβ and PPARγ mRNA levels to GAPDH were presented in Day 3 and (B) in Day 7 after the induction of osteogenic medium in the absence or presence of Sal B at different concentrations (0.1 μmol/L, 1 μmol/L and 10 μmol/L Sal B). (C) The oil red o staining reflected the lipid droplets in Day 14 after the treatment. (D) The relative protein levels of TAZ, C/EBPβ and PPARγ were presented in Day 3, in Day 7 and in Day 14 after the treatment. (E‐G) Bar graphs showed the means ± SD of the relative protein levels of TAZ, C/EBPβ and PPARγ to GAPDH from three independent experiments. (n = 3) *P < 0.05 vs the control group (cells cultured in adipogenic medium without Sal B)

Figure 4

TAZ knock‐down offset the Sal B induced osteogenesis in MC3T3‐E1 cells. (A‐C) Realtime RT‐PCR and Western blotting analysis suggested that the TAZ expression were significantly knocked down by the SiTAZ plasmid compared with the negative control and non‐transfected cells. (D‐H) TAZ knock‐down offset the Sal B induced up‐expression of RUNX2 and OCN in Day 3 after the introduction of osteogenic medium. (I, J) The quantification AR‐S results showed that TAZ knock‐down reduced the Sal B induced calcium droplets. Bar graphs showed the means ± SD from three independent experiments. (n = 3) ^# P < 0.05 vs the non‐transfection group; *P < 0.05 vs CON36, the negative control group; ^## P < 0.05 vs SiTAZ + Sal B group

Figure 5

TAZ knock‐down attenuated the Sal B inhibited adipogenesis in 3T3‐L1 cells. (A‐C) Real‐time RT‐PCR and Western blotting analysis suggested that the TAZ expression were significantly knocked down by the SiTAZ plasmid compared with the negative control and non‐transfected cells. (D‐H) TAZ knock‐down offset the Sal B decreased expression of C/EBPβ and PPARγ in Day 3 after the introduction of adipogenic medium. (I, J) The oil red o staining results showed that TAZ knock‐down attenuated the Sal B decreased lipid droplets. Bar graphs showed the means ± SD from three independent experiments. (n = 3) ^# P < 0.05 vs the non‐transfection group; *P < 0.05 vs CON36, the negative control group; ^## P < 0.05 vs SiTAZ + Sal B group

Figure 6

Sal B induced TAZ up‐expression was mediated by the MEK‐ERK signalling pathway during osteogenic differentiation in MC3T3‐E1 cells. (A) Real‐time RT‐PCR and (B) Western blotting analysis for the relative TAZ expression in Day 3 after the treatment. (C) Bar graphs showed the means ± SD from three independent experiments of the relative TAZ protein levels to GAPDH. (D, E) Western blotting showed that Sal B significantly lifted the p‐ERK/ERK ratio and U0126 blocked this regulation in Day 3 after the treatment. (F, G) The AR‐S quantification confirmed the involvement of MEK‐ERK signalling pathway in the facilitation of the osteogenic differentiation induced by Sal B in Day 14. (n = 3) *P < 0.05 vs the control group (cells cultured in osteogenic medium); ^# P < 0.05 vs the Sal B administration group

Figure 7

Sal B induced TAZ up‐expression was mediated by the MEK‐ERK signalling pathway during adipogenic differentiation in 3T3‐L1 cells. (A) Real‐time RT‐PCR and (B) Western blotting analysis for the relative TAZ expression in Day 3 after the treatment. (C) Bar graphs showed the means ± SD from three independent experiments of the relative TAZ protein levels to GAPDH. (D, E) Western blotting showed that Sal B significantly lifted the p‐ERK/ERK ratio and U0126 could blocked this regulation in Day 3 after the treatment. (F, G) The oil red o staining quantification confirmed the involvement of MEK‐ERK signalling pathway in the inhibition of the adipogenic differentiation induced by Sal B in Day 14. (n = 3) *P < 0.05 vs the control group (cells cultured in adipogenic medium); ^# P < 0.05 vs the Sal B administration group