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.

substances. 10 The structure of Sal B is shown in Figure S1 which consists of three molecules of Tanshinol and one molecule of caffeic acid. Its molecular formula is C36H30O16. Studies have shown that Sal B exerted neuro-protective effects and could alleviate liver fibrosis. 10,11 Recently, increasing attentions have been directed to the effects of Sal B on bone metabolism. 13,14 He et al 15 reported that Sal B promoted bone formation by increasing activity of alkaline phosphatase (ALP) in a rat tibia fracture model. However, the underlying mechanisms by which Sal B improves bone remodeling have not been well established.
TAZ, a transcriptional modulator, is one of such regulators that have key roles in cell proliferation, differentiation and function. 16,17 As reported, TAZ could interact with kinds of transcription factors to activate or repress specific gene expression, which might influence cell functions. 19, 20 Byun et al 19 have discovered that the Phorbaketal A increased the TAZ expression to promote osteogenic differentiation as well as inhibit adipogenic differentiation. Our team has also reported several researches focusing on TAZ induced osteogenic differentiation. 21 Thus, it attracted our interest that whether TAZ plays an important role in the Sal B-regulated cell differentiation.
In addition to the changes of the endogenous factors which are controlling the cell lineage-specific differentiation, this study indicated that Sal B administration altered the differential balance between osteogenesis and adipogenesis. Note that Sal B increased TAZ expression and facilitated the osteoblastogenesis at the expense of the reduced adipogenesis. We also pointed to a close link of MEK-ERK signalling transduction to the TAZ transcriptional networks during osteoblasts and adipocytes maturation. Taken together, this study further provides evidence for Sal B to be used as a potential therapeutic agent for the management of bone diseases clinically.

| Sal B administration
When the cells reached approximately 80% confluence, they were sub-cultured into a new culture flask and the growth medium were replaced by differential medium in the presence or absence of Sal B (0.1 mmol/L, 1 mmol/L, or 10 mmol/L). Then, we selected the best acting concentration of Sal B for subsequent experiments based on the expression of differential markers analysed by the real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) analysis, Western blotting analysis and staining after the induction of the differential medium, considering the toxicity effects of Sal B at different concentration on cell proliferation.

| Cell viability assay
Cells were seeded at a density of 5 × 10 4 cells/100 μL/well in 96well plates and incubated for cell viability assay. After 3 days' treatment, 20 μL of freshly prepared MTT (5 mg/mL; Solarbio) was added and the plates were incubated at 37°C for another 4 hours to form crystals. Then, 150 μL of dimethyl sulfoxide (DMSO) was added for 10 minutes to fully dissolve the crystals. Finally, the absorbance of each well was measured at a wavelength of 490 nm using the microplate spectrophotometer (BioTek Instruments, San Jose, CA).

| Cell cycle analysis
To identify the effect of Sal B on cell cycles, 3 days treated cells were harvested and fixed with 70% ethanol. After washed with PBS, cells were stained with propidium iodide (Sigma, USA) (5 mg/mL) for 30 minutes in the dark at 4°C. Fluorescence was measured with the flow cytometer equipped with a 570 nm argon ion laser (Epics XL, Beckman Coulter Corporation, FL) and the data were analysed using the Muticycle AV software.

| Plasmids transfection
The plasmid containing small interfering RNA (SiRNA) sequences against TAZ (SiTAZ) expression were designed and synthesized by Shanghai Genechem Corporation (Shanghai, China). Relatively

| Real-time reverse transcription-polymerase chain reaction
Total RNA was extracted using TRIzol ® reagent (Ambition). Total

| Inhibitor study
Ten micromoles per litre MEK-ERK inhibitor U0126 (Beyotime Institute of Biotechnology, China) and PI3K-Akt inhibitor LY294002 (Beyotime Institute of Biotechnology, China) were added simultaneously into differential medium after 24 hours' cell attachment.
Then the expression of TAZ was detected by real-time RT-PCR and Western blotting analyses.

| Statistics
Quantitative results were expressed as mean ± standard deviation (SD). All experiments were replicated at least three times.

| Sal B had no obvious toxicity effects on cell proliferation during differentiation
Initially, we tested whether or not Sal B at different concentrations

| Sal B inhibited adipogenic differentiation by increasing TAZ expression
As

| TAZ knock-down offset the Sal B induced osteogenesis
Transfected with plasmids containing SiTAZ sequences, the TAZ expression was knocked down in MC3T3-E1. GFP + cells were also observed with fluorescence microscopy ( Figure S2A). Transfection efficiency of the SiTAZ plasmid and its negative control plasmid (CON36) was high enough to be comparable among groups  Figure 4I,J).

| TAZ knock-down attenuated the Sal B inhibited adipogenesis
In order to confirm that TAZ was involeved in Sal B inhibited adi-   Figure 6A,B). Moreover, the Western blotting analysis in Day 3 after the treatment revealed that Sal B increased the p-ERK/ERK ratio to mediate the activation of MEK-ERK signalling pathway during osteoblastogenesis ( Figure 6D,E). Additionally, the AR-S quantification also confirmed that U0126 offset the facilitation of the calcium droplets induced by Sal B after 14 days' osteogenesis ( Figure 6F,G). In 3T3-L1 cells, the results were consistent with that in MC3T3-E1 cells ( Figure 7). In summary, we speculated that MEK-ERK signalling pathway was involved in the regulation of the TAZ up-expression induced with kinds of transcription factors to influence the stem cells fate determination. 37 As reported, TAZ binds strongly to the Pro-Pro-X-Tyr motif found within regulatory regions of RUNX2 and PPARγ. 10,21,38 It interacted with RUNX2 and co-activates RUNX2-dependent gene transcription, while interacting with PPARγ and repressing its downstream target gene expression. 38 With the progressive aging in general population, bone loss becomes a growing public problem. 46 Pressing requirements for the treatment of bone repair is to identify anabolic agents that can increase bone formation as well as decrease fat accumulation. This study confirms the positive role of Sal B in the facilitation of osteogenesis at the expense of the reduced adipogenesis by increasing TAZ expression, pointed a molecular link of MEK-ERK pathway to the TAZ-related switch of cells differentiation after Sal B administration, and provided evidence to use Sal B as a potential therapeutic agent for the management of bone repair clinically.

CO N FLI C T O F I NTE R E S T S
The authors confirm that there is no conflict of interests.