(-)-Epicatechin gallate (ECG) stimulates osteoblast differentiation via Runt-related transcription factor 2 (RUNX2) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated transcriptional activation

Abstract

Osteoporosis is a degenerative bone disease characterized by low bone mass and is caused by an imbalance between osteoblastic bone formation and osteoclastic bone resorption. It is known that the bioactive compounds present in green tea increase osteogenic activity and decrease the risk of fracture by improving bone mineral density. However, the detailed mechanism underlying these beneficial effects has yet to be elucidated. In this study, we investigated the osteogenic effect of (-)-epicatechin gallate (ECG), a major bioactive compound found in green tea. We found that ECG effectively stimulates osteoblast differentiation, indicated by the increased expression of osteoblastic marker genes. Up-regulation of osteoblast marker genes is mediated by increased expression and interaction of the transcriptional coactivator with PDZ-binding motif (TAZ) and Runt-related transcription factor 2 (RUNX2). ECG facilitates nuclear localization of TAZ through PP1A. PP1A is essential for osteoblast differentiation because inhibition of PP1A activity was shown to suppress ECG-mediated osteogenic differentiation. Taken together, the results showed that ECG stimulates osteoblast differentiation through the activation of TAZ and RUNX2, revealing a novel mechanism for green tea-stimulated osteoblast differentiation.

Keywords: Cell Differentiation; Mesenchymal Stem Cells; Molecular Cell Biology; Osteoblasts; Pharmacology.

FIGURE 1.

ECG stimulates the differentiation of osteoblasts. A, structure of ECG. B, ECG increases alkaline phosphatase activity in a dose-dependent manner. C3H10T1/2 cells were incubated in osteogenic differentiation medium in the presence of ECG at the indicated concentration. At 6 days after differentiation, alkaline phosphatase activity was visualized by staining to determine the osteogenic potential of ECG. A blue color indicates increased alkaline phosphatase activity. C, alkaline phosphatase activity in B was analyzed at the indicated time points. DMSO was used as a vehicle. **, p < 0.05 by Student's t test. D, ECG stimulates the expression of osteoblastic marker genes. C3H10T1/2 cells were incubated in osteoblastic differentiation medium in the presence of 10 μm ECG. After 6 days of differentiation, the cells were harvested, and total RNA was obtained. Using qRT-PCR, the expression of TAZ, Runx2, and Opn was analyzed. Their relative expression was calculated after normalization to the GAPDH level. *, p < 0.01; **, p < 0.05 by Student's t test. E, C3H10T1/2 cells were treated with ECG and induced to differentiate for 6 days. Whole-cell extracts were harvested, resolved by SDS-PAGE, and analyzed for TAZ, RUNX2, and β-actin levels using immunoblotting. Error bars, S.D.

FIGURE 2.

ECG stimulates RUNX2-mediated osteoblast differentiation. A, stimulation of RUNX2-driven gene expression by ECG. 293T cells were transfected with the RUNX2 expression plasmid (0.005 μg/well) and the 6xOSE2-luciferase reporter construct (0.05 μg/well), which contains six copies of the RUNX2-binding site in the osteocalcin promoter. After 24 h of transfection, the cells were incubated with 10 μm ECG. After 24 h, cell lysates were prepared to analyze the luciferase activities. Differences in the transfection efficiency were adjusted by normalizing the firefly luciferase activity to that of Renilla luciferase. The luciferase activity was calculated and expressed as -fold induction. *, p < 0.01, t test. B, 293T cells were transfected with HA-tagged RUNX2 and/or FLAG-tagged TAZ expression plasmids and incubated with 10 μm ECG for 24 h. Whole-cell lysates (WCL) were precipitated (IP) with FLAG-M2-agarose beads. The precipitates and whole-cell lysates were analyzed by immunoblot analysis (IB) with antibodies against HA and TAZ. C, C3H10T1/2 cells were incubated in the presence or absence of 10 μm ECG, and the cell lysates were immunoprecipitated with an IgG or anti-TAZ antibody. Endogenous TAZ bound to RUNX2 was analyzed by immunoblot analysis. D, TAZ structures. TAZ WW domain, which interacts with RUNX2, is deleted in TAZ ΔWW. aa, amino acids. E, FLAG-tagged TAZ wild type (T) and TAZ ΔWW-expressing (TΔWW) C3H10T1/2 cells were prepared by retrovirus. The TAZ expression of stable cell lines was analyzed by immunoblot analysis. The expression of β-actin was analyzed as a loading control. Bp, control cells. F, ECG increases recruitment of TAZ, not TAZ ΔWW, at the endogenous osteocalcin promoter. The above cells were treated for 4 days with osteogenic differentiation medium in the presence of 10 μm ECG, and enriched DNA immunoprecipitated using anti-FLAG antibodies was analyzed for osteocalcin promoter occupancy by PCR. ECG increases TAZ wild type recruitment, not TAZ ΔWW, into the osteocalcin promoter. G, the recruited TAZ in F were quantitatively analyzed by qRT-PCR. Recovered DNAs from input DNAs were quantified by analyzing the Ct value. *, p < 0.01 by Student's t test. Error bars, S.E.

FIGURE 3.

ECG increases nuclear localization of TAZ. A, C3H10T1/2 cells were incubated with 10 μm ECG. After 24 h, the cells were fixed, and the cellular location of TAZ was analyzed by immunocytochemistry. An FITC-conjugated secondary antibody was used for the green fluorescence signal. DAPI staining indicates the nuclei of the cells. The right-hand panel shows quantitative analysis of TAZ localization. The cellular distribution of TAZ was analyzed based on whether TAZ levels were higher in the nucleus (N > C), higher in the cytoplasm (N < C) or evenly distributed between the nucleus and cytoplasm (N = C). The percentage of cells in each category was determined after observing cells in five different microscopic fields. B and C, C3H10T1/2 cells were incubated with vehicle or 10 μm ECG for 24 h, and the cell lysates were prepared and fractionated into cytosol and nuclear extracts according to the indicated methods. TAZ (B) and RUNX2 (C) expression was analyzed by immunoblot analysis. D, C3H10T1/2 cells were treated for 2 days with osteogenic differentiation medium in the absence or presence of 10 μm ECG; cell lysates were prepared, and the phosphorylation status of TAZ at serine 89 was analyzed with a phospho-specific TAZ antibody, which was prepared with TAZ phosphopeptides (CHVRSHpSSPASL) at AbFrontier (Seoul, Korea). E, quantitative analysis of total and phosphorylated TAZ. The amount of total and phosphorylated TAZ at serine 89 from three independent experiments of D was analyzed with a densitometer, and relative -fold induction is shown here. *, p < 0.01; **, p < 0.05 by Student's t test. Error bars, S.E.

FIGURE 4.

ECG stimulates TAZ expression through PP1A. A, ECG stabilizes TAZ. C3H10T1/2 cells were incubated with 20 μg/ml cycloheximide in the presence or absence of 10 μm ECG. At the indicated time points, cell lysates were prepared, and the expression of TAZ was analyzed by immunoblot analysis. B, the PP1A inhibitor, okadaic acid, inhibits ECG-induced TAZ protein expression. C3H10T1/2 cells were treated with 10 μm ECG for 24 h, and 50 ng/ml okadaic acid was added for 3 h. TAZ protein and mRNA expression levels were analyzed using immunoblot and qRT-PCR, respectively (bottom). C, PP1A depletion leads to a decreased level of TAZ. C3H10T1/2 cells were transfected with 100 μmol of scrambled control siRNA (Con), mouse PP1A siRNA 1, or mouse PP1A siRNA 2 for 24 h and subsequently incubated in 10 μm ECG containing differentiation medium for 2 days. Next, cell lysates were prepared, and PP1A and TAZ expression levels were examined by immunoblot analysis. α-Tubulin expression was analyzed as a loading control. D, PP1A depletion decreases ECG-induced osteogenic differentiation. C3H10T1/2 cells in C were differentiated into osteoblasts, and alkaline phosphatase activity was analyzed at 8 days after differentiation. E, PP1A depletion decreases ECG-induced expression of osteogenic maker genes. After 8 days of differentiation, C3H10T1/2 cells in D were harvested, and total RNA was obtained. Using qRT-PCR, the expression of PP1A, Opn, Runx2, TAZ, and Oc were analyzed. Their relative expression was calculated after normalization to the GAPDH level. *, p < 0.01 by Student's t test. Error bars, S.E.

FIGURE 5.

ECG stimulates p38 MAPK for osteoblast differentiation. A, C3H10T1/2 cells were incubated with a vehicle DMSO, 10 μm ECG, and 2 ng/ml EGF. After 30 min, the cells were lysed, and the activity of cellular ERK, p38 MAPK, and JNK was analyzed by immunoblot analysis. The activation status of the kinases was analyzed using their phospho-specific (p-) antibodies. B, in the above condition, the activity of cellular AKT and GSK3β were analyzed by immunoblot analysis using phospho-specific antibodies. Error bars, S.E.

FIGURE 6.

ECG stimulates the osteogenic differentiation of human mesenchymal stem cells. A, bone marrow-derived human mesenchymal stem cells were treated with the indicated concentrations of ECG to induce osteoblast differentiation. After 12 days of differentiation, alkaline phosphatase activity was visualized to determine the osteogenic potential of ECG. The bottom panel shows quantitative alkaline phosphatase activity. B, qRT-PCR analysis of expression of the osteoblastic marker genes DLX5, Runx2, MSX2, and TAZ using total RNA prepared from the cells in A. *, p < 0.01; **, p < 0.05, Student's t test. C, hMSCs were incubated for 6 days with osteogenic differentiation medium in the presence of 10 μm ECG, and the expression of RUNX2 and TAZ was analyzed by immunoblot analysis. Error bars, S.E.

FIGURE 7.

Experimental model. ECG increases TAZ, Runx2, and PP1A expression. Increased PP1A, a phosphatase, facilitates dephosphorylation of TAZ, which inhibits 14-3-3 binding and proteasomal degradation, and facilitates the nuclear localization of TAZ. Under the activation of Hippo signal, the signaling component, Lats1/2 kinase, and casein kinase 1 can phosphorylate TAZ at serines 306 and 309, which can be recognized by proteasome complexes, and it induces the proteolytic degradation of TAZ. The phosphorylation of TAZ at serine 89 induces its interaction with 14-3-3, a scaffold protein, and the complexes are sequestered at the cytosol. When TAZ is dephosphorylated by PP1A, it moves into the nucleus and interacts with RUNX2 and stimulates the transcription of osteoblastic marker genes.