Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.
Abstract
Osteoblasts are derived from mesenchymal progenitors. Differentiation to osteoblasts and adipocytes is reciprocally regulated. Transcriptional coactivator with a PDZ-binding motif (TAZ) is a transcriptional coactivator that induces differentiation of mesenchymal cells into osteoblasts while blocking differentiation into adipocytes. To investigate the role of TAZ on bone metabolism in vivo, we generated transgenic mice that overexpress TAZ under the control of the procollagen type 1 promoter (Col1-TAZ). Whole body bone mineral density (BMD) of 6- to 19-week-old Col-TAZ mice was 4% to 7% higher than that of their wild-type (WT) littermates, whereas no difference was noticed in Col.1-TAZ female mice. Microcomputed tomography analyses of proximal tibiae at 16 weeks of age demonstrated a significant increase in trabecular bone volume (26.7%) and trabecular number (26.6%) with a reciprocal decrease in trabecular spacing (14.2%) in Col1-TAZ mice compared with their WT littermates. In addition, dynamic histomorphometric analysis of the lumbar spine revealed increased mineral apposition rate (42.8%) and the serum P1NP level was also significantly increased (53%) in Col.1-TAZ mice. When primary calvaria cells were cultured in osteogenic medium, alkaline phosphatase (ALP) activity was significantly increased and adipogenesis was significantly suppressed in Col1-TAZ mice compared with their WT littermates. Quantitative real-time polymerase chain reaction analyses showed that expression of collagen type 1, bone sialoprotein, osteocalcin, ALP, osterix, and Runx2 was significantly increased in calvaria cells from Col1-TAZ mice compared to their WT littermates. In vitro, TAZ enhanced Runx2-mediated transcriptional activity while suppressing the peroxisome proliferator-activated receptor gamma signaling pathway. TAZ also enhanced transcriptional activity from 3TP-Lux, which reflects transforming growth factor-beta (TGF-β)-mediated signaling. In addition, TAZ enhanced TGF-β-dependent nuclear translocation of Smad2/3 and Smad4. Taken together, these results suggest that TAZ positively regulates bone formation in vivo, which seems to be mediated by enhancing both Runx2 and TGF-β signaling.
Figures
Figure 1 Development of Col.1-TAZ transgenic mice. (A) Linearized structure of the Col.1–TAZ construct used for transgenic expression. The coding sequence of mouse TAZ was placed under the pro-α1 (I) collagen promoter. The 350 bp EcoRI-BamHI fragment was used as a probe in Southern blot analysis. The two-way arrows indicate the primers used for PCR genotyping. Both DNA probe and the 243 bp PCR product encompass the junction between the Flag tag and the 5′end of the TAZ reading frame. (B) Southern blot of the genomic DNA of a litter showing a 1.5 kb band product corresponding to the transgene. (C) Total protein extracts isolated from calvariae of either Col.1–TAZ mice or the WT littermate were separated by SDS-PAGE and blotted (Western blot) using an anti-Flag or anti-TAZ antibody. A band of 50 kDa, corresponding to the transgene, was detected by an anti-Flag antibody in the extracts from Col.1–TAZ mice but not from the WT littermate. Expression of the transgene was further confirmed by increased expression levels of TAZ in Col.1–TAZ mice detected by the TAZ antibody compared to the WT littermate.Figure 2 Body weight and bone phenotype in Col.1–TAZ mice. Total body BMD (A) was analyzed by dual energy X-ray absorptiometry (PixiMus). Body weight (B) was measured in male Col.1– TAZ mice and their WT littermates at 6, 10, 14, and 19 weeks (* p<0.05 vs. WT). (C) Representative μCT images of the proximal tibia from a 20-week-old male Col.1–TAZ mouse and its WT littermate. Static morphometric parameters including trabecular bone volume expressed as percentage of total tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp) were measured with the CT images. (D) Representative μCT images of the mid-femur from a 20-week-old male Col.1–TAZ mouse and its WT littermate. Cortical histomorphometric parameters were analyzed for cortical bone area (Ct.Ar) and average cortical thickness (Ct.Th) (* p<0.05 vs. WT littermates). Data are expressed as means ± SD.Figure 3 Histological analyses of male Col.1–TAZ mice and WT littermates at 20 weeks of age. (A) Representative von Kossa staining images of an undecalcified section of the lumbar spine (scale bar: 100 µm) and histomorphometric measurements for BV/TV and cortical shell thickness (Ct. Shell Th.). (B) Fluorescent images obtained from lumbar spine (scale bar: 100 µm) after calcein double labeling. Dynamic histomorphometric analyses of lumbar spine including bone formation rate (BFR) and mineral apposition rate (MAR). Serum levels of P1NP, a bone formation marker, at 8 weeks of age. (C) Representative TRAP staining images of a decalcified bone section from the distal femur (scale bar: 50 µm). Histomorphometric analyses of TRAP-stained bone sections including osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS). Data are expressed as means ± SD (* p<0.05 vs. WT littermates).Figure 4 TAZ overexpression enhances osteoblast differentiation but does not affect osteoclast formation in vitro. (A) Calvarial cells from Col.1–TAZ mice or their wild-type littermates (WT) were cultured for 6 days in osteogenic medium (OM) including ascorbic acid and β-glycerophosphate. ALP activity was measured in the cell layer and normalized to cellular protein content. Data are expressed as means ± SD (* p<0.05 vs. WT) (B) The same sets of cells were cultured for 10 days in adipogenic medium containing 10 µg/ml insulin, 0.1 µM dexamethasone, and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX), and stained with Oil Red O. (C) Total RNA was extracted from calvarial cells grown for 7 days in OM. The amount of mRNA for osteoblast marker gene was determined by real-time quantitative PCR and expressed as mRNA abundance in Col.1–TAZ mice relative to their WT littermates. (D) Total RNA was extracted from calvarial cells grown for 10 days in adipogenic medium. The amount of mRNA for adipogenic markers was determined by real-time quantitative PCR and expressed as mRNA abundance in Col.1–TAZ mice relative to their WT littermates. Data are expressed as means ± SD. * p<0.05 vs. WT (E) Quantitative RT-PCR analysis of RANKL and OPG gene expression and the ratio of RANKL∶OPG gene expression in calvarial cells. Data are expressed as means ± SD. (F) Representative TRAP-stained osteoclasts and the number of TRAP-positive multinucleated cells after 4∼5-day cultures of bone marrow macrophages (BMM) isolated from 6∼8-week old Col.1–TAZ mice and WT littermates in the presence of RANKL and M-CSF. Scale bar = 500 µm.Figure 5 Regulation of signaling pathways by TAZ in vitro. (A) C3H10T1/2 cells were transiently cotransfected with the p6OSE2-Luc plasmid and expression vector for TAZ (pEF-Flag-TAZ) or with the empty vector (pEF-BOS). The cells were also transfected with the expression vector for til (pcDNA3-til) or empty vector as indicated. (B) C3H10T1/2 cells transiently cotransfected with PPRE-Luc and the expression vector for TAZ (pEF-Flag-TAZ) or with the empty vector (pEF-BOS). The cells were also transfected the expression vector for PPARγ (pSPORT-PPARγ) or empty vector as indicated and were exposed to either the vehicle or 1 µM rosiglitazone. Forty-eight hours after transfection, the cells were harvested and used for luminometry. Luciferase activities were normalized for transfection efficiency using renilla activity and all values were expressed as a fold induction relative to basal promoter activity. The results shown are representative of three independent experiments. (C) Primary calvarial cells from Col.1-TAZ (T) or WT (W) mice were treated with 150 ng/ml rhBMP-2 for 2 days and harvested for chromatin immunoprecipitation assays. Soluble chromatins were prepared and precipitated using control IgG or anti-Flag antibodies. The Runx2 binding site in the mouse osteocalcin promoter was amplified by PCR. (D–F) C3H10T1/2 cells were transiently cotransfected with BRE-Luc, p3TP-Lux, and TOPflash and with the expression vector for TAZ (pEF-Flag-TAZ) or an empty vector (pEF-BOS). Cells were exposed to the vehicle or BMP-2, TGF-β, or Wnt-3A CM for BRE-Luc, p3TP-Lux, and TOPflash, respectively. Luciferase activity was measured as described above. Data are expressed as means ± S.D., and the graphical results are representative of three independent experiments, each performed in triplicate (*p<0.05 vs. control).Figure 6 TAZ enhances TGF-β-mediated Smad nuclear accumulation. (A) The UAMS-32 cells were transfected with pEF-BOS (BOS) or pEF-Flag-TAZ (TAZ). Endogenous Smad2/3 and Smad4 localization was determined following treatment with the vehicle or TGF-β. (B) Sub-cellular localization of Smad2/3 and Smad4. UAMS-32 cells were transiently transfected with pEF-BOS or pEF-Flag-TAZ, fractionated into nuclear and cytosolic fractions, and analyzed by immunoblotting for Smad2/3, Smad4, and Flag. The purity of nuclear and cytosolic fractions was confirmed by immunoblotting for Calnexin. (C) C3H10T1/2 cells transfected with pEF-BOS or pEF-Flag-TAZ were cultured for 8 days in osteogenic medium (OM) including ascorbic acid and β-glycerophosphate in the absence or presence of TGF-β inhibitors (SB-431542 or SB-525334). ALP activity was measured in the cell layer and normalized to cellular protein content. Data are expressed as means ± SD (* p<0.05 vs. pEF-BOS, # p<0.01 vs. vehicle) (D) Quantitative RT-PCR analysis of Col 1 and ALP in C3H10T1/2 cells (* p<0.05 vs. pEF-BOS, # p<0.01 vs. vehicle).
그림 출처: PubMed Central 공개 논문 (PMC3575506)
Conflict of interest statement
Competing Interests:The authors have declared that no competing interests exist.
