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. 2012 Nov;27(11):2344-58.
doi: 10.1002/jbmr.1694.

Loss of wnt/β-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes

Lige Song  1 , Minlin Liu, Noriaki Ono, F Richard Bringhurst, Henry M Kronenberg, Jun Guo
Affiliations

Loss of wnt/β-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes

Lige Song et al. J Bone Miner Res. 2012 Nov.

Abstract

Wnt signaling is essential for osteogenesis and also functions as an adipogenic switch, but it is not known if interrupting wnt signaling via knockout of β-catenin from osteoblasts would cause bone marrow adiposity. Here, we determined whether postnatal deletion of β-catenin in preosteoblasts, through conditional cre expression driven by the osterix promoter, causes bone marrow adiposity. Postnatal disruption of β-catenin in the preosteoblasts led to extensive bone marrow adiposity and low bone mass in adult mice. In cultured bone marrow-derived cells isolated from the knockout mice, adipogenic differentiation was dramatically increased, whereas osteogenic differentiation was significantly decreased. As myoblasts, in the absence of wnt/β-catenin signaling, can be reprogrammed into the adipocyte lineage, we sought to determine whether the increased adipogenesis we observed partly resulted from a cell-fate shift of preosteoblasts that had to express osterix (lineage-committed early osteoblasts), from the osteoblastic to the adipocyte lineage. Using lineage tracing both in vivo and in vitro we showed that the loss of β-catenin from preosteoblasts caused a cell-fate shift of these cells from osteoblasts to adipocytes, a shift that may at least partly contribute to the bone marrow adiposity and low bone mass in the knockout mice. These novel findings indicate that wnt/β-catenin signaling exerts control over the fate of lineage-committed early osteoblasts, with respect to their differentiation into osteoblastic versus adipocytic populations in bone, and thus offers potential insight into the origin of bone marrow adiposity.

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Conflict of interest statement

Disclosure

All authors state that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1. Progressively increased bone marrow fat in adult mice following postnatal knockout of β-catenin through the osterix promoter
(A–B) H&E staining of paraffin sections from proximal tibiae of 6-month old control (β-catf/f) and knockout (osx-cre;β-catf/f) mice treated with doxycycline (Dox, 1.5 mg/ml supplemented in drinking water) prenatally and until 4 months of age (A) or until 2 months of age (B). Treatment with Dox suppresses cre expression and thus prevents disruption of β-catenin. The earlier disruption of β-catenin by osx-cre at 2 months (B), led to more marrow adipose tissue and more bone loss in the knockout mice at 6 months. (C) μCT analysis of distal femurs and (D) von Kossa staining of plastic sections of proximal tibiae from 12-wk old mice treated with Dox prenatally and until 7 weeks of age.
Fig. 2
Fig. 2. Deletion of β-catenin causes increased adipogenesis and decreased osteogenesis
(A) Confocal images of BMSCs isolated from femurs and tibiae of (mT/mG);β-cat(f/f) mice at 8 weeks of age and infected with ade-GFP (upper panel) or ade-Cre (lower panel) at 10 days. Bar graph shows qRT-PCR analysis of mRNAs encoding β-catenin. (B) Representative images of Oil Red O and Von Kossa staining and quantification of Oil Red O and extracellular calcium content measurement (bar graphs) from infected BMSCs cultured in adipogenic or osteogenic medium for the indicated time. (C) Representative images of Oil Red O and Von Kossa staining and quantification of Oil Red O and extracellular calcium content measurement (bar graphs) from BMSCs isolated from 8-wk old femurs and tibiae of β-catf/f (CON) and osx-cre;β-catf/f (KO) mice treated with Dox until sacrifice and cultured in adipogenic or osteogenic medium for the indicated time. Data are plotted as mean ± SEM. Similar results were obtained from four independent experiments. *p < 0.05 vs control cells cultured for the same time.
Fig. 3
Fig. 3. Deletion of β-catenin in BMSCs increases adipogenic but suppresses osteogenic gene expression
(A–C) qRT-PCR analysis of mRNAs encoding β-catenin (β-cat) and axin2 (A), adipogenic genes (B) and osteogenic transcripts (C) in BMSCs isolated from 8-wk old femurs of β-catf/f mice, infected with adenoviral-Cre (Ade-cre) or with control adenovirus (Ade-GFP), and then cultured under either adipogenic or osteogenic medium for the indicated times. Data are plotted as mean ± SEM of fold expression of each mRNA relative to the expression level observed in the control infected cells cultured at the same intervals. *p < 0.05 vs control infected cells cultured for the same time. Similar results were obtained from four independent experiments.
Fig. 4
Fig. 4. Loss of β-catenin through the osterix-cre causes increased adipogenic and reduced osteogenic gene expression
(A–C) qRT-PCR analysis of mRNAs encoding wnt target genes (A), adipogenic genes (B) and osteogenic transcripts (C) in BMSCs isolated from 8-wk old tibiae and femurs of β-catf/f (CON) and osx-cre;β-catf/f (KO) mice treated with Dox until sacrifice, and cultured under either adipogenic or osteogenic condition for 14 or 21 days, as indicated. Data are plotted as mean ± SEM. *p < 0.05 vs CON-derived cells cultured for the same time. Results were obtained from four independent experiments (4 mice per genotype).
Fig 5
Fig 5. Tracing the preosteoblasts that had to express osterix with the double fluorescence reporter model
(A) Double fluorescence mT/mG reporter mice were mated to osx-cre mice. (B) Representative images by confocal microscopy of frozen sections from 4-wk old proximal tibia of mT/mG; osx-cre mice without administration of doxycycline; the lower row of images are higher-power views of the area delimited by the white box indicating the primary spongiosa. (C) Representative images first by fluorescence microscopy and then histology of same frozen sections from the same proximal tibia shown above; the upper row of images showing the secondary spongiosa and the lower row of images showing the cortical bone. All the images clearly demonstrate that the GFP-positive cells are restricted on bone but not in the marrow area.
Fig. 6
Fig. 6. Loss of β-catenin causes cell fate shift of preosteoblasts
Bone marrow derived cells were isolated from 8-wk old femurs and tibiae of mT/mG; osx-cre (CON) and mT/mG; osx-cre;β-catf/f (KO) mice treated with Dox until sacrifice, cultured under adipogenic condition for 21 days, and analyzed by Oil Red O staining and cellular immunofluorescent detection for FABP4 (red) and GFP (green). (A) Fluorescent microscope images show adipocytic cells that expressed both FABP4 and GFP at the same time (indicated by white arrows) in the cultured BMSCs exclusively from KO mice but not from CON mice at all, and bar graph quantitatively demonstrates a dramatic increase in number of FABP4 positive cells in the KO-derived culture. Data are plotted as mean ± SEM. *p < 0.05 vs CON-derived cells. (B) Representative confocal images of the cultured cells from KO mice (the lower row of images indicating magnified views of the cells delimited by the white box) show the adipocytic cells that strongly express both FABP4 and GFP at the same time (as indicated by arrowheads).
Fig. 7
Fig. 7. Confocal microscopy of adipocytic cells positive for FABP4 and either GFP or Tomato
Bone marrow derived cells were isolated from 8-wk old femurs and tibiae of mT/mG; osx-cre (CON) and mT/mG; osx-cre;β-catf/f (KO) mice treated with Dox until sacrifice, and cultured under adipogenic condition for 21 days. The cultured cells underwent immunofluorescent staining only for FABP4 and then were analyzed by confocal microscopy for FABP4 (red), GFP (green), tomato (pink) and DAPI (blue). (A) The confocal images show that in CON-derived BMSCs the FABP4-positive cells only express tomato and none express GFP. (B) The confocal images of the cultured cells from KO mice demonstrate FABP4-positive cells that also express either tomato (arrows) or GFP (arrowheads), but not both, at the same time.
Fig. 8
Fig. 8. In vivo lineage tracing and assessment of adiposity
(A) Confocal microscopy of 5-month old proximal tibiae from mT/mG; osx-cre (CON) and mT/mG; osx-cre;β-catf/f (KO) mice treated with Dox until 2-month old. “Grey” denotes the images without fluorescence. Many GFP-positive cells and cells double positive for GFP and FABP4 in the bone marrow were observed in KO but few in CON mice, and FABP4-positive cells were dramatically increased in KO mice. (B) Representative images from the KO confocal microscopy show some FABP4 positive cells that co-expressed GFP (as indicated by white arrows in the merged image of FABP4/GFP; Red: FABP4, Green: GFP and blue: DAPI stain for nucleus). (C) Bar graphs represent a quantitative assessment of FABP4 positive cells and the cells positive for both FABP4 and GFP in the metaphyseal trabecular region of the proximal tibia. Data are expressed as mean ± SEM. *p < 0.01 vs CON mice. We counted blindly the cells positive for both GFP and FABP4 from a total of approximately 1500 or 2100 FABP4 positive cells derived from four CON or four KO mice, respectively. (D) Representative micrographs of Oil Red O stain from the frozen sections of the proximal tibiae described in A and B. (E) qRT-PCR analysis of mRNAs encoding adipogenic genes; total RNA was isolated from one tibia of the same mice described in A and B. There were four mice in each group and data are plotted as mean ± SEM; *p < 0.05 vs CON mice.
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