Deficiency of GD3 Synthase in Mice Resulting in the Attenuation of Bone Loss with Aging

Figures

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  Figure 1. Synthetic pathway of gangliosides and the effects of targeted disruption of GD3 synthase. All b-series gangliosides (GD3, GD2, GD1b, GT1b, and GQ1b) in the box were deleted in GD3 synthase-knockout (GD3S KO) mice.

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  Figure 2. No detection of b-series gangliosides (GD3, GD2, GD1b, and GT1b) and GD3 synthase gene (St8sia1) in MC3T3 E1 cells. (A) Expression of b-series gangliosides in MC3T3 E1 cells on days 0 and 7 after induction to mature osteoblasts by flow cytometric analysis. Expression of GD3 in RAW264.7 cells without induction of osteoclastogenesis (Day 0). Red line: anti-gangliosides (GD3, GD2, GD1b, or GT1b) monoclonal antibodies (mAbs) (+), gray line: anti-gangliosides mAbs (-). (B) mRNA expression of St8sia1 in RAW264.7 cells and MC3T3 E1 cells on days 0, 7, 14, and 21 after induction to mature osteoblasts (n = 4). Data are expressed as mean ± SD. RAW264.7 cells without induction of osteoclastogenesis (Day 0) were used as a positive control in Figure 2A. Legend: ND, not detectable.

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  Figure 3. Expression of b-series gangliosides (GD3, GD2, GD1b, and GT1b) and GD3 synthase gene (St8sia1) in RAW264.7 cells and primary cultured pre-osteoclasts in the presence or absence of RANKL. (A) Expression of b-series gangliosides in RAW264.7 cells on days 0 and 2 after administration of RANKL (n = 3). (B) Expression of b-series gangliosides in primary cultured pre-osteoclasts on days 0 and 1 after administration of RANKL (n = 3). Red line: anti-gangliosides (GD3, GD2, GD1b, or GT1b) monoclonal antibodies (mAbs) (+), gray line: anti-gangliosides mAbs (-). The relative expression level (GD3, GD2, and GD1b) was defined as the ratio of the fluorescence intensity for the anti-gangliosides (GD3, GD2, or GD1b) monoclonal antibodies (mAbs) (+) to the fluorescence intensity for the anti-gangliosides mAbs (-). (C) mRNA expression of St8sia1 in RAW264.7 cells on days 0, 2, and 4 after administration of RANKL (n = 3). Data are expressed as mean ± SD. The single (*) and double asterisks (**) indicate p < 0.05 and p < 0.01, respectively.

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  Figure 4. Attenuation of loss of femoral cancellous bone mass with aging in GD3S KO mice. Analysis of bone densitometry of the distal region of the femur in wild type (WT) and GD3S KO mice by three-dimensional micro-computed tomography (3D-μCT). (A) 15-week-old mice (male, n = 12 for WT, n = 10 for GD3S KO). (B) 40-week-old mice (male, n = 12 for WT, n = 10 for GD3S KO). Bone volume/total volume (BV/TV, %), trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, 1/mm), and trabecular separation (Tb.Sp, μm) are presented. (C) Representative μCT images of femur in WT and GD3S KO mice at 40 weeks old. Results of WT mice (left) and GD3S KO mice (right) are shown. The scale bars are 1000 μm. Data are expressed as mean ± SD. The double asterisks (**) indicate p < 0.01.

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  Figure 5. No effects of GD3 synthase deficiency on osteoblast numbers shown in vivo. (A) 15-week-old mice (male, n = 12 for WT, n = 10 for GD3S KO). (B) 40-week-old mice (male, n = 12 for WT, n = 10 for GD3S KO). Osteoblast numbers/bone surface (Ob.N/BS, number/mm) and osteoblast surface/bone surface (Ob.S/BS, %) are presented. Data are expressed as mean ± SD.

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  Figure 6. Decrease in bone resorption parameters by GD3 synthase deficiency in vivo. (A) 15-week-old mice (male, n = 12 for WT, n = 10 for GD3S KO). (B) 40-week-old mice (male, n = 12 for WT, n = 10 for GD3S KO). Osteoclast numbers/bone surface (Oc.N/BS, number/mm), and osteoclast surface/bone surface (Oc.S/BS, %) are presented. (C) Images of tartrate-resistant acid phosphatase (TRAP) staining of femoral cancellous bone. Results for WT mice (upper) and GD3S KO mice (lower) are shown. Right images are higher magnifications of the boxed areas in the images on the left. The scale bars are 100 μm. Data are expressed as mean ± SD. The double asterisks (**) indicate p < 0.01.

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  Figure 7. Schematic illustration of the proposed regulation of bone metabolism by b-series gangliosides.