Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts
Abstract
:1. Introduction
2. Results
2.1. Expression of Globo-Series Glycosphingolipids (Gb3, Gb4, and Gb5), and Gb3 and Gb4 Synthase Genes in Osteoblasts
2.2. No Expression of Globo-Series Glycosphingolipids (Gb3, Gb4, and Gb5), and Gb3 and Gb4 Synthase Genes in RAW264.7 Cells and Primary Cultured Pre-Osteoclasts
2.3. Decrease in Femoral Cancellous Bone Mass in Gb3 Synthase-Knockout (Gb3S KO) Mice
2.4. Decrease in Bone Formation Parameters by Gb3 Synthase Deficiency
2.5. Decrease in Osteoblast Number in Femoral Cancellous Bone and Osteogenic Differentiation Marker’s Gene Expression (Runt-Related Transcription Factor 2, Alkaline Phosphatase, and Osteocalcin) in Long Bones by Gb3 Synthase Deficiency
2.6. No Differences in Osteoclast Number and Expression of Osteoclast Differentiation Marker’s Genes (Nuclear Factor of Activated T-Cells, Cytoplasmic 1 and Tartrate-Resistant Acid Phosphatase) between WT and Gb3S KO Mice
3. Discussion
4. Materials and Methods
4.1. Mice
4.2. Cell Culture
4.3. Antibodies
4.4. Induction to Mature Osteoblasts
4.5. In Vitro Osteoclast Induction
4.6. Flow Cytometry
4.7. Quantitative Real-Time PCR (qPCR)
4.8. Three-Dimensional Micro-Computed Tomography (3D-μCT Analysis)
4.9. Measurement of Bone Formation Rate Using Calcein Double Labeling
4.10. TRAP Staining
4.11. Bone Histomorphometric Analyses
4.12. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Target | Forward Primer | Backward Primer |
---|---|---|
A4galt | 5′-CCTGTTCCCATCTGGAGGAG-3′ | 5′-CCCTTTCATCAGCACAACCA-3′ |
B3galnt1 | 5′-TCTTGACTGCCCTTCCCAAT-3′ | 5′-GGAGCGTGAAGCGAAAGTCT-3′ |
Runx2 | 5′-CCCAGCCACCTTTACCTACA-3′ | 5′-TATGGAGTGCTGCTGGTCTG-3′ |
Alp | 5′-AACCCAGACACAAGCATTCC-3′ | 5′-GCCTTTGAGGTTTTTGGTCA-3′ |
osteocalcin | 5′-CCGGGAGCAGTGTGAGCTTA-3′ | 5′-AGGCGGTCTTCAAGCCATACT-3′ |
Nfatc1 | 5′-GGTGCTGTCTGGCCATAACT-3′ | 5′-GCGGAAAGGTGGTATCTCAA-3′ |
Trap | 5′-TCCTGGCTCAAAAAGCAGTT-3′ | 5′-ACATAGCCCACACCGTTCTC-3′ |
Gapdh | 5′-TGCACCACCAACTGCTTAG-3′ | 5′-GGATGCAGGGATGATGTTC-3′ |
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Hamamura, K.; Hamajima, K.; Yo, S.; Mishima, Y.; Furukawa, K.; Uchikawa, M.; Kondo, Y.; Mori, H.; Kondo, H.; Tanaka, K.; et al. Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts. Int. J. Mol. Sci. 2019, 20, 4619. https://doi.org/10.3390/ijms20184619
Hamamura K, Hamajima K, Yo S, Mishima Y, Furukawa K, Uchikawa M, Kondo Y, Mori H, Kondo H, Tanaka K, et al. Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts. International Journal of Molecular Sciences. 2019; 20(18):4619. https://doi.org/10.3390/ijms20184619
Chicago/Turabian StyleHamamura, Kazunori, Kosuke Hamajima, Shoyoku Yo, Yoshitaka Mishima, Koichi Furukawa, Makoto Uchikawa, Yuji Kondo, Hironori Mori, Hisataka Kondo, Kenjiro Tanaka, and et al. 2019. "Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts" International Journal of Molecular Sciences 20, no. 18: 4619. https://doi.org/10.3390/ijms20184619
APA StyleHamamura, K., Hamajima, K., Yo, S., Mishima, Y., Furukawa, K., Uchikawa, M., Kondo, Y., Mori, H., Kondo, H., Tanaka, K., Miyazawa, K., Goto, S., & Togari, A. (2019). Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts. International Journal of Molecular Sciences, 20(18), 4619. https://doi.org/10.3390/ijms20184619