Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis
Abstract
:1. Introduction
2. Results
2.1. Generation of Osteoblast-Specific Bcl2l1 Conditional Knock-Out Mice
2.2. Reduction in Bone Mass in Bcl2l1fl/flCre Mice
2.3. Increases in Osteoclast Parameters in Bcl2l1fl/flCre Mice in the Bone Histomorphometric Analysis
2.4. Increased Osteoblast Apoptosis and Serum TRAP5b Levels in Bcl2l1fl/flCre Mice
2.5. Osteoblastogenesis and Osteoclastogenesis In Vitro
2.6. Increases in Apoptosis and ATP Release by the Bcl2l1 Deletion
4. Materials and Methods
4.1. Mice
4.2. Real-Time RT-PCR and Western Blot Analyses
4.3. X-Gal Staining
4.4. Micro-CT Analysis
4.5. Histological Analysis
4.6. Serum Testing
4.7. Cell Culture
4.8. In Vitro Osteoclastogenesis
4.9. Measurement of ATP
4.10. Serum-Free Culture
4.11. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- O’Brien, C.A.; Jia, D.; Plotkin, L.I.; Bellido, T.; Powers, C.C.; Stewart, S.A.; Manolagas, S.C.; Weinstein, R.S. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology 2004, 145, 1835–1841. [Google Scholar] [CrossRef]
- Kousteni, S.; Bellido, T.; Plotkin, L.I.; O’Brien, C.A.; Bodenner, D.L.; Han, L.; Han, K.; DiGregorio, G.B.; Katzenellenbogen, J.A.; Katzenellenbogen, B.S.; et al. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: Dissociation from transcriptional activity. Cell 2001, 104, 719–730. [Google Scholar] [CrossRef]
- Stanislaus, D.; Yang, X.; Liang, J.D.; Wolfe, J.; Cain, R.L.; Onyia, J.E.; Falla, N.; Marder, P.; Bidwell, J.P.; Queener, S.W.; et al. In vivo regulation of apoptosis in metaphyseal trabecular bone of young rats by synthetic human parathyroid hormone (1-34) fragment. Bone 2000, 27, 209–218. [Google Scholar] [CrossRef]
- Gohel, A.; McCarthy, M.B.; Gronowicz, G. Estrogen prevents glucocorticoid-induced apoptosis in osteoblasts in vivo and in vitro. Endocrinology 1999, 140, 5339–5347. [Google Scholar] [CrossRef]
- Weinstein, R.S.; Jilka, R.L.; Parfitt, A.M.; Manolagas, S.C. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J. Clin. Investig. 1998, 102, 274–282. [Google Scholar] [CrossRef]
- Plotkin, L.I.; Weinstein, R.S.; Parfitt, A.M.; Roberson, P.K.; Manolagas, S.C.; Bellido, T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J. Clin. Investig. 1999, 104, 1363–1374. [Google Scholar] [CrossRef] [PubMed]
- Jilka, R.L.; Weinstein, R.S.; Bellido, T.; Roberson, P.; Parfitt, A.M.; Manolagas, S.C. Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J. Clin. Investig. 1999, 104, 439–446. [Google Scholar] [CrossRef]
- Tomkinson, A.; Reeve, J.; Shaw, R.W.; Noble, B.S. The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone. J. Clin. Endocrinol. Metab. 1997, 82, 3128–3135. [Google Scholar] [PubMed]
- Youle, R.J.; Strasser, A. The BCL-2 protein family: Opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 2008, 9, 47–59. [Google Scholar] [CrossRef] [PubMed]
- Tsujimoto, Y. Cell death regulation by the Bcl-2 protein family in the mitochondria. J. Cell. Physiol. 2003, 195, 158–167. [Google Scholar] [CrossRef]
- Yamashita, J.; Datta, N.S.; Chun, Y.-H.P.; Yang, D.-Y.; Carey, A.A.; Kreider, J.M.; Goldstein, S.A.; McCauley, L.K. Role of Bcl2 in Osteoclastogenesis and PTH Anabolic Actions in Bone. J. Bone Miner. Res. 2007, 23, 621–632. [Google Scholar] [CrossRef]
- Nagase, Y.; Iwasawa, M.; Akiyama, T.; Kadono, Y.; Nakamura, M.; Oshima, Y.; Yasui, T.; Matsumoto, T.; Hirose, J.; Nakamura, H.; et al. Anti-apoptotic molecule Bcl-2 regulates the differentiation, activation, and survival of both osteoblasts and osteoclasts. J. Biol. Chem. 2009, 284, 36659–36669. [Google Scholar] [CrossRef]
- Moriishi, T.; Kawai, Y.; Komori, H.; Rokutanda, S.; Eguchi, Y.; Tsujimoto, Y.; Asahina, I.; Komori, T. Bcl2 deficiency activates FoxO through Akt inactivation and accelerates osteoblast differentiation. PLoS ONE 2014, 9, e86629. [Google Scholar] [CrossRef]
- Moriishi, T.; Maruyama, Z.; Fukuyama, R.; Ito, M.; Miyazaki, T.; Kitaura, H.; Ohnishi, H.; Furuichi, T.; Kawai, Y.; Masuyama, R.; et al. Overexpression of Bcl2 in osteoblasts inhibits osteoblast differentiation and induces osteocyte apoptosis. PLoS ONE 2011, 6, e27487. [Google Scholar] [CrossRef]
- Boise, L.H.; Gonzalez-Garcia, M.; Postema, C.E.; Ding, L.; Lindsten, T.; Turka, L.A.; Mao, X.; Nunez, G.; Thompson, C.B. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993, 74, 597–608. [Google Scholar] [CrossRef]
- Gonzalez-Garcia, M.; Perez-Ballestero, R.; Ding, L.; Duan, L.; Boise, L.H.; Thompson, C.B.; Nunez, G. bcl-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria. Development 1994, 120, 3033–3042. [Google Scholar] [CrossRef]
- Fang, W.; Rivard, J.J.; Mueller, D.L.; Behrens, T.W. Cloning and molecular characterization of mouse bcl-x in B and T lymphocytes. J. Immunol. 1994, 153, 4388–4398. [Google Scholar] [CrossRef]
- Shiraiwa, N.; Inohara, N.; Okada, S.; Yuzaki, M.; Shoji, S.; Ohta, S. An additional form of rat Bcl-x, Bcl-xbeta, generated by an unspliced RNA, promotes apoptosis in promyeloid cells. J. Biol. Chem. 1996, 271, 13258–13265. [Google Scholar] [CrossRef]
- Yang, X.F.; Weber, G.F.; Cantor, H. A novel Bcl-x isoform connected to the T cell receptor regulates apoptosis in T cells. Immunity 1997, 7, 629–639. [Google Scholar] [CrossRef]
- Motoyama, N.; Wang, F.; Roth, K.A.; Sawa, H.; Nakayama, K.; Negishi, I.; Senju, S.; Zhang, Q.; Fujii, S.; Dennis, Y.; et al. Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science 1995, 267, 1506–1510. [Google Scholar] [CrossRef]
- Iwasawa, M.; Miyazaki, T.; Nagase, Y.; Akiyama, T.; Kadono, Y.; Nakamura, M.; Oshima, Y.; Yasui, T.; Matsumoto, T.; Nakamura, T.; et al. The antiapoptotic protein Bcl-xL negatively regulates the bone-resorbing activity of osteoclasts in mice. J. Clin. Investig. 2009, 119, 3149–3159. [Google Scholar] [CrossRef]
- Chekeni, F.B.; Elliott, M.R.; Sandilos, J.K.; Walk, S.F.; Kinchen, J.M.; Lazarowski, E.R.; Armstrong, A.J.; Penuela, S.; Laird, D.W.; Salvesen, G.S.; et al. Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 2010, 467, 863–867. [Google Scholar] [CrossRef]
- Buckley, K.A.; Hipskind, R.A.; Gartland, A.; Bowler, W.B.; Gallagher, J.A. Adenosine triphosphate stimulates human osteoclast activity via upregulation of osteoblast-expressed receptor activator of nuclear factor-kappa B ligand. Bone 2002, 31, 582–590. [Google Scholar] [CrossRef]
- Korcok, J.; Raimundo, L.N.; Ke, H.Z.; Sims, S.M.; Dixon, S.J. Extracellular nucleotides act through P2X7 receptors to activate NF-kappaB in osteoclasts. J. Bone Miner. Res. 2004, 19, 642–651. [Google Scholar] [CrossRef]
- Pellegatti, P.; Falzoni, S.; Donvito, G.; Lemaire, I.; Di Virgilio, F. P2X7 receptor drives osteoclast fusion by increasing the extracellular adenosine concentration. FASEB J. 2011, 25, 1264–1274. [Google Scholar] [CrossRef]
- Gallagher, J.A. ATP P2 receptors and regulation of bone effector cells. J. Musculoskelet. Neuronal Interact. 2004, 4, 125–127. [Google Scholar]
- Moriishi, T.; Fukuyama, R.; Miyazaki, T.; Furuichi, T.; Ito, M.; Komori, T. Overexpression of BCLXL in Osteoblasts Inhibits Osteoblast Apoptosis and Increases Bone Volume and Strength. J. Bone Miner. Res. 2016, 31, 1366–1380. [Google Scholar] [CrossRef]
- Komori, T. Cell Death in Chondrocytes, Osteoblasts, and Osteocytes. Int. J. Mol. Sci. 2016, 17, 2045. [Google Scholar] [CrossRef]
- Kennedy, O.D.; Laudier, D.M.; Majeska, R.J.; Sun, H.B.; Schaffler, M.B. Osteocyte apoptosis is required for production of osteoclastogenic signals following bone fatigue in vivo. Bone 2014, 64, 132–137. [Google Scholar] [CrossRef]
- Cheung, W.Y.; Fritton, J.C.; Morgan, S.A.; Seref-Ferlengez, Z.; Basta-Pljakic, J.; Thi, M.M.; Suadicani, S.O.; Spray, D.C.; Majeska, R.J.; Schaffler, M.B. Pannexin-1 and P2X7-Receptor Are Required for Apoptotic Osteocytes in Fatigued Bone to Trigger RANKL Production in Neighboring Bystander Osteocytes. J. Bone Miner. Res. 2016, 31, 890–899. [Google Scholar] [CrossRef]
- Lemaire, I.; Falzoni, S.; Zhang, B.; Pellegatti, P.; Di Virgilio, F. The P2X7 receptor and Pannexin-1 are both required for the promotion of multinucleated macrophages by the inflammatory cytokine GM-CSF. J. Immunol. 2011, 187, 3878–3887. [Google Scholar] [CrossRef]
- Zhou, Y.; Arredondo, H.M.; Wang, N. P2Y Receptors in Bone—Anabolic, Catabolic, or Both? Front. Endocrinol. 2021, 12, 818499. [Google Scholar] [CrossRef]
- Buckley, K.A.; Wagstaff, S.C.; McKay, G.; Gaw, A.; Hipskind, R.A.; Bilbe, G.; Gallagher, J.A.; Bowler, W.B. Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone. J. Biol. Chem. 2001, 276, 9565–9571. [Google Scholar] [CrossRef]
- Alvarenga, E.C.; Rodrigues, R.; Caricati-Neto, A.; Silva-Filho, F.C.; Paredes-Gamero, E.J.; Ferreira, A.T. Low-intensity pulsed ultrasound-dependent osteoblast proliferation occurs by via activation of the P2Y receptor: Role of the P2Y1 receptor. Bone 2010, 46, 355–362. [Google Scholar] [CrossRef]
- Syberg, S.; Brandao-Burch, A.; Patel, J.J.; Hajjawi, M.; Arnett, T.R.; Schwarz, P.; Jorgensen, N.R.; Orriss, I.R. Clopidogrel (Plavix), a P2Y12 receptor antagonist, inhibits bone cell function in vitro and decreases trabecular bone in vivo. J. Bone Miner. Res. 2012, 27, 2373–2386. [Google Scholar] [CrossRef]
- Sakai, K.; Miyazaki, J. A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission. Biochem. Biophys. Res. Commun. 1997, 237, 318–324. [Google Scholar] [CrossRef]
- Zhang, N.; He, Y.W. The antiapoptotic protein Bcl-xL is dispensable for the development of effector and memory T lymphocytes. J. Immunol. 2005, 174, 6967–6973. [Google Scholar] [CrossRef]
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Moriishi, T.; Kawai, Y.; Fukuyama, R.; Matsuo, Y.; He, Y.-W.; Akiyama, H.; Asahina, I.; Komori, T. Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis. Int. J. Mol. Sci. 2023, 24, 17319. https://doi.org/10.3390/ijms242417319
Moriishi T, Kawai Y, Fukuyama R, Matsuo Y, He Y-W, Akiyama H, Asahina I, Komori T. Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis. International Journal of Molecular Sciences. 2023; 24(24):17319. https://doi.org/10.3390/ijms242417319
Chicago/Turabian StyleMoriishi, Takeshi, Yosuke Kawai, Ryo Fukuyama, Yuki Matsuo, You-Wen He, Haruhiko Akiyama, Izumi Asahina, and Toshihisa Komori. 2023. "Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis" International Journal of Molecular Sciences 24, no. 24: 17319. https://doi.org/10.3390/ijms242417319