Zamani Ali, Decker Corinne, Cremasco Viviana, Hughes Lindsey, Novack Deborah V, Faccio Roberta
Department of Orthopaedics, Washington University School of Medicine, St. Louis, MO, USA.
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA.
J Bone Miner Res. 2015 Oct;30(10):1852-63. doi: 10.1002/jbmr.2533. Epub 2015 Aug 26.
Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue-specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGKζ, a highly expressed DGK isoform in osteoclasts (OCs). Strikingly, DGKζ(-/-) mice are osteoporotic because of a marked increase in OC numbers. In vitro, DGKζ(-/-) bone marrow macrophages (BMMs) form more numerous, larger, and highly resorptive OCs. Surprisingly, although increased DAG levels do not alter receptor activator of NF-κB (RANK)/RANK ligand (RANKL) osteoclastogenic pathway, DGKζ deficiency increases responsiveness to the proliferative and pro-survival cytokine macrophage colony-stimulating factor (M-CSF). We find that M-CSF is responsible for increased DGKζ(-/-) OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGKζ(-/-) cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGKζ/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLCγ2(+/-) BMMs, which have reduced DAG levels and form fewer OCs because of impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGKζ in PLCγ2(+/-) mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGKζ/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression.
在许多病理状况下，包括与骨质流失相关的病症中，都观察到二酰甘油（DAG）水平升高。然而，DAG积累对骨骼的影响从未被直接研究过。由于DAG受到组织特异性二酰甘油激酶（DGKs）的严格调控，我们试图通过基因敲除破骨细胞（OCs）中高表达的DGK异构体DGKζ，来研究DAG积累对骨稳态的生物学影响。令人惊讶的是，DGKζ(-/-)小鼠患有骨质疏松症，原因是OC数量显著增加。在体外，DGKζ(-/-)骨髓巨噬细胞（BMMs）形成的OC数量更多、体积更大且具有高度的吸收性。令人惊讶的是，尽管DAG水平升高不会改变核因子κB受体激活剂（RANK）/RANK配体（RANKL）破骨细胞生成途径，但DGKζ缺乏会增加对增殖和促生存细胞因子巨噬细胞集落刺激因子（M-CSF）的反应性。我们发现，M-CSF通过促进转录因子c-Fos的更高表达，导致DGKζ(-/-) OC分化增加，并且在DGKζ(-/-)培养物中敲低c-Fos会剂量依赖性地降低OC分化。使用缺乏TRE反应元件的c-Fos荧光素酶报告基因检测，我们还证明M-CSF通过DAG产生诱导最佳的c-Fos表达。最后，为了证明M-CSF/DGKζ/DAG轴在破骨细胞生成过程中对c-Fos调控的重要性，我们转向了PLCγ2(+/-) BMMs，它们由于破骨细胞生成主调节因子NFATc1和c-Fos的表达受损，DAG水平降低且形成的OC较少。令人惊讶的是，在PLCγ2(+/-)小鼠中基因敲除DGKζ可挽救OC形成并使c-Fos水平正常化，而不会改变NFATc1表达。据我们所知，这是第一份将M-CSF/DGKζ/DAG轴通过其对OC分化和c-Fos表达的作用，作为骨稳态关键调节因子的报告。
