Department of Diagnostic and Biological Sciences, University of Minnesota School of Dentistry, Minneapolis, MN 55455, USA.
Clinical and Translational Science Institute, University of Minnesota, Minneapolis, MN 55455, USA.
Int J Mol Sci. 2020 Feb 5;21(3):1056. doi: 10.3390/ijms21031056.
Balanced osteoclast and osteoblast activity is necessary for skeletal health, whereas unbalanced osteoclast activity causes bone loss in many skeletal conditions. A better understanding of pathways that regulate osteoclast differentiation and activity is necessary for the development of new therapies to better manage bone resorption. The roles of Protein Kinase D (PKD) family of serine/threonine kinases in osteoclasts have not been well characterized. In this study we use immunofluorescence analysis to reveal that PKD2 and PKD3, the isoforms expressed in osteoclasts, are found in the nucleus and cytoplasm, the mitotic spindle and midbody, and in association with the actin belt. We show that PKD inhibitors CRT0066101 and CID755673 inhibit several distinct aspects of osteoclast formation. Treating bone marrow macrophages with lower doses of the PKD inhibitors had little effect on M-CSF + RANKL-dependent induction into committed osteoclast precursors, but inhibited their motility and subsequent differentiation into multinucleated mature osteoclasts, whereas higher doses of the PKD inhibitors induced apoptosis of the preosteoclasts. Treating post-fusion multinucleated osteoclasts with the inhibitors disrupted the osteoclast actin belts and impaired their resorptive activity. In conclusion, these data implicate PKD kinases as positive regulators of osteoclasts, which are essential for multiple distinct processes throughout their formation and function.
成骨细胞和破骨细胞活性的平衡对于骨骼健康是必要的，而破骨细胞活性的失衡会导致许多骨骼疾病中的骨质流失。为了开发新的治疗方法来更好地控制骨质吸收，需要更好地了解调节破骨细胞分化和活性的途径。蛋白激酶 D (PKD) 家族丝氨酸/苏氨酸激酶在破骨细胞中的作用尚未得到很好的描述。在这项研究中，我们使用免疫荧光分析揭示了在破骨细胞中表达的 PKD2 和 PKD3，它们存在于细胞核和细胞质、有丝分裂纺锤体和中体中，并与肌动蛋白带相关联。我们表明，PKD 抑制剂 CRT0066101 和 CID755673 抑制破骨细胞形成的几个不同方面。用较低剂量的 PKD 抑制剂处理骨髓巨噬细胞对 M-CSF + RANKL 依赖性诱导成 committed 破骨细胞前体的作用很小，但抑制了它们的迁移和随后分化为多核成熟破骨细胞，而较高剂量的 PKD 抑制剂则诱导前破骨细胞凋亡。用抑制剂处理融合后的多核破骨细胞会破坏破骨细胞的肌动蛋白带并损害其吸收活性。总之，这些数据表明 PKD 激酶是破骨细胞的正调控因子，对于破骨细胞形成和功能的多个不同过程都是必不可少的。
