Regulation of Osteoclast Differentiation at Multiple Stages by Protein Kinase D Family Kinases

Int J Mol Sci. 2020 Feb 5;21(3):1056. doi: 10.3390/ijms21031056.

Abstract

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.

Keywords: actin cytoskeleton; bone resorption; cellular differentiation; osteoclasts; protein kinase D.

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / physiology
  • Azepines / pharmacology
  • Benzofurans / pharmacology
  • Bone Marrow Cells / drug effects
  • Bone Marrow Cells / metabolism
  • Bone Resorption / drug therapy
  • Bone Resorption / metabolism
  • Cell Differentiation / drug effects
  • Cell Differentiation / physiology*
  • Cells, Cultured
  • Female
  • Macrophage Colony-Stimulating Factor / metabolism
  • Macrophages / drug effects
  • Macrophages / metabolism
  • Male
  • Mice, Inbred C57BL
  • Osteoclasts / drug effects
  • Osteoclasts / metabolism*
  • Osteoclasts / physiology*
  • Protein Kinase C / metabolism*
  • Protein Kinase Inhibitors / pharmacology
  • Pyrimidines / pharmacology
  • Receptor Activator of Nuclear Factor-kappa B / metabolism

Substances

  • Azepines
  • Benzofurans
  • CID755673
  • CRT 0066101
  • Protein Kinase Inhibitors
  • Pyrimidines
  • Receptor Activator of Nuclear Factor-kappa B
  • Macrophage Colony-Stimulating Factor
  • protein kinase D
  • Protein Kinase C