Subcellular Localization of HDACs During Osteoclast Differentiation

Objectives: Histone deacetylases (HDACs) are negative regulators of transcription and have been shown to repress specific changes in gene expression. Class IIa HDACs, HDAC 4, 5, 7, and 9, have been shown in other cell types to be regulated by phosphorylation, which governs their subcellular localization and activity. Phosphorylation of class IIa HDACs shuttles the HDAC from the nucleus to the cytoplasm relieving the repression of gene expression. Previous studies from our lab and others have demonstrated that HDAC7 and HDAC9 are inhibitors of osteoclast differentiation. The objective of current study is to determine the subcellular location of class IIa HDACs during osteoclast differentiation.
Methods: Bone marrow macrophages (BMMs) were isolated from mouse femur and were differentiated into mature osteoclasts in the presence of RANK ligand (RANKL). Nuclear and cytoplasmic subcellular fractions of osteoclasts during differentiation were isolated on days 0-4 after treatment with RANKL. Additionally, osteoclasts were treated with kinase inhibitors present in a kinase inhibitor library (Enzo). The levels of total and phosphorylated HDACs were analyzed by Western blot.
Results: We hypothesize that class IIa HDACs shuttle to the nucleus of osteoclasts to repress gene expression necessary for osteoclast differentiation. Surprisingly, HDAC7 was detected in cytoplasmic fractions but not nuclear fractions of osteoclasts at all time points measured after RANKL. Additionally, we have determined that cytoplasmic-only and nuclear-only HDAC7 mutants can repress osteoclast differentiation as well as wildtype HDAC7. In contrast to HDAC7, HDAC4 was detected in both cytoplasmic and nuclear cell fractions at early time points after RANKL. Current work is determining HDAC phosphorylation status and the kinases responsible for the phosphorylation of HDACs during osteoclast differentiation.
Conclusions: Our current work suggests 1) class IIa HDACs have different subcellular localizations in osteoclasts and 2) activity of HDACs may be regulated by their subcellular localization.