Background TGF-βstimulation enhances the osteoclastgenesis, which is characteristiccally observed in inflamed joints of patients with rheumatoid arthritis. In some hematopoietic lineages including dendriticcells and T cells, TGF-βinduces PU.1 expression and subsequent its function. However, the relationship between PU.1 and TGF-βin osteoclastis largely unknown, even though PU.1 is known to be important for development of osteoclast.
Objectives To examine the involvement of PU.1 in TGF-β-mediated osteoclastdifferentiation.
Methods Osteoclasts were generated from whole mouse bone marrow cells by the cultivation with using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) for 3 days, and then adherent cells were further incubated with M-CSF and RANKL, in the presence or absence of TGF-β. To evaluate the effect of PU.1 in osteoclasts, PU.1 was knocked down using siRNAduring differentiation. Osteoclast formation was determined by TRAP staining, and the amount of mRNAs of the osteoclast marker genes was measured by quantitative real-time PCR. We analyzed the dynamics of PU.1 in TGF-β-mediated gene expression by using chromatin immunoprecipitation (ChIP) assay and Western blotting analysis.
Results The number of TRAP-positive multinucleated cells and the mRNA expression levels of osteoclast-specific molecules including NFATc1 or cathepsin K (Ctsk) were significantly reduced in PU.1 siRNA-introduced cells. TGF-β stimulation increased the mRNA levels of the osteoclast markers and the number of TRAP-positive multinucleated cells. ChIP assay showed that PU.1 bound the promoter regions of osteoclast marker genes, such as Acp5 or Ctsk, and the amount of PU.1 on these promoters was higher in TGF-β-stimulated cells. When the effect of TGF-β on PU.1 protein level was analyzed by Western blotting, PU.1 protein levels in whole cell and cytosolic fraction were reduced by TGF-β stimulation, whereas PU.1 was not detected in nucleus.
Conclusions TGF-βstimulation affects the expression and/or function of PU.1 in osteoclast-specific manner.
References RongHuets: Mol. Cell. Biol, 2007, 27, 4018-4027. Leonhard X. Heinz ets: Blood, 2006, 107, 1445-1453. Goswami. R ets: J Immunol, 2012, 188, 968-975
Disclosure of Interest None Declared