Gene array identification of osteoclast genes: differential inhibition of osteoclastogenesis by cyclosporin A and granulocyte macrophage colony stimulating factor.

Abstract

Treatment of adherent peripheral blood mononuclear cells (PBMCs) with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) stimulates the formation of multinucleate osteoclast-like cells. Treatment with M-CSF alone results in the formation of macrophage-like cells. Through the use of Atlas human cDNA expression arrays, genes regulated by RANKL were identified. Genes include numerous cytokines and cytokine receptors (RANTES and CSF2R proportional, variant ), transcription factors (nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and GA binding protein transcription factor alpha (GABPalpha)), and ribosomal proteins (60S L17 and 40S S20). Real-time PCR analysis showed significant correlation (R2 of 0.98 P < 0.01) with array data for all genes tested. Time courses showed differential activation patterns of transcription factors with early induction of FUSE binding protein 1 (FBP) and c-Jun, and later steady upregulation of NFATc1 and GABP by RANKL. Treatment with cyclosporin A, a known NFATc1 inhibitor, resulted in a blockade of osteoclast formation. The mononuclear cells resulting from high cyclosporin treatment (1,000 ng/ml) were cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) positive but expression of calcitonin receptor (CTR) was downregulated by more than 30-fold. Constant exposure of M-CSF- and RANKL-treated cells to GM-CSF resulted in inhibition of osteoclast formation and the downregulation of CTSK and TRAP implicating the upregulation of CSF2R in a possible feedback inhibition of osteoclastogenesis.