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.

WNT/β-Catenin Signaling Is Involved in Regulation of Osteoclast Differentiation by Human Immunodeficiency Virus Protease Inhibitor Ritonavir

“…Genes (n ϭ 4194) were affected in control versus RTV cultures on day 1 ( Figure 2A), whereas only 178 genes were so altered on day 5 (data not shown). This is consistent with the kinetics of transcriptional changes from microarray data obtained with murine 24,25 and human 16 OC precursors exposed to other drugs that impact OC formation. Major alterations took place before day 3 for 96% of genes in our human system, versus 91% of genes in a murine OC model.…”

“…In preliminary experiments we evaluated both M-CSF (100 ng/ml) and GM-CSF (100 ng/ml) as co-factors for RANKL (50 ng/ml) in the differentiation of OCs from primary adherent cell populations. This was done because although several in vitro and in vivo studies document a positive role for GM-CSF in osteoclastogenesis, 13,14 other experiments suggest that GM-CSF might inhibit OC formation driven by RANKL/M-CSF, 15,16 and our own initial studies with RTV used only RANKL/M-CSF. 9 In addition, circulating levels of GM-CSF are normal or elevated in HIV disease, whereas M-CSF levels might be suppressed.…”

FBPs Are Calibrated Molecular Tools To Adjust Gene Expression

“…The FBP level generally parallels c-myc expression (2); both are associated with proliferative states and are down-regulated upon differentiation. (Osteoclasts are an exception; during RANK ligand-induced osteoclast differentiation, FBP/FBP1 is up-regulated by over 40-fold [8].) Nuclear run-on demonstrates the transcriptional shutoff of FBP upon the differentiation of HL60 promyelomonocytic leukemia cells (2).…”

“…The wild type and mutant FBP NϩKH2ϩ3 (containing the N terminus fused to KH2ϩ3) were expressed with a six-His tag at their N termini and purified from the insoluble fraction using a His · Bind kit (Novagen) under 6 M-urea-denaturing conditions. Each pair of purified proteins was mixed with glutathione Sepharose in 1ϫ phosphate-buffered saline (PBS) containing 0.2% Triton X-100, incubated at 4°C for 1 h, washed three times using the same buffer, and eluted from beads into glutathione elution buffer (50 mM Tris-HCl [pH 8.0] and 10 mM reduced glutathione). The 29-mer from the FUSE noncoding strand (5Ј-GTATA TTCCC TCGGG ATTTT TTATT TTGT-3Ј) or the 40-mer from the FUSE coding strand (5Ј-AATAA CACAA AATAA AAAAT CCCGA GGGAA TATAC AT TAT-3Ј) was added to reaction mixtures at 500 nM where indicated in Fig.…”

Temperature-Regulated Microcolony Formation byBurkholderia pseudomalleiRequirespilAand Enhances Association with Cultured Human Cells

“…Bacterial cultures grown on agar were resuspended directly in 4 M guanidium isothiocyanate, 1% lauryl sarcosine solution. Following cell lysis, total RNA and first-strand cDNA were prepared as described previously (14). Quantitative real-time PCRs (Q-PCRs) were performed at an annealing temperature of 59°C or 62°C in 1ϫ SYBR green I supermix (Bio-Rad).…”