Interleukin-10 inhibits osteoclastogenesis by reducing NFATc1 expression and preventing its translocation to the nucleus

Abstract

Background: IL-10 has a potent inhibitory effect on osteoclastogenesis. In vitro and in vivo studies confirm the importance of this cytokine in bone metabolism, for instance IL-10-deficient mice develop the hallmarks of osteoporosis. Although it is known that IL-10 directly inhibits osteoclastogenesis at an early stage, preventing differentiation of osteoclast progenitors to preosteoclasts, the precise mechanism of its action is not yet clear. Several major pathways regulate osteoclastogenesis, with key signalling genes such as p38, TRAF6, NF-kappaB and NFATc1 well established as playing vital roles. We have looked at gene expression in eleven of these genes using real-time quantitative PCR on RNA extracted from RANKL-treated RAW264.7 monocytes.

Results: There was no downregulation by IL-10 of DAP12, FcgammaRIIB, c-jun, RANK, TRAF6, p38, NF-kappaB, Gab2, Pim-1, or c-Fos at the mRNA level. However, we found that IL-10 significantly reduces RANKL-induced NFATc1 expression. NFATc1 is transcribed from two alternative promoters in Mus musculus and, interestingly, only the variant transcribed from promoter P1 and beginning with exon 1 was downregulated by IL-10 (isoform 1). In addition, immunofluorescence studies showed that IL-10 reduces NFATc1 levels in RANKL-treated precursors and suppresses nuclear translocation. The inhibitory effect of IL-10 on tartrate-resistant acid phosphatase-positive cell number and NFATc1 mRNA expression was reversed by the protein kinase C agonist phorbol myristate acetate, providing evidence that interleukin-10 disrupts NFATc1 activity through its effect on Ca2+ mobilisation.

Conclusion: IL-10 acts directly on mononuclear precursors to inhibit NFATc1 expression and nuclear translocation, and we provide evidence that the mechanism may involve disruption of Ca2+ mobilisation. We detected downregulation only of the NFATc1 isoform 1 transcribed from promoter P1. This is the first report indicating that one of the ways in which IL-10 directly inhibits osteoclastogenesis is by suppressing NFATc1 activity.

Figure 1

Osteoclast formation is suppressed by IL-10. RAW264.7 cells were incubated with RANKL or RANKL plus IL-10 for 5 days. All cytokine concentrations were 50 ng/ml. Cells were fixed and stained for TRAP and the number of TRAP-positive mono and multinuclear osteoclasts quantified. Results are expressed as the mean ± SEM for each group from three separate experiments. IL-10 significantly suppressed osteoclast formation. * p < 0.05 versus control, ** p < 0.05 versus RANKL-treated group.

Figure 2

NFATc1 expression is suppressed by IL-10. RAW264.7 cells were incubated with cytokines (RANKL 50 ng/ml and IL-10 50 ng/ml) for 2 days and total RNA extracted. NFATc1 expression was assessed using quantitative PCR using specific primers recognising both isoforms (A) isoform 1 (B) or isoform 2 (C). Results are expressed as the mean ± SEM for each group from three separate experiments normalised for β-actin. * p < 0.05 versus control, ** p < 0.05 versus RANKL-treated group.

Figure 3

IL-10 reduces NFATc1 immunostaining intensity and nuclear translocation. NFATc1 staining was noted in the cytoplasm and nuclei of RANKL treated RAW264.7 cells (B and C), whereas only weak cytoplasmic staining was observed in RANKL + IL-10 treated cells (D.) No staining was noted in control cultures (A). Photographs taken at a magnification of × 400 A, B, D and ×1000 C.

Figure 4

PMA inhibits the suppressive effect of IL-10 on osteoclast formation. RAW264.7 cells were incubated with RANKL, IL-10 or PMA for 3 days. Cultures were washed to remove PMA and then incubated for a further two days with RANKL or RANKL + IL-10. Cells were fixed and stained for TRAP and the number of TRAP-positive mono and multinuclear osteoclasts quantified. IL-10 suppressed the number of TRAP positive cells and this was prevented by PMA. Results are expressed as the mean ± SEM for each group from three separate experiments n = 9. * p < 0.05 versus all other groups.