We showed that IL-20 was higher both in patients with osteoporotic bone loss and in OVX mice. IL-20 stimulates osteoclast differentiation and blocking IL-20 activity with the specific anti–IL-20 monoclonal antibody 7E has therapeutic potential for osteoporotic bone loss. The conventional wisdom is that M-CSF and RANKL are both necessary and sufficient for osteoclast differentiation. However, we showed that IL-20 endogenously secreted from HSCs is also essential for osteoclast differentiation; our evidence is that 7E completely blocked osteoclast differentiation in the presence of M-CSF and RANKL. In the correlation between IL-20 and the RANKL–RANK axis, we found that IL-20 up-regulated RANK expression on osteoclast precursors derived from bone marrow cells. In addition, IL-20 caused RANKL expression on osteoblasts. Even though TNF also promoted osteoclast formation, 7E potently inhibited osteoclast differentiation in the presence of TNF, which suggested that IL-20 may act independently on osteoclast differentiation. Treatment with 7E prevented IL-20 from inducing RANK and RANKL expression. IL-20–induced osteoclast differentiation was inhibited in IL-20R1−/− mouse-derived osteoclast precursor cells. We therefore conclude that IL-20R1 is important in IL-20–mediated osteoclastogenesis, and that IL-20 is an upstream activator of RANKL-RANK signaling. IL-20 is pivotal in osteoclast differentiation because it targeted not only osteoclast precursor cells but also osteoblasts.
Osteoclasts originate from hematopoietic progenitor cells, probably from CFU granulocyte-macrophages (GMs; Menaa et al., 2000
). CFU-GMs are different from multipotential progenitor cells, CFU-GEMMs. One study (Liu et al., 2003
) showed that IL-20 specifically promoted the in vitro and in vivo proliferation of CFU-GEMMs. We showed that M-CSF up-regulated IL-20 in osteoclast precursor cells. IL-20, in an autocrine manner, targeted osteoclast precursor cells and increased the expression of RANK. In addition, IL-20 activated osteoclastogenic signaling and 7E inhibited the activation of NFATc1, the master regulator for osteoclast differentiation. Therefore, we hypothesize that IL-20 contributes to osteoclastogenesis by promoting both the survival and the expansion of osteoclast precursor cells and increasing RANKL-induced activity. In the osteoclast differentiation assay, 7E blocked both early and later stage differentiation, which supports our hypothesis.
Cathepsin G is also a chemoattractant for osteoclast precursors by proteolytically activating PAR-1 (Wilson et al., 2009
). In the present study, IL-20 up-regulated the expression of cathepsin G in osteoclasts, which dose-dependently induced the cleavage of RANKL from the surface membrane of osteoblasts. Therefore, we speculate that IL-20 acts on osteoclasts to produce a large amount of cathepsin G in bone microenvironment. The IL-20–induced cathepsin G, in turn, recruits more osteoclast precursors that differentiate into mature osteoclasts and produce more cathepsin G, which leads to the generation of sRANKL.
The RANKL–RANK signaling mechanism is one pathway of osteoclast formation and activity (Kong et al., 1999
; Li et al., 2000
). We found that IL-20 increased RANKL mRNA and RANKL protein expression in osteoblasts, which suggested that IL-20 is an upstream regulator for RANKL expression. RANKL expression is also up-regulated in many malignant tumor cells, and is involved in cancer-associated bone destruction, tumor metastasis, and tumor-induced osteolysis (Park et al., 2007
; Wilson et al., 2008
). We previously (Hsing et al., 2006
) showed that IL-20 is expressed in squamous cell carcinoma of the skin, tongue, esophagus, and lung. However, the association of IL-20 with tumor progression and metastasis has not been clarified. It is therefore necessary to explore the effects of 7E on cancer-associated bone metastasis and bone erosion.
Soluble RANK antagonists and RANKL inhibitors such as osteoprotegerin and anti-RANKL antibody are inhibitors of bone resorption in clinical studies, presumably because of their effects on osteoclasts. One therapeutic drug to treat osteoporosis is denosumab, an anti-RANKL antibody (Kostenuik, 2005
; McClung, 2006
). Our results also showed that 7E protected OVX mice against osteoporotic bone loss and increased their BMD. In the in vitro assay, we showed that 7E reduced not only the number of TRAP+
osteoclasts, but also the expression of RANK in M-CSF–derived osteoclast precursor cells and of RANKL in osteoblasts. In addition, 7E almost totally prevented the RANKL-induced activation of TRAF6, c-Fos, NFATc1, and STAT3 because it down-regulated RANK expression in osteoclast precursors. Thus, 7E shows promise as a therapeutic drug for protection against bone destruction.
BMD is regulated by osteoclastogenic and osteoblastogenic activity (Walsh et al., 2005
). IL-20R1 deficiency protected mice from OVX-induced bone loss. This finding is consistent with our finding that serum IL-20 levels were higher in patients with osteopenia and osteoporosis than in healthy controls. This suggested that the osteoclastogenic activity of IL-20 might be activated only during pathological conditions such as estrogen deficiency or OVX-induced osteoporosis. To prove this, we treated healthy WT mice with 7E and found that it had no significant effect on their BMD compared with untreated mice. In addition, there was no significant difference in serum IL-20 levels between sham-operated mice treated with PBS and those treated with 7E, which indicated that serum IL-20 antibody from 7E treatment did not interfere with the ELISA analysis of IL-20 in 7E-treated OVX mice (unpublished data). Moreover, we still observed some in vitro osteoclast differentiation from osteoclast precursor cells of IL-20R1−/−
mice. This may be attributable to the alternative IL-20R2–IL-22R1 receptor complex signaling. Expression of all the three receptor subunits on osteoclast precursor cells suggested this possibility. In vitro osteoclast differentiation was inhibited in osteoclast precursor cells derived from IL-20R1−/−
mice with no effect of addition of IL-20. We hypothesize that the response threshold of IL-20 in IL-20R1−/−
mice is much higher than in WT mice because of different receptor complex signaling. No significant difference in BMD was observed in the 8-wk-old IL-20R1+/+
, and IL-20R1−/−
mice. However, IL-20R1−/−
mice had significantly higher BMD than did IL-20R1+/+
mice when they reached the age of 28 wk. This difference raised the possibility that an age-related estrogen deficiency might affect IL-20 levels, the activity of which was inhibited in IL-20R1−/−
mice. Additional studies are needed to explore the detailed molecular mechanism of estrogen’s regulation of IL-20. The intercross co-cultures of osteoclast precursors with osteoblastic cells from WT and IL-20R1−/−
mice demonstrated that the deficiency of IL-20R1 affected both osteoclast precursors and osteoblasts that impaired osteoclast differentiation. The co-cultures of IL-20R1−/−
osteoclast precursors with WT osteoblastic cells did not show as much impairment of osteoclast differentiation as the osteoblast-free system (). This may be attributed to IL-20–induced TNF production in the WT osteoblasts. Thus, IL-20 and IL-20R1 signaling are crucial not only in osteoclast differentiation, but also in the function of osteoblast. These findings support the hypothesis that IL-20 produced by osteoblasts and osteoclasts stimulate IL-20R1 signaling, which mediates the cross talk between osteoclasts and osteoblasts and regulates the bone remodeling. Furthermore, whether IL-20 is also involved in osteoblast differentiation is not well understood. It is necessary to further investigate the mechanism that IL-20 uses to maintain the balance between osteoclastogenesis and osteoblastogenesis in metabolic bone destruction and inflammatory bone diseases.
In summary, our findings provide evidence that IL-20 is an osteoclastogenic cytokine that up-regulates RANK on osteoclast precursor cells and RANKL on osteoblasts and affects the functions of both cells. We identified a pivotal role of IL-20 in osteoclast differentiation, and we conclude that using 7E to inhibit IL-20 is a potential therapeutic strategy for protecting against osteoporotic bone loss.