BACKGROUND
osteoclasts are terminally differentiated trap-positive cells derived from monocyte-macrophage lineage precursors and are responsible for bone resorption.
the process of osteoclast formation includes the proliferation and the differentiation of osteoclast progenitors into mononuclear preosteoclasts and the fusion of preosteoclasts into multinucleated osteoclasts.
excessive osteoclast activity plays a role in the development of several debilitating disorders such as osteoporosis, rheumatoid arthritis, and osteolytic metastases, and so it is vital that osteoclast development and function is properly regulated.
some of the signalling pathways that contribute to osteoclast differentiation have started to emerge.
signalling by rankl, an osteoblast-expressed member of the tumour necrosis factor superfamily, is essential for terminal differentiation of monocytes into osteoclasts.
resorptive stimuli initiate osteoclast formation by promoting the expression of osteoblastic rankl, which binds to its receptor rank.
binding of rankl to rank recruits several signalling intermediates including nf-κb, which is redistributed to the nucleus by traf family intermediates  <cit> , and nfatc <dig>  which induce the transcription of genes involved in osteoclast differentiation.
nfatc <dig> is critical to osteoclast formation  <cit> .
negative regulators of rankl signalling exist, including osteoprotegerin, a soluble decoy receptor for rankl, and ifn-β, a negative regulator of transcription factor c-fos expression  <cit> .
several cytokines, including ifn-γ, il- <dig>  and another tumour necrosis factor family member, trail, interfere with the ability of rankl to induce osteoclast differentiation.
there is rapid degradation of traf <dig> abolishing signalling in the case of ifn-γ  <cit> .
in the case of il- <dig>  signal transducer and activator of transcription  <dig> appears to be a key upstream regulatory molecule  <cit> .
in the case of trail, the mechanism is related to inhibition of the p38/mapk pathway via reduced p38/mapk phosphorylation  <cit> .
il- <dig> is a potent anti-inflammatory cytokine that suppresses both immunoproliferative and inflammatory responses.
it is produced primarily from t cells and activated macrophages and acts on the macrophage lineage.
animal studies implicate il- <dig> in the development and progression of arthritis  <cit>  and chronic colitis  <cit> .
il- <dig> has a critical role in the in vivo regulation of pro-inflammatory cytokine levels  <cit> .
it functions in a negative feedback loop, in which it suppresses the release of inflammatory cytokines and dampens the acute inflammatory response.
il- <dig> also has potent inhibitory effects on osteoclastogenesis  <cit> , but the molecular basis of its action is poorly understood.
il- <dig> inhibits the early stages of osteoclastogenesis, preventing differentiation of osteoclast progenitors to preosteoclasts.
it has been shown to mediate this action through both direct and indirect actions.
il- <dig> indirectly inhibits bone resorption by upregulating osteoprotegerin expression while downregulating expression of rankl and macrophage colony stimulating factor  <cit> .
il- <dig> also directly inhibits osteoclast formation  <cit> .
it was found that the inhibitory effect of il- <dig> on osteoclast formation is mediated through a direct action on osteoclast precursors  <cit> .
furthermore, enhanced osteoclastogenesis has been observed in cultures of bone marrow macrophages deficient in il- <dig> production  <cit> .
however, the molecular mechanism mediating this direct effect is unknown.
il-10-deficient mice are now available and results from these mice confirm the importance of this cytokine in bone metabolism.
they have been shown to develop the hallmarks of osteoporosis, namely, reduced bone mass, increased mechanical fragility, and suppressed bone formation  <cit> .
il- <dig> is an important endogenous suppressor of infection-stimulated bone resorption in vivo, and it appears that the anti-osteoclastic mechanisms induced by il- <dig> on bone are more effective than those mediated by il- <dig>  <cit> .
il-10-deficient mice exhibit significantly higher levels of periodontal alveolar bone loss than a matched control group  <cit> .
this increased alveolar bone loss appears to be a late onset condition, and analysis of serum levels of type i collagen c-telopeptide suggests that lack of il- <dig> may have a direct effect on bone homeostasis  <cit> .
clinical studies back up the theoretical importance of this work: an il- <dig> promoter gene polymorphism has been found that is associated with reduced bone mineral density and predisposes women to osteoporosis at the lumbar spine  <cit> .
in this paper we set out to learn more about how interleukin- <dig> directly inhibits the process of osteoclastogenesis by looking at its effects on selected gene expression in raw <dig> , a murine monocytic cell line.
RESULTS
il- <dig> suppresses osteoclast formation
three groups of raw <dig>  cells were set up – an untreated control group, a group treated with rankl, and a final group treated with rankl plus il- <dig>  as shown previously, after five days numerous mono and multinuclear trap-positive cells were seen in the rankl-treated cultures.
the percentage of trap-stained cells in each group was determined by light microscopy and it was found that il- <dig> significantly inhibits osteoclast formation but does not abolish it completely .
these results indicate that il- <dig> acts directly on monocytes to inhibit osteoclast differentiation.
nfatc <dig> is downregulated by il-10
rankl was found to induce the expression of nfatc <dig> mrna approximately 8-fold, and this was significantly suppressed by co-incubation with il- <dig> .
this is in keeping with the direct action of il- <dig> on osteoclast formation.
nfatc <dig> expression was not however completely abolished.
isoforms of nfatc <dig> are known to exist in both humans  <cit>  and mice  <cit> .
these isoforms are transcribed from two separate promoters, which can be arbitrarily designated p <dig> and p <dig>  transcripts beginning from p <dig> start with exon  <dig>  and are longer than those beginning from p <dig> and starting with exon  <dig> .
it is possible to design primers to differentiate between these variants and we did this.
we found that raw <dig>  cells express both nfatc <dig> isoforms  and rankl induced a significant  <dig> -fold increase in isoform  <dig> expression, which il- <dig> suppressed .
in contrast rankl and il- <dig> had little effect on the expression of isoform  <dig> .
therefore, it appears that il- <dig> may suppress osteoclastogenesis by blunting expression of nfatc <dig> isoform  <dig> rather than isoform  <dig>  because nfatc <dig> is central to osteoclast formation, any change in its expression would be expected to have a major effect on downstream gene expression.
expression of other osteoclast signalling genes is unaffected
none of the other osteoclast signalling genes tested – rank, traf <dig>  p <dig>  nf-κb, c-fos, c-jun, fcγriib, dap <dig>  gab <dig>  and pim <dig> – were upregulated as strongly as nfatc <dig> by rankl, and none of them were downregulated by il- <dig> .
the key signalling factors known to trigger nfatc <dig> expression are traf <dig> and c-fos  <cit> , and so the lack of response of these genes in our experiments suggests that the effect of il- <dig> on monocytes is not mediated through these pathways, in which case it must be mediated through an alternative mechanism.
il- <dig> alters cellular localisation of nfatc1
nfatc <dig> is the most strongly induced transcription factor gene following rankl stimulation  <cit> .
rankl also evokes ca2+ oscillations that lead to calcineurin-mediated activation of nfatc <dig>  in resting t cells nfatc <dig> is restricted to the cytoplasm.
following t-cell activation, a sustained increase in intracellular calcium activates the phosphatase calcineurin.
the activated calcineurin dephosphorylates nfatc <dig> and leads to increased nuclear accumulation.
therefore it was of interest to determine the cellular location of nfatc <dig> in the rankl- and il-10-treated monocytes.
in keeping with our qpcr results, rankl-treated cells showed intense cytoplasmic staining and nuclear accumulation was also noted .
similarly, again in keeping with our qpcr results, il- <dig> reduced the intensity of cytoplasmic staining and little nuclear accumulation was observed in these cultures.
this correlates with nfatc <dig> being highly active in the rankl-treated cells and less active in the presence of il- <dig>
il- <dig> may disrupt nfatc <dig> activity through its effect on ca2+ mobilisation
pkc plays a critical role in t cell receptor-induced nfat activation  <cit> .
deficiency of pkc primarily abrogates nfat transactivation.
this nfat defect appears to be secondary to reduced inositol  <dig> ,5-triphosphate generation and intracellular ca2+ mobilisation.
since we had not found any effect of il- <dig> on traf <dig> or c-fos, we wanted to see if we could find any evidence that il- <dig> is acting through its effects on ca2+ mobilisation, and this was done using the pkc agonist pma.
cells were treated with rankl, rankl plus il- <dig>  and rankl plus il- <dig> plus pma, and viewed under the microscope.
the suppressive effect of il- <dig> was abolished by pma treatment, as determined by extent of trap staining .
quantitative pcr results were in agreement, with pma reversing the inhibitory effect of il- <dig> on nfatc <dig> expression .
quantitative rt-pcr analysis of nfatc <dig> mrna expression in raw <dig>  cells.
cultures were treated with rankl, rankl and il- <dig> or rankl, il- <dig> and pma for  <dig> hours and total rna extracted.
quantitative pcr was performed on a techne quantica pcr machine using the dna binding dye sybr-green and specific primers for murine nfatc <dig> and β-actin.
for each sample the nfatc <dig> copy number was expressed as the relative copy number normalised to  <dig> β-actin copies.
samples were analysed in triplicate and are expressed as the mean of three separate experiments.
* p <  <dig>  versus rankl group.
discussion
il- <dig> is an anti-inflammatory cytokine that suppresses both immunoproliferative and inflammatory responses.
it is produced primarily from t cells and activated macrophages.
animal studies implicate il- <dig> in the development and progression of arthritis and chronic colitis.
il- <dig> has a critical role in the in vivo regulation of pro-inflammatory cytokine levels.
it functions in a negative feedback loop, in which it suppresses the release of inflammatory cytokines and dampens the acute inflammatory response.
clinical studies and il-10-deficient mice back up the theoretical importance of this work, as discussed in the introduction.
il- <dig> has been known for some time to have a potent inhibitory effect on osteoclastogenesis  <cit> .
it prevents differentiation of osteoclast progenitors to preosteoclasts, however its mechanism of action is still unclear.
we have studied the expression of eleven osteoclast signalling genes and have found that one of the ways in which il- <dig> directly inhibits osteoclastogenesis is by disrupting nfatc <dig> mrna expression and nuclear translocation.
nfatc <dig> is crucial for osteoclastogenesis, and so its downregulation may be expected to have major downstream effects.
we also provide evidence that this disruption to nfatc <dig> may be mediated through the effect of il- <dig> on ca2+ mobilisation.
in addition, it was found that only nfatc <dig> isoform  <dig> transcribed from promoter p <dig> was downregulated by il- <dig>
osteoclast formation was not abolished in the presence of il- <dig> and similarly nfatc <dig> levels did not revert to basal level in the il- <dig> treated cells.
several pathways contribute to induction of nfatc <dig> levels and it may well be the case that il- <dig> does not inhibit all of them.
the initial expression of nfatc <dig> is dependent on both the traf <dig> and c-fos pathways, which are unaffected by il- <dig>  rankl stimulation also results in the induction of ca2+ oscillations which activate nfatc <dig> via a calcineurin-dependent mechanism, targeting it to the nucleus  <cit> .
for the generation of osteoclasts it has been demonstrated that the high nfatc <dig> levels generated by autoregulation are necessary for cell lineage commitment .
therefore the reduction in levels, rather than complete abrogation, brought about by il- <dig> will be sufficient to cause the potent inhibition of osteoclastogenesis shown in these studies.
there are two variants of nfatc <dig> in mus musculus but only one of these, isoform <dig>  was downregulated by il- <dig>  it is not known why the shorter isoform  <dig> is not similarly modified by il- <dig>  this may in part be related to the relative inability of rankl to induce the expression of isoform  <dig> in raw cells, or it could be that the conformation of the dna is such that il-10-induced transcription factors only have access to the p <dig> promoter.
linked in with this, it is likely that the finding that nfatc <dig> expression was not completely abolished is due to a combination of residual expression from the p <dig> promoter and the inability of il- <dig> induced factors to suppress basal expression from the p <dig> promoter.
alternatively, it could also be that il- <dig> acts only on a subset of the various pathways that contribute to nfatc <dig> upregulation.
the activity of nfatc <dig> is not only dependent on its level of expression, but also its location within the cell.
it is active only when in the nucleus, as would be expected of a transcription factor.
this nuclear transport relies upon active calcineurin which triggers dephosphorylation of nfatc <dig>  therefore we used antibodies to establish the intracellular distribution of nfatc <dig>  the fact that very little staining, and in particular nuclear staining, was observed in the il- <dig> sample strengthens our evidence that the inhibitory effect of il- <dig> on osteoclastogenesis can be explained at least in part by its effect on nfatc <dig> expression and activity.
pkc plays a critical role in t cell receptor-induced nfat activation  <cit> .
deficiency of pkc primarily abrogates nfat transactivation.
this nfat transactivation defect appears to be secondary to reduced inositol  <dig> ,5-triphosphate generation and intracellular ca2+ mobilisation.
therefore, we set out to test whether il- <dig> is disrupting nfatc <dig> activity through its effect on ca2+ mobilisation.
we found that pma inhibits the suppressive effect of il- <dig> on osteoclast formation, as determined by trap staining and nfatc <dig> expression.
pma is an activator of pkc, and so if pma is overcoming the suppressive effects of il- <dig>  knowing how pma acts it would appear that il- <dig> is disrupting nfatc <dig> activity through its effect on ca2+ mobilisation.
therefore the effect of il- <dig> could involve pkc as pma reverses the suppressive action of il- <dig>
the mechanism by which il- <dig> interferes with osteoclast formation appears to be different from that used by other inhibitors, namely ifn-γ, il- <dig>  and trail.
il- <dig> also downregulates nfatc <dig> but it acts through suppression of c-fos  <cit> , we did not find any evidence for c-fos being downregulated although it remains possible that it could be inactivated by other means.
ifn-γ causes rapid degradation of traf <dig> protein; we found no difference in traf <dig> mrna levels but did not look at protein levels.
likewise, trail interferes with the p <dig> pathway and there was no downregulation of p <dig> mrna in il- <dig> treated cultures.
it is valuable to pinpoint the effect of il- <dig> on osteoclast signalling pathways since evidence is accumulating, particularly from studies of il-10-deficient mice, to illustrate the importance of this cytokine in bone homeostasis.
we have demonstrated that one of the ways by which il- <dig> directly inhibits osteoclast formation is to suppress nfatc <dig> expression and activation.
in vivo, il- <dig> may be expected to have an even more pronounced effect due to its indirect action of suppressing rankl expression  <cit>  which would further reduce nfatc <dig> activation through traf <dig>  this is the first report indicating that one of the ways in which il- <dig> inhibits osteoclastogenesis is by suppressing nfatc <dig> activity.
CONCLUSIONS
il- <dig> acts directly on mononuclear precursors to inhibit nfatc <dig> expression and nuclear translocation, and we provide evidence that the mechanism may involve disruption of ca2+ mobilisation.
we detected downregulation only of the nfatc <dig> isoform transcribed from promoter p <dig> and beginning with exon  <dig>  this is the first report indicating that one of the ways in which il- <dig> directly inhibits osteoclastogenesis is by suppressing nfatc <dig> activity.

 osteoclasts are terminally differentiated trap-positive cells derived from monocyte-macrophage lineage precursors and are responsible for bone resorption. the process of osteoclast formation includes the proliferation and the differentiation of osteoclast progenitors into mononuclear preosteoclasts and the fusion of preosteoclasts into multinucleated osteoclasts. excessive osteoclast activity plays a role in the development of several debilitating disorders such as osteoporosis, rheumatoid arthritis, and osteolytic metastases, and so it is vital that osteoclast development and function is properly regulated. some of the signalling pathways that contribute to osteoclast differentiation have started to emerge. signalling by rankl, an osteoblast-expressed member of the tumour necrosis factor superfamily, is essential for terminal differentiation of monocytes into osteoclasts. resorptive stimuli initiate osteoclast formation by promoting the expression of osteoblastic rankl, which binds to its receptor rank. binding of rankl to rank recruits several signalling intermediates including nf-κb, which is redistributed to the nucleus by traf family intermediates, and nfatc which induce the transcription of genes involved in osteoclast differentiation. nfatc is critical to osteoclast formation. negative regulators of rankl signalling exist, including osteoprotegerin, a soluble decoy receptor for rankl, and ifn-β, a negative regulator of transcription factor c-fos expression. several cytokines, including ifn-γ, il- and another tumour necrosis factor family member, trail, interfere with the ability of rankl to induce osteoclast differentiation. there is rapid degradation of traf abolishing signalling in the case of ifn-γ. in the case of il- signal transducer and activator of transcription appears to be a key upstream regulatory molecule. in the case of trail, the mechanism is related to inhibition of the p38/mapk pathway via reduced p38/mapk phosphorylation. il- is a potent anti-inflammatory cytokine that suppresses both immunoproliferative and inflammatory responses. it is produced primarily from t cells and activated macrophages and acts on the macrophage lineage. animal studies implicate il- in the development and progression of arthritis and chronic colitis. il- has a critical role in the in vivo regulation of pro-inflammatory cytokine levels. it functions in a negative feedback loop, in which it suppresses the release of inflammatory cytokines and dampens the acute inflammatory response. il- also has potent inhibitory effects on osteoclastogenesis, but the molecular basis of its action is poorly understood. il- inhibits the early stages of osteoclastogenesis, preventing differentiation of osteoclast progenitors to preosteoclasts. it has been shown to mediate this action through both direct and indirect actions. il- indirectly inhibits bone resorption by upregulating osteoprotegerin expression while downregulating expression of rankl and macrophage colony stimulating factor. il- also directly inhibits osteoclast formation. it was found that the inhibitory effect of il- on osteoclast formation is mediated through a direct action on osteoclast precursors. furthermore, enhanced osteoclastogenesis has been observed in cultures of bone marrow macrophages deficient in il- production. however, the molecular mechanism mediating this direct effect is unknown. il-10-deficient mice are now available and results from these mice confirm the importance of this cytokine in bone metabolism. they have been shown to develop the hallmarks of osteoporosis, namely, reduced bone mass, increased mechanical fragility, and suppressed bone formation. il- is an important endogenous suppressor of infection-stimulated bone resorption in vivo, and it appears that the anti-osteoclastic mechanisms induced by il- on bone are more effective than those mediated by il-. il-10-deficient mice exhibit significantly higher levels of periodontal alveolar bone loss than a matched control group. this increased alveolar bone loss appears to be a late onset condition, and analysis of serum levels of type i collagen c-telopeptide suggests that lack of il- may have a direct effect on bone homeostasis. clinical studies back up the theoretical importance of this work: an il- promoter gene polymorphism has been found that is associated with reduced bone mineral density and predisposes women to osteoporosis at the lumbar spine. in this paper we set out to learn more about how interleukin- directly inhibits the process of osteoclastogenesis by looking at its effects on selected gene expression in raw, a murine monocytic cell line.  il- suppresses osteoclast formation three groups of raw cells were set up – an untreated control group, a group treated with rankl, and a final group treated with rankl plus il- as shown previously, after five days numerous mono and multinuclear trap-positive cells were seen in the rankl-treated cultures. the percentage of trap-stained cells in each group was determined by light microscopy and it was found that il- significantly inhibits osteoclast formation but does not abolish it completely. these results indicate that il- acts directly on monocytes to inhibit osteoclast differentiation. nfatc is downregulated by il-10 rankl was found to induce the expression of nfatc mrna approximately 8-fold, and this was significantly suppressed by co-incubation with il-. this is in keeping with the direct action of il- on osteoclast formation. nfatc expression was not however completely abolished. isoforms of nfatc are known to exist in both humans and mice. these isoforms are transcribed from two separate promoters, which can be arbitrarily designated p and p transcripts beginning from p start with exon and are longer than those beginning from p and starting with exon. it is possible to design primers to differentiate between these variants and we did this. we found that raw cells express both nfatc isoforms and rankl induced a significant -fold increase in isoform expression, which il- suppressed. in contrast rankl and il- had little effect on the expression of isoform. therefore, it appears that il- may suppress osteoclastogenesis by blunting expression of nfatc isoform rather than isoform because nfatc is central to osteoclast formation, any change in its expression would be expected to have a major effect on downstream gene expression. expression of other osteoclast signalling genes is unaffected none of the other osteoclast signalling genes tested – rank, traf p nf-κb, c-fos, c-jun, fcγriib, dap gab and pim – were upregulated as strongly as nfatc by rankl, and none of them were downregulated by il-. the key signalling factors known to trigger nfatc expression are traf and c-fos, and so the lack of response of these genes in our experiments suggests that the effect of il- on monocytes is not mediated through these pathways, in which case it must be mediated through an alternative mechanism. il- alters cellular localisation of nfatc1 nfatc is the most strongly induced transcription factor gene following rankl stimulation. rankl also evokes ca2+ oscillations that lead to calcineurin-mediated activation of nfatc in resting t cells nfatc is restricted to the cytoplasm. following t-cell activation, a sustained increase in intracellular calcium activates the phosphatase calcineurin. the activated calcineurin dephosphorylates nfatc and leads to increased nuclear accumulation. therefore it was of interest to determine the cellular location of nfatc in the rankl- and il-10-treated monocytes. in keeping with our qpcr results, rankl-treated cells showed intense cytoplasmic staining and nuclear accumulation was also noted. similarly, again in keeping with our qpcr results, il- reduced the intensity of cytoplasmic staining and little nuclear accumulation was observed in these cultures. this correlates with nfatc being highly active in the rankl-treated cells and less active in the presence of il- il- may disrupt nfatc activity through its effect on ca2+ mobilisation pkc plays a critical role in t cell receptor-induced nfat activation. deficiency of pkc primarily abrogates nfat transactivation. this nfat defect appears to be secondary to reduced inositol,5-triphosphate generation and intracellular ca2+ mobilisation. since we had not found any effect of il- on traf or c-fos, we wanted to see if we could find any evidence that il- is acting through its effects on ca2+ mobilisation, and this was done using the pkc agonist pma. cells were treated with rankl, rankl plus il- and rankl plus il- plus pma, and viewed under the microscope. the suppressive effect of il- was abolished by pma treatment, as determined by extent of trap staining. quantitative pcr results were in agreement, with pma reversing the inhibitory effect of il- on nfatc expression. quantitative rt-pcr analysis of nfatc mrna expression in raw cells. cultures were treated with rankl, rankl and il- or rankl, il- and pma for hours and total rna extracted. quantitative pcr was performed on a techne quantica pcr machine using the dna binding dye sybr-green and specific primers for murine nfatc and β-actin. for each sample the nfatc copy number was expressed as the relative copy number normalised to β-actin copies. samples were analysed in triplicate and are expressed as the mean of three separate experiments. * p < versus rankl group. discussion il- is an anti-inflammatory cytokine that suppresses both immunoproliferative and inflammatory responses. it is produced primarily from t cells and activated macrophages. animal studies implicate il- in the development and progression of arthritis and chronic colitis. il- has a critical role in the in vivo regulation of pro-inflammatory cytokine levels. it functions in a negative feedback loop, in which it suppresses the release of inflammatory cytokines and dampens the acute inflammatory response. clinical studies and il-10-deficient mice back up the theoretical importance of this work, as discussed in the introduction. il- has been known for some time to have a potent inhibitory effect on osteoclastogenesis. it prevents differentiation of osteoclast progenitors to preosteoclasts, however its mechanism of action is still unclear. we have studied the expression of eleven osteoclast signalling genes and have found that one of the ways in which il- directly inhibits osteoclastogenesis is by disrupting nfatc mrna expression and nuclear translocation. nfatc is crucial for osteoclastogenesis, and so its downregulation may be expected to have major downstream effects. we also provide evidence that this disruption to nfatc may be mediated through the effect of il- on ca2+ mobilisation. in addition, it was found that only nfatc isoform transcribed from promoter p was downregulated by il- osteoclast formation was not abolished in the presence of il- and similarly nfatc levels did not revert to basal level in the il- treated cells. several pathways contribute to induction of nfatc levels and it may well be the case that il- does not inhibit all of them. the initial expression of nfatc is dependent on both the traf and c-fos pathways, which are unaffected by il- rankl stimulation also results in the induction of ca2+ oscillations which activate nfatc via a calcineurin-dependent mechanism, targeting it to the nucleus. for the generation of osteoclasts it has been demonstrated that the high nfatc levels generated by autoregulation are necessary for cell lineage commitment. therefore the reduction in levels, rather than complete abrogation, brought about by il- will be sufficient to cause the potent inhibition of osteoclastogenesis shown in these studies. there are two variants of nfatc in mus musculus but only one of these, isoform was downregulated by il- it is not known why the shorter isoform is not similarly modified by il- this may in part be related to the relative inability of rankl to induce the expression of isoform in raw cells, or it could be that the conformation of the dna is such that il-10-induced transcription factors only have access to the p promoter. linked in with this, it is likely that the finding that nfatc expression was not completely abolished is due to a combination of residual expression from the p promoter and the inability of il- induced factors to suppress basal expression from the p promoter. alternatively, it could also be that il- acts only on a subset of the various pathways that contribute to nfatc upregulation. the activity of nfatc is not only dependent on its level of expression, but also its location within the cell. it is active only when in the nucleus, as would be expected of a transcription factor. this nuclear transport relies upon active calcineurin which triggers dephosphorylation of nfatc therefore we used antibodies to establish the intracellular distribution of nfatc the fact that very little staining, and in particular nuclear staining, was observed in the il- sample strengthens our evidence that the inhibitory effect of il- on osteoclastogenesis can be explained at least in part by its effect on nfatc expression and activity. pkc plays a critical role in t cell receptor-induced nfat activation. deficiency of pkc primarily abrogates nfat transactivation. this nfat transactivation defect appears to be secondary to reduced inositol,5-triphosphate generation and intracellular ca2+ mobilisation. therefore, we set out to test whether il- is disrupting nfatc activity through its effect on ca2+ mobilisation. we found that pma inhibits the suppressive effect of il- on osteoclast formation, as determined by trap staining and nfatc expression. pma is an activator of pkc, and so if pma is overcoming the suppressive effects of il- knowing how pma acts it would appear that il- is disrupting nfatc activity through its effect on ca2+ mobilisation. therefore the effect of il- could involve pkc as pma reverses the suppressive action of il- the mechanism by which il- interferes with osteoclast formation appears to be different from that used by other inhibitors, namely ifn-γ, il- and trail. il- also downregulates nfatc but it acts through suppression of c-fos, we did not find any evidence for c-fos being downregulated although it remains possible that it could be inactivated by other means. ifn-γ causes rapid degradation of traf protein; we found no difference in traf mrna levels but did not look at protein levels. likewise, trail interferes with the p pathway and there was no downregulation of p mrna in il- treated cultures. it is valuable to pinpoint the effect of il- on osteoclast signalling pathways since evidence is accumulating, particularly from studies of il-10-deficient mice, to illustrate the importance of this cytokine in bone homeostasis. we have demonstrated that one of the ways by which il- directly inhibits osteoclast formation is to suppress nfatc expression and activation. in vivo, il- may be expected to have an even more pronounced effect due to its indirect action of suppressing rankl expression which would further reduce nfatc activation through traf this is the first report indicating that one of the ways in which il- inhibits osteoclastogenesis is by suppressing nfatc activity.  il- acts directly on mononuclear precursors to inhibit nfatc expression and nuclear translocation, and we provide evidence that the mechanism may involve disruption of ca2+ mobilisation. we detected downregulation only of the nfatc isoform transcribed from promoter p and beginning with exon this is the first report indicating that one of the ways in which il- directly inhibits osteoclastogenesis is by suppressing nfatc activity. 
il- has a potent inhibitory effect on osteoclastogenesis. the inhibitory effect of il- on tartrate-resistant acid phosphatase-positive cell number and nfatc mrna expression was reversed by the protein kinase c agonist phorbol myristate acetate, providing evidence that interleukin- disrupts nfatc activity through its effect on ca2+ mobilisation. il- acts directly on mononuclear precursors to inhibit nfatc expression and nuclear translocation, and we provide evidence that the mechanism may involve disruption of ca2+ mobilisation. in addition, immunofluorescence studies showed that il- reduces nfatc levels in rankl-treated precursors and suppresses nuclear translocation. although it is known that il- directly inhibits osteoclastogenesis at an early stage, preventing differentiation of osteoclast progenitors to preosteoclasts, the precise mechanism of its action is not yet clear. we detected downregulation only of the nfatc isoform transcribed from promoter p this is the first report indicating that one of the ways in which il- directly inhibits osteoclastogenesis is by suppressing nfatc activity. we have looked at gene expression in eleven of these genes using real-time quantitative pcr on rna extracted from rankl-treated raw monocytes. 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. nfatc is transcribed from two alternative promoters in mus musculus and, interestingly, only the variant transcribed from promoter p and beginning with exon was downregulated by il-.
 il- 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- 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 p traf nf-κb and nfatc 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 raw monocytes.  there was no downregulation by il- of dap fcγriib, c-jun, rank, traf p nf-κb, gab pim- or c-fos at the mrna level. however, we found that il- significantly reduces rankl-induced nfatc expression. nfatc is transcribed from two alternative promoters in mus musculus and, interestingly, only the variant transcribed from promoter p and beginning with exon was downregulated by il-. in addition, immunofluorescence studies showed that il- reduces nfatc levels in rankl-treated precursors and suppresses nuclear translocation. the inhibitory effect of il- on tartrate-resistant acid phosphatase-positive cell number and nfatc mrna expression was reversed by the protein kinase c agonist phorbol myristate acetate, providing evidence that interleukin- disrupts nfatc activity through its effect on ca2+ mobilisation.  il- acts directly on mononuclear precursors to inhibit nfatc expression and nuclear translocation, and we provide evidence that the mechanism may involve disruption of ca2+ mobilisation. we detected downregulation only of the nfatc isoform transcribed from promoter p this is the first report indicating that one of the ways in which il- directly inhibits osteoclastogenesis is by suppressing nfatc activity. 