Endogenous production of TGF-beta is essential for osteoclastogenesis induced by a combination of receptor activator of NF-kappa B ligand and macrophage-colony-stimulating factor

Differentiation of osteoclasts, the cells primarily responsible for bone resorption, is controlled by a variety of osteotropic hormones and cytokines. Of these factors, receptor activator of NF-kappaB (RANK) ligand (RANKL) has been recently cloned as an essential inducer of osteoclastogenesis in the presence of M-CSF. Here, we isolated a stroma-free population of monocyte/macrophage (M/Mphi)-like hemopoietic cells from mouse unfractionated bone cells that were capable of differentiating into mature osteoclasts by treatment with soluble RANKL (sRANKL) and M-CSF. However, the efficiency of osteoclast formation was low, suggesting the requirement for additional factors. The isolated M/Mphi-like hemopoietic cells expressed TGF-beta and type I and II receptors of TGF-beta. Therefore, we examined the effect of TGF-beta on osteoclastogenesis. TGF-beta with a combination of sRANKL and M-CSF promoted the differentiation of nearly all M/Mphi-like hemopoietic cells into cells of the osteoclast lineage. Neutralizing anti-TGF-beta Ab abrogated the osteoclast generation. These TGF-beta effects were also observed in cultures of unfractionated bone cells, and anti-TGF-beta blocked the stimulatory effect of 1, 25-dihydroxyvitamin D(3). Translocation of NF-kappaB into nuclei induced by sRANKL in TGF-beta-pretreated M/Mphi-like hemopoietic cells was greater than that in untreated cells, whereas TGF-beta did not up-regulate the expression of RANK, the receptor of RANKL. Our findings suggest that TGF-beta is an essential autocrine factor for osteoclastogenesis.     

Regulation of osteoclastogenesis and RANK expression by TGF-beta1

Transforming growth factor-beta (TGF-beta) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-beta on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-kappaB ligand (RANK-L) (50 ng/ml), TGF-beta1 (0.01-5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-beta1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-beta1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-beta1 resulted in a significant increase in the percentage of RANK+ RAW cells (P < 0.05), as well as an increase in the fluorescence intensity per cell (P < 0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-beta directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression.     

Influence of receptor activator of nuclear factor (NF)-κB ligand (RANKL), macrophage-colony stimulating factor (M-CSF) and fetal calf serum on human osteoclast formation and activity

Human osteoclast (OC) formation and activity was studied in cultures of peripheral blood mononuclear cells (PBMNC) from six healthy donors after stimulation with fetal calf serum (FCS), under the influence of the receptor activator of nuclear factor (NF)-κB ligand (RANKL) and the macrophage-colony stimulating factor (M-CSF). The results showed that selected FCS could stimulate OC formation without any medium supplementation with osteoclastogenic factors. The OC formation, investigated by quantification of multinucleated tartrate-resistant acid phosphatase-positive cells (TRAP+ cells), and the sensitivity of OC progenitors to RANKL and M-CSF, varied widely between individual donors. The OC resorption activity, measured in the “pit-assay” on dentine, was strictly dependent on the presence of RANKL and M-CSF in the medium and was also donor dependent. The considerable donor variability should be considered in culture studies investigating, e.g. the interactions of OC with biomaterials or the influence of cytokines, growth factors and drugs on osteoclastogenesis. An erratum to this article can be found at     

Ajulemic acid, a nonpsychoactive cannabinoid acid, suppresses osteoclastogenesis in mononuclear precursor cells and induces apoptosis in mature osteoclast-like cells

Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, prevents joint tissue injury in rats with adjuvant induced arthritis. Because activation of osteoclasts is central to the pathogenesis of bone erosion in patients with rheumatoid arthritis (RA), we investigated the influence of AjA on osteoclast differentiation and survival. Osteoclast cultures were established by stimulation of RAW264.7 cells and primary mouse bone marrow cultures with receptor activator of NF-kappaB ligand (RANKL). Simultaneous addition of AjA (15 and 30 microM) and RANKL to both culture systems significantly suppressed development of multinucleated osteoclasts (osteoclastogenesis) in a dose dependent manner, as determined by quantification of multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive cells. AjA impaired growth of RAW264.7 monocytes and prevented further osteoclast formation in cultures in which osteoclastogenesis had already begun. Reduction by AjA of both monocyte growth and osteoclast formation was associated with apoptosis, assayed by annexin V and propidium iodide staining, and caspase activity. The anti-osteoclastogenic effects of AjA did not require the continuous presence of AjA in the cell cultures. Based on these findings, we propose that AjA or other nonpsychoactive synthetic analogs of Cannabis constituents may be useful therapy for diseases such as RA and osteoporosis in which bone resorption is a central feature.     

Tumor necrosis factor-alpha mediates RANK ligand stimulation of osteoclast differentiation by an autocrine mechanism

Osteoblasts or bone marrow stromal cells are required as supporting cells for the in vitro differentiation of osteoclasts from their progenitor cells. Soluble receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF) is capable of replacing the supporting cells in promoting osteoclastogenesis. In the present study, using Balb/c-derived cultures, osteoclast formation in both systems-osteoblast/bone-marrow cell co-cultures and in RANKL-induced osteoclastogenesis-was inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha), and was enhanced by the addition of this cytokine. TNF-alpha itself promoted osteoclastogenesis in the presence of M-CSF. However, even at high concentrations of TNF-alpha the efficiency of this activity was much lower than the osteoclastogenic activity of RANKL. RANKL increased the level of TNF-alpha mRNA and induced TNF-alpha release from osteoclast progenitors. Furthermore, antibody to p55 TNF-alpha receptors (TNF receptors-1) (but not to p75 TNF-alpha receptors (TNF receptors-2) inhibited effectively RANKL- (and TNF-alpha() induced osteoclastogenesis. Anti-TNF receptors-1 antibody failed to inhibit osteoclastogenesis in C57BL/6-derived cultures. Taken together, our data support the hypothesis that in Balb/c, but not in C57BL/6 (strains known to differ in inflammatory responses and cytokine modulation), TNF-alpha is an autocrine factor in osteoclasts, promoting their differentiation, and mediates, at least in part, RANKL's induction of osteoclastogenesis.     

Human receptor activator of NF-κB ligand (RANKL) induces osteoclastogenesis of primates in vitro

Mouse receptor activator of NF-??B ligand (RANKL), which induces osteoclastogenesis from monocytes or macrophages, was independently cloned by three groups in 1997. Mouse osteoclasts have been induced from peripheral monocytes stimulated by RANKL and macrophage colony-stimulating factor (M-CSF) both in vitro and in vivo; however, the mechanism of primate osteoclastogenesis has not been studied. In addition, the effects of human RANKL on primate osteoclastogenesis remain to be elucidated. Here, we investigated the effect of human RANKL on the osteoclastogenesis of monocytes from five subspecies of primates. Human RANKL induced osteoclastogenesis of all the primates. In addition, human RANKL induced pit formation by osteoclasts from monocytes of the crab-eating macaque. We also demonstrated that the primate osteoclastogenesis was inhibited by a novel peptide, which inhibited human osteoclastogenesis in our previous study. Thus, these findings clearly demonstrated that human RANKL induces primate osteoclastogenesis in the presence of human M-CSF.     

Tumor necrosis factor stimulates osteoclastogenesis from human bone marrow cells under hypoxic conditions

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. In this study, we used human bone marrow cells (BMCs) to investigate the role of hypoxic exposure on human osteoclast (OC) formation in the presence of tumor necrosis factor (TNF). Exposing the BMCs to 3%, 5%, or 10% O₂ in the presence of receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, consistent with OCs. The addition of TNF under hypoxic conditions generated significantly greater numbers of mature OCs with more nuclei than OCs generated under normoxic conditions. Longer initial hypoxic exposure increased the number of OC precursor cells and facilitated the differentiation of OC precursor cells into multinucleated OCs. Quantitative RT-PCR analysis revealed that RANKL and TNFR1 were expressed at higher levels in non-OC cells from BMCs under hypoxic conditions than under normoxic conditions. Furthermore, to confirm the involvement of TNF-induced signaling, we examined the effects of blocking antibodies against TNFR1 and TNFR2 on OC formation under hypoxic conditions. The TNFR1 antibody was observed to significantly suppress OC formation. These results suggest that hypoxic exposure plays an important role in TNF-induced osteoclastogenesis from human BMCs.     

T cell-mediated increased osteoclast formation from peripheral blood as a mechanism for Crohn's disease-associated bone loss

The pathophysiology of osteoporosis in patients with Crohn's disease (CD) is still not completely elucidated. In this study, we evaluated osteoclastogenesis from peripheral blood cells of CD patients and studied the role of lymphocytes and inflammatory cytokines in this process. Peripheral blood mononuclear cells from seven patients with quiescent CD and matched healthy controls were isolated, and separated into T cells, B cells, and a T- and B-cell depleted fraction. In various culture combinations, osteoclast formation in the absence of the osteoclastogenic factors RANKL and M-CSF was assessed by scoring the number of tartrate-resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs). Cytokine levels in culture supernatants were measured. Formation of heterogeneous cell clusters in culture was noticed; a process that was inhibited by anti-LFA-1. In CD cultures, mean cluster area was up to threefold higher than in control cultures, and shown to be induced by T cells. Over tenfold higher numbers of TRACP(+) MNCs were found in CD cultures, but exclusively in cultures containing T cells. Formation of cell clusters correlated strongly with formation of TRACP(+) MNCs. Both cell cluster formation and osteoclast formation were related to IL-17 levels in vitro. In conclusion, osteoclastogenesis, preceded by cell cluster formation, is T cell-mediated and increased in patients with quiescent CD. Our findings suggest heterotypic interactions between osteoclast precursors and T cells to be a triggering step in osteoclast formation in CD. Furthermore, our results propose a possible role for IL-17 in osteoclastogenesis in CD patients, and as such in CD-associated bone loss.     

Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPalpha-interaction

Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPalpha) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPalpha-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPalpha strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)+ osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47-/- mice were strongly reduced, and bones of CD47-/- mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPalpha interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47-/- mice.     

Effects of prostaglandin E2 and lipopolysaccharide on osteoclastogenesis in RAW 264.7 cells

INTRODUCTION: Prostaglandins (PGs) can act on both hematopoietic and osteoblastic lineages to enhance osteoclast formation. METHODS: We examined PGE2 stimulated osteoclastogenesis in RAW 264.7 cells and the role of endogenous PGE2 in lipopolysaccharide (LPS) stimulated osteoclastogenesis. RESULTS: RANKL (1-100 ng/ml) increased formation of osteoclasts, defined as tartrate resistant acid phosphatase multinucleated cells, with peak effects at 30 ng/ml. Addition of PGE2 (0.01-1.0 microM) to RANKL (30 ng/ml) dose dependently increased osteoclast number 30-150%. Use of NS-398 (0.1 microM) or indomethacin (Indo, 1.0 micro M) to block endogenous PG synthesis had little effect on the response to RANKL alone but significantly decreased the response to PGE2. Addition of LPS (100 ng/ml) to RANKL increased osteoclast number 50%, and this response was significantly decreased by NS-398 and Indo. RANKL and PGE2 produced small, additive increases in COX-2 mRNA levels, while LPS produced a larger increase. PG release into the medium was not increased by RANKL and PGE2 but markedly increased by LPS. CONCLUSION: We conclude that RANKL stimulated osteoclastogenesis can be enhanced by PGE2 and LPS though direct effects on the hematopoietic cell lineage and that these effects may be mediated in part by induction of COX-2 and enhanced intracellular PG production.     

Modulation of Osteoclastogenesis with Macrophage M1- and M2-Inducing Stimuli

Macrophages are generated through the differentiation of monocytes in tissues and they have important functions in innate and adaptive immunity. In addition to their roles as phagocytes, macrophages can be further differentiated, in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), into osteoclasts (multinucleated giant cells that are responsible for bone resorption). In this work, we set out to characterize whether various inflammatory stimuli, known to induce macrophage polarization, can alter the type of multinucleated giant cell obtained from RANKL differentiation. Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated. Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics. Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.     

Human interleukin-1-induced murine osteoclastogenesis is dependent on RANKL, but independent of TNF-alpha

Although interleukin-1 (IL-1) has been implicated in the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are largely unknown. Recently, a cytokine-driven, stromal cell-free mouse osteoclastogenesis model was established. A combination of macrophage colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL) was proven to be sufficient in inducing differentiation of bone marrow hematopoietic precursor cells to bone-resorbing osteoclasts in the absence of stromal cells or osteoblasts. This study utilizes this model to examine the impact of human IL-1beta on in vitro osteoclastogenesis of bone marrow progenitor cells. We found that osteoclast precursor cells failed to undergo osteoclastogenesis when treated with IL-1 alone. In contrast, IL-1 dramatically up-regulated osteoclastogenesis by 2.5- to 4-folds in the presence of RANKL and M-CSF. The effect can be significantly blocked by IL-1 receptor antagonist (p < 0.01). Tumor necrosis factor-alpha (TNF-alpha) was undetectable in the culture medium of differentiating osteoclasts induced by IL-1. Adding exogenous TNF-alpha neutralizing antibody had no influence on the IL-1-induced effect as well. These results show that in the absence of stromal cells, IL-1 exacerbates osteoclastogenesis by cooperating with RANKL and M-CSF, while TNF-alpha is not involved in this IL-1-stimulated osteoclast differentiation pathway.     

Cross-talk between RANKL and FRP-1/CD98 Systems: RANKL-mediated osteoclastogenesis is suppressed by an inhibitory anti-CD98 heavy chain mAb and CD98-mediated osteoclastogenesis is suppressed by osteoclastogenesis inhibitory factor

The two pathways to osteoclastogenesis, RANKL-mediated and CD98-mediated osteoclastogenesis, have recently been reported. RANKL, OCIF, and TIMP-3 mRNAs are not found in monocytes freshly isolated or incubated with anti-FRP-1/CD98hc antibody. RANK, TACE, and M-CSF mRNAs can be detected in these cells. Interestingly, the expressed amount of RANK mRNA increases by cultivation of monocytes with anti-CD98hc antibody and maximal expression is observed in osteoclast-like cells. CD98-mediated cell aggregation and multinucleated giant cell formation are blocked by OCIF. OCIF also suppressed the CD98-mediated induction of Sp1 and c-src mRNAs in monocytes. Soluble RANK shows no effect on CD98-mediated cell aggregation and multinucleated giant cell formation. When blood monocytes were incubated with RANKL and M-CSF, c-src and Sp1 mRNAs were first found in blood monocytes incubated with these cytokines for 7 days. On the contrary, c-src mRNA could be detected 3 h after treatment of blood monocytes with anti-CD98hc mAb. LAT-1 mRNA was not found, and the expression levels of Y(+)LAT-1 and Y(+)LAT-2 mRNAs were not changed in monocytes stimulated without or with anti-CD98hc mAb or RANKL and M-CSF. An inhibitory mAb directed against CD98hc, HBJ 127, shows a suppressive effect on RANKL-mediated cell aggregation and cell fusion. Thus, there is cross-talk between these two pathways.     

Gingival fibroblasts are better at inhibiting osteoclast formation than periodontal ligament fibroblasts

Various studies indicate that periodontal ligament fibroblasts (PLF) have some similarities to osteoblasts, for example they have the capacity to induce the formation of osteoclast-like cells. Here, we investigated whether a second population of tooth-associated fibroblasts, gingival fibroblasts (GF), has similar osteoclastogenesis properties. PLF and GF were co-cultured with peripheral blood mononuclear cells (PBMC) in the presence and absence of dexamethasone and 1alpha,25dihydroxycholecalciferol (dex + vit D(3)) on plastic and on cortical bone slices. Tartrate resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs) were more abundant in co-cultures with PLF than in GF-PBMC co-cultures, more abundant on plastic compared to bone and more abundant in the presence of dex + vit D(3). In line with these findings was an inhibition of MNC formation and not inhibition of existing osteoclasts by medium conditioned by GF. We next investigated whether expression of molecules important for osteoclastogenesis differed between the two types of fibroblasts and whether these molecules were regulated by dex + vit D(3). OPG was detected at high levels in both fibroblast cultures, whereas RANKL could not be detected. Resorption of bone did not occur by the MNCs formed in the presence of either fibroblast subpopulation, suggesting that the fibroblasts secrete inhibitors of bone resorption or that the osteoclast-like cells were not functional. The incapacity of the MNCs to resorb was abolished by culturing the fibroblast-PBMC cultures with M-CSF and RANKL. Our results suggest that tooth-associated fibroblasts may trigger the formation of osteoclast-like cells, but more importantly, they play a role in preventing bone resorption, since additional stimuli are required for the formation of active osteoclasts.     

Inhibition of histone deacetylase suppresses osteoclastogenesis and bone destruction by inducing IFN-beta production

Osteoclasts are bone-resorptive multinucleated cells that are differentiated from hemopoietic cell lineages of monocyte/macrophages in the presence of receptor activator of NF-kappaB ligand (RANKL) and M-CSF. Downstream signaling molecules of the receptor of RANKL, RANK, modulate the differentiation and the activation of osteoclasts. We recently found that histone deacetylase inhibitors (HDIs), known as anticancer agents, selectively suppressed osteoclastogenesis in vitro. However, the molecular mechanism underlying inhibitory action of HDIs in osteoclastogenesis and the effect of HDIs on pathological bone destruction are still not remained to be elucidated. In this study, we show that a depsipeptide, FR901228, inhibited osteoclast differentiation by not only suppressing RANKL-induced nuclear translocation of NFATc1 but also increasing the mRNA level of IFN-beta, an inhibitor of osteoclastogenesis. The inhibition of osteoclast formation by FR901228 was abrogated by the addition of IFN-beta-neutralizing Ab. In addition, treatment of adjuvant-induced arthritis in rats revealed that FR901228 inhibited not only disease development in a prophylactic model but also bone destruction in a therapeutic model. Furthermore, immunostaining of the joints of therapeutically treated rats revealed significant production of IFN-beta in synovial cells. Taken together, these data suggest that a HDI inhibits osteoclastogenesis and bone destruction by a novel action to induce the expression of osteoclast inhibitory protein, IFN-beta.     

Prostaglandin E2 induced the differentiation of osteoclasts in mouse osteoblast-depleted bone marrow cells

Prostaglandin (PG) E(2) is a known bone absorbing agent that acts on osteoblasts to facilitate osteoclastogenesis by increasing the secretion of RANKL. In the present study, we investigated the direct action of PGE(2) on osteoclastic progenitors that differentiate into TRAP-positive multinucleated cells. The hematopoietic stem cell obtained from murine bone marrow was purified by a Sephadex G-10 column, and cultured in the presence of CSF-1 and RANKL to facilitate cell differentiation. The introduction of low-density PGE(2) into the culture resulted in a drastic increase of TRAP-positive multinucleated cells, whereas the addition of high-density PGE(2) had the opposite effect. PCR analysis revealed increased level of EP3 mRNA in undifferentiated cells and reduced level after the development of osteoclast; EP1, EP2 and EP4 were constitutively expressed throughout the differentiation. Investigation of intracellular signaling verified that low-density PGE(2) suppressed PKA activity in undifferentiated cells, suggesting that PGE(2) acts on the osteoclastic cell lineage to facilitate cell differentiation by suppressing PKA in the presence of RANKL.     

Characterisation of the osteoclastogenic potential of human osteoblastic and fibroblastic conditioned media

Although M‐CSF and RANKL are sufficient to promote in vitro osteoclastogenesis, in vivo this is a complex process which requires the action of many signalling molecules and cellular crosstalks. In this work, isolated or combined conditioned media, obtained from human adult skin fibroblast and bone marrow cells, were tested for their osteoclastogenic potential, through an indirect co‐culture system, in the absence of recombinant M‐CSF and RANKL. Osteoclastogenesis was assessed on human peripheral blood mononuclear cells (PBMC) and CD14+ cell cultures by quantification of total protein content, tartrate‐resistant acid phosphatase (TRAP) activity, presence of multinucleated cells positive for TRAP, RT‐PCR of TRAP, CATK, CA2, c‐myc and c‐src and presence of multinucleated cells displaying actin rings, vitronectin and calcitonin receptors. Cultures supplemented with M‐CSF and RANKL were used as positive controls. It was observed that the conditioned medium from dexamethasone osteogenic‐induced bone marrow cell cultures displayed the highest osteoclastogenic potential, with similar behaviour to that observed in the presence of both M‐CSF and RANKL. Comparatively, fibroblastic conditioned medium elicited a slightly lower osteoclastogenic response. Combination of both conditioned media resulted in a significant increase of TRAP activity. On the other hand, conditioned medium from non‐osteogenic‐induced bone marrow cell cultures presented the lowest osteoclastogenic potential. These results were observed for both PBMC and CD14+ cell cultures, suggesting that fibroblast and osteoblast cells are able to modulate osteoclastogenesis in the absence of physical cell–cell interactions. In addition, osteoclastogenic potential of bone marrow cells increases with their osteoblastic differentiation. J. Cell. Biochem. 109: 205–216, 2010. © 2009 Wiley‐Liss, Inc.     

Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase

Background

Phenylketonuria (PKU) is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques.

Methodology

Peripheral blood mononuclear cell (PBMC) cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL). Flow cytometry was utilized to analyze osteoclast precursors (OCPs) and T cell phenotype. Tumour necrosis factor α (TNF-α), RANKL and osteoprotegerin (OPG) were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated.

Principal Findings

Several in vitro studies in PKU patients'' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS). This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia.

Conclusions

Our results indicate that PKU spontaneous osteoclastogenesis depends on the circulating OCP increase and the activation of T cells. Osteoclastogenesis correlates with clinical parameters, suggesting its value as a diagnostic tool for an early assessment of an increased bone resorption in PKU patients.