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

ABSTRACT: 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 NF?B ligand (RANKL). Flow cytometry was utilized to analyze osteoclast precursors (OCPs) and T cell phenotype. Tumour necrosis factor ? (TNF-?), RANKL and osteoprotegerin (OPG) were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-? 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-? 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.

SUBMITTER: Roato I

PROVIDER: S-EPMC2994752 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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

Roato Ilaria I Porta Francesco F Mussa Alessandro A D'Amico Lucia L Fiore Ludovica L Garelli Davide D Spada Marco M Ferracini Riccardo R

PloS one 20101130 11

<h4>Background</h4>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.<h4>Methodology</h4>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 analy ...[more]

PMID: 21152388

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Project description:ObjectiveActivation of osteoclastogenesis at the bone site in rheumatoid arthritis (RA) is well established. The mechanisms by which circulating osteoclast precursors contribute are still unclear. Peroxisome proliferator-activated receptor ? coactivator 1? (PGC-1?) is implicated in transcriptional regulation of osteoclastogenesis in mouse models. This study was undertaken to investigate the contribution of PGC-1? to circulating osteoclast precursors and its link to bone destruction in RA.MethodsPGC-1? expression in RA peripheral blood CD14+ monocytes was increased and showed correlation with joint destruction shown on radiographs. Cells from RA patients or healthy controls were transfected with a lentivirus vector for PGC-1? gene silencing or overexpression and cultured with macrophage colony-stimulating factor and RANKL. Bone resorption activity, bone-degrading enzymes, and signaling molecules were measured in these mature osteoclasts.ResultsIncreased nuclear accumulation of PGC-1? was observed in RA peripheral blood CD14+ monocytes, and these cells had stronger osteoclastogenesis than in healthy controls. PGC-1? protein expression was positively correlated with radiographic joint destruction (r = 0.396-0.413; all P < 0.05). PGC-1? knockdown suppressed (51-82% reduction) the expression of cathepsin K, tartrate-resistant acid phosphatase (TRAP), and matrix metalloproteinase 9 (MMP-9), as well as osteoclast differentiation and bone resorption activity. Conversely, PGC-1? overexpression increased these markers (by 1.5-1.8-fold) and osteoclastogenesis. VIVIT, an inhibitor of NFATc1 activation, inhibited the effect of overexpressed PGC-1? by reducing cathepsin K, TRAP, and MMP-9 expression. Chromatin immunoprecipitation assay and dual-luciferase reporter gene assay showed PGC-1? bound to NFATc1 promoter, leading to transcriptional activation.ConclusionActivation of the PGC-1?/NFATc1 pathway in circulating osteoclast precursors was associated with bone destruction in RA. This may represent a new treatment target.

Project description:Osteoclasts are the only somatic cells with bone-resorbing capacity and, as such, they have a critical role not only in normal bone homeostasis (called 'bone remodelling') but also in the pathogenesis of bone destructive disorders such as rheumatoid arthritis and osteoporosis. A major focus of research in the field has been on gene regulation by osteoclastogenic cytokines such as receptor activator of NF-kappaB-ligand (RANKL, also known as TNFSF11) and TNF-alpha, both of which have been well documented to contribute to osteoclast terminal differentiation. A crucial process that has been less well studied is the trafficking of osteoclast precursors to and from the bone surface, where they undergo cell fusion to form the fully differentiated multinucleated cells that mediate bone resorption. Here we report that sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood, induces chemotaxis and regulates the migration of osteoclast precursors not only in culture but also in vivo, contributing to the dynamic control of bone mineral homeostasis. Cells with the properties of osteoclast precursors express functional S1P(1) receptors and exhibit positive chemotaxis along an S1P gradient in vitro. Intravital two-photon imaging of bone tissues showed that a potent S1P(1) agonist, SEW2871, stimulated motility of osteoclast precursor-containing monocytoid populations in vivo. Osteoclast/monocyte (CD11b, also known as ITGAM) lineage-specific conditional S1P(1) knockout mice showed osteoporotic changes due to increased osteoclast attachment to the bone surface. Furthermore, treatment with the S1P(1) agonist FTY720 relieved ovariectomy-induced osteoporosis in mice by reducing the number of mature osteoclasts attached to the bone surface. Together, these data provide evidence that S1P controls the migratory behaviour of osteoclast precursors, dynamically regulating bone mineral homeostasis, and identifies a critical control point in osteoclastogenesis that may have potential as a therapeutic target.

Project description:Cells with monocyte/macrophage lineage expressing receptor activator of NF-?B (RANK) differentiate into osteoclasts following stimulation with the RANK ligand (RANKL). Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition) or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition). BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS) and tumor necrosis factor -?TNF-? activated the NF-?B-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6) in BMMs induced their differentiation into osteoclasts even under the non-adherent condition. These results suggest that cell adhesion signaling regulates RANK expression in osteoclast precursors.

Dataset Information

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

Publications

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

Similar Datasets

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