Role of tartrate-resistant acid phosphatase (TRAP) in long bone development.

Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.

Tartrate-resistant acid phosphatase deficiency causes a bone dysplasia with autoimmunity and a type I interferon expression signature.

We studied ten individuals from eight families showing features consistent with the immuno-osseous dysplasia spondyloenchondrodysplasia. Of particular note was the diverse spectrum of autoimmune phenotypes observed in these individuals (cases), including systemic lupus erythematosus, Sjögren's syndrome, hemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynaud's disease and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13, and linkage analysis yielded a combined multipoint log(10) odds (LOD) score of 3.6. Sequencing of ACP5, encoding tartrate-resistant acid phosphatase, identified biallelic mutations in each of the cases studied, and in vivo testing confirmed a loss of expressed protein. All eight cases assayed showed elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I interferon signature. Our findings reveal a previously unrecognized link between tartrate-resistant acid phosphatase activity and interferon metabolism and highlight the importance of type I interferon in the genesis of autoimmunity.

VEGF and its role in the early development of the long bone epiphysis.

In long bones of murine species, undisturbed development of the epiphysis depends on the generation of vascularized cartilage canals shortly after birth. Despite its importance, it is still under discussion how this event is exactly regulated. It was suggested previously that, following increased hypoxia in the epiphyseal core, angiogenic factors are expressed and hence stimulate the ingrowth of the vascularized canals. In the present study, we tested this model and examined the spatio-temporal distribution of two angiogenic molecules during early development in mice. In addition, we investigated the onset of cartilage hypertrophy and mineralization. Our results provide evidence that the vascular endothelial growth factor is expressed in the epiphyseal resting cartilage prior to the moment of canal formation and is continuously expressed until the establishment of a large secondary ossification centre. Interestingly, we found no expression of secretoneurin before the establishment of the canals although this factor attracts blood vessels under hypoxic conditions. Epiphyseal development further involves maturation of the resting chondrocytes into hypertrophic ones, associated with the mineralization of the cartilage matrix and eventual death of the latter cells. Our results suggest that vascular endothelial growth factor is the critical molecule for the generation of the epiphyseal vascular network in mice long bones. Secretoneurin, however, does not appear to be a player in this event. Hypertrophic chondrocytes undergo cell death by a mechanism interpreted as chondroptosis.

Acid phosphatase 5 is responsible for removing the mannose 6-phosphate recognition marker from lysosomal proteins.

Most newly synthesized proteins destined for the lysosome reach this location via a specific intracellular pathway. In the Golgi, a phosphotransferase specifically labels lysosomal proteins with mannose 6-phosphate (Man-6-P). This modification is recognized by receptors that target the lysosomal proteins to the lysosome where, in most cell types, the Man-6-P recognition marker is rapidly removed. Despite extensive characterization of this pathway, the enzyme responsible for the removal of the targeting modification has remained elusive. In this study, we have identified this activity. Preliminary investigations using a cell-based bioassay were used to follow a dephosphorylation activity that was associated with the lysosomal fraction. This activity was high in the liver, where endogenous lysosomal proteins are efficiently dephosphorylated, but present at a much lower level in the brain, where the modification persists. This observation, combined with an analysis of the expression of lysosomal proteins in different tissues, led us to identify acid phosphatase 5 (ACP5) as a candidate for the enzyme that removes Man-6-P. Expression of ACP5 in N1E-115 neuroblastoma cells, which do not efficiently dephosphorylate lysosomal proteins, significantly decreased the steady state levels of Man6-P glycoproteins. Analysis of ACP5-deficient mice revealed that levels of Man-6-P glycoproteins were highly elevated in tissues that normally express ACP5, and this resulted from a failure to dephosphorylate lysosomal proteins. These results indicate a central role for ACP5 in removal of the Man-6-P recognition marker and open up new avenues to investigate the importance of this process in cell biology and medicine.

Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy.

Tartrate-resistant acid phosphatase (TRAP), once considered to be just a histochemical marker of osteoclasts is now recognised to be a molecule of widespread occurrence with functions in both the skeleton and the immune system. TRAP is expressed by osteoclasts, macrophages, dendritic cells and a number of other cell types. It has a critical role in many biological processes including skeletal development, collagen synthesis and degradation, the mineralisation of bone, cytokine production by macrophages and dendritic cells, macrophage recruitment, dendritic cell maturation and a role in the development of Th1 responses. TRAP is able to degrade skeletal phosphoproteins including osteopontin (OPN), identical to the T-cell cytokine, Eta-1. In this review, we discuss the role of TRAP in bone and immune cells and suggest that TRAP may be implicated in autoimmune disorders regulated by Th1 inflammatory responses as well as certain cancers.

Altered collagen in tartrate-resistant acid phosphatase (TRAP)-deficient mice: a role for TRAP in bone collagen metabolism.

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein that is highly expressed by osteoclasts, macrophages, and dendritic cells. The enzyme is secreted by osteoclasts during bone resorption, and serum TRAP activity correlates with resorptive activity in disorders of bone metabolism. TRAP is essential for normal skeletal development. In knockout mice lacking TRAP, bone shape and modeling is altered with increased mineral density. Here, we report the effect of TRAP on the biochemical and biomechanical properties of collagen, the major protein constituting the bone matrix, using these mice. Femurs from TRAP-/- and wild-type mice were used in these studies. The biomechanical properties were investigated using a three-point bending technique. Collagen synthesis was determined by measuring cross-link content using high-performance liquid chromatography and amino acid analysis. Collagen degradation was determined by measuring matrix metalloproteinase-2 (MMP-2) activity. The rates of collagen synthesis and degradation were significantly greater in bones from TRAP-/- mice compared with wild type. At 8 weeks, there was an increase in the intermediate cross-links but no significant difference in animals aged 6 months. There was a significant increase in mature cross-links at both ages. A significant increase in MMP-2 production both pro and active was observed. A significant increase in ultimate stress and Young's modulus of elasticity was needed to fracture the bones from mice deficient in TRAP. We conclude that both synthesis as well as degradation of collagen are increased when TRAP is absent in mice at 8 weeks and 6 months of age, showing that TRAP has an important role in the metabolism of collagen.

Human breast cancer cell lines and tissues express tartrate-resistant acid phosphatase (TRAP).

Tartrate-resistant acid phosphatase (TRAP) is expressed by osteoclasts, macrophages and dendritic cells. TRAP has been identified in a wide variety of tissues, however, its biological function is not fully understood. Serum TRAP is a marker of diseases involving excessive bone resorption including metastatic bone disease in breast cancer patients and can be used to monitor responses to treatment. Our aim in this study was to determine whether TRAP is expressed by human breast tumours. Four breast cancer cell lines were assayed for TRAP activity. MDA-MB-435, the most tumourigenic line, had an activity twofold higher than the other cell lines. Immunohistochemistry using a TRAP specific antibody confirmed that both cell lines and human breast tumours express TRAP. Expression was absent in benign tissues and abundant in more aggressive tumours. This work suggests that tumour derived TRAP contributes to the raised enzyme activity found in the serum of breast cancer patients.

TRACP Influences Th1 pathways by affecting dendritic cell function.

UNLABELLED: TRACP, a marker of osteoclasts, is also expressed by cells of the immune system. We identified a novel function for TRACP in the dendritic cell. DCs from TRACP knockout mice have impaired maturation and trigger reduced Th1 responses in vivo. We postulate that TRACP has an important role in the presentation of antigens to T cells. INTRODUCTION: TRACP is highly expressed by osteoclasts, activated macrophages, and dendritic cells (DCs). Knockout mice lacking TRACP have an intrinsic defect in osteoclastic resorption and macrophages that display abnormal immunomodulatory responses and cytokine secretion profiles. Our aim in this study was to investigate the significance of TRACP in the inductive phase of the immune response by examining dendritic cells from TRACP(-/-) mice. MATERIALS AND METHODS: Maturational state and function of leukocyte subsets in mice was assessed by flow cytometry. The ability of the immune system to respond to nonspecific activation and to specific antigen was assessed by delayed type hypersensitivity and the presence of isotype-specific serum antibody in vivo and T-cell proliferation and cytokine production in vitro. RESULTS: The ability of lipopolysaccharide (LPS) to upregulate MHC II and CD80 in DCs from TRACP(-/-) mice was reduced compared with wildtype mice, although production of IL-10 by DCs from TRACP-deficient animals was increased. T- and B-cell responses not involving antigen presentation (anti-CD3, TNP-ficoll) were normal in TRACP(-/-) mice, but responses to T-dependent antigens were impaired. Specifically, TRACP(-/-) mice had defective delayed hypersensitivity responses to picryl chloride and reduced proliferative responses to ovalbumin compared with wildtype mice. In response to ovalbumin, but not anti-CD3, T cells from TRACP(-/-) mice produced less interferon-gamma (IFN-gamma), but there was no difference in IL-4 production: TRACP(-/-) mice also produced less ovalbumin (OVA)-specific IgG2a after immunization. CONCLUSIONS: The finding that DCs from TRACP(-/-) mice have impaired maturation and defective Th1 responses shows that TRACP is important for polarizing responses in naïve T cells to antigen-presented dendritic cells.

Tartrate-resistant acid phosphatase: a potential target for therapeutic gold.

Gold compounds are disease-modifying agents for the treatment of rheumatoid arthritis. They act on the immune system but the mechanism is not fully understood. Gold has been shown to affect antigen processing by T-cells and also reduces expression of cytokines in macrophages. Tartrate-resistant acid phosphatase (TRAP), expressed by osteoclasts, macrophages and dendritic cells is an enzyme with roles in skeletal metabolism and the immune response. TRAP is able to degrade skeletal phosphoproteins including osteopontin, identical to the T-cell cytokine, Eta-1; we thus propose that TRAP regulates the Eta-1 pathway common to the immune system and skeleton. We compared the distribution of osteopontin and TRAP in sections of 18-day-old embryonic mice by immunohistochemistry. Both proteins occurred in the same locations. To determine whether gold compounds exert their effects by modification of TRAP activity, we examined the action of gold chloride and the prodrugs, aurothioglucose and aurothiomalate on the dephosphorylation of osteopontin by TRAP. Aurothioglucose and aurothiomalate had little effect on phosphatase activity; gold chloride was a potent non-competitive inhibitor (Ki < 47 x 10(-9) M). These findings indicate a possible molecular mechanism for the action of therapeutic gold and further implicate TRAP in the control of immunity.

Tartrate-resistant acid phosphatase knockout mice.

TRACP is a lysosomal enzyme found in diverse tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages. To investigate the function of TRACP in vivo, we have generated mice in which the gene-encoding TRACP has been selectively disrupted by targeted homologous recombination in murine embryonic stem cells. Homozygous TRACP "knockout" mice have progressive foreshortening and deformity of the long bones and axial skeleton suggesting a role for TRACP in endochondral ossification. There is increased mineralization reflecting a mild osteopetrosis caused by reduced osteoclast modeling activity. These knockout mice also display an impairment of macrophage function with abnormal immunomodulatory cytokine responses. Superoxide formation and nitrite production were enhanced in stimulated macrophages lacking TRACP as was the secretion of the proinflammatory cytokines TNF-alpha, interleukin (IL)-1beta, and IL-12. TRACP knockout mice showed delayed clearance of the microbial pathogen Staphylococcus aureus after sublethal intraperitoneal inoculation. The macrophages lacking TRACP showed an increase in tartrate-sensitive lysosomal acid phosphatase activity (LAP). The TRACP knockout mice were bred with mice lacking LAP. Mice lacking both TRACP and LAP had even shorter bones than the TRACP single knockouts. Osteopontin, identical to the T-cell cytokine eta-1, accumulated adjacent to actively resorbing osteoclasts suggesting that both phosphatases are important for processing this protein. We propose that TRACP may be an important regulator of osteopontin/eta-1 activity common to both the immune system and skeleton.