Tartrate-resistant acid phosphatase type 5/ACP5 promotes cell cycle entry of 3T3-L1 preadipocytes by increasing IGF-1/Akt signaling.

Tartrate-resistant acid phosphatase (TRAP, encoded by ACP5)-overexpressing mice exhibit hyperplastic obesity. As the molecular mechanism remains elusive, the aims were to characterize the effect of TRAP on preadipocyte proliferation. We investigated cell cycle entry and signal transduction, that is, insulin-like growth factor 1 (IGF-1)/ insulin receptor substrate 1 (IRS-1) and the Akt signaling pathways, in 3T3-L1 preadipocytes treated with the TRAP 5a isoform. Results show that TRAP 5a increases S-phase entry. TRAP 5a stimulation increases IGF-1 mRNA and IRS-1 activation, indicative of insulin-like growth factor 1 receptor (IGF1R) activation. Furthermore, TRAP 5a stimulation resulted in Akt signaling pathway activation and subsequent increased nuclear translocation of ß-catenin. In conclusion, TRAP 5a increases proliferation of preadipocytes in a dose-dependent fashion by promoting entry into S-phase. Part of this effect is likely due to increased IGF-1 signaling through the Akt signaling pathway.

Intensive weight gain therapy in patients with anorexia nervosa results in improved serum tartrate-resistant acid phosphatase (TRAP) 5a and 5b isoform protein levels.

AIM: Tartrate-resistant acid phosphatase (TRAP) exists as isoforms 5a and 5b. TRAP 5a is a biomarker of chronic inflammation and influences adipose tissue and 5b associates with bone metabolism/pathologies. The aim was to investigate the association of serum TRAP 5a/5b isoforms with fat and bone markers and anthropometric parameters in patients with anorexia nervosa (AN) during weight gain therapy. METHODS: Twenty-five Swedish female AN patients, age 16-24 years, were treated for 12 weeks with a high-energy diet with six meals daily. Serum TRAP 5a/5b, markers of fat/glucose metabolism, markers of bone resorption and formation were measured. Parameters of bone and body composition were assessed by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. RESULTS: BMI increased from median 15.4 kg/m2 to 19.0 kg/m2, p < 0.0001. TRAP 5a and 5a/5b ratio increased but TRAP 5b decreased during the study. TRAP Δ5a and Δ5b correlated with Δinsulin and Δadiponectin, respectively. TRAP 5b correlated with trabecular density at start but not at week 12. At 12 weeks, TRAP 5b correlated with CTX, and Δ decrease in TRAP 5b correlated to Δ increase in bone-specific alkaline phosphatase. CONCLUSIONS: This clinical interventional study resulted in increased BMI in patients with AN. The decreased TRAP 5b protein levels confirm a role for TRAP 5b as a marker of bone resorption, whereas increased TRAP 5a seemed to derive from systemic changes in bone as well as metabolic changes. The combined detection of TRAP 5a and TRAP 5b in serum could be an indicator of improved bone metabolism. LEVEL OF EVIDENCE: Level III, prospective interventional cohort study.

A Novel Sandwich ELISA for Tartrate-Resistant Acid Phosphatase 5a and 5b Protein Reveals that Both Isoforms are Secreted by Differentiating Osteoclasts and Correlate to the Type I Collagen Degradation Marker CTX-I In Vivo and In Vitro.

Tartrate-resistant acid phosphatase type 5 (TRAP) exists as two isoforms, 5a and 5b. 5b is a marker of osteoclast number and 5a of chronic inflammation; however, its association with bone resorption is unknown. In this study, a double-TRAP 5a/5b sandwich ELISA measuring 5a and 5b protein in the same sample was developed. TRAP 5a and 5b protein levels were evaluated as osteoclast differentiation/activity markers in serum and in culture, and their correlation to the resorption marker CTX-I was examined. Serum TRAP 5a and 5b concentrations in healthy men were 4.4 ± 0.6 ng/ml and 1.3 ± 0.2 ng/ml, respectively, and they correlated moderately to each other suggesting that their secretion is coupled under healthy conditions. A correlation was also observed between serum TRAP 5a and 5b with CTX-I, suggesting that both TRAP isoforms associate with osteoclast number. During osteoclast differentiation on plastic/bone, predominantly 5b increased in media/lysate from M-CSF/RANKL-stimulated CD14+ PBMCs. However, substantial levels of 5a were detected at later stages suggesting that both isoforms are secreted from differentiating OCs. More TRAP 5b was released on bone indicating a connection to osteoclast resorptive activity, and a peak in TRAP 5b/5a-ratio coincided with rapid CTX-I release. At the end of the culture period of M-CSF + RANKL-stimulated CD14+ PBMCs, there was a correlation between the secretion of TRAP 5a and 5b proteins with CTX-I. The correlation of not only 5b but also 5a with collagen degradation, both in serum and osteoclast cultures indicates that a considerable proportion of the TRAP 5a originates from osteoclasts and may reflect a hitherto undisclosed regulatory mechanism during bone resorption and bone remodeling.

Peripheral blood monocytes show increased osteoclast differentiation potential compared to bone marrow monocytes.

Bone marrow (BM) and peripheral blood (PB) derived mononuclear cells are precursors of in vitro osteoclast differentiation. However, few studies have compared the phenotypic and functional properties of osteoclasts generated from these sources and the effects of different growth factors on osteoclastogenesis. Both cell types differentiated into functional osteoclasts, but culturing the cells with or without transforming growth factor beta (TGF-ß) and dexamethasone revealed differences in their osteoclastogenic capacity. When receptor activator for nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were used for differentiation, we did not observe differences in bone resorption activity or expression of osteoclastogenic genes calcitonin receptor (CR) and nuclear factor of activated T-cells (NFATc1) between the osteoclasts formed from the two sources. Addition of TGF-ß and dexamethasone led to higher number of nuclei in multinuclear cells and increased expression of tartrate resistant acid phosphatase (TRACP) 5a and 5b, CR and NFATc1 in PB- derived osteoclasts depicting the higher osteoclastogenic potential and responsiveness to TGF-ß and dexamethasone in PB monocytes. These results conclude that the choice of the osteoclast precursor source as well as the choice of osteoclastogenic growth factors are essential matters in determining the phenotypic characteristics of heterogeneous osteoclast populations.

Increased amount of phosphorylated proinflammatory osteopontin in rheumatoid arthritis synovia is associated to decreased tartrate-resistant acid phosphatase 5B/5A ratio.

BACKGROUND: Osteopontin (OPN) is an immunoregulatory protein which production increases in both rheumatoid arthritis (RA) and osteoarthritis (OA). Phosphorylated osteopontin (Phospho-OPN) is known to increase macrophage and osteoclast activation, this process is controlled by extracellular tartrate-resistant acid phosphatase (TRAcP), also a biomarker for RA. Here, we evaluated the phosphorylation status of OPN in RA and OA synovia, as well as its correlation with TRAcP isoforms. METHODS: Synovial tissue and fluid were obtained from 24 RA (14 seropositive and 10 seronegative) and 24 OA patients. Western blotting was used to analyze the extent of OPN phosphorylation. TRAcP isoforms were measured in synovial fluid using ELISA; immunohistochemistry assessed the distribution of OPN and TRAcP expressing cells in the synovial tissue, especially distinguishing between the TRAcP isoforms. RESULTS: Full-length OPN was more phosphorylated in RA than in OA (p<0.05). The thrombin cleaved C-terminal end of OPN was also more phosphorylated in RA (p<0.05). RA patients had a lower concentration of TRAcP 5B and higher concentration of less active 5A in their synovial fluid compared to OA patients. The TRAcP 5B/5A ratio was decreased in RA and correlated negatively with the amount of phospho-OPN (p<0.05). TRAcP positive cells for both isoforms were found all along the synovial lining; OPN antibody staining was localized in the extracellular matrix. CONCLUSION: Our data suggests that in RA the synovial fluid contains insufficient amounts of TRAcP 5B which increase levels of the proinflammatory phospho-OPN. This may lead to increased macrophage and osteoclast activation, resulting in the increased local inflammation and bone resorption present in RA joints.

The adipokine tartrate-resistant acid phosphatase 5a in serum correlates to adipose tissue expansion in obesity.

PURPOSE: Tartrate-resistant acid phosphatase (TRAP) exists as two isoforms, 5a and 5b. TRAP 5a is elevated in adipose tissue of obese women, interacts with pre-adipocytes and is linked to insulin-sensitive hyperplastic obesity when overexpressed in mice. The aim of this study was to investigate the relation between serum TRAP 5a, adiposity indices and metabolic syndrome risk markers in lean and obese women, using a newly developed TRAP 5a-specific ELISA. MATERIALS AND METHODS: A TRAP 5a sandwich ELISA was optimized using TRAP 5a-specific monoclonal antibodies and tested in sera of healthy males. TRAP 5a levels were quantitated in sera from healthy males and lean and obese women. RESULTS: Serum TRAP 5a protein levels were lower in obese women in comparison with lean. In obese, but not in lean women, serum TRAP 5a correlated positively to % fat mass, BMI, waist- and hip circumference, waist-to-hip ratio and PAI, while no correlations to serum leptin, HOMA, glucose, insulin, FFA, HDL, TG, APO-A1 and APO-B were observed. CONCLUSIONS: TRAP 5a serum levels correlated positively to anthropometric obesity parameters but not to metabolic syndrome risk factors, indicating that serum TRAP 5a is associated with pathological adipose tissue expansion.

The growth factor-like adipokine tartrate-resistant acid phosphatase 5a interacts with the rod G3 domain of adipocyte-produced nidogen-2.

The adipokine tartrate resistant acid phosphatase (TRAP) 5a isoform exerts a growth factor-effect on pre-adipocytes. This study aimed to identify potential TRAP 5a interacting proteins in pre-adipocytes using pull down assays in combination with mass spectrometry. Nidogen-2, a protein shown to be expressed intracellularly and for secretion by pre-adipocytes, was shown to interact, through its globular G3 domain, with TRAP 5a in vitro. In vivo, TRAP 5a interacted with nidogen-2 in cultured 3T3-L1 mouse pre-adipocytes, as well as with transforming growth factor-ß (TGF-ß) interacting protein (TRIP-1), which is a protein that has previously been suggested to interact with TRAP in bone. In addition, TRAP 5a and nidogen-2 co-localized in adipose tissue cells in situ. These results indicate that TRAP 5a interacts with nidogen-2 and TRIP-1 in pre-adipocytic cells.

Caveolae-mediated endocytosis of the glucosaminoglycan-interacting adipokine tartrate resistant acid phosphatase 5a in adipocyte progenitor lineage cells.

Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo. However its molecular interaction with pre-adipocytes remains unknown. Therefore, this study aimed to investigate membrane interaction of TRAP and its endocytosis routes in pre-adipocytes. Confocal and/or electron microscopy were used to detect TRAP in untreated or TRAP 5a/b treated pre-adipocytes under conditions that allow or inhibit endocytosis in combination with co-staining of endocytotic vesicles. TRAP interaction with heparin/heparan sulfate was verified by gel filtration. It could be shown that TRAP 5a, but not 5b, binds to the membrane of pre-adipocytes where it co-localizes with heparin-sulfate proteoglycan glypican-4. Also in vitro, TRAP 5a exhibited affinity for both heparin and heparan sulfate with heparin inhibiting its enzyme activity. Upon caveolae-mediated endocytosis of saturating levels of TRAP 5a, TRAP 5a co-localized intracellularly with glypican-4 and late endosomal marker Rab-7 positive vesicles. The protein was also located in multivesicular bodies (MVBs) but did not co-localize with lysosomal marker LAMP-1. TRAP 5a endocytosis was also detectable in pre-osteoblasts, but not fibroblasts, embryonic MSCs or mature adipocytes. These results indicate that TRAP 5a exhibits binding to cell surface, endocytosis and affinity to glucosaminoglycans (GAGs) in pre-adipocyte and pre-osteoblast lineage cells in a manner similar to other heparin-binding growth factors.