Endogenous expression and endocytosis of tartrate-resistant acid phosphatase (TRACP) by osteoblast-like cells.

Tartrate-resistant acid phosphatase (TRACP) is produced by macrophages and other cells of the monohistiocytic lineage. In particular, osteoclasts are characterized for a high expression of this enzyme. Yet, several data suggest that other bone cell types, such as osteocytes and osteoblasts, may also express activity of this enzyme. This is particularly obvious at sites were osteoclasts resorb bone, suggesting that osteoclasts (or their precursors) somehow induce TRACP activity in osteoblasts. In the present study, we investigated this by culturing human osteoblast-like cells with and without conditioned medium (MCM) from human blood monocytes (as a source of osteoclast precursors). High levels of TRACP activity were found in osteoblast-like cells cultured with MCM. Depletion of TRACP from this medium resulted in the absence of its activity in osteoblast-like cells, thus suggesting that the TRACP activity in these cells was the result of endocytosed TRACP that was released by the monocytes in the MCM. Osteoblast-like cells cultured in control (non-conditioned) medium contained very low levels of TRACP-like activity. However, the cells expressed TRACP mRNA and incubation of extracts of these cells with active cathepsin B did induce activity of a TRACP-like enzyme. Inhibition of the activity of cysteine proteinases in general and of cathepsin B in particular, completely blocked TRACP activity of the osteoblast-like cells. This TRACP-like enzyme but not the alleged endocytosed fraction of TRACP was inhibited by fluoride, suggesting that the fractions may be different isoenzymes. Our data seem to indicate that osteoblast-like cells may contain two different fractions of TRACP, one that is released by monocytes and subsequently endocytosed by osteoblast-like cells and a second endogenous fraction that is present in an inactive proform. We hypothesize that the capacity of osteoblast-like cells to endocytose TRACP is important for the removal of this enzyme during or following the bone resorptive activity of the osteoclast.

Serum tartrate-resistant acid phosphatase 5b, but not 5a, correlates with other markers of bone turnover and bone mineral density.

Human serum contains two isoforms of tartrate-resistant acid phosphatase (TRACP) known as TRACP 5a and TRACP 5b with pH optima of 5.0 and 5.8, respectively. Preliminary data suggest that serum TRACP 5b is derived from osteoclasts and serum TRACP 5a from some other cells. It has been reported that heparin inhibits TRACP 5a but has no effect on the activity of TRACP 5b. Here we show that heparin has no effect on serum TRACP activity, as determined using our previously published immunoassay, suggesting that the immunoassay does not detect TRACP 5a. The change of serum TRACP 5b activity after 6 months HRT, determined by this immunoassay, correlated significantly with the changes of all markers of bone turnover determined, including serum N- and C-terminal propeptides of type I collagen and urinary-free deoxypyridinoline. Serum TRACP 5b activity was significantly elevated in patients with osteoporosis and had a significant negative correlation with bone mineral density (BMD). Serum TRACP 5a activity, determined by an immunoassay, showed no correlation with serum TRACP 5b activity, with BMD, or with any of the markers of bone turnover. These results show that serum TRACP 5b, but not 5a, reflects the bone resorption rate, and that our TRACP 5b immunoassay may be a specific method for the determination of the bone resorption rate from serum samples.

Estrogen receptor alpha, but not estrogen receptor beta, is involved in the regulation of the OPG/RANKL (osteoprotegerin/receptor activator of NF-kappa B ligand) ratio and serum interleukin-6 in male mice.

Estrogens are important for the male skeleton. Osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), interleukin-6 (IL-6), IL-1 and tumor necrosis factor alpha (TNFalpha) have been suggested to be involved in the skeletal effects of estrogen. We treated orchidectomized mice with estradiol for 2 weeks and observed a 143% increase in the trabecular bone mineral density of the distal metaphysis of femur that was associated with a decreased OPG/RANKL mRNA ratio in vertebral bone. A similar decreased OPG/RANKL ratio was also seen after estrogen treatment of ovariectomized female mice. The effect of estrogen receptor (ER) inactivation on the OPG/RANKL ratio was dissected by using intact male mice lacking ER alpha (ERKO), ER beta (BERKO) or both receptors (DERKO). The expression of OPG was increased in ERKO and DERKO but not in BERKO male mice, resulting in an increased OPG/RANKL ratio. Furthermore, serum levels of IL-6 and tartrate-resistant acid phosphatase 5b (TRAP 5b) were decreased in ERKO and DERKO, but not in BERKO male mice. These results demonstrate that ER alpha, but not ER beta, is involved in the regulation of the vertebral OPG/RANKL ratio, serum levels of IL-6 and TRAP 5b in male mice.

Estrogen receptor specificity in the regulation of the skeleton in female mice.

There are two known estrogen receptors, estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta), which may mediate the actions of estrogen. The aim of the present study was to compare fat content, skeletal growth and adult bone metabolism in female mice lacking ER alpha (ERKO), ER beta (BERKO) or both ERs (DERKO). We demonstrate that endogenous estrogens decrease the fat content in female mice via ER alpha and not ER beta. Interestingly, the longitudinal bone growth was decreased in ERKO, increased in BERKO, but was intermediate in DERKO females, demonstrating that ER alpha and ER beta exert opposing effects in the regulation of longitudinal bone growth. The effects on longitudinal bone growth were correlated with similar effects on serum levels of IGF-I. A complex regulation of the trabecular bone mineral density (BMD), probably caused by a disturbed feedback regulation of estrogen and testosterone, was observed in female ER-inactivated mice. Nevertheless, a partial functional redundancy for ER alpha and ER beta in the maintenance of the trabecular BMD was observed in the female mice at 60 days of age. Thus, ER alpha and ER beta may have separate effects (regulation of fat), opposing effects (longitudinal bone growth) or partial redundant effects (trabecular BMD at 60 days of age), depending on which parameter is studied.

Rapid screening method for osteoclast differentiation in vitro that measures tartrate-resistant acid phosphatase 5b activity secreted into the culture medium.

BACKGROUND: Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) 5b into the circulation. We studied the release of TRAP 5b from osteoclasts using a mouse in vitro osteoclast differentiation assay. METHODS: We developed and characterized a polyclonal antiserum in rabbits, using purified human osteoclastic TRAP 5b as antigen. The antiserum was specific for TRAP in Western analysis of mouse osteoclast culture medium and was used to develop an immunoassay. We cultured mouse bone marrow-derived osteoclast precursor cells for 3-7 days with or without clodronate in the presence of vitamin D and analyzed the number of osteoclasts formed and the amount of TRAP 5b activity released into the culture medium. RESULTS: TRAP 5b activity was not secreted from osteoclast precursor cells. Addition of clodronate-containing liposomes decreased in a dose-dependent manner the number of osteoclasts and TRAP 5b activity released in 6-day cultures. The amount of TRAP 5b activity in the medium detected by the immunoassay correlated significantly with the number of osteoclasts formed (r = 0.94; P<0.0001; n = 120). CONCLUSIONS: The TRAP 5b immunoassay can be used to replace the laborious and time-consuming microscopic counting of osteoclasts in the osteoclast differentiation assay and to test the effects of potential therapeutic agents on osteoclast differentiation, enabling fast screening of large amounts of potential therapeutic agents.

Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption.

Human serum contains two forms of tartrate-resistant acid phosphatase (TRAP), 5a and 5b. Of these, 5a contains sialic acid and 5b does not. We show here that antigenic properties and pH optimum of TRAP purified from human osteoclasts are identical to those of serum TRAP 5b and completely different from those of serum TRAP 5a, suggesting that 5b would be derived from osteoclasts and 5a from some other source. We developed a novel immunoassay specific for 5b using a monoclonal antibody O1A as capture antibody. O1A did not bind acid phosphatase derived from platelets and erythrocytes. Western analysis showed that O1A was specific for TRAP in both human bone and serum. We measured bound TRAP activity at pH 6.1, where 5b is highly active and 5a almost completely inactive. The immunoassay detected more than 90% of the initial TRAP 5b activity after 8-h incubation of serum samples at 25 degrees C and after 3 days incubation at 4 degrees C. Serum TRAP 5b activity decreased significantly after 6 months of hormone replacement therapy (HRT) of postmenopausal women compared with the change observed in postmenopausal women receiving placebo (p < 0.0001). Instead, no significant differences were observed between the changes in the placebo and HRT groups in total serum TRAP amount. These results show that serum TRAP 5b is a specific and sensitive marker for monitoring antiresorptive treatment. Instead, total serum TRAP cannot be used for that purpose. These findings may turn out to be a significant improvement in using serum TRAP as a resorption marker.