Jussi M. Halleen

Biochemical Markers of Bone Metabolism and Prediction of Fracture in Elderly Women

We studied the ability of various markers of bone turnover to predict fracture in 1040 randomly recruited 75‐year‐old women. A total of 178 of the women sustained at least one fracture during follow‐up (mean, 4.6 years). In elderly women, TRACP5b and urinary fragments of osteocalcin are promising new markers for prediction of fracture, in particular, vertebral fracture.

Intracellular Machinery for Matrix Degradation in Bone-Resorbing Osteoclasts†

In osteoclasts, TRACP co‐localized with cathepsin K in transcytotic vesicles and was activated by cathepsin K in vitro, suggesting that TRACP may degrade organic matrix components in transcytotic vesicles in an event regulated by cathepsin K.

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.

Serum TRACP 5b is a useful marker for monitoring alendronate treatment: comparison with other markers of bone turnover

We studied clinical performance of serum TRACP 5b and other bone turnover markers, including S‐CTX, U‐DPD, S‐PINP, S‐BALP, and S‐OC, for monitoring alendronate treatment. TRACP 5b had higher clinical sensitivity, area under the ROC curve, and signal‐to‐noise ratio than the other markers.

Estrogen Reduces the Depth of Resorption Pits by Disturbing the Organic Bone Matrix Degradation Activity of Mature Osteoclasts

Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-term...

Alendronate disturbs vesicular trafficking in osteoclasts.

The nitrogen-containing bisphosphonate alendronate inhibits osteoclast-mediated bone resorption through inhibition of the mevalonate pathway. This results in impaired protein prenylation and may affect the function of small GTPases in osteoclasts. Since these proteins are important regulators of vesicle transport in cells, we investigated the possible interference of alendronate with these processes in isolated rat osteoclasts. We show here that alendronate-induced inhibition of bone resorption coincides with accumulation of tartrate-resistant acid phosphatase- and electron dense material-containing tubular vesicles in osteoclasts. Alendronate-induced changes in osteoclasts also included widening of the sealing zone areas and incomplete organization of tight attachments and ruffled borders. Osteoclasts also appeared partially detached from the bone surface, and organic matrix was typically dissolved only at the edges of the resorption pits on alendronate-coated bone slices. In contrast, resorption pits on the control and clodronate-coated bone slices were thoroughly resorbed. Inhibition of bone resorption by alendronate was not, however, related to a decrease in osteoclast number. In conclusion, our findings suggest that alendronate inactivates osteoclasts by mechanisms that impair their intracellular vesicle transport, apoptosis being only a secondary phenomenon to this.

Serum tartrate-resistant acid phosphatase isoforms in rheumatoid arthritis

OBJECTIVES Our objective was to evaluate the significance and source of serum tartrate-resistant acid phosphatase (TRACP) in patients with rheumatoid arthritis (RA). METHODS Thirty-five RA, 32 osteoarthritis (OA) and 16 control subjects were studied. Serum TRACP-5b activity and total TRACP protein were determined by immunoassay. TRACP isoforms were analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Serum bone alkaline phosphatase (BAP), cross-linked N-terminal telopeptides (NTx), and C-terminal telopeptides (ICTP) of type I collagen were estimated as markers of bone turnover. C-reactive protein (CRP) was measured as a marker of chronic inflammation. Macrophages and dendritic cells (DC) were developed from peripheral blood monocytes. Cell lysates and culture supernatants were analyzed for TRACP isoforms by immunoassay and PAGE. RESULTS In RA, mean TRACP-5b activity was normal, but median total TRACP protein was increased twofold (p<0.001). In OA, TRACP-5b activity and protein were normal. In RA, TRACP-5b activity correlated weakly with ICTP (r=0.56) while TRACP protein levels correlated weakly with NTx (r=0.43). Additionally, TRACP protein, but not TRACP-5b activity correlated significantly with CRP (r=0.42). Macrophage and DC lysates contained TRACP-5b, while tissue culture supernatants contained TRACP-5a. CONCLUSIONS Increased total TRACP protein in RA sera was probably due to TRACP-5a and not derived from osteoclasts. Rather, it could be a secreted product of inflammatory macrophages and DC.

Two Different Pathways for the Maintenance of Trabecular Bone in Adult Male Mice

Androgens may regulate the male skeleton either directly via activation of the androgen receptor (AR) or indirectly via aromatization of androgens into estrogen and, thereafter, via activation of estrogen receptors (ERs). There are two known estrogen receptors, ER‐α and ER‐β. The aim of this study was to investigate the relative roles of ER‐α, ER‐β, and AR in the maintenance of trabecular bone in male mice. Seven‐month‐old male mice, lacking ER‐α (ERKO), ER‐β (BERKO), or both receptors (DERKO), were orchidectomized (orx) and treated for 3 weeks with 0.7 μg/mouse per day of 17β‐estradiol or vehicle. No reduction in trabecular bone mineral density (BMD) was seen in ERKO, BERKO, or DERKO mice before orx, showing that neither ER‐α nor ER‐β is required for the maintenance of a normal trabecular BMD in male mice. After orx, there was a pronounced decrease in trabecular BMD, similar for all groups, resulting in equal levels of trabecular BMD in all genotypes. This reduction was reversed completely in wild‐type (WT) and BERKO mice treated with estrogen, and no significant effect of estrogen was found in ERKO or DERKO mice. In summary, the trabecular bone is preserved both by a testicular factor, presumably testosterone acting via AR and by an estrogen‐induced activation of ER‐α. These results indicate that AR and ER‐α are redundant in the maintenance of the trabecular bone in male mice. In contrast, ER‐β is of no importance for the regulation of trabecular bone in male mice.

Properties and expression of human tartrate-resistant acid phosphatase isoform 5a by monocyte-derived cells

Human serum tartrate‐resistant acid phosphatase exists as two enzyme isoforms (TRACP 5a and 5b), derived by differential, post‐translational processing of a common gene product. Serum TRACP 5b is from bone‐resorbing osteoclasts (OC) and becomes elevated in diseases of increased bone resorption. TRACP 5a is secreted by macrophages (MΦ) and dendritic cells (DC) and is increased in many patients with rheumatoid arthritis. Our purpose was to fully characterize the properties of human TRACP isoforms and to produce an antibody specific to TRACP 5a for use as a biomarker in chronic inflammatory diseases. Partially purified, natural serum TRACP isoforms and recombinant TRACP 5a (rTRACP 5a) were compared with respect to specific activity and subunit structure and presence of sialic acid. Mice were immunized with rTRACP 5a, and resulting hybridomas were screened for monoclonal antibody to serum TRACP 5a. One antibody, 220, was tested for its epitope specificity and use in various immunological techniques. rTRACP 5a had properties identical to serum TRACP 5a. Antibody 220 was specific for the trypsin‐sensitive epitope in the loop peptide, present only in TRACP 5a. Antibody 220 was effective for specific immunoprecipitation, immunoassay, and immunoblot of TRACP 5a. Intact TRACP was present in MΦ, DC, and OC. TRACP 5a was the predominant isoform secreted by MΦ and DC, whereas TRACP 5b was the predominant isoform secreted by OC. TRACP isoforms 5a and 5b may have different functions inside and outside of monocyte‐derived cells. Antibody 220 is an important resource for studies of the biosynthetic relationship among TRACP isoforms and of the significance of serum TRACP 5a as a marker in diseases of bone metabolism and inflammation.

Potential function for the ROS-generating activity of TRACP.

TRACP is an enzyme with unknown biological function. It is expressed primarily in bone‐resorbing osteoclasts and activated macrophages. In addition to its phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS). In resorbing osteoclasts, TRACP is localized in transcytotic vesicles transporting bone matrix degradation products from the resorption lacuna to a functional secretory domain in the basolateral membrane. ROS generated by TRACP are capable of destroying organic bone matrix components, suggesting that they may be targeted to further destroy initial matrix degradation products in the transcytotic vesicles. The transcytotic route of osteoclasts is analogous with the antigen presentation route of macrophages transporting endocytosed foreign material into cell surface for presentation to other cells of the immune system. Macrophages overexpressing TRACP have elevated levels of intracellular ROS. In alveolar macrophages, TRACP is colocalized with endocytosed Staphylococcus aureus, a pathogen whose clearance is reduced in TRACP‐deficient mice, suggesting that ROS generated by TRACP may be targeted to destroy foreign material in the antigen presentation route of macrophages. These data suggest that the ROS generating activity of TRACP may have an important role both in bone resorption and in the immune defense system.