A COX-2 Inhibitor Does Not Interfere With the Bone-Protective Effects of Loading in Male Mice With Arthritis.

Mechanical loading enhances bone strength and counteracts arthritis-induced inflammation-mediated bone loss in female mice. It is unknown whether nonsteroidal anti-inflammatory drugs (NSAIDs; eg, COX-2 inhibitors) can reduce inflammation without affecting the loading-associated bone formation in male mice. The aim of this study was to investigate if loading combined with a COX-2 inhibitor (NS-398) could prevent arthritis-induced bone loss and inflammation in male mice. Four-month-old male C57BL/6J mice were subjected to axial tibial mechanical loading three times/week for 2 weeks. Local mono-arthritis was induced with a systemic injection of methylated bovine serum albumin on the first day of loading, followed by a local injection in one knee 1 week later. The arthritis induction, knee swelling, bone architecture, and osteoclast number were evaluated in the hind limbs. C-terminal cross-links as a marker for osteoclast activity was measured in serum. Compared with loading and arthritis alone, loading of the arthritic joint enhanced swelling that was partly counteracted by NS-398. Loading of the arthritic joint enhanced synovitis and articular cartilage damage compared with loading alone. Loading increased cortical bone and counteracted the arthritis-induced decrease in epiphyseal bone. NS-398 did not alter the bone-protective effects of loading. C-terminal cross-links, a bone resorption marker, was increased by arthritis but not loading. In conclusion, loading prevented arthritis-induced epiphyseal and metaphyseal bone loss, and NS-398 reduced knee swelling without affecting the bone-protective effects of loading. If our results can be extrapolated to the human situation, specific COX-2 inhibitors could be used in combination with loading exercise to prevent pain and swelling of the joint without influencing the bone-protective effects of loading. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Cathepsin K regulates localization and secretion of Tartrate-Resistant Acid Phosphatase (TRAP) in TRAP-overexpressing MDA-MB-231 breast cancer cells.

BACKGROUND: Tartrate-resistant acid phosphatase (TRAP/ ACP5) belongs to the binuclear metallophosphatase family and is present in two isoforms. The primary translation product is an uncleaved TRAP 5a isoform with low phosphatase activity. TRAP 5a can be post-translationally processed to a cleaved TRAP 5b isoform with high phosphatase activity by e.g. cysteine proteinases, such as Cathepsin K (CtsK). The relevance of the phosphatase activity of TRAP 5b has been demonstrated for proliferation, migration and invasion of cancer cells. TRAP-overexpressing MDA-MB-231 breast cancer cells displayed higher levels of TRAP 5a and efficient processing of TRAP 5a to TRAP 5b protein, but no changes in levels of CtsK when compared to mock-transfected cells. In TRAP-overexpressing cells colocalization of TRAP 5a and proCtsK was augmented, providing a plausible mechanism for generation of TRAP 5b. CtsK expression has been associated with cancer progression and has been pharmacologically targeted in several clinical studies. RESULTS: In the current study, CtsK inhibition with MK-0822/Odanacatib did not abrogate the formation of TRAP 5b, but reversibly increased the intracellular levels of a N-terminal fragment of TRAP 5b and reduced secretion of TRAP 5a reversibly. However, MK-0822 treatment neither altered intracellular TRAP activity nor TRAP-dependent cell migration, suggesting involvement of additional proteases in proteolytic processing of TRAP 5a. Notwithstanding, CtsK was shown to be colocalized with TRAP and to be involved in the regulation of secretion of TRAP 5a in a breast cancer cell line, while it still was not essential for processing of TRAP 5a to TRAP 5b isoform. CONCLUSION: In cancer cells multiple proteases are involved in cleaving TRAP 5a to high-activity phosphatase TRAP 5b. However, CtsK-inhibiting treatment was able to reduce secretion TRAP 5a from TRAP-overexpressing cancer cells.

Bones in human CYP26B1 deficiency and rats with hypervitaminosis A phenocopy Vegfa overexpression.

Angulated femurs are present prenatally both in CYP26B1 deficient humans with a reduced capacity to degrade retinoic acid (RA, the active metabolite of vitamin A), and mice overexpressing vascular endothelial growth factor a (Vegfa). Since excessive ingestion of vitamin A is known to induce spontaneous fractures and as the Vegfa-induced femur angulation in mice appears to be caused by intrauterine fractures, we analyzed bones from a CYP26B1 deficient human and rats with hypervitaminosis A to further explore Vegfa as a mechanistic link for the effect of vitamin A on bone. We show that bone from a human with CYP26B1 mutations displayed periosteal osteoclasts in piles within deep resorption pits, a pathognomonic sign of hypervitaminosis A. Analysis of the human angulated fetal femur revealed excessive bone formation in the marrow cavity and abundant blood vessels. Normal human endothelial cells showed disturbed cell-cell junctions and increased CYP26B1 and VEGFA expression upon RA exposure. Studies in rats showed increased plasma and tissue Vegfa concentrations and signs of bone marrow microhemorrhage on the first day of excess dietary vitamin A intake. Subsequently hypervitaminosis A rats displayed excess bone formation, fibrosis and an increased number of megakaryocytes in the bone marrow, which are known characteristics of Vegfa overexpression. This study supports the notion that the skeletal phenotype in CYP26B1 deficient human bone is caused by excess RA. Our findings suggest that an initial part of the vitamin A mechanism causing bone alterations is mediated by excess Vegfa and disturbed bone marrow microvessel integrity.

Bone Alkaline Phosphatase and Tartrate-Resistant Acid Phosphatase: Potential Co-regulators of Bone Mineralization.

Phosphorylated osteopontin (OPN) inhibits hydroxyapatite crystal formation and growth, and bone alkaline phosphatase (BALP) promotes extracellular mineralization via the release of inorganic phosphate from the mineralization inhibitor inorganic pyrophosphate (PPi). Tartrate-resistant acid phosphatase (TRAP), produced by osteoclasts, osteoblasts, and osteocytes, exhibits potent phosphatase activity towards OPN; however, its potential capacity as a regulator of mineralization has not previously been addressed. We compared the efficiency of BALP and TRAP towards the endogenous substrates for BALP, i.e., PPi and pyridoxal 5'-phosphate (PLP), and their impact on mineralization in vitro via dephosphorylation of bovine milk OPN. TRAP showed higher phosphatase activity towards phosphorylated OPN and PPi compared to BALP, whereas the activity of TRAP and BALP towards PLP was comparable. Bovine milk OPN could be completely dephosphorylated by TRAP, liberating all its 28 phosphates, whereas BALP dephosphorylated at most 10 phosphates. OPN, dephosphorylated by either BALP or TRAP, showed a partially or completely attenuated phosphorylation-dependent inhibitory capacity, respectively, compared to native OPN on the formation of mineralized nodules. Thus, there are phosphorylations in OPN important for inhibition of mineralization that are removed by TRAP but not by BALP. In conclusion, our data indicate that both BALP and TRAP can alleviate the inhibitory effect of OPN on mineralization, suggesting a potential role for TRAP in skeletal mineralization. Further studies are warranted to explore the possible physiological relevance of TRAP in bone mineralization.

Aminothiazoles inhibit RANKL- and LPS-mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells.

Periodontitis is characterized by chronic inflammation and osteoclast-mediated bone loss regulated by the receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase-1 (mPGES-1) on RANKL- and lipopolysaccharide (LPS)-mediated osteoclastogenesis and prostaglandin E2 (PGE2 ) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4-([4-(2-naphthyl)-1,3-thiazol-2-yl]amino)phenol (TH-848) or 4-(3-fluoro-4-methoxyphenyl)-N-(4-phenoxyphenyl)-1,3-thiazol-2-amine (TH-644). Aminothiazoles significantly decreased the number of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells in cultures of RANKL- and LPS-stimulated RAW 264.7 cells, as well as reduced the production of PGE2 in culture supernatants. LPS-treatment induced mPGES-1 mRNA expression at 16 hrs and the subsequent PGE2 production at 72 hrs. Conversely, RANKL did not affect PGE2 secretion but markedly reduced mPGES-1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL-activated RAW 264.7 cells, TH-848 and TH-644 down-regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE2 production was also confirmed in LPS-stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS- and RANKL-mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.

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.

Fluid pressure induces osteoclast differentiation comparably to titanium particles but through a molecular pathway only partly involving TNFα.

In contrast to the well-understood inflammatory pathway driven by TNFα, by which implant-derived particles induce bone resorption, little is known about the process in which loosening is generated as a result of force-induced mechanical stimulus at the bone-implant interface. Specifically, there is no knowledge as to what cells or signaling pathways couple mechanical stimuli to bone resorption in context of loosening. We hypothesized that different stimuli, i.e., fluid flow versus wear particles, act through different cytokine networks for activation and localization of osteoclasts. By using an animal model in which osteoclasts and bone resorption were induced by fluid pressure or particles, we were able to detect distinct differences in osteoclast localization and inflammatory gene expression between fluid pressure and titanium particles. Fluid pressure recruits and activates osteoclasts with bone marrow contact away from the fluid pressure exposure zone, whereas titanium particles recruit and activate osteoclasts in areas in direct contact to particles. Fluid pressure induced weaker expression of the selected inflammatory related genes, although the eventual degree of osteoclast induction was similar in both models. Using TNFαRa (4 mg/kg) (Enbrel) and dexamethasone (2 mg/kg) as specific and more general suppressors of inflammation we showed that the TNFαRa failed to generate statistically impaired osteoclast generation while dexamethasone was much more potent. These results demonstrate that fluid pressure induces osteoclasts at a different localization than titanium particles by a molecular pathway less associated with TNFα and the innate system, which open up for other pathways controlling pressure induced osteoclastogenesis.

Localization and expression of prothrombin in rodent osteoclasts and long bones.

The serum protein prothrombin (PT) is proteolytically converted to thrombin during the coagulation cascade by the cell-associated prothrombinase complex. In vitro, RANKL-differentiated osteoclasts express tissue factor and coagulation factor Xa, which convert PT to thrombin (Karlstrom et al. Biochem Biophys Res Commun 394:593-599, 2010). The present study investigated the localization of PT in bone as well as the expression of PT mRNA in bone and osteoclasts. Herein, immunoblot analysis detected PT and smaller proteolytically cleaved fragments with sizes consistent with the action of prothrombinase in a protein fraction extracted with guanidine-HCl EDTA from mouse tibia. Light microscopic and ultrastructural immunohistochemistry demonstrated the presence of PT in the newly formed bone matrix of the metaphysis. Furthermore, fluorescent immunohistochemistry on metaphyseal trabecular bone showed that PT colocalized with MMP-9-expressing subepiphyseal osteoclasts, whereas cathepsin K-expressing osteoclasts were closely associated with PT of the bone matrix. RT-qPCR analysis revealed that PT mRNA was detected in tibia. Expression of PT mRNA in the tibia was 0.2% of the level in the liver. In addition, PT mRNA expression was increased by RANKL-induced differentiation of bone marrow macrophages to osteoclasts. The results demonstrate that PT is synthesized and proteolytically processed in bone. Furthermore, PT is present mainly in the newly formed bone matrix of the metaphyseal trabecular bone compartment in close association to osteoclasts. In addition, MMP-9-positive osteoclasts contain PT, and PT expression is increased during osteoclastogenesis.

Resveratrol-conjugated poly-ε-caprolactone facilitates in vitro mineralization and in vivo bone regeneration.

Incorporation of osteoinductive factors in a suitable scaffold is considered a promising strategy for generating osteogenic biomaterials. Resveratrol is a polyphenol found in parts of certain plants, including nuts, berries and grapes. It is known to increase DNA synthesis and alkaline phosphatase (ALP) activity in osteoblasts and to prevent femoral bone loss in ovariectomized (OVX) rats. In the present study resveratrol was coupled through a hydrolysable covalent bond with the carboxylic acid groups in porous poly-ε-caprolactone (PCL) surface grafted with acrylic acid (AA). The osteogenic effect of this new scaffold was evaluated in mesenchymal cell culture and in the rat calvarial defect model. We found that the incorporation of resveratrol caused increased ALP activity of rat bone marrow stromal cells and enhanced mineralization of the cell-scaffold composites in vitro. After 8 weeks the calvarial defects implanted with resveratrol-conjugated PCL displayed a higher X-ray density than the defects implanted with control PCL. Bone-like structures, positively immunostained for bone sialoprotein, were shown to be more extensively formed in the resveratrol-conjugated PCL. These results show that incorporation of resveratrol into the AA-functionalized porous PCL scaffold led to a significant increase in osteogenesis.

Osteoclast polarization is not required for degradation of bone matrix in rachitic FGF23 transgenic mice.

Hypophosphatemic transgenic (tg) mice overexpressing FGF23 in osteoblasts display disorganized growth plates and reduced bone mineral density characteristic of rickets/osteomalacia. These FGF23 tg mice were used as an in vivo model to examine the relation between osteoclast polarization, secretion of proteolytic enzymes and resorptive activity. Tg mice had increased mRNA expression levels of the osteoblast differentiation marker Runx2 and mineralization-promoting proteins alkaline phosphatase and bone sialoprotein in the long bones compared to wild type (wt) mice. In contrast, expression of alpha1(I) collagen, osteocalcin, dentin matrix protein 1 and osteopontin was unchanged, indicating selective activation of osteoblasts promoting mineralization. The number of osteoclasts was unchanged in tg compared to wt mice, as determined by histomorphometry, serum levels of TRAP 5b activity as well as mRNA expression levels of TRAP and cathepsin K. However, tg mice displayed elevated serum concentrations of C-terminal telopeptide of collagen I (CTX) indicative of increased bone matrix degradation. The majority of osteoclasts in FGF23 tg mice lacked ultrastructural morphological signs of proper polarization. However, they secreted both cathepsin K and MMP-9 at levels comparable to osteoclasts with ruffled borders. Mineralization of bone matrix thus appears essential for inducing osteoclast polarization but not for secretion of osteoclast proteases. Finally, release of CTX by freshly isolated osteoclasts was increased on demineralized compared to mineralized bovine bone slices, indicating that the mineral component limits collagen degradation. We conclude that ruffled borders are implicated in acidification and subsequent demineralization of the bone matrix, however not required for matrix degradation. The data collectively provide evidence that osteoclasts, despite absence of ruffled borders, effectively participate in the degradation of hypomineralized bone matrix in rachitic FGF23 tg mice.

Proteolytic processing and polarized secretion of tartrate-resistant acid phosphatase is altered in a subpopulation of metaphyseal osteoclasts in cathepsin K-deficient mice.

Tartrate-resistant acid phosphatase (TRAP) is an enzyme highly expressed in osteoclasts and thought to participate in osteoclast-mediated bone turnover. Cathepsin K (Ctsk) is the major collagenolytic cysteine proteinase expressed in osteoclasts and has recently been shown to be able to proteolytically process and activate TRAP in vitro. In this study, 4-week-old Ctsk(-/-) mice were analysed for TRAP expression at the mRNA, protein and enzyme activity levels to delineate a role of cathepsin K in TRAP processing in osteoclasts in vivo. The absence of cathepsin K in osteoclasts was associated with increased expression of TRAP mRNA, monomeric TRAP protein and total TRAP activity. Proteolytic processing of TRAP was not abolished but prematurely arrested at an intermediate stage without changing enzyme activity, a finding confirmed with RANKL-differentiated osteoclast-like cell line RAW264.7 treated with the cysteine proteinase inhibitor E-64. Thus, the increase in total TRAP activity was mainly due to increased cellular content of monomeric TRAP. The increase in monomeric TRAP expression was more pronounced in osteoclasts of the distal compared to the proximal part of the metaphyseal trabecular bone, suggesting a site-dependent role for cathepsin K in TRAP processing. Moreover, intracellular localization of monomeric TRAP was altered in distal metaphyseal osteoclasts from Ctsk(-/-) mice. Additionally, TRAP was secreted into the ruffled border as the processed form in osteoclasts of Ctsk(-/-) mice, unlike in osteoclasts from wild-type mice which secreted TRAP to the resorption lacuna as the monomeric form. The results demonstrate that cathepsin K is not only involved in proteolytic processing but also affects the intracellular trafficking of TRAP, particularly in osteoclasts of the distal metaphysis. However, contribution by other yet unidentified protease(s) to TRAP processing must also be invoked since proteolytic cleavage of TRAP is not abolished in Ctsk(-/-) mice. Importantly, this study highlights functional differences between bone-resorbing clasts within the trabecular metaphyseal bone, suggesting potentially important differences in the regulation of differentiation and activation depending on the precise anatomical localization of the clast population.

Cholesterol-sensing receptors, liver X receptor alpha and beta, have novel and distinct roles in osteoclast differentiation and activation.

UNLABELLED: The liver X receptor (alpha,beta) is responsible for regulating cholesterol homeostasis in cells. However, our studies using the LXRalpha-/-, LXRbeta-/-, and LXRalpha-/-beta-/- mice show that both LXRalpha and beta are also important for bone turnover, mainly by regulating osteoclast differentiation/activity. INTRODUCTION: The liver X receptors (alpha,beta) are primarily responsible for regulating cholesterol homeostasis within cells and the whole body. However, as recent studies show that the role for this receptor is expanding, we studied whether the LXRs could be implicated in bone homeostasis and development. MATERIALS AND METHODS: pQCT was performed on both male and female LXRalpha-/-, LXRbeta-/-, LXRalpha-/-beta-/-, and WT mice at 4 months and 1 year of age. Four-month-old female mice were additionally analyzed with reference to qPCR, immunohistochemistry, histomorphometry, transmission electron microscopy, and serum bone turnover markers. RESULTS: At the mRNA level, LXRbeta was more highly expressed than LXRalpha in both whole long bones and differentiating osteoblast-like MC3T3-E1 and osteoclast-like RAW 264.7 cells. Four-month-old female LXRalpha-/- mice had a significant increase in BMD because of an increase in all cortical parameters. No difference was seen regarding trabecular BMD. Quantitative histomorphometry showed that these mice had significantly more endosteal osteoclasts in the cortical bone; however, these cells appeared less active than normal cells as suggested by a significant reduction in serum levels of cross-linked carboxyterminal telopeptides of type I collagen (CTX) and a reduction in bone TRACP activity. Conversely, the female LXRbeta-/- mice exhibited no change in BMD, presumably because a significant decline in the number of the trabecular osteoclasts was compensated for by an increase in the expression of the osteoclast markers cathepsin K and TRACP. These mice also had a significant decrease in serum CTX, suggesting decreased bone resorption; however, in addition presented with an increase in the expression of osteoblast associated genes, bone formation markers, and serum leptin levels. CONCLUSIONS: Our findings show that both LXRs influence cellular function within the bone, with LXRalpha having an impact on osteoclast activity, primarily in cortical bone, whereas LXRbeta modulates trabecular bone turnover.

Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts.

Tartrate-resistant acid phosphatase (TRAP) is a metallophosphoesterase participating in osteoclast-mediated bone turnover. Activation of TRAP is associated with the redox state of the di-iron metal center as well as with limited proteolytic cleavage in an exposed loop domain. The cysteine proteinases cathepsin B, L, K, and S as well as the matrix metalloproteinase-2, -9, -13, and -14 are expressed by osteoclasts and/or other bone cells and have been implicated in the turnover of bone and cartilage. To identify proteases that could act as activators of TRAP in bone, we report here that cathepsins K and L, in contrast to the matrix metalloproteinases, efficiently cleaved and activated recombinant TRAP in vitro. Activation of TRAP by cathepsin K/L was because of increases in catalytic activity, substrate affinity, and sensitivity to reductants. Processing by cathepsin K occurred sequentially by an initial excision of the loop peptide Gly(143)-Gly(160) followed by the removal of a Val(161)-Ala(162) dipeptide at the N terminus of the C-terminal 16-kDa TRAP subunit. Cathepsin L initially released a shorter Gln(151)-Gly(160) peptide and completed processing at Ser(145) or Gly(143) at the C terminus of the N-terminal 23-kDa TRAP subunit and at Arg(163) at the N terminus of the C-terminal 16-kDa TRAP subunit. Mutation of Ser(145) to Ala partly mimicked the effect of proteolysis on catalytic activity, identifying Ser(145) as well as Asp(146) (Funhoff, E. G., Ljusberg, J., Wang, Y., Andersson, G., and Averill, B. A. (2001) Biochemistry 40, 11614-11622) as repressive amino acids of the loop region to maintain the TRAP enzyme in a catalytically latent state. The C-terminal sequence of TRAP isolated from rat bone was consistent with cathepsin K-mediated processing in vivo. Moreover, cathepsin K, but not cathepsin L, co-localized with TRAP in osteoclast-resorptive compartments, supporting a role for cathepsin K in the extracellular processing of monomeric TRAP in the resorption lacuna.

N-glycosylation influences the latency and catalytic properties of mammalian purple acid phosphatase.

Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase or uteroferrin, contains two potential consensus N-glycosylation sites at Asn(97) and Asn(128). In this study, endogenous rat bone PAP was found to possess similar N-glycan structures as rat recombinant PAP heterologously expressed in baculovirus-infected Sf9 insect cells. PAP from Sf9 cells was shown to contain two N-linked oligosaccharides, whereas PAP expressed by mammalian CHO-K1 cells was less extensively glycosylated. The extent of N-glycosylation affected the catalytic properties of the enzyme, as N97Q and N128Q mutants, containing a single oligosaccharide chain, exhibited a lower substrate affinity and catalytic activity compared to those of the fully glycosylated PAP in the native, monomeric state. The differences in substrate affinity and catalytic activity were abolished and partially restored, respectively, by proteolytic cleavage in the loop domain, indicating that the extent of N-glycosylation influences the interaction of the repressive loop domain with catalytically important residues.

The effects of colony-stimulating factor-1 (CSF-1) on the development of osteoclasts and their expression of tartrate-resistant acid phosphatase (TRAP) in toothless (tl-osteopetrotic) rats.

The osteopetrotic mutation toothless (tl) in the rat is characterized by a limited number of osteoclasts with reduced amounts and/or activity of tartrate-resistant acid phosphatase (TRAP). Treatment of tl/tl mutants with the cytokine colony-stimulating factor (CSF)-1 increases both osteoclast number and enzyme activity, consistent with a loss-of-function mutation in the CSF-1 gene recently detected in this mutant. We have pursued these observations to demonstrate that there is a dose-dependent increase in osteoclast number, but not to normal levels. Osteoclasts in CSF-1-treated tl/tl mutants are large, have well-developed clear zones and ruffled borders, and secrete TRAP into resorption lacunae. The expression of TRAP mRNA, protein, and enzyme activity per bone appear normal after CSF-1 treatment. However, in contrast to the predominantly apical intracellular distribution in normal osteoclasts, an enrichment of TRAP enzyme activity in osteoclasts of CSF-1-treated tl/tl mutants is observed in the basal part of the cell. Our observations suggest that the CSF-1-treated mutant bones contain an abundance of mature osteoclasts, actively expressing markers for osteoclasts such as TRAP, cathepsin K, and matrix metalloproteinase (MMP)-9. Accumulation of TRAP at the end of the endocytic pathway in mature osteoclasts formed during CSF-1 treatment suggests that the TRAP enzyme has a rapid turnover in these highly active cells and uses a transcytotic pathway.

TRACP as an osteopontin phosphatase.

TRACP is synthesized as a latent proenzyme requiring proteolytic processing to attain maximal phosphatase activity. Excision of an exposed loop domain abolishes the interaction between the loop residue Asp146 and a ligand to the redox-sensitive iron of the active site, most likely Asn91, providing a mechanism for the enzyme repression. Both cathepsin K and L efficiently cleave in the loop domain and activate the latent enzyme, and we propose that cathepsin K acts as a physiological activator of TRACP in osteoclasts, whereas cathepsin L might fulfill a similar role in different types of macrophages. Considering the rather broad substrate specificity of TRACP, a tight regulation of its activity in the cell appears warranted. Besides proteolytic cleavage, the enzyme should need a specific local environment with a slightly acidic pH and reducing equivalents to keep the enzyme fully active. Cellular subcompartments where these required conditions prevail are potential subcellular site(s) of TRACP action. Of bone phosphoproteins shown to be substrates for TRACP, both osteopontin and bone sialoprotein are colocalized with TRACP in the resorption lacuna of the osteoclasts, and dephosphorylation of OPN impair its ability to promote adhesion as well as migration of osteoclasts in vitro. A role for TRACP as an osteopontin phosphatase in bone is therefore suggested. The expression of TRACP as well as OPN in other tissues with possible interactions between the two could suggest a more general function for TRACP as a regulator of OPN phosphorylation and bioactivity.