Macrophage expressed tartrate-resistant acid phosphatase 5 promotes pulmonary fibrosis progression.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder involving scarring of pulmonary tissue and a subsequent decrease in respiratory capacity, ultimately resulting in death. Tartrate resistant acid phosphatase 5 (ACP5) plays a role in IPF but the exact mechanisms are yet to be elucidated. In this study, we have utilized various perturbations of the bleomycin mouse model of IPF including genetic knockout, RANKL inhibition, and macrophage adoptive transfer to further understand ACP5's role in pulmonary fibrosis. Genetic ablation of Acp5 decreased immune cell recruitment to the lungs and reduced the levels of hydroxyproline (reflecting extracellular matrix-production) as well as histological damage. Additionally, gene expression profiling of murine lung tissue revealed downregulation of genes including Ccl13, Mmp13, and Il-1α that encodes proteins specifically related to immune cell recruitment and macrophage/fibroblast interactions. Furthermore, antibody-based neutralization of RANKL, an important inducer of Acp5 expression, reduced immune cell recruitment but did not decrease fibrotic lung development. Adoptive transfer of Acp5-/- bone marrow-derived monocyte (BMDM) macrophages 7 or 14 days after bleomycin administration resulted in reductions of cytokine production and decreased levels of lung damage, compared to adoptive transfer of WT control macrophages. Taken together, the data presented in this study suggest that macrophage derived ACP5 plays an important role in development of pulmonary fibrosis and could present a tractable target for therapeutic intervention in IPF.

RANKL-dependent osteoclast differentiation and gene expression in bone marrow-derived cells from adult mice is sexually dimorphic.

Sex-specific differences in bone integrity and properties are associated with age as well as the number and activity of cells involved in bone remodeling. The aim of this study was to investigate sex-specific differences in adhesion, proliferation, and differentiation of mouse bone marrow derived cells into osteoclasts. The adherent fraction of bone marrow- derived cells from 12-week-old male and female C57BL/6J mice were assessed for their adhesion, proliferation, and receptor activator of nuclear factor κB (RANKL)-induced differentiation into osteoclasts. Female bone marrow derived macrophages (BMDMs) displayed higher adhesion and proliferation ratio upon macrophage colony stimulating factor (M-CSF) (day 0) and M-CSF + RANKL (day 4) treatment, respectively. On the contrary, male BMDMs differentiated more efficiently into osteoclasts upon RANKL-treatment compared to females (day 5). To further understand these sex-specific differences at the gene expression level, BMDMs treated with M-CSF (day 0) and M-CSF + RANKL (day 4), were assessed for their differential expression of genes through RNA sequencing. M-CSF treatment resulted in 1106 differentially expressed genes, while RANKL-treatment gave 473 differentially expressed genes. Integrin, adhesion, and proliferation-associated genes were elevated in the M-CSF-treated female BMDMs. RANKL-treatment further enhanced the expression of the proliferation- associated genes, and of genes associated with inhibition of osteoclast differentiation in the females, while RANK-signaling-associated genes were upregulated in males. In conclusion, BMDM adhesion, proliferation and differentiation into osteoclasts are sex-specific and may be directed by the PI3K-Akt signaling pathway for proliferation, and the colony stimulating factor 1-receptor and the RANKLsignaling pathway for the differentiation.

Tartrate resistant acid phosphatase 5 (TRAP5) mediates immune cell recruitment in a murine model of pulmonary bacterial infection.

Introduction: During airway infection, upregulation of proinflammatory cytokines and subsequent immune cell recruitment is essential to mitigate bacterial infection. Conversely, during prolonged and non-resolving airway inflammation, neutrophils contribute to tissue damage and remodeling. This occurs during diseases including cystic fibrosis (CF) and COPD where bacterial pathogens, not least Pseudomonas aeruginosa, contribute to disease progression through long-lasting infections. Tartrate-resistant acid phosphatase (TRAP) 5 is a metalloenzyme expressed by alveolar macrophages and one of its target substrates is the phosphoglycoprotein osteopontin (OPN). Methods: We used a knockout mouse strain (Trap5-/-) and BALB/c-Tg (Rela-luc)31Xen mice paired with siRNA administration or functional protein add-back to elucidate the role of Trap5 during bacterial infection. In a series of experiments, Trap5-/- and wild-type control mice received intratracheal administration of P.aerugniosa (Xen41) or LPS, with mice monitored using intravital imaging (IVIS). In addition, multiplex cytokine immunoassays, flow cytometry, multispectral analyses, histological staining were performed. Results: In this study, we found that Trap5-/- mice had impaired clearance of P. aeruginosa airway infection and reduced recruitment of immune cells (i.e. neutrophils and inflammatory macrophages). Trap5 knockdown using siRNA resulted in a decreased activation of the proinflammatory transcription factor NF-κB in reporter mice and a subsequent decrease of proinflammatory gene expression. Add-back experiments of enzymatically active TRAP5 to Trap5-/- mice restored immune cell recruitment and bacterial killing. In human CF lung tissue, TRAP5 of alveolar macrophages was detected in proximity to OPN to a higher degree than in normal lung tissue, indicating possible interactions. Discussion: Taken together, the findings of this study suggest a key role for TRAP5 in modulating airway inflammation. This could have bearing in diseases such as CF and COPD where excessive neutrophilic inflammation could be targeted by pharmacological inhibitors of TRAP5.

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.

A Sub-Clone of RAW264.7-Cells Form Osteoclast-Like Cells Capable of Bone Resorption Faster than Parental RAW264.7 through Increased De Novo Expression and Nuclear Translocation of NFATc1.

The murine macrophage cell line RAW264.7 is extensively used as a progenitor to study osteoclast (OC) differentiation. RAW264.7 is a heterogeneous cell line, containing sub-clones with different abilities to form OCs. The aim of this study was to identify characteristics within the heterogeneous RAW264.7 cells that define sub-clones with an augmented ability to form bone-resorbing OCs (H9), as well as sub-clones representing non-OCs (J8). RAW264.7 sub-clones were isolated by single cell cloning. Selection was based on TRAP/cathepsin K expression in sub-clone cultures without added RANKL. Sub-clones before and after differentiation with RANKL were assayed for multiple OC-characteristics. Sub-clone H9 cells presented a higher expression of OC-markers in cultures without added RANKL compared to the parental RAW264.7. After 6 days of RANKL stimulation, sub-clone H9 cells had equal expression levels of OC-markers with RAW264.7 and formed OCs able to demineralize hydroxyapatite. However, sub-clone H9 cells displayed rapid differentiation of OC already at Day 2 compared to Day 4 from parental RAW264.7, and when cultured on plastic and on bone they were more efficient in resorption. This rapid differentiation was likely due to high initial expression/nuclear translocation of OC master transcription factor, NFATc1. In contrast to H9, J8 cells expressed initially very low levels of OC-markers, and they did not respond to RANKL-stimulation by developing OC-characteristics/OC-marker expression. Hence, H9 is an additional clone suitable for experimental setup requiring rapid differentiation of large numbers of OCs.

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.

Cyclin-dependent kinase 8/19 inhibition suppresses osteoclastogenesis by downregulating RANK and promotes osteoblast mineralization and cancellous bone healing.

Cyclin-dependent kinase 8 (CDK8) is a mediator complex-associated transcriptional regulator that acts depending on context and cell type. While primarily under investigation as potential cancer therapeutics, some inhibitors of CDK8-and its paralog CDK19-have been reported to affect the osteoblast lineage and bone formation. This study investigated the effects of two selective CDK8/19 inhibitors on osteoclastogenesis and osteoblasts in vitro, and further evaluated how local treatment with a CDK8/19 inhibitor affects cancellous bone healing in rats. CDK8/19 inhibitors did not alter the proliferation of neither mouse bone marrow-derived macrophages (BMMs) nor primary mouse osteoblasts. Receptor activator of nuclear factor κΒ (NF-κB) ligand (RANKL)-induced osteoclastogenesis from mouse BMMs was suppressed markedly by inhibition of CDK8/19, concomitant with reduced tartrate-resistant acid phosphatase (TRAP) activity and C-terminal telopeptide of type I collagen levels. This was accompanied by downregulation of PU.1, RANK, NF-κB, nuclear factor of activated T-cells 1 (NFATc1), dendritic cell-specific transmembrane protein (DC-STAMP), TRAP, and cathepsin K in RANKL-stimulated BMMs. Downregulating RANK and its downstream signaling in osteoclast precursors enforce CDK8/19 inhibitors as anticatabolic agents to impede excessive osteoclastogenesis. In mouse primary osteoblasts, CDK8/19 inhibition did not affect differentiation but enhanced osteoblast mineralization by promoting alkaline phosphatase activity and downregulating osteopontin, a negative regulator of mineralization. In rat tibiae, a CDK8/19 inhibitor administered locally promoted cancellous bone regeneration. Our data indicate that inhibitors of CDK8/19 have the potential to develop into therapeutics to restrict osteolysis and enhance bone regeneration.

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.

Transgenic overexpression of tartrate-resistant acid phosphatase is associated with induction of osteoblast gene expression and increased cortical bone mineral content and density.

Bone remodeling is a central event in the maintenance of skeletal tissue, and involves cycles of resorption followed by the formation of bone tissue. The activity of osteoclasts and osteoblasts during these cycles is tightly regulated by systemic and local factors coupling the action of these cells. Tartrate-resistant acid phosphatase (TRAP) is predominantly expressed in bone by osteoclasts but has also been detected in osteoblasts and osteocytes. Moreover, TRAP can stimulate the differentiation of mesenchymal lineage cells, i.e. progenitors of osteoblasts and adipocytes. In order to further explore the effects of TRAP on bone turnover, the structural and molecular phenotypes of osteoclasts and osteoblasts were assessed in TRAP-overexpressing transgenic mice. Transgenic mice of both sexes display increased cortical bone mineral content and density, which cannot be accounted for by decreased bone resorption since osteoclast numbers and resorptive activity do not differ from wild-type mice. Examination of the osteoblast phenotype revealed that markers of bone formation, i.e. procollagen type I N-terminal propeptides, and osteoblast lineage markers as well as the TRAP 1B mRNA transcript are increased in TRAP-overexpressing mice. Expression of the osteoclast-selective TRAP 1C mRNA is not increased in TRAP transgenic mice. Elevated expression of TRAP mRNA and protein were detected in osteoblasts, osteocytes and in the bone matrix of TRAP transgenic mice, suggesting that TRAP overexpression in osteoblast lineage cells is associated with increased cortical bone mineral content and density. The data presented here support the hypothesis that TRAP overexpression in the osteoblastic cell lineage stimulates the differentiation and/or activation of these cells.

Induction and cellular expression of tartrate resistant acid phosphatase during dextran sodium sulphate induced colitis in rats.

The aim of this study was to investigate the cellular and molecular expression of tartrate resistant acid phosphatase (TRAP) as a marker of activated macrophages in macrophage dependent dextran sulphate sodium (DSS)-induced colitis in rats. In normal colon, TRAP+/CX(3)CR(1)+ macrophages were located in the upper part of the lamina propria. In the early stage (day 1-3) of acute colitis prior to histopathological changes, induction of the cytokines TNFalpha, IL-12 and IFN gamma occurred concomitant with increased mRNA and enzyme activity of TRAP along with a slight increase of TRAP immunolabelling in macrophages of the upper lamina propria, suggesting induction of TRAP in resident macrophages. Among these cytokines, TNFalpha up-regulated TRAP expression in the RAW 264.7 monocyte/macrophage cell line. In a later phase (day 7) with fulminant colitis, a massive infiltration of macrophages including recruited TRAP+/CCR2+ cells was observed also in the lower part of the lamina propria as well as in the submuscular layer. Additionally, differentiated cellular expression of pro- and mature TRAP also suggest that mucosal macrophages in the lower part of lamina propria bordering the sub-mucosa provide a source of replenishment of macrophages situated in the upper lamina propria. In conclusion, induction of TRAP provides an early sign of macrophage responsiveness in DSS induced colitis.

Monomeric tartrate resistant acid phosphatase induces insulin sensitive obesity.

BACKGROUND: Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP) is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer. PRINCIPAL FINDINGS: Using mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity. CONCLUSION: Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity.

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.

Myosin-X provides a motor-based link between integrins and the cytoskeleton.

Unconventional myosins are actin-based motors with a growing number of attributed functions. Interestingly, it has been proposed that integrins are transported by unidentified myosins to facilitate cellular remodelling. Here we present an interaction between the unconventional myosin-X (Myo10) FERM (band 4.1/ezrin/radixin/moesin) domain and an NPXY motif within beta-integrin cytoplasmic domains. Importantly, knock-down of Myo10 by short interfering RNA impaired integrin function in cell adhesion, whereas overexpression of Myo10 stimulated the formation and elongation of filopodia in an integrin-dependent manner and relocalized integrins together with Myo10 to the tips of filopodia. This integrin relocalization and filopodia elongation did not occur with Myo10 mutants deficient in integrin binding or with a beta(1)-integrin point mutant deficient in Myo10 binding. Taken together, these results indicate that Myo10-mediated relocalization of integrins might serve to form adhesive structures and thereby promote filopodial extension.

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.