Copyright ©The Histochemical Society, Inc.

Eccentric Localization of Osteocytes Expressing Enzymatic Activities, Protein, and mRNA Signals for Type 5 Tartrate-resistant Acid Phosphatase (TRAP)

Yukiko Nakano, Satoru Toyosawa and Yoshiro Takano

Biostructural Science, Graduate School of Tokyo Medical and Dental University, Tokyo, Japan (YN,YT), and Department of Oral Pathology, Graduate School of Dentistry, Osaka University, Osaka, Japan (ST)

Correspondence to: Prof. Yoshiro Takano, Biostructural Science, Graduate School of Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail: takanoy.bss{at}tmd.ac.jp


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Enzymatic activity of type 5 tartrate-resistant acid phosphatase (TRAP) has been regarded as one of the reliable markers for osteoclasts and their precursors. The presence of TRAP activity in osteocytes near the bone resorbing surface has also been pointed out in some reports. However, the significance of TRAP reactions in osteocytes remains controversial and, in fact, there is no agreement as to whether the histochemical enzyme reactions in osteocytes represent the TRAP enzyme generated by the respective osteocytes or is a mere diffusion artifact of the reaction products derived from the nearby osteoclasts. Current histochemical, immunohistochemical, and in situ hybridization studies of rat and canine bones confirmed TRAP enzyme activity, TRAP immunoreactivity, and the expression of Trap mRNA signals in osteocytes located close to the bone-resorbing surface. TRAP/Trap- positive osteocytes thus identified were confined to the areas no further than 200 µm from the bone-resorbing surface and showed apparent upregulation of TRAP/Trap expression toward the active osteoclasts. Spatial and temporal patterns of TRAP/Trap expression in the osteocytes should serve as a valuable parameter for further analyses of biological interactions between the osteocytes and the osteoclasts associated with bone remodeling.

(J Histochem Cytochem 52:1475–1482, 2004)

Key Words: tartrate-resistant acid phosphatase (TRAP) • osteocyte • osteoclast • immunohistochemistry • in situ hybridization • bone


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
BONE REMODELING is a series of complex processes of bone matrix formation, mineralization, and its resorption performed by the three types of bone cells; osteoblasts, osteoclasts, and osteocytes (Buckwalter and Cooper 1987Go; Marks and Popoff 1988Go). Accumulating data indicate important roles played by the osteoblasts in differentiation and function of the osteoclasts via RANK/RANKL interactions (Anderson et al. 1997Go; Wong et al. 1997Go; Yasuda et al. 1998Go). The osteocytes are former osteoblasts embedded in the mineralized bone matrix, which form functional units with the osteoblasts via gap junctions of their cytoplasmic processes. Unlike the osteoblasts, however, the putative roles of osteocytes in the regulation of bone remodeling, if any, have been largely unknown. In fact, in the process of bone remodeling, the osteocytes may appear merely excavated by the osteoclasts, freed from the osteocytic lacunae, or phagocytosed by the osteoclasts (Palumbo 1986Go; Boabaid et al. 2001Go; Bronckers et al. 2003Go). In recent studies, however, Zhao et al. (2002)Go indicated accelerated formation and activation of osteoclasts by the osteocyte-like cells in vitro. Smit et al. (2002)Go suggested that the alteration of viability of osteocytes under the influence of strain-induced fluid flow in bone fluid compartments may relate to the alignment of the osteons in long bones.

In bone tissues, the presence of two types of acid phosphatases (ACPase, EC 3.1.3.2); tartrate-sensitive ACPase, and tartrate-resistant ACPase was confirmed by biochemical analysis (Anderson and Toverud 1977Go). The latter was further characterized according to its electrophoretic mobility and was designated as type 5 tartrate-resistant acid phosphatase (TRAP), type 5 ACPase, or purple ACPase (Yam et al. 1971Go; Anderson and Toverud 1982Go,1986Go; Andersson et al. 1984Go). Since Minkin (1982)Go indicated exclusive localization of this enzyme in osteoclasts, TRAP has been widely used as one of the reliable histochemical and/or functional markers for osteoclasts. However, histochemical TRAP reactions have also been found in the osteoblasts and osteocytes located near the intensely TRAP-positive osteoclasts (Wergedal and Baylink 1969Go; Bianco et al. 1988Go; Yamamoto and Nagai 1998Go; Irie et al. 2000Go; Bonucci et al. 2001Go; Bonucci and Nanci 2001Go). Among these studies, Irie et al. (2000)Go proposed a functional correlation of the TRAP activity in osteocytes with osteocytic osteolysis. Bonucci et al. (2001)Go noted enhanced TRAP reactions in the osteocytes in Ca-depleted rats by the Azo-dye histochemical staining method, and suggested a correlation between the TRAP activity of osteocytes and the calcium levels in the body fluid. However, the authors did not rule out the possibility that artifactual diffusion of the enzymatic reaction products from the nearby osteoclasts toward the osteocytes might have caused the seemingly enhanced TRAP reactions in the osteocytes (Wergedal and Baylink 1969Go; Bland and Ashhurst 1998Go). At present, therefore, the significance of histochemical reactions for TRAP in osteocytes remains elusive. If the enzyme reactions in osteocytes represent the TRAP enzyme generated by the respective osteocytes, the modulated appearance of this enzyme in osteocytes should provide a clue to elucidate the role of the osteocytes in bone remodeling.

Here we sought to clarify the actual localization of TRAP/Trap in the osteocytes by enzyme histochemistry, immunohistochemistry (IHC), and in situ hybridization (ISH).


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Tissue Preparation
Protocols for animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University. All experiments were carried out according to the Guidelines for Animal Experimentation at Tokyo Medical and Dental University.

Semithin Sectioning of Fresh-frozen Freeze-substituted Specimens
Three-week-old Wistar rats were anesthetized by ether inhalation followed by an IP injection of 8% chloral hydrate solution (400 mg/kg bw) and killed by phlebotomy. Maxillas, mandibles, tibias and femurs were immediately excised, cut into small pieces, and quenched in liquid propane cooled with liquid nitrogen for rapid freezing. The frozen pieces were freeze-substituted with absolute ethanol at –80C for 4 days, gradually brought to 4C, and embedded in Technovit 7100 (Heraeus Kulzer; Werrhein, Germany) at 0C. Two-µm-thick sections were cut by glass knives or a Histoknife (Diatome; Bienne, Switzerland) attached to the ultramicrotome (Leica ULTRA CUT; Leica Aktiengesellschaft, Vienna, Austria) and adhered to the glass slides without heating.

Paraffin- or Cryosectioning of Chemically Fixed Specimens
Five-week-old Wistar rats were anesthetized as described above and perfused via the left cardiac ventricle with 4% paraformaldehyde (PFA) in 0.1 M cacodylate buffer (pH 7.4). Upper and lower jaws and limbs were excised and further immersed in the fixative for 1 day at 4C.

A canine (beagle, 1.5 years old) was anesthetized with sodium thiopental (25 mg/kg bw) and perfused via the abdominal aorta with 4% PFA in 0.1 M cacodylate buffer (pH 7.4). Femora and tibiae were excised and further immersed in the fixative for 1 day at 4C. The diaphyses of excised bones of rats and canine were processed for either cryosectioning, paraffin embedding, or Technovit embedding preceded by 10% EDTA decalcification at 4C for 10 days. The specimens for cryosectioning were immersed overnight in a 30% sucrose/PBS solution for cryoprotection, frozen in cold hexane (–90C), and cut into 6-µm-thick frozen sections. Others were dehydrated through a graded series of ethanol and embedded routinely in paraffin or Technovit 7100.

Enzyme Histochemistry of TRAP
For histochemical localization of TRAP activities, the Azo-dye method was performed according to Burstone (1962)Go after slight modifications. Briefly, the sections were incubated in a medium composed of 1.5 mM naphthol AS-MX phosphate as substrate, 0.5 mM Fast Red Violet LB salt as capture agent, and 20 mM L(+)-tartrate in 0.1 M acetate buffer (pH 5.2) at 37C for 10–20 min. The final pH of the incubation medium was adjusted to pH 5.2.

Immunohistochemistry
The paraffin sections or frozen sections of rat bones were subjected to microwave treatment in 10 mM citrate buffer (pH 6.0) for 20 min. Then the sections were immersed in a solution of 0.3% H2O2 in absolute methanol to inhibit endogenous peroxidase activity. After a blocking treatment with 10% goat serum, mouse anti-human TRAP antibody (ready to use) (Zymed Laboratories; South San Francisco, CA) was applied for 12 hr at room temperature (RT). The incubation with the secondary antibody was carried out for 1 hr using Histofine simple stain AP (M) or Histofine simple stain rat MAX-PO (Nichirei; Tokyo, Japan) at RT. The site of immunoreaction was visualized by treating sections with a Vector Blue kit (Vector Laboratories; Burlingame, CA) or a 2% 3'3-diaminobenzidine (DAB) solution supplemented with 0.002% H2O2. Normal mouse serum was used as negative control of primary antibodies.

After H2O2 treatment, some paraffin sections of rat and canine bones were subjected to a digestion treatment with 2.5 mM trypsin in 5 mM Tris-HCl buffer (pH 7.3) supplemented with 2.25 mM CaCl2 for 20 min at 37C. Preceded by a blocking treatment with 10% swine serum, the sections treated with trypsin were incubated with rabbit anti-rat dentin matrix protein 1 (DMP1) antibody (1:100–400) (Toyosawa et al. 2001Go) and the sections without trypsin treatment were incubated with rabbit anti-human TRAP (1:1000) [polyclonal antibody against human TRAP peptide (CTYIEASGKSLFKTRLPRRARP) supplied by TaKaRa Bio, Shiga, Japan] for 12 hr at RT and then with biotinylated swine anti-rabbit Igs (1:400) (Dako Cytomation; Glostrup, Denmark) for 1 hr at RT. After further treatment with HRP-conjugated streptavidin (Dako Cytomation; Carpinteria, CA) for 30 min at RT, the site of immunoreaction was visualized by a DAB solution as described for TRAP immunostaining. Some of the immunostained sections were incubated in the Azo-dye medium before DAB treatment for a dual demonstration of enzymatic activity and IHC localization of TRAP. Normal rabbit serum was used as negative control of primary antibodies.

In Situ Hybridization
After deparaffinization, the paraffin sections of rat bones were treated with 0.2 N HCl for 20 min at RT and digested with 5 µg/ml proteinase K at 37C for 15 min. They were then postfixed with 4% PFA/PBS solution, immersed in 2 mg/ml glycine/PBS for 30 min, and kept in 40% deionized formamide in 4 x SSC until hybridization. Hybridization was carried out at 42C for 15 hr with FITC-conjugated oligo-cDNA (52-mer) for Trap type 5 [GenBank M76110 (Ek-Rylander et al. 1991Go)]. After a series of rinses with 2 x SSC, the site of reaction was visualized with a GenPoint Fluorescein kit (Dako Cytomation; Carpinteria, CA) according to the manufacturer's instructions. A sense probe was used as the negative control.

Measurement of the Extent of TRAP/Trap-positive Osteocytes
The distance between the bone resorption surface and the osteocytes positive for the enzymatic or IHC reactions or those expressing mRNA signals for TRAP/Trap was measured using Adobe Photoshop software (Adobe Systems; Tucson, AZ) to determine the extent of the area of the osteocytes positive for the respective parameters. The statistical significance was determined by Student's t-test and p less than 0.05 was considered significant.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Enzymatic Reactions and Protein Localization of TRAP
The Azo-dye method revealed intense granular histochemical reactions of enzymatic activity of TRAP in the cytoplasm of osteoclasts, and moderate reaction in the cytoplasm of some osteocytes located close to the bone resorption surface in both rat (Figures 1a and 1b) and canine (Figures 1c and 1d) bones. Extracellular TRAP reactions were also detected along the bone resorbing surface as well as in the ruffled border regions of the osteoclasts. The TRAP-positive osteocytes contained some granular reactions in the weakly positive homogeneous cytoplasm. The intensity of histochemical reactions of TRAP in osteocytes decreased drastically in the slightly remote areas and became undetectable in those located no farther than 200 µm away from the bone surface undergoing resorption. The staining patterns of TRAP reactions in the osteocytes were identical in the sections of all types of specimens processed by different methods and also in both long bones and the alveolar bone.



View larger version (173K):
[in this window]
[in a new window]
 
Figure 1

Histochemical reactions of TRAP activity on Technovit sections of fresh-frozen, freeze-substituted undecalcified specimen of rat alveolar bone (a,b), and aldehyde-fixed and decalcified specimen of canine femur (diaphysis) (c,d). In addition to intensely TRAP-positive multinucleated osteoclasts, some osteocytes close to the resorption surface also display TRAP reactions (a,c, arrows). At higher magnification, TRAP-positive osteocytes exhibit intense granular reactions in the slightly TRAP-positive cytoplasm in both rat (b) and canine (d) bones. Bars = 20 µm. (e) Panoramic view of a paraffin section of aldehyde-fixed, decalcified specimen of canine femur (diaphysis) showing distribution of TRAP-positive and TRAP-negative osteocytes in the cortical bone. In addition to the osteocytes near the cutting cone (cc), groups of osteocytes at left of figure also display TRAP reactions, although they have no connection with osteoclasts. Bar = 250 µm.

 
When the longitudinal sections of the cortical bone of canine femurs were examined, the areas of TRAP-positive osteocytes were clearly visible near the intensely TRAP-positive cutting cone of the osteon, as expected (Figure 1e). Interestingly, however, there were also areas of TRAP-positive osteocytes in which not a single osteoclast was visible nearby. A careful examination of the adjacent sections confirmed the existence of a cutting cone underneath the seemingly osteoclast-free areas of TRAP-positive osteocytes (data not shown).

IHC Localization of TRAP
To clarify the distribution of TRAP enzyme as proteins, we examined the IHC localization of TRAP in bone cells using mouse monoclonal and rabbit polyclonal antibodies raised against human TRAP. In both rat and canine bones, distinct TRAP immunoreactions were confirmed in the cytoplasm of the osteoclasts undergoing bone resorption. TRAP immunoreactions were also observed in the osteocytes closely associated with the resorption surface (Figure 2) . The immunoreactions in the osteocytes were weaker than those in the osteoclasts and were confined to the granular structures in the cytoplasm (Figure 2b; Figure 2c, inset).



View larger version (103K):
[in this window]
[in a new window]
 
Figure 2

Immunolocalization of TRAP proteins on frozen section of aldehyde-fixed, decalcified specimen of rat alveolar bone (a,b) and canine femur (c). (a) In addition to intensely immunopositive osteoclasts (oc), osteocytes near the resorption surface also show positive immunoreactions for TRAP (arrowheads). Bar = 25 µm. (b) Higher magnification of an immunopositive osteocyte showing TRAP immunoreactions confined to cytoplasmic granules. Bar = 10 µm. (c) Osteoclasts immunopositive for TRAP protein (oc) and immunopositive osteocytes located around the front edge of the cutting cone (arrowheads) in the diaphysis of canine bone. Bar = 50 µm. (Inset) Higher magnification of a TRAP-positive osteocyte showing intensely immunopositive granular structures in the cytoplasm. Bar = 10 µm.

 
Expression of Trap mRNA Signals
Intense Trap mRNA signals appeared in the cytoplasm of osteoclasts of rat bones (Figure 3) . As in the case of enzyme histochemical and IHC observations of TRAP, modest but significant signals for Trap mRNA were observed in the cytoplasm of the osteocytes located close to the osteoclasts (Figure 3, arrows). No signal was detected by the sense probe.



View larger version (166K):
[in this window]
[in a new window]
 
Figure 3

Demonstration of ISH signals for Trap mRNA on a paraffin section of aldehyde-fixed, decalcified specimen of rat humerus. Distinct Trap signals (brown) are confirmed in the cytoplasm of osteocytes close to the osteoclasts (oc) expressing intense Trap signals. Osteocytes at some distance from bone resorption surface do not show Trap mRNA. Bar = 25 µm.

 
DMP1 Immunoreactivity
In contrast to the eccentric localization of TRAP/Trap-positive osteocytes, the immunoreactions for DMP1 were localized along the inner surface of the osteocytic lacunae and bone canaliculi throughout the cortical and trabecular bones of both rat and canine bones (Figure 4a) . Some osteocytes close to the osteoclasts showed histochemical TRAP reactions in the DMP1 immunopositive lacunae in doubly stained sections (Figure 4b, arrows).



View larger version (148K):
[in this window]
[in a new window]
 
Figure 4

Immunolocalization of DMP1 on a paraffin section of aldehyde-fixed, decalcified specimen of canine femur (diaphysis). (a) Immunoreactions are evenly located in the osteocytes and its lacunae throughout the whole area of bone. (Inset) Higher magnification of immunopositive osteocytes showing DMP1 immunoreactions along the inner surface of osteocyte lacunae and bone canaliculi. cc, cutting cone. (b) Fluorescent micrograph of a section showing histochemical TRAP reactions (red) and DMP1 immunoreactions (brown). Arrows indicate TRAP/DMP1 double-positive osteocytes near the resorption surface of a cutting cone. oc, osteoclast Bars: a = 250 µm, inset = 25 µm; b = 25 µm.

 
The Extent of TRAP/Trap-positive Osteocytes
The extent of the areas of TRAP/Trap-positive osteocytes was measured in rat bones (humerus and alveolar bones) as the distance from the bone resorbing surface. The areas of TRAP/Trap-positive osteocytes were significantly narrower than that of the osteocytes expressing histochemical TRAP reactions. There was no statistically significant difference between the areas of the immunopositive osteocytes and the mRNA-expressing osteocytes (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1

Extent of the areas of TRAP/Trap-expressing osteocytes from the resorption surface of the rat bone determined by three histochemical parameters*

 

    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The validity of the TRAP reactions in osteocytes detected by enzyme histochemistry has been controversial. The following interpretations may be applicable concerning the significance of the enzymatic reactions of TRAP in osteocytes. (a) The enzyme reactions in osteocytes are the consequence of artifactual diffusion of histochemical reaction products of TRAP from the actual reaction site (bone resorbing surface), which occurred during histochemical processing. (b) The enzyme reactions in osteocytes are not histochemical diffusion artifact but true reactions representing the TRAP proteins in the respective osteocytes, which have diffused from the bone resorbing surface through the bone canaliculi under physiological conditions in vivo. (c) The enzyme reactions in osteocytes represent the TRAP enzyme proteins generated by the respective osteocytes. Thus far, none of these hypotheses has been tested.

In our present observations of canine bone sections, we noted areas of TRAP-positive osteocytes that had no connection with the osteoclasts (cutting cone) (Figure 1e). As already mentioned, the area of TRAP-positive osteocytes and the cutting cone are spatially closely located in vivo. In histological sections, however, these two areas are often divided into separate sections and hence are totally isolated from each other when the sections are subjected to enzyme histochemical staining. It is therefore safe to state that the TRAP enzyme reactions in the osteocytes are not a diffusion artifact of the reaction products derived from the nearby osteoclasts, either in the cutting cones or in other bone resorbing surfaces. The observation of TRAP immunoreactions in osteocytes provides firm evidence for the presence of TRAP enzyme proteins in these cells and further supports the intrinsic enzyme activity of osteocytes. However, the presence of TRAP proteins in osteocytes does not conclusively indicate that the enzyme is the product of the respective cells. The TRAP proteins being released from the functioning osteoclasts into the resorption lacunae may diffuse through bone canaliculi and further into osteocytic lacunae, and may be taken up by the osteocytes. Such internalized TRAP proteins may also show granular immunoreactivity as well as histochemical enzymatic reactions in the cytoplasm of osteocytes, as shown in Figures 1b, 1d, and 2b. In some cases, weak histochemical TRAP reactions could be seen in some bone canaliculi near the resorption surface (data not shown). In this context, the expression of Trap mRNA signals in osteocytes, as shown by ISH, could finally confirm the origin of TRAP enzyme proteins in osteocytes. The difference in the extent of the areas of histochemically TRAP-reactive osteocytes and TRAP/Trap-positive osteocytes (Table 1) may simply be attributed to the difference in the sensitivity of the individual methods used for detection: enzyme histochemistry, ISH, and IHC. Taking our findings together, we suggest that the osteocytes in the local environment near the bone resorption surface synthesize TRAP proteins and show histochemical TRAP reactions in the cytoplasmic granular structures. The possible diffusion of some TRAP proteins derived from osteoclasts through bone canaliculi toward the TRAP-positive osteocytes cannot be excluded.

A number of authors have studied the function of TRAP in biological systems (Drexler and Gignac 1994Go; Fleckenstein and Drexler 1997Go; Lamp and Drexler 2000Go). However, the actual role(s) of this enzyme remains to be explored. In osteoclasts, TRAP has been suggested to generate free radicals and to directly participate in bone resorption (Hayman and Cox 1994Go), to dephosphorylate osteopontin and bone sialoprotein, and to serve as an osteoclast detachment factor (Ek-Rylander et al. 1994Go). TRAP is also suggested to serve as a protein tyrosine phosphatase (Halleen et al. 1998Go) and to modulate intracellular vesicular transport (Hollberg et al. 2002Go). Obviously, in osteocytes, the functional significance of TRAP is undetermined. Bianco et al. (1988)Go reported punctate TRAP enzyme reactions in the cytoplasm of young osteocytes in the upper part of the metaphyseal trabecular bones of rats and its upregulation under hypocalcemic conditions (Bonucci et al. 2001Go). In the diaphysis of rat bones lacking secondary osteons, Mason et al. (1996)Go stated that osteocytes do not constitutively express Trap signals. Our present observations confirmed that, at least in rat and canine bones, osteocytes do not generally express TRAP/Trap activities and signals but do so when they come into close proximity to the osteoclastic resorption surface under physiological conditions. Therefore, the site-specific distribution of TRAP/Trap-positive and -negative osteocytes appears to indicate the presence of groups of osteocytes at different functional states in a single bone. The site-specific localization of TRAP/Trap-positive osteocytes in trabecular and cortical bones undergoing remodeling appears to indicate some contribution of osteocytes to the regulation of osteoclastic bone resorption and subsequent bone formation.

DMP1, a member of the family of non-collagenous bone matrix proteins, has been reported to be located along the inner surface of osteocytic lacunae and bone canaliculi and its mRNA signal to be expressed by osteocytes (Toyosawa et al. 2001Go). Our observations (Figure 4a) reconfirmed the characteristic localization patterns of this protein in both rat and canine bones. These previous (Toyosawa et al. 2001Go) and current data appear to indicate that the osteocytes expressing TRAP/Trap are viable and continue to maintain osteocytic functions. It is not known whether the upregulated TRAP/Trap expression in osteocytes is an indication of a cellular response to progressive bone resorption by osteoclasts or is a sign of elevated expression of putative regulatory factors in local osteocytes that may control the direction of osteoclastic bone resorption (Figure 5) . However, the TRAP/Trap expression in osteocytes may serve as a reliable functional parameter of osteocytes for analysis of cellular interactions during bone resorption and remodeling. Analyses of molecular expression in TRAP/Trap-positive osteocytes and -negative osteocytes are in progress in our laboratory.



View larger version (55K):
[in this window]
[in a new window]
 
Figure 5

Summary diagram of TRAP/Trap expression in osteocytes and its spatial relation to osteoclasts. Characteristic expressions of TRAP/Trap in osteocytes may reflect positive roles of osteocytes that generate putative regulatory factors on osteoclastic bone resorption (arrow 1), or may be a mere sign of regressive cellular response to destructive bone resorption (arrow 2).

 

    Acknowledgments
 
Supported in part by Grants-in-Aid for Scientific Research (15791036; 14370577) from the Japan Society for the Promotion of Science.


    Footnotes
 
Received for publication May 9, 2004; accepted June 25, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, Teepe MC, et al. (1997) A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175–179[CrossRef][Medline]

Anderson TR, Toverud SU (1977) Chromatographic separation of two acid phosphatases from rat bone. Calcif Tissue Res 24:187–190[Medline]

Anderson TR, Toverud SU (1982) Effect of Fe2+ and ascorbic acid on acid phosphatases from rat bone. Calcif Tissue Int 34:54–58[Medline]

Anderson TR, Toverud SU (1986) Purification and characterization of purple acid phosphatase from developing rat bone. Arch Biochem Biophys 247:131–139[Medline]

Andersson G, Ek-Rylander B, Hammarstrom L (1984) Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone. Arch Biochem Biophys 228:431–438[Medline]

Bianco P, Ballanti P, Bonucci E (1988) Tartrate-resistant acid phosphatase activity in rat osteoblasts and osteocytes. Calcif Tissue Int 43:167–171[Medline]

Bland YS, Ashhurst DE (1998) Is acid phosphatase activity present in bone matrix at sites of endochondral ossification in rabbit fracture callus? Histochem J 30:405–411[CrossRef][Medline]

Boabaid F, Cerri PS, Katchburian E (2001) Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats. Tissue Cell 33:318–325[Medline]

Bonucci E, Mocetti P, Silvestrini G, Ballanti P, Zalzal S, Fortin M, Nanci A (2001) The osteoblastic phenotype in calcium-depleted and calcium-repleted rats: a structural and histomorphometric study. J Electron Microsc 50:333–347[Abstract/Free Full Text]

Bonucci E, Nanci A (2001) Alkaline phosphatase and tartrate-resistant acid phosphatase in osteoblasts of normal and pathologic bone. Ital J Anat Embryol 106(suppl 1):129–133

Bronckers AL, Sasaguri K, Engelse MA (2003) Transcription and immunolocalization of Runx2/Cbfa1/Pebp2alphaA in developing rodent and human craniofacial tissues: further evidence suggesting osteoclasts phagocytose osteocytes. Microsc Res Tech 61:540–548[CrossRef][Medline]

Buckwalter JA, Cooper RR (1987) Bone structure and function. Instr Course Lect 36:27–48[Medline]

Burstone MS (1962) Enzyme Histochemistry. New York, Academic Press

Drexler HG, Gignac SM (1994) Characterization and expression of tartrate-resistant acid phosphatase (TRAP) in hematopoietic cells. Leukemia. 8:359–368[Medline]

Ek-Rylander B, Bill P, Norgard M, Nilsson S, Andersson G (1991) Cloning, sequence, and developmental expression of a type 5, tartrate-resistant, acid phosphatase of rat bone. J Biol Chem 266:24684–24689[Abstract/Free Full Text]

Ek-Rylander B, Flores M, Wendel M, Heinegard D, Andersson G (1994) Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. Modulation of osteoclast adhesion in vitro. J Biol Chem 269:14853–14856[Abstract/Free Full Text]

Fleckenstein E, Drexler HG (1997) Tartrate-resistant acid phosphatase: gene structure and function. Leukemia 11:10–13[CrossRef]

Halleen JM, Kaija H, Stepan JJ, Vihko P, Vaananen HK (1998) Studies on the protein tyrosine phosphatase activity of tartrate-resistant acid phosphatase. Arch Biochem Biophys. 352:97–102[CrossRef][Medline]

Hayman AR, Cox TM (1994) Purple acid phosphatase of the human macrophage and osteoclast. Characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells. J Biol Chem 269:1294–1300[Abstract/Free Full Text]

Hollberg K, Hultenby K, Hayman A, Cox T, Andersson G (2002) Osteoclasts from mice deficient in tartrate-resistant acid phosphatase have altered ruffled borders and disturbed intracellular vesicular transport. Exp Cell Res 279:227–238[CrossRef][Medline]

Irie K, Ozawa H, Yajima T (2000) The histochemical and cytochemical changes from formative to resorptive osteocytes. Acta Histochem Cytochem 33:385–391[CrossRef]

Lamp EC, Drexler HG (2000) Biology of tartrate-resistant acid phosphatase. Leuk Lymphoma 39:477–484[Medline]

Marks SC Jr, Popoff SN (1988) Bone cell biology: the regulation of development, structure, and function in the skeleton. Am J Anat 183:1–44[Medline]

Mason DJ, Hillam RA, Skerry TM (1996) Constitutive in vivo mRNA expression by osteocytes of beta-actin, osteocalcin, connexin-43, IGF-I, c-fos and c-jun, but not TNF-alpha nor tartrate-resistant acid phosphatase. J Bone Miner Res 11:350–357[Medline]

Minkin C (1982) Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int 34:285–290[Medline]

Palumbo C (1986) A three-dimensional ultrastructural study of osteoid-osteocytes in the tibia of chick embryos. Cell Tissue Res 246:125–131[Medline]

Smit TH, Burger EH, Huyghe JM (2002) A case for strain-induced fluid flow as a regulator of BMU-coupling and osteonal alignment. J Bone Miner Res 17:2021–2029[Medline]

Toyosawa S, Shintani S, Fujiwara T, Ooshima T, Sato A, Ijuhin N, Komori T (2001) Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts. J Bone Miner Res 16:2017–2026[Medline]

Wergedal JE, Baylink DJ (1969) Distribution of acid and alkaline phosphatase activity in undemineralized sections of the rat tibial diaphysis. J Histochem Cytochem 17:799–806[Medline]

Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, Kalachikov S, et al. (1997) TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 272:25190–25194[Abstract/Free Full Text]

Yam LT, Li CY, Lam KW (1971) Tartrate-resistant acid phosphatase isoenzyme in the reticulum cells of leukemic reticuloendotheliosis. N Engl J Med 284:357–360[Medline]

Yamamoto T, Nagai H (1998) Ultrastructural localization of tartrate-resistant acid phosphatase activity in rat osteoblasts. J Electron Microsc 47:659–663[Medline]

Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, et al. (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602[Abstract/Free Full Text]

Zhao S, Zhang YK, Harris S, Ahuja SS, Bonewald LF (2002) MLO-Y4 osteocyte-like cells support osteoclast formation and activation. J Bone Miner Res 17:2068–2079[Medline]





This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Nakano, Y.
Articles by Takano, Y.
Articles citing this Article
PubMed
PubMed Citation
Articles by Nakano, Y.
Articles by Takano, Y.


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]