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Volume 52 (11): 1475-1482, 2004
Copyright ©The Histochemical Society, Inc.
Eccentric Localization of Osteocytes Expressing Enzymatic Activities, Protein, and mRNA Signals for Type 5 Tartrate-resistant Acid Phosphatase (TRAP)
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
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Summary |
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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
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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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 1987
; Marks and Popoff 1988). Accumulating data indicate important roles played by the osteoblasts in differentiation and function of the osteoclasts via RANK/RANKL interactions (Anderson et al. 1997; Wong et al. 1997; Yasuda et al. 1998). 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 1986; Boabaid et al. 2001; Bronckers et al. 2003). In recent studies, however, Zhao et al. (2002) indicated accelerated formation and activation of osteoclasts by the osteocyte-like cells in vitro. Smit et al. (2002) 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 1977). 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. 1971; Anderson and Toverud 1982,1986; Andersson et al. 1984). Since Minkin (1982) 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 1969; Bianco et al. 1988; Yamamoto and Nagai 1998; Irie et al. 2000; Bonucci et al. 2001; Bonucci and Nanci 2001). Among these studies, Irie et al. (2000) proposed a functional correlation of the TRAP activity in osteocytes with osteocytic osteolysis. Bonucci et al. (2001) 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 1969; Bland and Ashhurst 1998). 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).
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Materials and Methods |
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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) 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. 2001) 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. 1991)]. 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.
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Results |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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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.
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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).
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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.
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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).
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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).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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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 1994; Fleckenstein and Drexler 1997; Lamp and Drexler 2000). 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 1994), to dephosphorylate osteopontin and bone sialoprotein, and to serve as an osteoclast detachment factor (Ek-Rylander et al. 1994). TRAP is also suggested to serve as a protein tyrosine phosphatase (Halleen et al. 1998) and to modulate intracellular vesicular transport (Hollberg et al. 2002). Obviously, in osteocytes, the functional significance of TRAP is undetermined. Bianco et al. (1988) 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. 2001). In the diaphysis of rat bones lacking secondary osteons, Mason et al. (1996) 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. 2001). Our observations (Figure 4a) reconfirmed the characteristic localization patterns of this protein in both rat and canine bones. These previous (Toyosawa et al. 2001) 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.
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Acknowledgments |
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Supported in part by Grants-in-Aid for Scientific Research (15791036; 14370577) from the Japan Society for the Promotion of Science.
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Footnotes |
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Received for publication May 9, 2004; accepted June 25, 2004
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