Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome

We have used temperature and nocodazole blocks in an in vivo basolateral to apical transcytosis assay to dissociate the early transcytotic steps occurring during the formation of transcytotic vesicles and their microtubule-dependent translocation into the apical region, from the late steps when transcytotic cargo is delivered into the apical media. We found that polarized MDCK cells transfected with rabbit polymeric IgA receptor (pIgA-R) internalize basolaterally added pIgA-R ligand ([Fab]2 fragment of IgG against the receptor's ectodomain) at 17 degrees C but do not deliver it to the apical PM. Instead, the ligand accumulates in an apically localized transcytotic compartment, distal to the basolateral endosome and the microtubule- requiring translocation step. We have characterized this compartment and show that it is distinct from basolateral transferrin recycling endosomes, basolateral early endosomes or late endosomes or lysosomes. The apical transcytotic compartment colocalizes with the compartment containing apically recycling membrane markers (ricin and apically internalized pIgA-R ligand) but is distinct from the compartment receiving apically internalized fluid phase marker (BSA). This compartment is an intermediate station of the overall pathway since transcytotic ligand can exit the compartment and be released into the apical medium when cells preloaded at 17 degrees C are subsequently incubated at 37 degrees C. We have used this system to examine the effect of Brefeldin A (BFA) and the involvement of trimeric GTPases in the late (post apical transcytotic compartment) steps of the transcytotic pathway. We found that addition of BFA or cholera toxin, a known activator of Gs alpha, to cells preloaded with transcytotic ligand at 17 degrees C significantly inhibits the exit of ligand from the apical transcytotic compartment. General structure and function of the apical endosome are not affected since neither BFA nor cholera toxin inhibit the recycling of apically internalized membrane markers (ricin and pIgA-R ligand) from the same compartment. The data suggest that transcytosis connects the "membrane-sorting" sub-domain of the basolateral endosome with a homologous sub-domain of the apical endosome and that exit of transcytosing cargo from the apical endosome is controlled by a BFA and trimeric G protein sensitive mechanism, distinct from that used for recycling of apically internalized proteins (ricin or pIgA-R).     

Osteoclastic tartrate-resistant acid phosphatase (Acp 5): its localization to dendritic cells and diverse murine tissues.

Tartrate-resistant acid phosphatase (TRAP) is a histochemical marker of the osteoclast. It is also characteristic of monohistiocytes, particularly alveolar macrophages, and is associated with diverse pathological conditions, including hairy cell leukemia and AIDS encephalopathy. To study the biology of this enzyme, we investigated its expression and activity in mouse tissues. Confocal fluorescence studies showed that TRAP is localized to the lysosomal compartment of macrophages. In adult mice, high activities of the enzyme were demonstrated in bone, spleen, liver, thymus, and colon, with lower amounts in lung, stomach, skin, brain, and kidney. Trace amounts were detected in testis, muscle, and heart. Expression of TRAP mRNA was investigated in tissue sections by in situ hybridization and protein expression was monitored by histochemical staining or immunohistochemically. TRAP is widely expressed in many tissues, where it is associated with cells principally originating from the bone marrow, including those of osteoclast/macrophage lineage. The cellular distribution of TRAP mRNA and enzyme antigen in the tissues corresponds closely to that of cells staining with an antibody directed to the CD80 (B7) antigen. Therefore, to confirm its putative localization in dendritic cells, isolated bone marrow dendritic cells were matured in culture. These co-stained strongly for TRAP protein and the CD80 antigen. These studies demonstrate that TRAP is a lysosomal enzyme that is found in diverse murine tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages, as previously shown. (J Histochem Cytochem 48:219-227, 2000)     

Characterization of the MAL2-positive compartment in oligodendrocytes

Oligodendrocytes (OLs), the myelin-producing cells of the central nervous system, segregate different surface subdomains at the plasma membrane as do other differentiated cells such as polarized epithelia and neurons. To generate the complex membrane system that characterizes myelinating OLs, large amounts of membrane proteins and lipids need to be synthesized and correctly targeted. In polarized epithelia, a considerable fraction of apical proteins are transported by an indirect pathway involving a detour to the basolateral membrane before being internalized and transported across the cell to the apical membrane by a process known as transcytosis. The apical recycling endosome (ARE) or its equivalent, the subapical compartment (SAC), of hepatocytes is an intracellular trafficking station involved in the transcytotic pathway. MAL2, an essential component of the machinery for basolateral-to-apical transcytosis, is an ARE/SAC resident protein. Here, we show that, after differentiation, murine oligodendrocyte precursor and human oligodendroglioma derived cell lines, Oli-neu and HOG, respectively, up-regulate the expression of MAL2 and accumulate it in an intracellular compartment, exhibiting a peri-centrosomal localization. In these oligodendrocytic cell lines, this compartment shares some of the main features of the ARE/SAC, such as colocalization with Rab11a, sensitivity to disruption of the microtubule cytoskeleton with nocodazole, and lack of internalized transferrin. Therefore, we suggest that the MAL2-positive compartment in oligodendrocytic cells could be a structure analogous to the ARE/SAC and might have an important role in the sorting of proteins and lipids for myelin assembly during oligodendrocyte differentiation.     

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non- transcytosing molecules.     

Tartrate-resistant acid phosphatase facilitates hydroxyl radical formation and colocalizes with phagocytosed Staphylococcus aureus in alveolar macrophages

Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed specifically in osteoclasts and activated macrophages, two phagocytosing cell types originating from the same hematopoietic stem cells. TRAP contains a binuclear iron centre which has been shown to generate reactive oxygen species (ROS). In this study murine macrophage like cell line RAW-264 overexpressing TRAP was shown to produce elevated levels of hydroxyl radicals compared to parental cells. TRAP transfected cells also had reduced growth rate indicating harmful effects of excessive intracellular ROS levels. Using TRAP specific antibody TRAP protein was shown in alveolar macrophages partially colocalize with late endosomal/lysosomal markers Rab7, Lamp 1 and MHC II molecules that bind antigenic peptides. TRAP also colocalized into compartments where Staphylococcus aureus were phagocytosed. These results suggest that TRAP may have an important biological function in the defence mechanism of macrophages by generating intracellular ROS which would be targeted to destroy phagocytosed foreign material.     

Morphological and histochemical comparison of the cells elicited by ectopic bone implants and tibial osteoclasts.

Pellets of mineralized and demineralized bone and a composite mixture of mineralized and demineralized, devitalized bone particles were implanted subcutaneously on the dorsal body wall of young adult rats. Two weeks post-implantation, the pellets were removed and processed for histochemical and morphological analyses. Rat proximal tibia was also processed for evaluation. The levels of tartrate-resistant acid phosphatase (TRAP) activity in the multinucleated giant cells (MNGCs) from each of the three implants and from osteoclasts were assessed using an image analyzer. The osteoclasts from the proximal tibia and the majority of MNGCs from the demineralized implants demonstrated high levels of TRAP activity. MNGCs from the mineralized implants showed either a low level or absence of TRAP activity. Most MNGCs from the composite implants exhibited a low level of TRAP activity; however, there was a population of cells that demonstrated a high level of reaction product, similar to that seen in the tibia and demineralized implant. Morphologically, osteoclasts from the proximal tibia and from the osteogenic demineralized implant exhibited ruffled borders. A small population of MNGCs from the composite implant also revealed osteoclastic features. In summary, MNGCs from the mineralized implant did not exhibit a level of TRAP reaction product or morphology similar to osteoclasts, while the majority of cells from the demineralized implant and a subpopulation of the MNGCs elicited by the composite implant did demonstrate TRAP expression and morphology similar to osteoclasts. The expression of osteoclastic characteristics in cells at an ectopic site may be dependent on accessory signals from the skeletal microenvironment; such signals appear to be absent from or incomplete in the mineralized implants but appear to be present when demineralized bone particles are implanted.     

Polarized Sphingolipid Transport from the Subapical Compartment: Evidence for Distinct Sphingolipid Domains

In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study, evidence is provided that demonstrates that these sphingolipids constitute separate membrane domains at the luminal side of the SAC membrane. Furthermore, as revealed by the use of various modulators of membrane trafficking, such as calmodulin antagonists and dibutyryl-cAMP, it is shown that the fate of these separate sphingolipid domains is regulated by different signals, including those that govern cell polarity development. Thus under conditions that stimulate apical plasma membrane biogenesis, SM is rerouted from a SAC-to-basolateral to a SAC-to-apical pathway. The latter pathway represents the final leg in the transcytotic pathway, followed by the transcytotic pIgR-dIgA protein complex. Interestingly, this pathway is clearly different from the apical recycling pathway followed by glucosylceramide, further indicating that randomization of these pathways, which are both bound for the apical membrane, does not occur. The consequence of the potential coexistence of separate sphingolipid domains within the same compartment in terms of "raft" formation and apical targeting is discussed.     

Cellubrevin is present in the basolateral endocytic compartment of hepatocytes and follows the transcytotic pathway after IgA internalization

The endocytic compartment of polarized cells is organized in basolateral and apical endosomes plus those endocytic structures specialized in recycling and transcytosis, which are still poorly characterized. The complexity of the various populations of endosomes has been demonstrated by the exquisite repertoire of endogenous proteins. In this study we examined the distribution of cellubrevin in the endocytic compartment of hepatocytes, since its intracellular location and function in polarized cells are largely unknown. Highly purified rat liver endosomes were isolated from estradiol-treated rats, and the early/sorting endosomal fraction was further subfractionated in a multistep sucrose density gradient, and studied. Analysis of dissected endosomal fractions showed that cellubrevin was located in early/sorting endosomes, with Rab4, annexins II and VI, and transferrin receptor, but in a specific subpopulation of these early endosomes with the same density range as pIgA and Raf-1. Interestingly, only in those isolated endosomal fractions, endosomes enriched in transcytotic structures (of livers loaded with IgA), the polymeric immunoglobulin receptor specifically co-immunoprecipitated with cellubrevin. In addition, confocal and immuno-electron microscopy identification of cellubrevin in tubular structures underneath the sinusoidal plasma membrane together with the re-organization of cellubrevin, in the endocytic compartment, after the IgA loading, strongly suggest the involvement of cellubrevin in the transcytosis of pIgA.     

Trans-Golgi network and subapical compartment of HepG2 cells display different properties in sorting and exiting of sphingolipids

In HepG2 cells, the subapical compartment (SAC) is involved in the biogenesis of membrane polarity. By contrast, direct apical transport originating from the trans-Golgi network (TGN), which may contribute to polarity establishment, has been poorly defined in these cells. Thus, although newly synthesized sphingolipids can be directly transported from the TGN to the apical membrane, numerous apical resident proteins are traveling via the transcytotic route. Here, we developed an in vitro transport assay and compared the molecular sorting of 6-[N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)amino] hexanoyl-sphingomyelin (C(6)NBD-SM) and C(6)NBD-glucosylceramide (C(6)NBD-GlcCer) in TGN and SAC. SM is released from both TGN and SAC in the lumenal leaflet of transport vesicles. This holds also for GlcCer released from the SAC but not for a substantial fraction that departed from the Golgi. Distinct transport vesicles, enriched in either SM or GlcCer are released from SAC, consistent with their rigid sorting in this compartment. Different vesicle populations could not be recovered from TGN, although in situ experiments reveal that GlcCer is preferentially transported to the apical membrane, reflecting different transport mechanisms. The results indicate that in HepG2 cells sphingolipids are mainly sorted in the SAC membrane and that the release of SM from SAC and TGN is differentially regulated.     

Acid phosphatase activity is detected preferentially in the osteoclastic lineage by pre-treatment with cyanuric chloride

We previously reported a simple method to detect osteoid matrices in decalcified bone sections by pre-treatment with cyanuric chloride. We have applied this technique to identify osteoclasts and their precursors in rats. In JB-4 sections prepared from untreated bone tissues with cyanuric chloride, both acid phosphatase (ACP) and tartrate-resistant acid phosphatase (TRAP) were found not only in osteoclasts and bone marrow mononuclear cells but also in osteoblasts. In contrast, treatment of bones with cyanuric chloride resulted in staining ACP preferentially in osteoclasts and mononuclear cells adjacent to the bone surface. In the osteoclasts and most of the ACP-positive mononuclear cells, autoradiography showed calcitonin binding. Decalcification with EDTA did not affect the staining for ACP activity in bones treated with cyanuric chloride. It was possible to simultaneously identify ACP and osteoid matrix in a decalcified section. In soft tissues without treatment with cyanuric chloride, both ACP and TRAP were detected in splenic macrophages, alveolar macrophages, and proximal convoluted ducts in kidney. Neither ACP nor TRAP was found in these cell types in the tissues treated with cyanuric chloride. This procedure provides a new, simple method to identify a more restricted population in the osteoclastic lineage than that detected by TRAP staining.     

Polarized expression of type I phosphodiesterases in epithelial cells]

Type I phosphodiesterases are differently expressed by different cell types. Three members have been identified, PC-1, B10 and autotaxin. They are between 40 and 50% identical at the amino acid sequence level. Hepatocytes express both B10 and PC-1 at their plasma membrane. However, B10 is exclusively expressed at the apical pole whereas PC-1 is located at the basolateral pole. Studies of the biosynthetic route of B10 in hepatocytes shows that B10 is first transported to the basolateral surface and secondarily reaches the basolateral surface. The transcytotic step between the basolateral and apical surface occurs through a tubular endosomal compartment identical to the transcytotic compartment of the polymeric IgA receptor. Transfected in the polarized cell lines MDCK and Caco-2 of renal and intestinal origin, B10 and PC-1 are expressed at the apical and basolateral poles respectively, as in hepatocytes. However, the biosynthetic transport of B10 occurs directly in MDCK cells and both directly and by transcytosis in Caco-2 cells. Truncation of the cytoplasmic domain of PC-1 generates an apical protein indicating that the basolateral signal of PC-1 is likely to be in the cytoplasmic domain. The nature of the apical targeting signal of B10 is under investigation.     

Quantification of Tartrate Resistant Acid Phosphatase Activity Using a Computerized Image Analysis System

Tartrate resistant acid phosphatase (TRAP) has been accepted as a marker for identification of osteoclasts. A method is reported here for quantitating TRAP using an image analysis system. The amount of the enzyme specific to osteoclasts can be used to differentiate osteoclasts from other cells capable of TRAP expression. TRAP expression characteristic of the osteoclast was compared with that of multinucleated giant cells (MNGC)s recruited to the site of subcutaneously implanted mineralized bone matrix. Two weeks post-implantation, the pellets were removed and processed for the demonstration of TRAP along with rat proximal tibiae. A large amount of TRAP was consistently expressed by the in situ osteoclasts. The MNGCs associated with the mineralized bone implants expressed little if any TRAP reaction product. Using this system, the amount of TRAP reaction product or any other enzyme reaction product expressed can be objectively and reproducibly quantitated.     

Quantification of Tartrate Resistant Acid Phosphatase Activity Using a Computerized Image Analysis System

Tartrate resistant acid phosphatase (TRAP) has been accepted as a marker for identification of osteoclasts. A method is reported here for quantitating TRAP using an image analysis system. The amount of the enzyme specific to osteoclasts can be used to differentiate osteoclasts from other cells capable of TRAP expression. TRAP expression characteristic of the osteoclast was compared with that of multinucleated giant cells (MNGC)s recruited to the site of subcutaneously implanted mineralized bone matrix. Two weeks post-implantation, the pellets were removed and processed for the demonstration of TRAP along with rat proximal tibiae. A large amount of TRAP was consistently expressed by the in situ osteoclasts. The MNGCs associated with the mineralized bone implants expressed little if any TRAP reaction product. Using this system, the amount of TRAP reaction product or any other enzyme reaction product expressed can be objectively and reproducibly quantitated.     

Human breast cancer cell lines and tissues express tartrate-resistant acid phosphatase (TRAP)

Tartrate-resistant acid phosphatase (TRAP) is expressed by osteoclasts, macrophages and dendritic cells. TRAP has been identified in a wide variety of tissues, however, its biological function is not fully understood. Serum TRAP is a marker of diseases involving excessive bone resorption including metastatic bone disease in breast cancer patients and can be used to monitor responses to treatment. Our aim in this study was to determine whether TRAP is expressed by human breast tumours. Four breast cancer cell lines were assayed for TRAP activity. MDA-MB-435, the most tumourigenic line, had an activity twofold higher than the other cell lines. Immunohistochemistry using a TRAP specific antibody confirmed that both cell lines and human breast tumours express TRAP. Expression was absent in benign tissues and abundant in more aggressive tumours. This work suggests that tumour derived TRAP contributes to the raised enzyme activity found in the serum of breast cancer patients.     

Tartrate-resistant acid phosphatase (Acp 5): identification in diverse human tissues and dendritic cells.

Histochemical demonstration of tartrate-resistant acid phosphatase (TRAP) is used for the specific identification of osteoclasts. The enzyme, which we have shown to be critical for normal bone development in mice, is also characteristic of monohistiocytes, including alveolar macrophages, and is associated with diverse pathological conditions such as Gaucher's disease and hairy cell leukemia. TRAP activity is enhanced in serum when bone resorption is increased, and the activity is used routinely to monitor treatment responses in Gaucher's disease. We have lately shown widespread expression of the enzyme in murine tissues with particular reference to the skin, thymus, gut epithelia, and isolated dendritic cells, suggesting a possible role in immunity. To further clarify the significance of TRAP in human physiology, we have examined its distribution in non-skeletal human tissues and in CD34+ -derived human dendritic cells. TRAP mRNA determined by Northern blotting analysis was expressed abundantly in spleen, liver, colon, lung, small intestine, kidney, stomach, testis, placenta, lymph node, thymus, peripheral blood leukocyte, bone marrow, and fetal liver. Expression of TRAP protein was investigated by immunohistochemistry, with which the enzyme was identified in multiple tissues. Histochemical staining detected enzymatically active protein in spleen, lung, skin, colon, stomach, and ileum. Active TRAP was identified in CD34+ -derived immature dendritic cells and co-localized to intracellular CD63 positive organelles. When these cells were matured by induction with LPS, the TRAP activity increased fivefold and remained within the cell during the phase associated with CD63 surface expression. Our findings demonstrate widespread expression of TRAP in human tissues. Its abundant expression in epithelia and dendritic cells suggests a potential role in antigen processing and in immune responses.     

Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture

Certain epithelial cells synthesize the polymeric immunoglobulin receptor (pIgR) to transport immunoglobulins (Igs) A and M into external secretions. In polarized epithelia, newly synthesized receptor is first delivered to the basolateral plasma membrane and is then, after binding the Ig, transcytosed to the apical plasma membrane, where the receptor-ligand complex is released by proteolytic cleavage. In a previous work (Ikonen et al., 1993), we implied the existence of a dendro-axonal transcytotic pathway for the rabbit pIgR expressed in hippocampal neurons via the Semliki Forest Virus (SFV) expression system. By labeling surface-exposed pIgR in live neuronal cells, we now show (a) internalization of the receptor from the dendritic plasma membrane to the dendritic early endosomes, (b) redistribution of the receptor from the dendritic to the axonal domain, (c) inhibition of this movement by brefeldin A (BFA) and (d) stimulation by the activation of protein kinase C (PKC) via phorbol myristate acetate (PMA). In addition, we show that a mutant form of the receptor lacking the epithelial basolateral sorting signal is directly delivered to the axonal domain of hippocampal neurons. Although this mutant is internalized into early endosomes, no transcytosis to the dendrites could be observed. In epithelial Madin-Darby Canine Kidney (MDCK) cells, the mutant receptor could also be internalized into basolaterally derived early endosomes. These results suggest the existence of a dendro-axonal transcytotic pathway in neuronal cells which shares similarities with the basolateral to apical transcytosis in epithelial cells and constitute the basis for the future analysis of its physiological role.     

Nonpolarized cells selectively sort apical proteins from cell surface to a novel compartment,but lack apical retention mechanisms

Membrane trafficking is central to establishing and maintaining epithelial cell polarity. One open question is to what extent the mechanisms regulating membrane trafficking are conserved between nonpolarized and polarized cells. To answer this question, we examined the dynamics of domain-specific plasma membrane (PM) proteins in three classes of hepatic cells: polarized and differentiated WIF-B cells, nonpolarized and differentiated Fao cells, and nonpolarized and nondifferentiated Clone 9 cells. In nonpolarized cells, mature apical proteins were uniformly distributed in the PM. Surprisingly, they were also in an intracellular compartment. Double labeling revealed that the compartment contained only apical proteins. By monitoring the dynamics of antibody-labeled molecules in nonpolarized cells, we further found that apical proteins rapidly recycled between the compartment and PM. In contrast, the apical PM residents in polarized cells showed neither internalization nor return to the basolateral PM from which they had originally come. Cytochalasin D treatment of these polarized cells revealed that the retention mechanisms are actin dependent. We conclude from these data that both polarized and nonpolarized cells selectively sort apical proteins from the PM and transport them to specific, but different cellular locations. We propose that the intracellular recycling compartment in nonpolarized cells is an intermediate in apical surface formation.     

Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.     

Eccentric localization of osteocytes expressing enzymatic activities, protein, and mRNA signals for type 5 tartrate-resistant acid phosphatase (TRAP).

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 microm 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.