Immunolocalization of vacuolar-type H+-ATPase, cathepsin K, matrix metalloproteinase-9, and receptor activator of NFkB ligand in odontoclasts during physiological root resorption of human deciduous teeth
Department of Orthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ohta-ku, Tokyo 145-8515, Japan
Department of Oral Histology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Physiological root resorption during shedding of human deciduous teeth is mediated mainly by multinucleated giant cells, odontoclasts (Boyde and Lester, 1967; Furseth, 1968; Morita et al., 1970; Freilich, 1971; Ten Cate and Anderson, 1986; Sasaki et al., 1988a,b, 1989, 1990a; Sasaki and Ueno-Matsuda, 1992; Matsuda, 1992; Sahara et al., 1994, 1996; Sahara, 1998). Odontoclastic root resorption is also a significant clinical issue in association with orthodontic tooth movement (Brudvick and Rygh, 1994; Sato et al., 2000a,b). Odontoclasts are responsible for resorption of such dental hard tissues as cementum, dentine, and enamel, and physiological root resorption by odontoclasts has been suggested to be regulated by a mechanism different from that of bone remodeling, including bone resorption (Furseth, 1968; Freilich, 1971). However, the precise cellular mechanisms whereby root resorption takes place have not been fully established. Although no difference has ever been recognized between odontoclasts and osteoclasts in structural and histochemical properties including tartrate-resistant acid phosphatase (Ten Cate and Anderson, 1986; Sasaki et al., 1988a,b; Sasaki and Ueno-Matsuda, 1992; Matsuda, 1992; Sahara et al., 1994, 1996; Sahara, 1998), it is still uncertain whether odontoclasts involved in physiological root resorption possess functional features identical to those of osteoclasts.
In bone resorption, osteoclasts are the principal cells directly responsible for solubilization of mineral components (i.e., calcium hydroxyapatite) and the subsequent degradation of insoluble type-1 collagen of the bone matrix and are characterized structurally by a prominent ruffled border-clear zone complex facing the resorptive bone surfaces (see reviews by Vaes, 1988; Delaisse and Vaes, 1992; Sasaki, 1996). In the initial phase of osteoclastic bone resorption, hydroxyapatite crystals are decalcified by acidification of the subosteoclastic microenvironment created by vacuolar-type H+-ATPase, which is highly localized in the ruffled border membranes of osteoclasts (Baron et al., 1985; Vaes, 1988; Sundquist et al., 1990; Vaananen et al., 1990; Sasaki et al., 1994; Yokoya et al., 1997). Resorption lacunae are then formed by degradation of bone type-I collagen, mediated mainly by cathepsin K, a lysosomal cysteine proteinase, which is highly and selectively expressed in osteoclasts (Tezuka et al., 1994; Bromme and Okamoto, 1995; Inaoka et al., 1995; Li et al., 1995; Saneshige et al., 1995; Bossard et al., 1996; Bromme et al., 1996; Drake et al., 1996; Inui et al., 1997; Littlewood-Evans et al., 1997; Kamiya et al., 1998; Yazawa et al., 1998). In concert with cysteine proteinase, acid-soluble and -insoluble type I collagens are thought to be further degraded in the subosteoclastic microenvironment by matrix metalloproteinase-9 (MMP-9, 92-kDa gelatinase/type IV collagenase = gelatinase B) produced by osteoclasts (Everts et al., 1992; Reponen et al., 1994; Wucherpfennig et al., 1994; Okada et al., 1995). However, the expression of these enzyme proteins in human odontoclasts during root resorption remains to be clarified.
In addition, receptor activator of NFKB ligand (RANKL) has been identified recently as an important regulator of osteoclastogenesis (Lacey et al., 1998; Matsuzaki et al., 1998; Tsukii et al., 1998; Yasuda et al., 1998). RANKL expressed in the plasma membranes of osteoblasts/stromal cells was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced osteoclast formation from osteoclast progenitors (Lacey et al., 1998; Matsuzaki et al., 1998; Tsukii et al., 1998; Yasuda et al., 1998; Takami et al., 1999). Although RANKL is one of the key regulatory molecules in osteoclast formation and function, the presence and involvement of RANKL in physiological root resorption has not yet been examined. To gain further insight into the cellular mechanisms of physiological root resorption, the authors examined the immunocytochemical expression of vacuolar-type H+-ATPase, cathepsin K, MMP-9 and RANKL in odontoclasts of human deciduous teeth undergoing physiological root resorption.
MATERIALS AND METHODS
Human deciduous incisors and molars used in this study were extracted in orthodontic treatment, because of prolonged retention, or both. Immediately after extraction, the teeth were fixed by immersion in a mixture of 4% formaldehyde and 0.1% glutaraldehyde in 0.1-M sodium cacodylate buffer (pH 7.3). They were then decalcified in 10% EDTA solution for approximately 4 weeks, washed in a 0.1 M sodium cacodylate buffer, and through dehydration with graded series of ethanol, embedded in LR white resin (London resin, Basingstoke, UK), which was polymerized at –20°C with ultraviolet rays. Ultrathin sections were cut by using a diamond knife on a Reichert-Jung Ultracut OmU-4 and mounted on Formvar-coated nickel grids. The sections were then processed for immunocytochemical localization of vacuolar-type H+ -ATPase, cathepsin K, MMP-9, and RANKL as described below.
To block nonspecific binding, the sections were first treated with 10% bovine serum albumin (BSA) in 0.01 M phosphate-buffered saline (PBS) for 1 hr. The sections were then incubated with rabbit antiserum raised against vacuolar-type H+-ATPase (1:500 dilution with 1%BSA/PBS) (kindly provided by Dr. Moriyama, Hiroshima University; Moriyama and Nelson, 1989), cathepsin K (1:500 dilution with 1%BSA/PBS) (kindly provided by Dr. Sakai, Nagasaki University; Kamiya et al., 1998), or RANKL (1:100 dilution with 1%BSA/PBS) (Kartsogiannis et al., 1999) overnight at 4°C. Other sections were incubated with mouse antiserum raised against human MMP-9 (Fuji Chemical Co., Ltd., Japan), diluted 1:100 with 1% BSA/PBS, overnight at 4°C. Control sections were incubated with either nonimmune rabbit serum (1:500 or 1:100 dilution with 1%BSA/PBS) or nonimmune mouse serum (1:100 dilution with 1%BSA/PBS). After incubation, the sections were rinsed with PBS and incubated with goat anti-rabbit IgG for localization of vacuolar-type H+-ATPase, cathepsin K, and RANKL and with goat anti-mouse IgG for MMP-9, both conjugated with 10-nm colloidal gold particles (BioCell Research Laboratories, Cardiff, UK) diluted 1:100 with PBS for 1 hr. All of these procedures were carried out in a moisture chamber at room temperature. After rinsing with PBS and distilled water, the sections were stained with 2% uranyl acetate for 4 min and examined with a Hitachi HU-12A electron microscope at 75kV.
For light microscopy, decalcified tissues were embedded in paraffin, and 4-μm-thick sections were routinely prepared and processed for localization of vacuolar-type H+-ATPase, cathepsin K, MMP-9, and RANKL by a biotin-streptavidin-horseradish peroxidase method, by means of a Histofine SAB-PO kit (Nichirei Co., Ltd., Tokyo). The sections were incubated for 2 hr with the primary antibody diluted 1:500 (H+-ATPase and cathepsin K) or 1:100 (MMP-9 and RANKL) with PBS at room temperature. After being immunostained, sections were lightly stained with hematoxylin and examined with an AHBS3 light microscope (Olympus, Tokyo).
In human deciduous teeth undergoing root resorption, odontoclasts on the resorbing root dentine were strongly immunostained for vacuolar-type H+-ATPase, particularly at the ruffled border areas facing the resorption lacunae (Fig. 1, inset). Ultrastructurally, most of these odontoclasts exhibited well-developed ruffled borders consisting of deep and regular membrane infoldings toward the cytoplasm, and the accumulation of numerous pale vacuoles in the cytoplasm proximal to the ruffled borders (Fig. 1). Immunoelectron microscopic localization of vacuolar-type H+-ATPase demonstrated deposition of numerous immunogold particles mainly along the limiting membranes of pale vacuoles and along the ruffled border membranes of these odontoclasts (Fig. 1).
Odontoclasts were strongly immunostained for cathepsin K, mainly in the ruffled border areas facing the resorption lacunae (Fig. 2, inset). Immunostaining was also detected along the resorbing root dentine surfaces (Fig. 2, inset). Ultrastructural localization of cathepsin K demonstrated intense deposition of immunogold particles within pale vacuoles and the extracellular canals of the ruffled borders in odontoclasts (Fig. 2). Deposition of immunogold particles in the resorbing root dentine surfaces was variable (data not shown).
Odontoclasts were also immunostained for MMP-9, mainly in the ruffled border areas facing the resorption lacunae. Immunostaining was also detected along the resorbing root dentine surfaces (Fig. 3, inset). Ultrastructural localization of MMP-9 demonstrated deposition of immunogold particles within pale vacuoles and the extracellular canals of the ruffled borders in odontoclasts (Fig. 3). Deposition of immunogold particles in the resorbing dentine surfaces was also evident (Fig. 3).
RANKL immunostaining was observed in odontoclasts and in some of the adjacent mononuclear stromal cells located on resorbing root dentine surfaces (Fig. 4, inset). At the ultrastructural level, immunogold particles were localized in the odontoclast cytoplasm and along the ruffled border membranes (Fig. 4). In all these immunocytochemical examinations, the negative control sections showed that the replacement of the primary antibody with nonimmune normal serum resulted in sparse immunoreaction over the tissue sections (Fig. 5).
Past scanning electron microscopic observations have indicated that decalcification of the root dentine surfaces in the resorption lacunae preceded the removal of the organic materials (Boyde et al., 1984; Jones et al., 1984; Sasaki et al., 1988a, 1990b; Matsuda, 1992). Namely, exposure of collagen fibrils at the resorbing cementum and dentine surfaces, dissolution of the peritubular matrix around dentinal tubules in the resorbing dentine surfaces, and dissolution of the enamel rods and/or their peripheral areas at the resorbing enamel surfaces were clearly demonstrated (Sasaki et al., 1988a; Matsuda, 1992). Therefore, it is apparent that decalcification of the matrices occurs at the resorbing surfaces of these dental hard tissues. In this regard, Matsuda (1992) localized the enzymatic activity of H+-K+-ATPase along the ruffled border membranes and the lysosomal limiting membranes of odontoclasts. It is well known that the acid release by osteoclasts during bone resorption is mediated by vacuolar-type H+-ATPase localized in the ruffled border membranes and is involved in dissolution of apatite crystals (Sundquist et al., 1990; Vaananen et al., 1990; Sasaki et al., 1994). The present study first clarified the subcellular localization of this vacuolar-type H+-ATPase in the ruffled border membranes of human odontoclasts during physiological root resorption.
Concerning the subsequent organic matrix degradation, as in osteoclastic bone resorption (Goto et al., 1993, 1994), lysosomal cysteine proteinase such as cathepsin B was previously immunolocalized in human odontoclasts (Sasaki and Ueno-Matsuda, 1992). In the current observation, odontoclasts were strongly immunostained for cathepsin K, which is also highly expressed in osteoclasts. Localization of cathepsin K and MMP-9 and their functions in osteoclastic degradation of bone type-I collagen have been well documented (Everts et al., 1992; Reponen et al., 1994; Wucherpfennig et al., 1994; Okada et al., 1995; Inui et al., 1997; Littlewood-Evans et al., 1997; Kamiya et al., 1998; Yazawa et al., 1998). We herein found identical subcellular localization of both cathepsin K and MMP-9 in human odontoclasts, which suggests the involvement of both enzymes in the extracellular degradation of dentine collagens during root resorption. In addition, secretion of nonspecific acid phosphatase toward the resorbing dentine matrix by human odontoclasts also suggested the extracellular degradation of various matrix constituents (Sasaki et al., 1988a,b). From these results, it is suggested that, as in osteoclastic bone resorption, odontoclasts are directly involved in decalcification of apatite crystals in dental hard tissues by active extrusion of proton ions mediated by vacuolar-type H+-ATPase and the extracellular degradation of dentine type-I collagens mainly by cathepsin K and MMP-9.
The presence of cementoblast-like or fibroblast-like stromal cells in association with odontoclasts has been reported in root resorption of deciduous teeth (Sasaki et al., 1990a,b; Tanaka et al., 1990; Sahara, 1998). Similarity of the tooth resorption-repair sequence (i.e., root resorption and cementum formation) to the bone remodeling sequence has also been pointed out (Sasaki et al., 1990a,b; Sahara, 1998). Although mononuclear stromal cells adjacent to odontoclasts have been suggested to play roles in the root resorption-repair phenomenon, the cell-to-cell interactions between odontoclasts and mononuclear stromal cells remain to be clarified. In this regard, it was a very interesting finding that immunoreaction of RANKL, the regulator of osteoclast differentiation, was detected in odontoclasts and some of the adjacent mononuclear stromal cells located on resorbing root dentine surfaces.
RANKL has been shown to be expressed as a membrane-associated factor by osteoblastic cells (Anderson et al., 1997) and to be involved in osteoclast function as well as osteoclast differentiation (Kong et al., 1999). In bone remodeling, the cell–cell interaction between osteoblasts/stromal cells and osteoclast progenitors is reported to be essential for osteoclast formation (Yasuda et al., 1998). Our results suggest that, as in osteoblasts in bone resorption, RANKL may be produced by mononuclear stromal cells in root resorbing tissues. RANKL immunoreaction was also detected in the odontoclast cytoplasm. Recently, Kartsogiannis et al. (1999) showed that RANKL mRNA and RANKL protein were expressed in the osteoclast cytoplasm located within resorption lacunae. They suggested that, as well as a supportive function of osteoblastic stromal cells for osteoclast differentiation, RANKL detected in osteoclasts correlated with the resorptive capability of osteoclasts and osteoclast survival (also suggested by Fuller et al., 1998, and Takami et al., 1999). It has also been reported that RANKL induces pit-forming activity of cultured osteoclasts (Lacey et al., 1998; Matsuzaki et al., 1998; Yasuda et al., 1998). In fact, by using a fetal mouse long bone culture system, Tsukii et al. (1998) demonstrated that a genetically engineered soluble-form RANKL elicited bone resorption assessed by 45Ca release. Thus, RANKL seems to enhance bone-resorbing activity of osteoclasts. Although our results seem to coincide well with these studies and suggest the involvement of RANKL in odontoclast differentiation and activity during physiological root resorption, the mechanisms whereby osteo/odontoclastic RANKL regulates their differentiation and functions remain to be elucidated. However, from all these results, the cellular mechanisms of root resorption appear to be quite similar to those of osteoclastic bone resorption. At present, except for the expression of calcitonin receptors, which have still not been detected in odontoclasts, there seem to be no cytologic differences between osteoclasts and odontoclasts. With the detection of calcitonin receptor expression, it may be elucidated that, even though involved in physiological root resorption of deciduous teeth, odontoclasts are multinucleated giant cells identical to osteoclasts.