Immunocytochemical Localization of Caveolin-3 in the Synoviocytes of the Rat Temporomandibular Joint During Development.код для вставкиСкачать
THE ANATOMICAL RECORD 291:233–241 (2008) Immunocytochemical Localization of Caveolin-3 in the Synoviocytes of the Rat Temporomandibular Joint During Development MASAHIRO NIWANO,1,2 KAYOKO NOZAWA-INOUE,1* AKIKO SUZUKI,1 NOBUYUKI IKEDA,2 RITSUO TAKAGI,2 AND TAKEYASU MAEDA1 1 Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 2 Division of Oral Maxillofacial Surgery, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan ABSTRACT Caveolins—caveolin-1, -2, -3 (Cav1, 2, 3)—are major components of caveolae, which have diverse functions. Our recent study on the temporomandibular joint (TMJ) revealed expressions of Cav1 and muscle-speciﬁc Cav3 in some synovial ﬁbroblast-like type B cells with well-developed caveolae. However, the involvement of Cav3 expression in the differentiation and maturation of type B cells remains unclear. The present study therefore examined the chronological alterations in the localization of Cav3 in the synovial lining cells of the rat TMJ during postnatal development by immunocytochemical techniques. Observations showed immature type B cells possessed a few caveolae with Cav1 but lacked Cav3 protein at postnatal day 5 (P5). At P14, Cav3-immunopositive type B cells ﬁrst appeared in the synovial lining layer. They increased in number and immunointensity from P14 to P21 as occlusion became active. In immunoelectron microscopy and double immunolabeling with heat shock protein 25 (Hsp25) and Cav3, coexpressed type B cells developed rough endoplasmic reticulum and numerous caveolae, while the Cav3-immunonegative type B cell with Hsp25 immunoreaction possessed few of these. Results suggest that Cav3 expression, which is closely related to added functional stimuli, reﬂects the differentiation of the type B synoviocytes. Anat Rec, 291:233– 241, 2008. Ó 2008 Wiley-Liss, Inc. Key words: temporomandibular joint; rat; caveolin; development Caveolae are 50- to 100-nm ﬂask-shaped microdomains of the plasma membrane that play important roles in various cellular functions, including signal transduction (for review, see Quest et al., 2004), transcytosis (for review, see Stan, 2002), cholesterol transport (Smart et al., 1996), and tumor suppression (Lee et al., 1998). Caveolin, a major structural protein of caveolae (Rothberg et al., 1992), has three subtypes—caveolin-1 (Cav1), -2 (Cav2), and -3 (Cav3; for review, see Cohen et al., 2004). The colocalization of Cav1 and Cav2 has been reported in most cell types such as adipocytes, endothelial cells, and ﬁbroblasts (for review, see Quest et al., Ó 2008 WILEY-LISS, INC. synovial membrane; Grant sponsor: MEXT (Ministry of Education, Culture, Sports, Science and Technology, Japan); Grant number: 18791346. *Correspondence to: Kayoko Nozawa-Inoue, Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan. Fax: 18125-223-6499. E-mail: [email protected] Received 18 September 2007; Accepted 5 December 2007 DOI 10.1002/ar.20655 Published online in Wiley InterScience (www.interscience.wiley. com). 234 NIWANO ET AL. 2004), but Cav3 is essentially restricted to striated (cardiac and skeletal) muscle cells (Tang et al., 1996; Hagiwara et al., 2002). The expression of Cav3 has been also reported in smooth muscle cells, astroglial cells (Ikezu et al., 1998), sinus endothelial cells in spleen (Uehara and Miyoshi, 2002), ciliated airway epithelial cells of the trachea and bronchial tree (Krasteva et al., 2007), and chondrocytes in the limb cartilage (Schwab et al., 1999, 2000). Although the precise function of Cav3 remains unclear, mutations or alterations of Cav3expression are responsible for speciﬁc diseases such as cardiomyopathy, myodystrophy, and Alzheimer’s disease (for review, see Quest et al., 2004). The temporomandibular joint (TMJ) is a bilateral diarthrosis between the mandibular fossa of the temporal bone and the mandibular condyle. The TMJ is covered with a thick ﬁbrous capsule, which is largely divided into two parts: an outer ﬁbrous layer and an inner synovial membrane. The synovial membrane is involved in the production, secretion, and absorption of viscous synovial ﬂuids that make smooth jaw movement possible and that supply oxygen and nutrition to both the articular cartilage and articular disk. The synovial membrane in the TMJ consists of a surface synovial lining layer and a connective sublining layer. In addition, the synovial lining layer contains two types of synovial lining cells: macrophage-like type A and ﬁbroblast-like type B cells (for review, see Nozawa-Inoue et al., 2003). The ﬁbroblast-like type B cells have important functions in the production/secretion of type I and II collagens, ﬁbronectin, and glycosaminoglycans—including hyaluronic acid—into the synovial interstitium and ﬂuids. In particular, hyaluronic acid plays an essential role in maintaining the viscosity of synovial ﬂuids. Therefore, previous studies have focused on the origin and functions of the ﬁbroblast-like type B cells (Nozawa-Inoue et al., 2003, 2006, 2007; Ikeda et al., 2004; Suzuki et al., 2005, 2006). Electron microscopically, the ﬁbroblast-like type B cell is characterized by a well-developed rough endoplasmic reticulum (rER), a nucleus with less heterochromatin, long cytoplasmic projections, and a surrounding basement membrane-like structure in addition to numerous caveolae in the cell membrane. On the other hand, the macrophage-like type A cell possesses a nucleus with rich heterochromatin, lysosomes, vacuoles, and ﬁlopodia-like processes. In our developmental study (Ikeda et al., 2004), the immature type B cells were ﬁrst detectable at embryonic day 19 (E19) in rat TMJ, and exhibited postnatal maturation in close relationship with the formation of the articular cavity thereafter. However, the detailed maturation process of these type B cells remains unclear due to lack of a reliable cell marker for them. Our recent study reported an intense expression of Cav1 in all ﬁbroblast-like type B cells of the adult rat TMJ (Nozawa-Inoue et al., 2006). In addition, type B synoviocytes that possessed well-developed cell organelles expressed muscle-speciﬁc Cav3 in their caveolae as well, whereas a few type B synoviocytes that had relatively poor cell organelles lacked Cav3 immunoreactions in the adult rat TMJ (Nozawa-Inoue et al., 2007). These data suggested that Cav3-expression reﬂects differences in their differentiation stages or functions among the type B cells. However, the involvement of Cav3 expression in the differentiation and maturation of type B cells remains to be clariﬁed. The present study therefore examined the chronological alterations in the localization of Cav3 in the synovial lining cells of postnatal rat TMJ by immunocytochemical techniques at both the light and electron microscopic levels. Furthermore, the colocalization of Cav3 and either Cav1 or heat shock protein 25 (Hsp25)—a marker of the type B cells—were demonstrated to conﬁrm the formation of caveolae in developing type B cells. MATERIALS AND METHODS All experiments were performed under the guidelines of the Niigata University Intramural Animal Use and Care Committee (approval number 700). Animals and Tissue Preparation Thirty male Wistar rats were obtained at postnatal day 1 (P1), P3, P5, P7, P14, and P21 (n 5 5 each). We deﬁned the day of birth (P1) as 24–48 hr after birth according to our previous reports (Ikeda et al., 2004; Suzuki et al., 2005). Under anesthesia by an intraperitoneal injection of 8% chloral hydrate (400 mg/kg), the animals were perfused with a ﬁxative containing 4% paraformaldehyde and 0.025% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4). The removed heads were decalciﬁed with a 10% ethylene diamine tetra-acetic acid disodium (EDTA-2Na) solution at 48C. After decalciﬁcation, the TMJ were removed en bloc, equilibrated in a 30% sucrose solution for cryoprotection, and embedded in OCT compound (Tissue-Tek1; Sakura Finetechnical, Tokyo, Japan). Serial sagittal sections including the TMJ were cut at a thickness of 8 mm in a cryostat (CM3050S; Leica Microsystems, Nussloch, Germany) and mounted onto silane-coated glass slides. Immunocytochemistry The cryostat sections were processed for immunocytochemistry using a commercially available avidin–biotin complex (ABC) kit (Vector Lab. Inc., Burlingame, CA). The sections were reacted overnight at 48C with a monoclonal antibody to Cav3 (1: 1,500; BD Transduction Laboratory, San Diego, CA), which recognizes Cav3 from the rat and mouse (manufacturer’s instruction). In addition, a monoclonal antibody for Cav1 (1:600; BD Transduction Laboratory), which recognizes this from the human, mouse, rat, dog, and chick (manufacturer’s instruction) was used as well. The bound primary antibody was then localized using a biotinylated anti-mouse IgG (1:100; Vector Lab. Inc.) and subsequently with ABC conjugated with peroxidase (Vector Lab. Inc.) for 90 min each at room temperature. Final visualization used 0.04% 3,30 diaminobenzidine tetrahydrochloride and 0.002% H2O2 in a 0.05 M Tris-HCl buffer (pH 7.6). Immunoreacted sections were counterstained with 0.03% methylene blue. Some immunostained sections without counterstaining were post-ﬁxed in 1% OsO4 reduced with 1.5% potassium ferrocyanide for 1 hr at 48C, dehydrated in an ascending series of ethanol, and ﬁnally embedded in epoxy resin (Epon 812; Taab, Berkshire, UK). Plastic sections (1 mm thick) were stained with 0.03% methylene blue. Ultrathin sections (70 nm thick) were brieﬂy double-stained with uranyl acetate and lead citrate and examined in an H-7000 transmission electron microscope (Hitachi Co. Ltd, Tokyo, Japan). DEVELOPMENT OF SYNOVIAL LINING CELLS 235 Double-labeling Immunocytochemistry for Cav3 and Either Cav1 or Hsp25 For ﬂuorescent double-labeling immunocytochemistry, sections were incubated with the mouse monoclonal antibody to Cav3, followed by ﬂuorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:100; Vector Lab. Inc.). They were further reacted with a rabbit polyclonal antisera against either Cav1 (1:600 Santa Cruz Biotechnology Inc., Santa Cruz, Canada) or Hsp25 (1:1,000; Stressgen Biotechnologies, Victoria, Canada), and subsequently by Texas RedTM-conjugated anti-rabbit IgG (1:100; Vector Lab. Inc.). After rinsing, the double-labeled sections were coverslipped with a Vectashield1 mounting medium with 40 ,6-diamidino-2-phenylindole (DAPI; Vector Lab. Inc.) and ﬁnally examined in a ﬂuorescent microscope (AxioImager M; Carl Zeiss, Oberkochen, Germany). Semiquantitative Evaluation The numbers of the Cav3- or Cav1-immunopositive type B synoviocytes were counted on each double-immunostained sections (n 5 10) with the anti-Hsp25 antibody. The sections were randomly selected both in the same animals and each developmental period. Fluorescent images containing whole views of the posterosuperior portion of the synovial membrane were captured at a magniﬁcation of 3400, and two observers in a blind counted cell numbers within the area 4 3 104 mm2 (200 mm 3 200 mm) from the tip of the synovial fold based on the major axis. Finally, the distribution patterns of Cav1- or Cav3-immunopositive cells in the type B synoviocytes were evaluated semiquantitatively and classiﬁed into ﬁve groups arbitrarily: no reactivity, weak reactivity on a part of the cells (50–60%), weak reactivity on the majority of the cells (80–90%), strong reactivity on the majority of the cells, and all cells with strong reactivity. Immunocytochemical Controls Immunocytochemical controls were performed by: (1) an absorption test using primary antibodies with the corresponding antigens of each antibody, (2) replacing the primary antiserum with phosphate-buffered saline, (3) or omitting the anti-mouse/rabbit IgG. These control sections did not reveal any immunoreaction. The speciﬁcity of the antibodies for Cav3 and Cav1 was checked by cross-preabsorption tests as well according to our previous report (Nozawa-Inoue et al., 2007). RESULTS P1 to P3 At P1, the upper and lower articular cavities had already formed (Fig. 1a), becoming expanded by P3. At P3, a part of the synovial membrane had begun to protrude into the upper articular cavity at the posterior portion forming a small synovial fold (Fig. 1c). Several cells existed in the surface of synovial membrane, but the lining cell layer was not discriminated from the sublining connective tissue (Fig. 1b,c). Immunohistochemistry revealed that intense Cav3 immunoreactions were localized in the sarcolemma of the skeletal muscles, but they were absent in the other cellular element in the Fig. 1. a: Cav3-immunoreactions in the rat temporomandibular joint (TMJ) at postnatal day 1 (P1). A frozen sagittal section counterstained with methylene blue. An arrow indicates the anterior direction. The upper and lower articular cavity has been formed. Intense Cav3 expressions are localized in the skeletal muscles (M). The cells in the temporal bone (T), the articular disk (D), and mandibular condyle (C) do not exhibit any Cav3 immunoreactivity. b,c: Higher magniﬁcation of the posterior portion of the upper articular cavity at P1 (b: boxed area in a) and P3 (c). The surface of the synovial membrane at these stages is composed of ﬂat and round cells, which are difﬁcult to distinguish from the connective sublining cells. c: The budding of a synovial fold is observed in the posterosuperior portion at P3. No synovial cell shows Cav3 immunoreaction. Asterisk indicates articular cavity. Scale bars 5 200 mm in a, 50 mm in b,c. TMJ (Fig. 1a). Throughout the developmental period of this study, the cellular elements in the temporal bone, articular disk, and mandibular condyle failed to exhibit any Cav3 immunoreaction. P5 to P7 The synovial fold in the posterosuperior portion had grown further at P5 to P7 (Fig. 2a–c). The superﬁcial lining cell layer was clearly distinguishable from the sublining layer of the synovial membrane. The synovial lining cells showed a tendency to be arranged with a single cell layer at the wall of synovial recess, while they formed two or three layers at the tip of the fold (Fig. 2c). Despite the increased immunointensity of Cav3 in the skeletal muscle at this stage, no Cav3-immunoreactive cells existed in the TMJ (Fig. 2a). Two types of typical synovial lining cells—the macrophage-like type A cell and ﬁbroblast-like type B cell—could be detected at P5 under the electron microscope (Fig. 2d). The ﬁbroblast-like type B cells at this stage had clear nuclei, several expanded rER, cytoplasmic processes, and a few caveolae without Cav3 immunoreaction (Fig. 2d,e). In addition, immunoelectron microscopic observation demonstrated Cav1-reactive products in these caveolae of the ﬁbroblast-like type B cells at P5 (Fig. 2f,g). Double immunolabeling at P5 showed that Hsp25- or Cav1-positive B 236 NIWANO ET AL. Fig. 2. a–g: Light (a–c) and immunoelectron (d–g) micrographs of synovial lining cells for Cav3 at postnatal day 5 (P5; (a,b,d,e) and P7 (c), and Cav1 at P5 (f,g). Photomicrographs of e and g are higher magniﬁcations of boxed areas in d, f, respectively. a: The synovial fold in the posterosuperior portion has grown further. The skeletal muscle (M) shows a higher intensity of immunoreactivity than the previous stage. b,c: The lining cell layer (arrowheads) is clearly distinguished from the connective sublining layer. No Cav3-immunopositive cell exists in the synovial fold at this stage as well. d,e: The synovial lining cells cover the surface of the synovial membrane with their cytoplas- mic projections (arrow). The macrophage-like type A cell (A) has a nucleus with rich heterochromatin, lysosomes, vacuoles, and ﬁlopodialike processes. The ﬁbroblast-like type B cells (B) at this stage have clear nuclei, several expanded rER, and several caveolae (e) without Cav3 immunoreaction. f,g: Immunoreactivities for Cav1 appear as electron-dense materials on the cell membrane. At P5, the ﬁbroblastlike type B cell possesses caveolae with Cav1 protein in their cell membrane (arrowheads in g). Asterisk indicates articular cavity. Scale bars 5 500 mm in a, 50 mm in b,c, 4 mm in d, 0.4 mm in e, 2 mm in f, 0.2 mm in g. synoviocytes, slender and round in shape, covered the synovial surface and lacked Cav3 immunoreaction (Fig. 5a,b). Numerous ﬁbroblasts and endothelial cells also exhibited Cav1 immunoreaction (Fig. 5b). conﬁned to the caveolae of certain lining cells; they possessed cytoplasmic processes (Fig. 3b), well-developed rER (Fig. 3c), and more abundant caveolae (Fig. 3d) than at the previous stage, all morphological features similar to the mature type B synoviocytes. P14 The synovial lining layer consisting of one- to threelayered lining cells became identical to that in adults as shown in our previous study at the light microscopic level (Nozawa-Inoue et al., 2006). Immunocytochemistry for Cav3 ﬁrst demonstrated a signiﬁcant Cav3 expression in the synovial lining layer of the synovial membrane at P14 (Fig. 3a,b). Cav3 immunoreactions were P21 Several synovial folds developed in the posterosuperior portion at P21 (Fig. 4a). The expression of Cav3 in the synovial lining cells had increased in quantity and immunointensity beyond those at P14 (Fig. 4a,b). Ultrastructurally, almost all type B synoviocytes forming characteristic thick and long cytoplasmic projections 237 DEVELOPMENT OF SYNOVIAL LINING CELLS Fig. 3. a–d: Light (a,b) and immunoelectron (c,d) micrographs of synovial lining cells for Cav3 at postnatal day 14 (P14). a: Cav3 expressions (arrowheads) are restricted to the surface of the synovial membrane. b: A plastic section counterstained with methylene blue. Immunoreactivities of Cav3 are localized in the cell membrane of certain lining cells (arrowheads) with cytoplasmic processes (arrows). c: expressed Cav3 immunoreactions in their caveolae. On the other hand, a few type B cells which had poor cell organelles—such as a few rER—lacked Cav3-immunoreactions (Fig. 4c,d). In contrast, the macrophage-like type A cells did not show any detectable Cav3-expression (Fig 4c). In a double immunostaining for Cav3 and either Hsp25 or Cav1, the type B synoviocytes with Hsp25 expressions formed a continuous covering on the synovial lining layer (Fig. 5c). Other either Hsp25- or Cav1positive cells such as endothelial cells sparsely existed in the sublining layer (Fig. 5c,d,f); however, Cav3 immunoreactions were restricted to the synovial lining cells (Fig. 5c,e,f). Most of the Hsp25- or Cav1-positive type B cells coexpressed Cav3 immunoreaction, whereas a few type B cells lacked this (Fig. 5c,f). The chronological expressions in immunoreactivity for Cav1 and Cav3 in type B synoviocytes during development of the rat TMJ are summarized in Table 1. Higher magniﬁcation of the boxed area in b. A Cav3-immunopositive type B cell (B) has developed rough endoplasmic reticulum (rER) and more abundant caveolae than at the previous stage. d: Cav3 immunoreactions are conﬁned to the caveolae. Asterisk indicates articular cavity. Scale bars 5 50 mm in a, 20 mm in b, 2 mm in c, 0.2 mm in d. DISCUSSION A series of ultrastructural surveys has agreed that the synovial lining cell layer consists of two kinds of synoviocytes—including macrophage-like type A and ﬁbroblastlike type B cells—which respectively function in the resorption and synthesis/secretion of synovial ﬂuids (for reviews, Iwanaga et al., 2000; Nozawa-Inoue et al., 2003). However, immunoelectron microscopy pointed out the possibility that type B synoviocytes in the TMJ could be further divided into some phenotypes by their immunocytochemical properties (Nozawa-Inoue et al., 1999). In addition, the lack of reliable and speciﬁc markers for each type of synoviocyte has caused a delay in clarifying the characteristic biological features of TMJ, whose phylogeny and ontogeny considerably differ from those in other systemic joints (Suzuki et al., 2005, 2006), despite several markers for synovial lining cells (Iwanaga et al., 238 NIWANO ET AL. Fig. 4. Light (a,b) and immunoelectron (c,d) micrographs of synovial lining cells for Cav3 at postnatal day 21 (P21). a: Several welldeveloped synovial folds are observed in the postero-superior portion. b: The expression of Cav3 has increased in number and the intensity more than those at the previous stage. c: An immunoelectron micrograph of the boxed area in b. The Cav3-immunoreaction pattern in most of the type B cells (B) appears as a dotted line on its entire cell membrane. The type B cells with Cav3 immunoreactions (B) possess well-developed rough endoplasmic reticulum (rER), thick and long cytoplasmic projection (arrows), and more caveolae than those at previous stages. A few type B cells (white B) without Cav3 immunoreactivity have poor cell organelles. A macrophage-like type A cell (A) does not show any immunoreaction. d: Higher magniﬁcation of the boxed area in c. The type B cell has Cav3-positive (arrowheads) and Cav3negative caveolae (arrows). Asterisk indicates articular cavity. Scale bars 5 50 mm in a, 20 mm in b, 4 mm in c, 0.5 mm in d. DEVELOPMENT OF SYNOVIAL LINING CELLS 239 Fig. 5. a,b: Merged ﬂuorescent images of the synovial membrane at postnatal day 5 (P5) stained with Cav3 (green) and heat shock protein 25 (Hsp25, red in a) or Cav1(red in b). a: The slender and round shaped type B cells with Hsp25 immunoreaction are arranged in the synovial lining layer. No Cav3 reactivity is detected. b: The ﬁbroblastlike type B cells, the endothelial cells, and sublining ﬁbroblasts express Cav1 immnoreactions, but none of them shows Cav3 immunoreactivities. c: Double labeling for Cav3 (green) and Hsp25 (red) in the synovial membrane at P21. Hsp25-immunopositive type B cells extend their cytoplasmic projections, covering the surface of the synovial membrane. Most of the type B cells with Hsp25 immunoreaction coexpress Cav3 (arrowheads). A few type B cells with Hsp25 immunoreaction lack Cav3 immunoreactivity (arrows). d–f: Double labeling for Cav1 (d), Cav3 (e), and merged image (f) obtained from same sections at P21. Immunoreactions for Cav1 and Cav3 colocalize along the cell membrane of the ﬁbroblast-like type B cells (arrowheads). A few Cav1-immunopositive lining cells (arrow) are devoid of Cav3 expression. Asterisk indicates articular cavity. Scale bars 5 20 mm. 2000). Our research group has reported intense immunoreactions of Hsp25 and Cav1 in the ﬁbroblast-like type B cells of the rat TMJ (Nozawa-Inoue et al., 1999, 2006). In particular, Hsp25 immunocytochemistry disclosed their detailed morphological conﬁgurations (Nozawa-Inoue et al., 1999) and developmental processes (Ikeda et al., 2004; Suzuki et al., 2005). However, these immunohistochemical markers have the disadvantage of recognizing the same antigens in other cellular components such as endothelial cells of the TMJ synovial membrane. The present study ﬁnding on the speciﬁc immunoreaction indicates that Cav3 is a useful speciﬁc marker for the mature type B cells, as shown in our recent study. The formation process of the TMJ, in the particular synovial lining cell layer and articular cavity, has been controversial. By the use of immunocytochemistry for Hsp25 at light and electron microscopic levels (Ikeda et al., 2004), the arrangement and morphological matu- 240 NIWANO ET AL. TABLE 1. Stage-speciﬁc expression pattern of Cav1- and Cav3-immunopositive type B synoviocytes during developmenta Hsp25 Cav1 Cav3 P3 P5 P7 P14 P21 111 2 2 111 1 2 111 1 2 111 111 6 111 111 11 2, no reactivity; 1/2, weak reactivity on a part of the type B cells (50–60%); 1, weak reactivity on the majority of the cells (80–90%); 11, strong reactivity on the majority of the cells; 111, all the type B cells with strong reactivity. P, postnatal day; Hsp25, heat shock protein 25. a ration of type B cells have been conﬁrmed to be closely related to the formation of the articular cavity. In one study, both immature and mature type B synoviocytes expressed Hsp25-immunoreaction, as predicted by a close functional relationship with actin dynamics and Hsp25 (Lavoie et al., 1993; Huot et al., 1996). In contrast, current observations revealed that Cav3-immunoreactive type B cells had morphological conﬁgurations of the mature cell, and there was a time lag between Hsp25 and Cav3 immunoexpression in type B cells as shown in Figure 5. This ﬁnding suggests that Cav3 expression is closely related to the differentiation stage of the synovial type B cells rather than the developmental stage of the synovial membrane. The functional signiﬁcance of Cav3 in the ﬁbroblast-like type B cells remains unclear, although caveolin proteins are necessary for the formation of caveolae. In the present study, it was at P14 when the ﬁbroblast-like type B synoviocytes came to exhibit Cav3 immunoreaction. Acquisition of Cav3 protein in the ﬁbroblast-like type B cells after forming caveolae with Cav1 immunoreaction indicates differing functions between Cav1 and Cav3 in the type B synoviocytes, suggesting that Cav3 plays further functions in addition to forming caveolae as speculated in the muscle cells (Kogo et al., 2006). The involvement of Cav3 has been reported in cell proliferation and differentiation because Cav3 was not expressed in proliferating myoblasts but increased with progress in myocyte differentiation (Galbiati et al., 1999), comparable with experimental data that indicated that Cav3 functions as a negative regulator of muscle cell proliferation (Ratajczak et al., 2005). This idea is acceptable with our present ﬁndings in which an exhibition of Cav3 immunoreactivity was recognizable in the synovial type B cells after completion of the proliferation of lining cells, when the synovial membrane showed a multiple epithelial-like arrangement. Previous reports have suggested that the formation of the synovial membrane and articular cavity in TMJ is closely related to the commencement of active jaw movement after birth (cf. Ikeda et al., 2004). The onset of Cav3 expression in the ﬁbroblastic type B cells (P14) is regarded as a stage during which mandibular movement becomes active and complicated (Shimizu et al., 1996). Indeed, occlusion was established between the rat ﬁrst molars at P14. At this stage, a drastic increase in type B cells serving to produce synovial ﬂuids, which make smooth jaw movement possible, has been reported in the rat TMJ (Tsuyama et al., 1995; Ikeda et al., 2004). Our investigations revealed that the mechanoreceptors increased drastically in number and showed their matu- ration at P15 to P24 in the periodontal ligament, which serves as a sensory apparatus of the masticatory system (Nakakura-Ohshima et al., 1993, 1995; Igarashi et al., 2007), suggesting the involvement of added functional stimuli such as occlusal force in the development and maturation of the masticatory apparatus. Taking these ﬁndings together, it is conceivable that the expression of Cav3 in the type B cells is closely related to jaw movement because TMJ might be inﬂuenced by mechanical stress due to occlusal loading. Further investigations are necessary to determine the functions of Cav3 in the ﬁbroblast-like type B cells using some experimental models or methods, such as cell culture, breeding animals under unphysiologic conditions, or arthritis models. 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