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Immunocytochemical Localization of Caveolin-3 in the Synoviocytes of the Rat Temporomandibular Joint During Development.

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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: nozawa@dent.niigata-u.ac.jp
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.
ACKNOWLEDGMENTS
The authors thank Mr. Masaaki Hoshino and Mr. Kiichi Takeuchi, Division of Oral Anatomy, Department of
Oral Biological Science, Niigata University Graduate
School of Medical and Dental Sciences, for their technical assistance.
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development, synoviocytes, temporomandibular, caveolin, joint, localization, rat, immunocytochemical
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