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Immunolocalization of Caveolin-1 and Caveolin-3 in Monkey Skeletal, Cardiac and Uterine Smooth Muscles

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Immunolocalization of Caveolin-1 and Caveolin-3 in Monkey Skeletal, Cardiac and Uterine Smooth Muscles CELL STRUCTURE AND FUNCTION 27: 375–382 (2002) © 2002 by Japan Society for Cell Biology Immunolocalization of Caveolin-1 and Caveolin-3 in Monkey Skeletal, Cardiac and Uterine Smooth Muscles ∗ Yasuko Hagiwara 1 , Yasushi Nishina2, Hiroshi Yorifuji2, and Tateki Kikuchi1 1Department of Animal Models for Human Disease, National Institute of Neuroscience, National Center of Neu- rology and Psychiatry, Kodaira, Tokyo 187-8502, Japan, and 2Department of Anatomy II, National Defence Medical College, Tokorozawa, Saitama 359-8513, Japan ABSTRACT. Caveolin, a 20–24 kDa integral membrane protein, is a principal component of caveolar domains. Caveolin-1 is expressed predominantly in endothelial cells, fibroblasts, and adipocytes, while the expression of caveolin-3 is confined to muscle cells. However, their localization in various muscles has not been well documented. Using double-immunofluorescence labeling and confocal laser microscopy, we examined the localization of caveolins-1 and 3 in adult monkey skeletal, cardiac and uterine smooth muscles and the co-immunolocalization of these caveolins with dystrophin, which is a product of the Duchenne muscular dystrophy gene. In the skeletal muscle tissue, caveolin-3 was localized along the sarcolemma except for the transverse tubules, and co- immunolocalized with dystrophin, whereas caveolin-1 was absent except in the blood vessels of the muscle tissue. In cardiac muscle cells, caveolins-1 and -3 and dystrophin were co-immunolocalized on the sarcolemma and transverse tubules. In uterine smooth muscle cells, caveolin-1, but not caveolin-3, was co-immunolocalized with dystrophin on the sarcolemma. Key words: caveolin/skeletal muscle/cardiac muscle/smooth muscle/dystrophin Caveolin is a major component of the caveolae which are caveolin-1 or -3. Recently, caveolin-3 was detected in tissue small invaginations of the plasma membrane. Three distinct homogenate of the small intestine but not of the uterus in mammalian caveolins have been identified to date: caveo- mice (Li et al., 2001). lin-1, -2, and -3 (Way and Parton, 1995; Scherer et al., Caveolae are abundant in endothelial cells, adipocytes 1996; Tang et al., 1996). Caveolins-1 and 3 are closely re- and muscle cells and have been implicated in a variety of lated based on primary sequence homology, showing 65% cellular functions, including endothelial transcytosis, calci- identity and 85% similarity (Tang et al., 1996). Caveolins-1 um uptake and signal transduction (Fujimoto, 1993; Lisanti and 2 are co-expressed in many cell types, such as et al., 1994; Parton, 1996; Couet et al., 1997; Anderson, endothelial cells, fibroblasts and adipocytes, where they 1998; Kurzchlia and Parton, 1999). In skeletal muscle cells, form hetero-oligomers (Scherer et al., 1996). In contrast, caveolae are often associated with the opening of transverse the expression of caveolin-3 is muscle-specific (Tang et al., (T-) tubules and have been implicated in their formation. 1996). In smooth muscle cells of the taenia coli, caveolin Based on studies of cultured chicken skeletal muscle cells has been reported to be localized on the sarcolemma (North (Ezerman and Ishikawa, 1967; Ishikawa, 1968) and devel- et al., 1993). However, it has not been well documented oping mouse cardiac muscle cells (Ishikawa and Yamada, whether the caveolin expressed in smooth muscle cells is 1975), caveolae are proposed to play a role in the formation of the T-tubule system during muscle development. The T- *To whom correspondence should be addressed: Department of Animal tubules appear first as caveolae that form tubules through Models for Human Disease, National Institute of Neuroscience, National caveolar proliferation and into interconnected arrays of Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi-cho, Kodaira- three-dimensional networks. These previous observations shi, Tokyo 187-8502, Japan. Tel: +81–42–341–2711, Fax: +81–42–346–1746 are supported by a recent study that caveolin-3 is associated E-mail: [email protected] with T-tubules at early stages of development, while caveo- Abbreviations: T-tubules, transverse tubules; DAP, dystrophin-associat- lin-3 is no longer observed on the T-tubules in adult skeletal ed proteins; PBS, phosphate-buffered saline; FITC, fluorescein isothio- cyanate; TRITC, tetra-methyl-rhodamine isothiocyanate. muscles (Parton et al., 1997). However, in caveolin-3 375 Y. Hagiwara et al. knockout mice, T-tubules can be formed, although some for C-terminus was purchased from Novocastra Laboratories abnormalities in the T-tubule have been reported (Galbiati et (Newcastle, UK). al., 2001). After caveolin-3 was first characterized, Song et al. Immunoblot analysis (1996) biochemically observed the presence of dystrophin Immunoblot analysis was performed as described previously in a caveolin-3-rich fraction. Dystrophin is a protein associ- (Hagiwara et al., 1995). Briefly, cryostat sections of the muscle ated with actin filaments, which form the membrane cyto- tissue were suspended with 10 volumes of 70 mM Tris-HCl (pH skeletal network, and with β-dystroglycan (Hoffman et al., 6.7), 10% SDS, 10 mM ethylene-diamine tetraacetic acid and 5% 1987; Suzuki et al., 1992). Subsequently, McNally et al. β-mercaptoethanol. The suspensions were boiled for 5 min, and (1998) reported that caveolin-3 is present in a preparation of then centrifuged at 15,000 rpm for 15 min at 4°C. The supernatants the complex of dystrophin and dystrophin-associated pro- were subjected to 14% polyacrylamide gel electrophoresis in the teins (DAP). These authors considered caveolin-3 to be a presence of 1% SDS according to the method of Laemmli (1970). new member of the DAP family. Furthermore, transgenic After electrophoresis, the proteins were electroblotted onto an overexpression of caveolin-3 in skeletal muscle cells caused immobilon-P membrane (Millipore, Bedford, MA, USA) according downregulation of dystrophin (Garbiati et al., 2000). How- to the method of Kyhse-Anderson (1984). The membrane was in- ever, Crosbie et al. (1998) observed that the amount of cubated in a blocking solution and then allowed to react with the caveolin-3 recovered decreases with the progress of purifi- primary antibody at 37°C for 1 h. Horse radish peroxidase-labeled cation of the dystrophin-glycoprotein complex and that they IgG, a secondary antibody, was applied to the membrane at room did not consider caveolin-3 as a DAP. temperature for 30 min. Signals were detected using an ECL detec- Mutations of the caveolin-3 gene have been reported to tion kit (Amersham Pharmacia Biotech, Little Chalfont, UK). be involved in a type of limb-girdle muscular dystrophy inherited in an autosomal dominant manner (Minetti et al., Immunohistochemistry 1998). However, because some apparent mutations of µ caveolin-3 gene have turned out to be harmless, the problem Cryosections of 6 m thickness were analyzed by immunofluo- is raised whether deficiency of caveolin-3 causes dystrophic rescence staining using specific antibodies as described pre- phenotypes or not. Recently, we developed caveolin-3-null viously (Hagiwara et al., 1998). Briefly, the sections were dried mice that have a mild muscular dystrophic phenotype, al- and pretreated with chilled acetone for 10 min. After incubation in a blocking solution containing 0.1% casein/0.1% gelatin in though the disease is inherited in an autosomal recessive ° manner (Hagiwara et al., 2000). This was followed by a phosphate-buffered saline (PBS, pH 7.4) for 1 h at 4 C, the sections were incubated overnight with the primary antibodies at similar report (Galbiati et al., 2001). These findings clearly ° show that loss of caveolin-3 causes dystrophic changes. 4 C. The bound antibodies were detected with fluorescein To better understand the roles of caveolins in muscle isothiocyanate (FITC)-conjugated goat F (ab’) anti-rabbit IgG (G+L) cells, we compared the localization of caveolins-1 and 3 in (TAGO Inc., Burlingame, CA, USA) or tetra-methyl-rhodamine skeletal, cardiac and smooth muscle and the cellular colo- isothiocyanate (TRITC)-conjugated mouse IgG (H+L) (KPL, Inc., calization of the caveolins and dystrophin by immuno- Gaithersburg, MD, USA) as the secondary antibodies. Confocal histochemistry. laser scanning microscopy was performed using an Axioplan 2 microscope on an LSM 510 system (Carl Zeiss, Oberkochen, Germany) and M system (Leica, Heidelberg, Germany). Materials and Methods Tissues Results Limb skeletal, heart ventricular and uterine muscles were obtained Homogenates of limb skeletal, heart ventricular and uterine from adult monkeys (Macaca fascicularis) and immediately frozen muscle from a monkey were examined by immunoblot anal- in isopentane cooled by liquid nitrogen and stored in deep-freezer ysis using antibodies against caveolins-1 and 3. As shown in at –80°C until use. These tissues were provided from the National Figure 1, caveolin-1 was detected in all of the tissues exam- Institute for Infectious Diseases, Japan. ined (Fig. 1A), whereas caveolin-3 was detected in skeletal and heart muscle but not in uterine muscle (Fig. 1B). Antibodies The skeletal muscle was examined immunohistochemi- cally using anti-caveolin-1 and -3 antibodies. In the longitu- Rabbit polyclonal and mouse monoclonal antibodies against a dinal and transverse sections of the limb skeletal muscle, the sequence of rat caveolin-3, TEEHTDLEARIIKDIHCKEIDL (aa. sarcolemma but not the T-tubules was stained by the caveo- 3–24), and against an 11.1-kDa protein fragment corresponding to lin-3 antibody (Figs.
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