ARTICLE |
Correspondence to: Sumio Nishikawa, Dept. of Biology, Tsurumi U. School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan. E-mail: nishikawa-s@tsurumi-u.ac.jp
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Summary |
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CREB-binding protein (CBP) was examined in ameloblasts and in other enamel organ-derived cells of the rat incisor, using Western blotting analysis and immunocytochemistry by specific antibodies. Western blotting of labial tissues, including ameloblasts of the incisors, detected a single band with a molecular weight equivalent to the reported value of CBP. In immunocytochemistry, CBP was localized in ameloblast nuclei in the maturation zone but not in the secretion and transition zones. The nuclei of the other enamel organ-derived cells were also positive. Because this protein is suggested to take part in c-Jun-mediated transcription, the present study and the results of a previous report showing c-Jun localization in the nuclei of enamel organ-derived cells suggest that the enamel organ-derived cells, including maturation ameloblasts, undergo active transcriptional regulation. (J Histochem Cytochem 50:14551460, 2002)
Key Words: CBP, ameloblasts, enamel maturation, Western blotting, immunohistochemistry
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Introduction |
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IN THE MATURATION ZONE of amelogenesis, ameloblasts play roles in increase of mineral content in enamel as well as in matrix resorption (
It was shown in transfection experiments that influxes of calcium by opening of L-type calcium channels represent a pathway for c-Jun activation and that stimulation of c-Jun-mediated transcription requires CREB-binding protein (CBP) function (
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Materials and Methods |
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Sixteen male Wistar rats (78 weeks, 160260 g) (Jcl Wistar; Clea Japan, Tokyo, Japan; institutional guidelines were followed) were used for immunocytochemistry. The animals were perfused under sodium pentobarbital (Nembutal; Abbott, North Chicago, IL) anesthesia with 4% paraformaldehyde in 0.1 M phosphate buffer solution (PB), pH 7.2, via the left ventricle at room temperature (RT) for 10 min. They were immersed in the same fixative at 4C for 2 hr and the maxillary and mandibular incisors were dissected. After being washed with 0.1 M PB, the teeth were demineralized with 5% disodium EDTA solution adjusted to pH 7.3 with sodium hydroxide solution and were placed at 4C for 34 weeks. Demineralized incisors were cut transversely into two segments. Both segments were immersed in 25% sucrose in PBS overnight, rapidly frozen, and then cut longitudinally (68 µm thick) using a cryotome.
Two different antibodies against CBP were used in this study: a monoclonal antibody (MAb CBP(C-1) against the carboxy terminus of human CBP, and a polyclonal antibody (PAb) CBP(A-22) against the amino terminus of human CBP. The closely related protein p300 was also examined by a PAb p300(C-20). Because CBP is known to be a CREB-binding protein, CREB immunocytochemistry was performed with a PAb CREB-1(C-21) (Table 1). All were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal c-jun/AP-1(Ab-1) antibody was purchased from Oncogene Research Products (Cambridge, MA) (Table 1).
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Cryosections on glass microscopic slides were incubated for 20 min with 0.6% H2O2 in 80% methanol to inhibit endogenous peroxidase activity. These were labeled with the appropriate antibody, as described above, diluted with 1% bovine serum albumin in PBS (BSAPBS) at RT for 1 hr. They were then labeled with HRP-conjugated anti-rabbit or anti-mouse IgG (Cappel; West Chester, PA) diluted 1:100 with 1% BSA-PBS for 30 min. In addition, some specimens were labeled with FITC-conjugated anti-mouse IgG (Cappel). HRP-labeled sections were incubated with a solution composed of 0.5 mg/ml diaminobenzidine (DAB) and 2 mg/ml (NH4)2 Ni(SO4)2 6H2O in Tris-HCl (pH 7.6) for 10 min, then with the DAB solution containing 0.005% H2O2 for 212 min. Some FITC-labeled sections were further labeled with rhodaminephalloidin (Molecular Probes; Eugene, OR) to detect RA and SA (
For Western blotting analysis, maxillary and mandibular incisors were dissected from three adult male Wistar rats (710 months, 480580 g) under diethyl ether inhalation. The labial surface of the incisors containing ameloblasts and other enamel organ epithelia was scraped with a razor blade. Tissues including enamel organ epithelia were dissolved in a sample buffer composed of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 25% glycerin, 1% SDS, and 5% 2-mercaptoethanol, and then they were incubated at 100C for 5 min. They were subjected to SDS-PAGE using a 520% gradient slab gel and the procedure of
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Results |
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In Western blotting analysis using the anti-CBP (A-22) antibody and epithelial tissues of rat lower incisors, including ameloblasts, a single band was detected slightly above 250 kD on the membrane (Fig 1), which is equivalent to the reported electrophoretic value of CBP (265 kD) (
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In immunocytochemistry, CBP (C-1) was detected in the ameloblast nuclei of the maturation zone (Fig 2 Fig 3 Fig 4), but not of the secretion and transition zones (Fig 4b), although CBP (A-22) showed slight labeling in the secretion zone (Fig 4a) and the maturation zone. Because the overall labeling intensity of anti-CBP (C-1) was higher than that of anti-CBP (A-22), anti-CBP (C-1) was used for immunocytochemistry. In some specimens the labeling intensity was not uniform throughout the entire maturation zone. The intensity in the SA region tended to be stronger than in the adjacent RA region (Fig 2 and Fig 3). Comparison of two neighboring sections of the same tooth, labeled either with anti-CBP (C-1) or anti-c-Jun antibody, showed that the labeling pattern of either antibody was complementary to that of the other. SA had stronger CBP reaction and weaker c-Jun reaction, whereas RA had stronger c-Jun reaction and weaker CBP reaction (Fig 2a2d). Even in an RA zone, CBP labeling varied considerably in the ameloblast nuclei but exhibited no regular pattern. To confirm the preference of the CBP labeling in the SA zone, double labeling with anti-CBP (C-1) and rhodaminephalloidin (RhPh) was performed. The results showed that stronger CBP reactivity in the nuclei was related to ameloblasts with strong basal fluorescence of RhPh (Fig 3a and Fig 3b), which has been previously shown to be SA (
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Another transcriptional co-activator, p300, closely related to CBP, was examined immunocytochemically but no specific labeling was detected in the ameloblasts and the other enamel organ-derived cells. The prototypical target transcription factor CREB was not detected immunocytochemically in the ameloblasts and the other enamel organ-derived cells (data not shown). Because the antibody used also reacted to CREM and ATF-1, these two CREB family proteins were also below detectable limits in the ameloblasts. Control sections did not show any specific labeling.
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Discussion |
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In the maturation zone of amelogenesis, there is strong immunoreactivity of several AP-1 members (
CBP is a transcriptional co-activator that binds to CREB phosphorylated by protein kinase A (B, and nuclear receptors, leading a role of CBP as a more general co-activator than was previously expected (
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Acknowledgments |
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Supported in part by a grant-in-aid for scientific research (no. 11671824) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan to promote 2001-Multidisciplinary Research Projects in 20012005.
I thank Drs T. Tanabe and M. Fukae (Department of Biochemistry, Tsurumi University School of Dental Medicine) for Western blotting analysis.
Received for publication March 11, 2002; accepted May 29, 2002.
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