Using in vitrotooth germ cultures and analysis by confocal microscopy, ameloblasts treated with sodium fluoride were found to have elevated amounts of filamentous actin. Because this response is reduced by inhibitors of the Rho/ROCK signaling pathway, we generated mice that express dominant negative RhoA (RhoADN) in ameloblasts for in vivo analysis. Expression of the EGFP-RhoADN fusion protein was evaluated by RT-PCR and immunohistochemistry, and teeth were analyzed by scanning electron microscopy. The 3 strains expressed at either low (TgEGFP-RhoADN-8), intermediate (TgEGFP-RhoADN-2), or high (TgEGFP-RhoADN-13) levels, and the molar teeth from the 3 strains had enamel hypoplasia and surface defects. We conclude that RhoADN expressed in ameloblasts interferes with normal enamel development through the pathway that is induced by sodium fluoride.

Keywords:
Enamel, Transgenic mice, RhoA, Ameloblasts
1.
Biz, M.T., R. Marques, V.O. Crema, A.S. Moriscot, M.F. dos Santos (2010) GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts. Cell Tissue Res 340: 459–470.
2.
Chalfie, M. (1995) Green fluorescent protein. Photochem Photobiol 62: 651–658.
3.
Chen, E., Z.A. Yuan, J.T. Wright, S.P. Hong, Y. Li, P.M. Collier, B. Hall, M. D’Angelo, S. Decker, R. Piddington, W.R. Abrams, A.B. Kulkarni, C.W. Gibson (2003) The small bovine amelogenin LRAP fails to rescue the amelogenin null phenotype. Calcif Tissue Int 73: 487–495.
4.
Davies, S.P., H. Reddy, M. Caivano, P. Cohen (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105.
5.
Gibson, C.W., Z.A. Yuan, Y. Li, B. Daly, C. Suggs, M.A. Aragon, F. Alawi, A.B. Kulkarni, J.T. Wright (2007) Transgenic mice that express normal and mutated amelogenins. J Dent Res 86: 331–335.
6.
Hatakeyama, J., S. Fukumoto, T. Nakamura, N. Haruyama, S. Suzuki, Y. Hatakeyama, L. Shum, C.W. Gibson, Y. Yamada, A.B. Kulkarni (2009) Synergistic roles of amelogenin and ameloblastin. J Dent Res 88: 318–322.
7.
Li, Y., S. Decker, Z.A. Yuan, P.K. DenBesten, M.A. Aragon, K. Jordan-Sciutto, W.R. Abrams, J Huh, C. McDonald, E. Chen, M. MacDougall, C.W. Gibson (2005) Effects of sodium fluoride on the actin cytoskeleton of murine ameloblasts. Arch Oral Biol 50: 681–688.
8.
Maekawa, M., T. Ishizaki, S. Boku, N. Watanabe, A. Fujita, A. Iwamatsu, et al. (1999) Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285: 895–898.
9.
Nishikawa, S., K. Fujiwara, H. Kitamura (1988) Formation of the tooth enamel rod pattern and the cytoskeletal organization in secretory ameloblasts of the rat incisor. Eur J Cell Biol 47: 222–232.
10.
Nishikawa, S., K. Josephsen (1987) Cyclic localization of actin and its relationship to junctional complexes in maturation ameloblasts of the rat incisor. Anat Rec 219: 21–31.
11.
Nishikawa, S., H. Kitamura (1986) Localization of actin during differentiation of the ameloblast, its related epithelial cells and odontoblasts in the rat incisor using NBD-phallacidin. Differentiation 30: 237–243.
12.
Nishikawa, S., S. Tsukita, S. Tsukita, S. Sasa (1990) Localization of adherens junction proteins along the possible sliding interface between secretory ameloblasts of the rat incisor. Cell Struct Funct 15: 245–249.
13.
Pugach, M.K., Y. Li, C. Suggs, J.T. Wright, M.A. Aragon, Z.A. Yuan, D. Simmons, A.B. Kulkarni, C.W. Gibson (2010) The amelogenin C-terminus is required for enamel development. J Dent Res 89: 165–169.
14.
Qiu, R.G., J. Chen, F. McCormick, M. Symons (1995) A role for Rho in Ras transformation. Proc Natl Acad Sci USA 92: 11781–11785.
15.
Rasband, W.S. (1997–2009) ImageJ, US National Institutes of Health. http://rsb.info.nih.gov/ij/.
16.
Ridley, A.J., A. Hall (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389–399.
17.
Robinson, C., S. Connell, J. Kirkham, S.J. Brookes, R.C. Shore, A.M. Smith (2004) The effect of fluoride on the developing tooth. Caries Res 38: 268–276.
18.
Smith, C.E. (1979) Ameloblasts: secretory and resorptive functions. J Dent Res 58 (Spec Issue B): 695–707.
19.
Smith, C.E., A. Nanci (1995) Overview of morphological changes in enamel organ cells associated with major events in amelogenesis. Int J Dev Biol 39: 153–161.
20.
Smith, C.E., A. Nanci, P. DenBesten (1993) Effects of chronic fluoride exposure on morphometric parameters defining the stages of amelogenesis and ameloblast modulation in rat incisors. Anat Rec 237: 243–258.
21.
Vincent, S., J. Settleman (1999) Inhibition of RhoGAP activity is sufficient for the induction of Rho-mediated actin reorganization. Eur J Cell Biol 78: 539–548.
22.
Yuan, Z.A., P.M. Collier, J. Rosenbloom, C.W. Gibson (1996) Analysis of amelogenin mRNA during bovine tooth development. Arch Oral Biol 41: 205–213.
© 2011 S. Karger AG, Basel
2011
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.