Title: The Receptor Activator of Nuclear Factor-κB Ligand-mediated Osteoclastogenic Pathway Is Elevated in Amelogenin-null Mice
Abstract: Amelogenins, major components of developing enamel, are predominantly involved in the formation of tooth enamel. Although amelogenins are also implicated in cementogenesis, their precise spatial expression pattern and molecular role are not clearly understood. Here, we report for the first time the expression of two alternate splice forms of amelogenins, M180 and the leucine-rich amelogenin peptide (LRAP), in the periodontal region of mouse tooth roots. Lack of M180 and LRAP mRNA expression correlated with cementum defects observed in the amelogenin-null mice. The cementum defects were characterized by an increased presence of multinucleated cells, osteoclasts, and cementicles. These defects were associated with an increased expression of the receptor activator of the nuclear factor-κB ligand (RANKL), a critical regulator of osteoclastogenesis. These findings indicate that the amelogenin splice variants, M180 and LRAP, are critical in preventing abnormal resorption of cementum. Amelogenins, major components of developing enamel, are predominantly involved in the formation of tooth enamel. Although amelogenins are also implicated in cementogenesis, their precise spatial expression pattern and molecular role are not clearly understood. Here, we report for the first time the expression of two alternate splice forms of amelogenins, M180 and the leucine-rich amelogenin peptide (LRAP), in the periodontal region of mouse tooth roots. Lack of M180 and LRAP mRNA expression correlated with cementum defects observed in the amelogenin-null mice. The cementum defects were characterized by an increased presence of multinucleated cells, osteoclasts, and cementicles. These defects were associated with an increased expression of the receptor activator of the nuclear factor-κB ligand (RANKL), a critical regulator of osteoclastogenesis. These findings indicate that the amelogenin splice variants, M180 and LRAP, are critical in preventing abnormal resorption of cementum. Ameloblasts synthesize and secrete amelogenins into the dental enamel matrix that undergo systematic proteolysis during enamel mineralization. Numerous mutations were found in the amelogenin coding sequences in patients with the most common genetic disorder affecting enamel, amelogenesis imperfecta (1Wright J.T. Butler W.T. J. Dent. Res. 1989; 68: 1328-1330Crossref PubMed Scopus (29) Google Scholar, 2Lagerstrom M. Dahl N. Nakahori Y. Nakagome Y. Backman B. Landegren U. Pettersson U. Genomics. 1991; 10: 971-975Crossref PubMed Scopus (230) Google Scholar, 3Salido E.C. Yen P.H. Koprivnikar K. Yu L.C. Shapiro L.J. Am. J. Hum. Genet. 1992; 50: 303-316PubMed Google Scholar, 4Wright J.T. Hall K.I. Yamauche M. Arch. Oral Biol. 1997; 42: 149-159Crossref PubMed Scopus (45) Google Scholar, 5Collier P.M. Sauk J.J. Rosenbloom S.J. Yuan Z.A. Gibson C.W. Arch. Oral Biol. 1997; 42: 235-242Crossref PubMed Scopus (122) Google Scholar). The targeted disruption of the amelogenin gene locus in mice also showed a hypoplastic enamel phenotype similar to amelogenesis imperfecta, confirming an important role of amelogenins in enamel formation (6Gibson C.W. Yuan Z.A. Hall B. Longenecker G. Chen E. Thyagarajan T. Sreenath T. Wright J.T. Decker S. Piddington R. Harrison G. Kulkarni A.B. J. Biol. Chem. 2001; 276: 31871-31875Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). In addition to their role in enamel formation, amelogenins are also believed to play a key role in the formation of root cementum, a mineralized layer on the surface of root dentin (7Diekwisch T.G. Int. J. Dev. Biol. 2001; 45: 695-706PubMed Google Scholar, 8Zeichner-David M. Matrix Biol. 2001; 20: 307-316Crossref PubMed Scopus (79) Google Scholar). During cementogenesis, Hertwig's epithelial root sheath dissociates to form cell aggregates (epithelial rests of Malassez) that are located between the alveolar bone and the tooth root. The mesenchyme-derived cementoblasts secrete cementum matrix onto the root surface to form cementum. The presence of amelogenins was detected on the tooth root surface close to the site of acellular cementum (9Hamamoto Y. Nakajima T. Ozawa H. Uchida T. Oral Surg. Oral. Med. Oral Pathol. Oral Radiol. Endod. 1996; 81: 703-709Abstract Full Text PDF PubMed Scopus (51) Google Scholar) and in the epithelial remnants of the root sheath in rat molars (10Fong C.D. Hammarstrom L. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2000; 90: 218-223Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), indicating their potential role during cementogenesis. Interestingly, amelogenins were also detected in Hertwig's epithelial root sheath cells and the epithelial rests of Malassez (11Slavkin H.C. J. Periodontol. 1976; 47: 249-255Crossref PubMed Scopus (103) Google Scholar, 12Slavkin H.C. Bringas Jr., P. Bessem C. Santos V. Nakamura M. Hsu M.Y. Snead M.L. Zeichner-David M. Fincham A.G. J. Periodontal. Res. 1989; 24: 28-40Crossref PubMed Scopus (120) Google Scholar, 13Slavkin H.C. Bessem C. Fincham A.G. Bringas Jr., P. Santos V. Snead M.L. Zeichner-David M. Biochim. Biophys. Acta. 1989; 991: 12-18Crossref PubMed Scopus (100) Google Scholar). Therapeutic application of an enamel matrix derivative (EMDOGAIN®, Biora AB, Malmö, Sweden) rich in amelogenins resulted in regeneration of cementum, the surrounding alveolar bone, and periodontal ligament (PDL) 1The abbreviations used are: PDL, periodontal ligament; LRAP, leucine-rich amelogenin peptide; CM, cementoblast; RANK, receptor activator of nuclear factor κB; RANKL, RANK ligand; PBS, phosphate-buffered saline; TRAP, tartrate-resistant acid phosphatase; RT, reverse transcription; OPG, osteoprotegerin; TRAF 6, tumor necrosis factor receptor-associated factor 6. in the experimental treatment of periodontitis (14Hammarstrom L. Heijl L. Gestrelius S. J. Clin. Periodontol. 1997; 24: 669-677Crossref PubMed Google Scholar, 15Gestrelius S. Andersson C. Johansson A.C. Persson E. Brodin A. Rydhag L. Hammarstrom L. J. Clin. Periodontol. 1997; 24: 678-684Crossref PubMed Google Scholar, 16Cardaropoli G. Leonhardt A.S. J. Periodontol. 2002; 73: 501-504Crossref PubMed Scopus (32) Google Scholar, 17Velasquez-Plata D. Scheyer E.T. Mellonig J.T. J. Periodontol. 2002; 73: 433-440Crossref PubMed Scopus (92) Google Scholar). However, it is not clear from these studies whether the amelogenin or non-amelogenin components of an enamel matrix derivative regulate regeneration of cementum and periodontal tissues. The present study was undertaken to investigate the expression of various alternate splice forms of amelogenins in tooth roots and correlate their expression with the cementum defects observed in amelogenin-null mice teeth. Herein, we report the expression of two mRNA splice forms of amelogenin, M180 and LRAP, in cementoblast (CM)/PDL cells of wild-type mice. Interestingly, a progressive deterioration of cementum was observed in the amelogenin-null mice, suggesting a possible function for the specific amelogenin splice forms. These defects were also associated with an increased receptor activator of nuclear factor-κB ligand (RANKL) expression near the cementum, suggesting that the amelogenins may play a key role in the maintenance of cementum through the RANKL/RANK-mediated osteoclastogenic pathway. Amelogenin-null Mice—Amelogenin-null mice were generated by gene targeting (6Gibson C.W. Yuan Z.A. Hall B. Longenecker G. Chen E. Thyagarajan T. Sreenath T. Wright J.T. Decker S. Piddington R. Harrison G. Kulkarni A.B. J. Biol. Chem. 2001; 276: 31871-31875Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar), housed in a pathogen-free animal facility, and fed a dough diet (Bio-Serv, Holton Industries Co., Frenchtown, NJ) and autoclaved water ad libitum. Standard National Institutes of Health guidelines were followed to monitor the health status of the mice and for housing and breeding practices. Preparation of Tissue Sections—Amelogenin-null mice and wild-type controls 1-day-, 6-month-, and 1-year-old were used for the present study. Eight mice in each group were anesthetized and perfused with 4% paraformaldehyde in 0.1 m phosphate-buffered saline (PBS), pH 7.4. After dissection, the skulls were fixed in 4% paraformaldehyde for 24 h, decalcified in 10% EDTA and 0.01 m PBS (pH 7.4) for 4–6 weeks at 4 °C, dehydrated in a graded series of ethanol, embedded in paraffin, and serially sectioned into coronal sections at 8-μm thickness. The sections were stained with hematoxylin and eosin using standard protocols. Frozen sections from EDTA-decalcified skulls of three mice in each group were stained for tartrate-resistant acid phosphatase (TRAP) activity with the leukocyte acid phosphatase kit (Sigma). Immunohistochemistry—Frozen sections from the wild-type and amelogenin-null skulls were immunostained for RANKL using goat polyclonal antibodies against mouse RANKL (R&D Systems, Minneapolis, MN) overnight at 4 °C at a dilution of 1:100. After washing in PBS, the sections were incubated with peroxidase-conjugated mouse antibodies against goat IgG (Vector Laboratories, Burlingame, CA). To visualize the immunoreactant, the sections were treated with diaminobenzidine substrate and counterstained with hematoxylin for light microscopy. Quantitative Micrograph Analysis—Light micrographs of the sections stained with hematoxylin and eosin were used for the histomorphometric measurements. Twenty slides, each with three sections of the first two mandibular molars from each mouse, were used for counting defects in cementum and dentin (e.g. multinuclear cells and cementicle numbers). Each number on the histogram represents the mean ± S.D. of observations from either the first or second mandibular molar from a total of four mice. Scanning Electron Microscopy Analysis of Molar Teeth—Molars from the wild-type and amelogenin-null mice were photographed using scanning electron microscopy at 20 kV (Jeol JSM T330A, Jeol, Inc., Peabody, MA). RNA Isolation and Gene Expression Analysis by RT-PCR—CM/PDL cell populations were established from cells lining the tooth root surface of 6-month-old wild-type and amelogenin-null mice. Mandibular first molars were extracted by dissecting the molars with adherent PDL from surrounding alveolar bone. The CM/PDL cells were isolated from the surface of the mandibular molars as described (18D'Errico J.A. MacNeil R.L. Takata T. Berry J. Strayhorn C. Somerman M.J. Bone. 1997; 20: 117-126Crossref PubMed Scopus (157) Google Scholar, 19D'Errico J.A. Ouyang H. Berry J.E. MacNeil R.L. Strayhorn C. Imperiale M.J. Harris N.L. Goldberg H. Somerman M.J. Bone. 1999; 25: 39-47Crossref PubMed Scopus (112) Google Scholar). Briefly, molars were placed in a 1.5-ml centrifuge tube containing PBS with 1 mg/ml collagenase (Worthington, Lakewood, NJ) and 0.25% trypsin-EDTA (Invitrogen) and incubated for 2 h at 37 °C. Mandibular first molar tooth germs from 1 day-old wild-type and amelogenin-null mice were dissected from surrounding tissues. Total RNA was isolated using the RNA isolation kit (Stratagene) and treated with DNase. The RNA samples (1 μg each) were subjected to first strand cDNA synthesis using the SuperScript™ first strand synthesis system for the RT-PCR kit (Invitrogen). RT-PCR was performed using gene-specific primers as described in Table I. All PCR reactions were carried out in a PerkinElmer gene PCR system 600. PCR products were cloned into a T-vector (Promega, Madison, WI), and the nucleotide sequences were determined by cycle sequencing.Table IPrimers used for RT-PCR analysisSpecificityOligonucleotide sequence (5′-3′)ResiduesGenBank™ accession no.Product size (bp)Annealing temperatureCycle no.AmelogeninForwardAATGGGGACCTGGATTTTGTTTG59-81D31768—aProduct sizes of major bands are 601, 576, 496, 256, and 221 bp.64°C30ReverseTCCCGCTTGGTCTTGTCTGTCGCT614-637EnamelinForwardCATCCTTATTCCACTACATCCCCTG3115-3139NM0174685445826ReverseGGCGTGTTTTTTGGCTGAGAAG3638-3659RANKLForwardGGTCGGGCAATTCTGAATT957-976AF0537138125830ReverseGGGAATTACAAAGTGCACCAG1749-1769OPGForwardACCTCACCACAGAGCAGCTT1159-1178AB138982696026ReverseGTGCAGGAACCTCATGGTCT1409-1428TRAF 6ForwardGAGGAGATCCAGGGCTACGA410-429NM0094242925830ReverseATGTACTTGATGATCCTCGA702-721GAPDHbGAPDH, glyceraldehyde-3-phosphate dehydrogenase.ForwardCCATCACCATCTTCCAGGAG258-276XM1935323236026ReverseGCATGGACTGTGGTCATGAG562-581a Product sizes of major bands are 601, 576, 496, 256, and 221 bp.b GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Open table in a new tab Amelogenins Are Expressed in CM/PDL Cells of Wild-type Mice—RT-PCR was performed using amelogenin exon 2- and exon 6-specific primers to identify the expression of mRNAs resulting from alternate splicing in the CM/PDL cells. Amelogenin transcripts were detected in the periodontal tissue (CM/PDL cells) from 6-month-old wild-type mice and tooth bud from 1-day-old wild-type mice (Fig. 1A). Six different amplified products of amelogenin mRNA were detected in tooth bud, whereas only two products were present in CM/PDL cells. All PCR products were cloned, and their nucleotide sequences were determined. The two amplified products (576 and 221 bp) that were detected in the CM/PDL cells contained exons 2, 3, 5, and 6 and exons 2, 3, 5, and partial 6, respectively (Fig 1B). These sequences were in agreement with the reported amelogenin splice forms (20Lau E.C. Simmer J.P. Bringas Jr., P. Hsu D.D. Hu C.C. Zeichner-David M. Thiemann F. Snead M.L. Slavkin H.C. Fincham A.G. Biochem. Biophys. Res. Commun. 1992; 188: 1253-1260Crossref PubMed Scopus (105) Google Scholar, 21Simmer J.P. Hu C.C. Lau E.C. Sarte P. Slavkin H.C. Fincham A.G. Calcif. Tissue Int. 1994; 55: 302-310Crossref PubMed Scopus (85) Google Scholar, 22Gibson C.W. Golub E. Ding W.D. Shimokawa H. Young M. Termine J. Rosenbloom J. Biochem. Biophys. Res. Commun. 1991; 174: 1306-1312Crossref PubMed Scopus (92) Google Scholar). The amelogenins derived from these spliced forms were identified previously as M180 and M59 (LRAP) in the ameloblasts. Enamelin, one of the enamel matrix proteins expressed in ameloblasts, was expressed in wild-type and the null tooth buds but not in the CM/PDL cell populations. Increased Cementum and Dentin Defects in Amelogenin-null Mice—The wild-type mice did not show any significant difference in the cementum thickness or abnormalities in the pulp and surrounding bone regions (Fig. 2A). However, the cementum of the null mice displayed resorptive lacunae at sites where periodontal ligaments attach to the cementum surface (Fig. 2, B and D). Multiple intrusive attachments of PDL extended through the cementum into the root dentin of the amelogenin-null mice. Furthermore, we examined the surface of the root cementum of the amelogenin-null mice by scanning electron microscopy. The molar root surface of the wild-type mice appeared smooth and without any gross indentations (Fig. 2E). However, the amelogenin-null mice displayed distinct indentations on the surface of the cementum (Fig. 2F). Unlike the wild-type tooth roots (Fig. 2G), the fractured sections of the amelogenin-null mouse tooth roots showed resorptive lacunae extending deep into the cementum (Fig. 2H). We further quantitated the cementum and dentin defects in the null mice (Fig. 3B) and compared them with the wild-type mice (Fig. 3A). As described under “Experimental Procedures,” 20 sagittal (serial) sections from three wild-type and three amelogenin-null mice, each at 6 months and 1 year of age, were stained with hematoxylin and eosin and counted for all resorptive lacunae. The first and second molars both showed a pattern of progressive increase in cementum defects (Fig. 3C). The amelogenin-null mice displayed four times more cementum defects at both 6 months and 1 year of age as compared with wild-type mice. Similarly, in the first and second molars, 13 times more root dentin defects were observed at both 6 months and 1 year of age as compared with wild-type mice (Fig. 3D). Increased Presence of Multinucleated Cells in Amelogenin-null Mouse Teeth—As compared with the wild-type mouse tooth roots (Fig. 4C), many multinucleated cells were observed in the cementum and dentin regions of the null tooth roots (Fig. 4D). The amelogenin-null mice displayed a 2-fold increase in number of the multinucleated cells as compared with wild-type mice at both 6 months and 1 year of age (Fig. 4E). Although the null mice had significantly more multinucleated cells than the wild-type mice, they did not show any progressive increase in number with age. Interestingly, these cells were stained positive for TRAP activity, indicative of their osteoclastic/odontoclastic nature (Fig. 4B), whereas wild-type mice did not display similar TRAP activity (Fig. 4A). Increased Osteoclastogenesis Near the Roots of Amelogenin-null Mouse Teeth—To correlate tooth root defects with the osteoclastogenic deregulation in CM/PDL cells of amelogenin-null mice, we examined the expression of RANKL, osteoprotegerin (OPG), and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF 6) by RT-PCR and immunohistochemical analysis. RT-PCR analysis revealed that RANKL and TRAF 6 mRNA levels were significantly elevated in CM/PDL cells of the null mice (Fig. 5A). However, the expression of OPG, an orphan receptor for RANKL, was not altered. The distribution of RANKL in the periodontal tissue of amelogenin-null mice was also examined by immunohistochemical analysis. RANKL immunoreactivity was not detected in the PDL cells of wild-type teeth (Fig. 5B). In contrast, the amelogenin-null PDL cells showed more intense staining for RANKL near the bone and cementum surface (Fig. 5C). These observations suggest that the abnormal localization of osteoclasts close to the tooth root correlates with the accelerated resorption of cementum in the amelogenin-null mice. Increased Cementicles at the Periodontal Ligament Space in Amelogenin-null Mice—The progressive occurrence of cementicles adhering to the cementum surface in the amelogenin-null mice indicates a defect in periodontal tissue and possibly a “hypercementosis-like” condition (Fig. 6B). The amelogenin-null mice displayed 2–4 times more cementicles near the molar tooth root at 6 and 12 months of age (Fig. 6C). The presence of cementicles is well documented in the periodontal spaces in pathological conditions as well as in aging humans. The increased presence of these cementicles confirms abnormal cementum in amelogenin-null mice. Amelogenins, highly conserved proteins that constitute 90% of the enamel organic matrix, are produced by ameloblasts shortly before tooth eruption. Numerous experimental approaches have indicated that amelogenins play an important role in amelogenesis (6Gibson C.W. Yuan Z.A. Hall B. Longenecker G. Chen E. Thyagarajan T. Sreenath T. Wright J.T. Decker S. Piddington R. Harrison G. Kulkarni A.B. J. Biol. Chem. 2001; 276: 31871-31875Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 23Diekwisch T. David S. Bringas Jr., P. Santos V. Slavkin H.C. Development. 1993; 117: 471-482PubMed Google Scholar, 24Lyngstadaas S.P. Risnes S. Sproat B.S. Thrane P.S. Prydz H.P. EMBO J. 1995; 14: 5224-5229Crossref PubMed Scopus (125) Google Scholar). Although specific amelogenin splice products have been implicated in tissue-specific epithelial-mesenchymal signaling during tooth development (20Lau E.C. Simmer J.P. Bringas Jr., P. Hsu D.D. Hu C.C. Zeichner-David M. Thiemann F. Snead M.L. Slavkin H.C. Fincham A.G. Biochem. Biophys. Res. Commun. 1992; 188: 1253-1260Crossref PubMed Scopus (105) Google Scholar, 21Simmer J.P. Hu C.C. Lau E.C. Sarte P. Slavkin H.C. Fincham A.G. Calcif. Tissue Int. 1994; 55: 302-310Crossref PubMed Scopus (85) Google Scholar, 22Gibson C.W. Golub E. Ding W.D. Shimokawa H. Young M. Termine J. Rosenbloom J. Biochem. Biophys. Res. Commun. 1991; 174: 1306-1312Crossref PubMed Scopus (92) Google Scholar, 25Veis A. Tompkins K. Alvares K. Wei K. Wang L. Wang X.S. Brownell A.G. Jengh S.M. Healy K.E. J. Biol. Chem. 2000; 275: 41263-41272Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), the distribution of specific splice forms and the precise functions associated with the individual peptides are still unclear. The implied but undefined role of amelogenins in cementogenesis formed the basis of the present study. During tooth development, at least nine different mRNA splice forms are generated from the amelogenin gene as a result of alternate splicing (26Veis A. Cell. Mol. Life Sci. 2003; 60: 38-55Crossref PubMed Scopus (138) Google Scholar). Interestingly, the presence of amelogenins in the tooth root region has been detected only by immunohistochemical and in situ hybridization studies (9Hamamoto Y. Nakajima T. Ozawa H. Uchida T. Oral Surg. Oral. Med. Oral Pathol. Oral Radiol. Endod. 1996; 81: 703-709Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 10Fong C.D. Hammarstrom L. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2000; 90: 218-223Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 27Fukae M. Tanabe T. Yamakoshi Y. Yamada M. Ujiie Y. Oida S. J. Bone Miner. Metab. 2001; 19: 236-243Crossref PubMed Scopus (18) Google Scholar). However, these studies could not identify the presence of individual alternate splice forms of mRNA or their translational products. Unlike unerupted molar teeth, the CM/PDL cells from the adult wild-type mice displayed expression of two specific mRNA splice forms (M180 and LRAP) of amelogenin. Expression of M180 and LRAP in CM/PDL indicate their potential role in cementum formation and perhaps in cementum maintenance. A detailed analysis of the amelogenin-null mice revealed normal cementogenesis but poor maintenance of the cementum, as observed by the increased presence of tooth root resorption. Dentin, cementum, and enamel of permanent teeth normally do not undergo resorption (28Gunraj M.N. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 1999; 88: 647-653Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Under certain clinical conditions such as chronic inflammation of the pulp and pulp necrosis (29Borum M.K. Andreasen J.O. Endod. Dent. Traumatol. 1998; 14: 31-44Crossref PubMed Google Scholar) and physical trauma during orthodontic tooth movement (30Brezniak N. Wasserstein A. Angle Orthod. 2002; 72: 175-179PubMed Google Scholar, 31Leach H.A. Ireland A.J. Whaites E.J. Br. Dent. J. 2001; 190: 16-22Crossref PubMed Scopus (65) Google Scholar), the permanent teeth undergo resorption. The tooth resorption seen in the amelogenin-null mice could be explained as a result of physical trauma due to enamel hypoplasia and attrition of molar cusps, similar to orthodontics movements. 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Recent reports showed calcified bodies known as psammoma-like ossicles and cementicles in the osteoblastoma and juvenile ossifying fibroma of the craniofacial skeleton (44El-Mofty S. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2002; 93: 296-304Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 45Yagishita H. Taya Y. Kanri Y. Matsuo A. Nonaka H. Fujita H. Aoba T. J. Oral Pathol. Med. 2001; 30: 499-503Crossref PubMed Scopus (19) Google Scholar). In rare pathological conditions, a large number of cementicles may fuse together to give rise to an odontogenic tumor (45Yagishita H. Taya Y. Kanri Y. Matsuo A. Nonaka H. Fujita H. Aoba T. J. Oral Pathol. Med. 2001; 30: 499-503Crossref PubMed Scopus (19) Google Scholar, 46Althoff J. Koch W. Reichart P. J. Oral Pathol. 1986; 15: 11-15Crossref PubMed Scopus (2) Google Scholar). These cementicles were also observed in human aging. 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It appears from these experiments that amelogenins may be essential in regulating the critical balance between osteoblastic and osteoclastic activity in bone remodeling. The expression of only M180 and LRAP in the periodontal region further supports the hypothesis that, in addition to enamel mineralization, amelogenins may have other functions. Increased RANKL pathway expression in the absence of amelogenins in the periodontal region indicates that amelogenins play a key role in the regulation of the osteoclastogenic pathway. Unlike clinical orthodontic movements, the resorptive phenomenon observed in the amelogenin-null tooth roots is more preferential toward cementum than alveolar bone. The restricted expression of amelogenins in the periodontal region between the alveolar bone and the cementum by the epithelial rests of Malassez indicate that amelogenins may prevent abnormal resorption of cementum. We thank Drs. Mary Jo Danton, Hynda Kleinman, Pamela Robey, Yoshihiko Yamada, and Marian Young for critical reading of the manuscript and Drs. Mirei Chiba, Satoshi Fukumoto, Kenn Holmbeck, Yoko Iwamatsu, Yasuyuki Sasano, Haruhiko Takada, and Ichiro Takahashi for helpful discussions.