Enamelysin (Matrix Metalloproteinase 20)-deficient Mice Display an Amelogenesis Imperfecta Phenotype
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m209100200
ISSN1083-351X
AutoresJohn J. Caterina, Ziedonis Skobe, Joanne Shi, Yanli Ding, James P. Simmer, Henning Birkedal‐Hansen, John D. Bartlett,
Tópico(s)Oral microbiology and periodontitis research
ResumoEnamelysin is a tooth-specific matrix metalloproteinase that is expressed during the early through middle stages of enamel development. The enamel matrix proteins amelogenin, ameloblastin, and enamelin are also expressed during this same approximate developmental time period, suggesting that enamelysin may play a role in their hydrolysis. In support of this interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant amelogenin at virtually all of the precise sites known to occur in vivo. Thus, enamelysin is likely an important amelogenin-processing enzyme. To characterize the in vivobiological role of enamelysin during tooth development, we generated an enamelysin-deficient mouse by gene targeting. Although mice heterozygous for the mutation have no apparent phenotype, the enamelysin null mouse has a severe and profound tooth phenotype. Specifically, the null mouse does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ morphology as development progresses. Our findings demonstrate that enamelysin activity is essential for proper enamel development. Enamelysin is a tooth-specific matrix metalloproteinase that is expressed during the early through middle stages of enamel development. The enamel matrix proteins amelogenin, ameloblastin, and enamelin are also expressed during this same approximate developmental time period, suggesting that enamelysin may play a role in their hydrolysis. In support of this interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant amelogenin at virtually all of the precise sites known to occur in vivo. Thus, enamelysin is likely an important amelogenin-processing enzyme. To characterize the in vivobiological role of enamelysin during tooth development, we generated an enamelysin-deficient mouse by gene targeting. Although mice heterozygous for the mutation have no apparent phenotype, the enamelysin null mouse has a severe and profound tooth phenotype. Specifically, the null mouse does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ morphology as development progresses. Our findings demonstrate that enamelysin activity is essential for proper enamel development. Dental enamel covers the crown of the tooth and is unique among mineralized tissues because of its high mineral content, large crystals, and organized prism pattern. Other mineralized tissues such as bone, dentin, and cementum are composed of ∼20% organic material. In contrast, mature enamel has less than 1% organic matter by weight (1LeFerve M. Manly R. J. Am. Dent. Assoc. 1932; 24: 233-242Google Scholar, 2Deakins M. Volker J.F. J. Dent. Res. 1941; 20: 117-121Crossref Scopus (30) Google Scholar). Moreover, enamel crystallites possess a volume that is 100 times greater than the volume of crystallites found in other mineralizing tissues. These enamel crystallites form enamel rods that, in turn, form a unique interlacing (decussating) prism pattern. As a result, dental enamel is the hardest substance in the body. Its hardness is intermediate between that of iron and carbon steel, yet it also has a high elasticity (3Cole A.S. Eastoe J. Biochemistry and Oral Biology. 2nd Ed. Butterworth & Co. LTD, London1988: 460-474Crossref Google Scholar). Although mature enamel is a very hard protein-free tissue, it does not start this way. Enamel development (amelogenesis) consists of several stages that include the secretory, transition, and maturation stages. During the secretory stage, enamel crystallites elongate into long thin ribbons that are only a few apatitic unit cells in thickness (about 10 nm) with a width of ∼30 nm (4Daculsi G. Kerebel B. J. Ultrastruct. Res. 1978; 65: 163-172Crossref PubMed Scopus (133) Google Scholar, 5Cuisinier F.J. Steuer P. Senger B. Voegel J.C. Frank R.M. Calcif. Tissue Int. 1992; 51: 259-268Crossref PubMed Scopus (43) Google Scholar). The ribbons are evenly spaced, are oriented parallel to each other, and grow in length but very little in width and thickness. Ultimately, enamel crystal length determines the final thickness of the enamel layer as a whole (for review, see Ref.6Smith C.E. Crit. Rev. Oral Biol. Med. 1998; 9: 128-161Crossref PubMed Scopus (572) Google Scholar). It is during the secretory stage that the columnar-shaped ameloblast cells, located adjacent to the forming enamel, secrete specialized enamel proteins into the enamel matrix. These proteins include amelogenin (7Eastoe J.E. J. Dent. Res. 1979; 58: 753-764Crossref PubMed Google Scholar), ameloblastin (8Krebsbach P.H. Lee S.K. Matsuki Y. Kozak C.A. Yamada K.M. Yamada Y. J. Biol. Chem. 1996; 271: 4431-4435Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), and enamelin (9Hu C.C. Fukae M. Uchida T. Qian Q. Zhang C.H. Ryu O.H. Tanabe T. Yamakoshi Y. Murakami C. Dohi N. Shimizu M. Simmer J.P. J. Dent. Res. 1997; 76: 1720-1729Crossref PubMed Scopus (119) Google Scholar). Amelogenin is the predominant component and comprises ∼90% of total enamel matrix protein (10Termine J.D. Belcourt A.B. Christner P.J. Conn K.M. Nylen M.U. J. Biol. Chem. 1980; 255: 9760-9768Abstract Full Text PDF PubMed Google Scholar). Interestingly, the full-length enamel proteins are found only at the mineralizing front, suggesting that they participate in crystal elongation (11Smith C.E. Pompura J.R. Borenstein S. Fazel A. Nanci A. Anat. Rec. 1989; 224: 292-316Crossref PubMed Scopus (88) Google Scholar, 12Hu C.C. Fukae M. Uchida T. Qian Q. Zhang C.H. Ryu O.H. Tanabe T. Yamakoshi Y. Murakami C. Dohi N. Shimizu M. Simmer J.P. J. Dent. Res. 1997; 76: 648-657Crossref PubMed Scopus (143) Google Scholar, 13Fukae M. Tanabe T. Murakami C. Dohi N. Uchida T. Shimizu M. Adv. Dent. Res. 1996; 10: 111-118Crossref PubMed Scopus (81) Google Scholar, 14Fukae M. Tanabe T. Shimizu M. Tsurumi Shigaku. 1977; 3: 15-17PubMed Google Scholar, 15Robinson C. Briggs H.D. Kirkham J. Atkinson P.J. Arch. Oral Biol. 1983; 28: 993-1000Crossref PubMed Scopus (39) Google Scholar, 16Aoba T. Fukae M. Tanabe T. Shimizu M. Moreno E.C. Calcif. Tissue Int. 1987; 41: 281-289Crossref PubMed Scopus (152) Google Scholar, 17Robinson C. Shore R.C. Kirkham J. Stonehouse N.J. J Biol. Buccale. 1990; 18: 355-361PubMed Google Scholar, 18Ryu O.H. Hu C.C. Simmer J.P. Adv. Dent. Res. 1996; 10: 150-158Crossref PubMed Scopus (12) Google Scholar, 19Murakami C. Dohi N. Fukae M. Tanabe T. Yamakoshi Y. Wakida K. Satoda T. Takahashi O. Shimizu M. Ryu O.H. Simmer J.P. Uchida T. Histochem. Cell Biol. 1997; 107: 485-494Crossref PubMed Scopus (45) Google Scholar). In contrast, the protein cleavage products are found throughout the enamel layer, suggesting that they prevent crystallite growth in width and thickness (16Aoba T. Fukae M. Tanabe T. Shimizu M. Moreno E.C. Calcif. Tissue Int. 1987; 41: 281-289Crossref PubMed Scopus (152) Google Scholar). Enamelysin is a member of the matrix metalloproteinase family, and its mRNA has been cloned from pig (20Bartlett J.D. Simmer J.P. Xue J. Margolis H.C. Moreno E.C. Gene. 1996; 183: 123-128Crossref PubMed Scopus (176) Google Scholar), human (21Llano E. Pendas A.M. Knauper V. Sorsa T. Salo T. Salido E. Murphy G. Simmer J.P. Bartlett J.D. Lopez-Otin C. Biochemistry. 1997; 36: 15101-15108Crossref PubMed Scopus (186) Google Scholar), cow (22Den Besten P.K. Punzi J.S. Li W. Eur. J. Oral Sci. 1998; 106: 345-349Crossref PubMed Scopus (30) Google Scholar), and mouse (23Caterina J. Shi J. Sun X. Qian Q. Yamada S. Liu Y. Krakora S. Bartlett J.D. Yamada Y. Engler J.A. Birkedal-Hansen H. Simmer J.P. J. Dent. Res. 2000; 79: 1697-1703Crossref PubMed Google Scholar). Enamelysin is secreted into the enamel matrix during the secretory stage through transition stage of enamel development (24Bartlett J.D. Ryu O.H. Xue J. Simmer J.P. Margolis H.C. Connect. Tissue Res. 1998; 39: 101-109Crossref PubMed Scopus (42) Google Scholar, 25Bartlett J.D. Simmer J.P. Crit. Rev. Oral Biol. Med. 1999; 10: 425-441Crossref PubMed Scopus (231) Google Scholar, 26Fukae M. Tanabe T. Uchida T. Lee S.K. Ryu O.H. Murakami C. Wakida K. Simmer J.P. Yamada Y. Bartlett J.D. J. Dent. Res. 1998; 77: 1580-1588Crossref PubMed Scopus (90) Google Scholar, 27Bègue-Kirn C. Krebsbach P.H. Bartlett J.D. Butler W.T. Eur. J. Oral Sci. 1998; 106: 963-970Crossref PubMed Scopus (249) Google Scholar). Because enamelysin is present in the mineralizing front, it is thought to participate in the early cleavage events that allow the crystals to grow in length but not in width or thickness (25Bartlett J.D. Simmer J.P. Crit. Rev. Oral Biol. Med. 1999; 10: 425-441Crossref PubMed Scopus (231) Google Scholar). Previously, recombinant enamelysin was demonstrated to cleave recombinant amelogenin at virtually all of the precise cleavage sites that were demonstrated to occur in vivo (28Ryu O.H. Fincham A.G. Hu C.C. Zhang C. Qian Q. Bartlett J.D. Simmer J.P. J. Dent. Res. 1999; 78: 743-750Crossref PubMed Scopus (171) Google Scholar). Thus, enamelysin was identified as a predominant amelogenin-processing enzyme. As the secretory stage ends and the transition stage begins, the ameloblasts shrink in size and down-regulate protein release into the enamel matrix. These changes are associated with an end of the elongation of enamel crystals. The transition stage is followed by the maturation stage, where enamelysin expression is eliminated, and the crystallites grow in width and thickness but no longer in length. The remaining proteins within the enamel matrix are degraded by an enamel matrix serine proteinase (Kallikrein-4) before their export out of the enamel (25Bartlett J.D. Simmer J.P. Crit. Rev. Oral Biol. Med. 1999; 10: 425-441Crossref PubMed Scopus (231) Google Scholar, 29Simmer J.P. Fukae M. Tanabe T. Yamakoshi Y. Uchida T. Xue J. Margolis H.C. Shimizu M. DeHart B.C. Hu C.C. Bartlett J.D. J. Dent. Res. 1998; 77: 377-386Crossref PubMed Scopus (143) Google Scholar, 30Scully J.L. Bartlett J.D. Chaparian M.G. Fukae M. Uchida T. Xue J. Hu C.C. Simmer J.P. Connect. Tissue Res. 1998; 39: 111-122Crossref PubMed Scopus (25) Google Scholar, 31Hu J.C. Zhang C. Sun X. Yang Y. Cao X. Ryu O. Simmer J.P. Gene. 2000; 251: 1-8Crossref PubMed Scopus (68) Google Scholar, 32Hu J.C. Ryu O.H. Chen J.J. Uchida T. Wakida K. Murakami C. Jiang H. Qian Q. Zhang C. Ottmers V. Bartlett J.D. Simmer J.P. J. Dent. Res. 2000; 79: 70-76Crossref PubMed Scopus (46) Google Scholar). Enamel attains its final hardened form at the completion of the maturation stage. These general features of amelogenesis are remarkably consistent among different species (33Robinson C. Kirkham J. Weatherell J.A. Richards A. Josephsen K. Fejerskov O. Caries Res. 1988; 22: 321-326Crossref PubMed Scopus (37) Google Scholar). Enamelysin is unique among the MMP 1The abbreviations used are: MMP, matrix metalloproteinase; AI, amelogenesis imperfecta; bp, base pairs; kb, kilobase pair(s); TEMED, N,N,N‘,N‘-tetramethylethylenediamine family members because of its highly restricted pattern of expression. One study assessed 51 different cell lines for enamelysin expression, but none were positive (34Grant G.M. Giambernardi T.A. Grant A.M. Klebe R.J. Matrix Biol. 1999; 18: 145-148Crossref PubMed Scopus (57) Google Scholar). Conversely, enamelysin expression was observed in pathologic tissues such as in ghost cells of calcifying odontogenic cysts (35Takata T. Zhao M. Nikai H. Uchida T. Wang T. Histochem. J. 2000; 32: 223-229Crossref PubMed Scopus (28) Google Scholar), odontogenic tumors (36Takata T. Zhao M. Uchida T. Wang T. Aoki T. Bartlett J.D. Nikai H. J. Dent. Res. 2000; 79: 1608-1613Crossref PubMed Scopus (47) Google Scholar), and human tongue carcinoma cells (37Väänänen A. Srinivas R. Parikka M. Palosaari H. Bartlett J.D. Iwata K. Grenman R. Stenman U.H. Sorsa T. Salo T. J. Dent. Res. 2001; 80: 1884-1889Crossref PubMed Scopus (36) Google Scholar). Recently, enamelysin expression was also observed in bradykinin-treated granulosa cells isolated from the follicles of porcine ovaries (38Kimura A. Kihara T. Ohkura R. Ogiwara K. Takahashi T. Biol. Reprod. 2001; 65: 1462-1470Crossref PubMed Scopus (26) Google Scholar). However, with the exception of the ameloblasts of the enamel organ and the odontoblasts of the dental papilla (20Bartlett J.D. Simmer J.P. Xue J. Margolis H.C. Moreno E.C. Gene. 1996; 183: 123-128Crossref PubMed Scopus (176) Google Scholar, 21Llano E. Pendas A.M. Knauper V. Sorsa T. Salo T. Salido E. Murphy G. Simmer J.P. Bartlett J.D. Lopez-Otin C. Biochemistry. 1997; 36: 15101-15108Crossref PubMed Scopus (186) Google Scholar, 22Den Besten P.K. Punzi J.S. Li W. Eur. J. Oral Sci. 1998; 106: 345-349Crossref PubMed Scopus (30) Google Scholar, 23Caterina J. Shi J. Sun X. Qian Q. Yamada S. Liu Y. Krakora S. Bartlett J.D. Yamada Y. Engler J.A. Birkedal-Hansen H. Simmer J.P. J. Dent. Res. 2000; 79: 1697-1703Crossref PubMed Google Scholar, 24Bartlett J.D. Ryu O.H. Xue J. Simmer J.P. Margolis H.C. Connect. Tissue Res. 1998; 39: 101-109Crossref PubMed Scopus (42) Google Scholar, 26Fukae M. Tanabe T. Uchida T. Lee S.K. Ryu O.H. Murakami C. Wakida K. Simmer J.P. Yamada Y. Bartlett J.D. J. Dent. Res. 1998; 77: 1580-1588Crossref PubMed Scopus (90) Google Scholar, 27Bègue-Kirn C. Krebsbach P.H. Bartlett J.D. Butler W.T. Eur. J. Oral Sci. 1998; 106: 963-970Crossref PubMed Scopus (249) Google Scholar), no other intact physiologically normal tissue has been demonstrated to express enamelysin. Therefore, to date enamelysin is considered a tooth-specific MMP. To characterize the in vivo role of enamelysin during amelogenesis, we have generated a mouse with a null mutation that eliminates enamelysin activity. This mouse has a severe and profound phenotype that includes altered amelogenin processing, enamel that delaminates from the dentin, hypoplastic enamel, a disorganized prism pattern, and a deteriorating tooth morphology as enamel development progresses. These results demonstrate that enamelysin plays a critical protein-processing role during enamel development. All animals used in this work were housed in Association for Assessment and Accreditation of Laboratory Animal Care-approved facilities, and all operations were performed in accord with protocols approved by each Institute's Institutional Animal Care and Use Committees. The enamelysin catalytic domain targeting vector was constructed using gene sequences cloned from a 129-strain mouse genomic library in the Lambda Fix II vector (Stratagene, La Jolla, CA). The targeting vector was pBluescript SK+ including a 4.6-kb 5′ homology-spanning sequence from a PstI site in the 3′ region of intron 2 to the PstI site at the 5′ end of intron 4. The 1.1-kb 3′ homology arm encompassed the sequence from theBamHI site at the end of exon 5 to the XbaI site near the 3′ end of intron 5. The sequence from the PstI site in intron 4 through the EcoRI site at the 5′ end of intron 5 were replaced with a phosphoglycerate kinase promoter-driven hypoxanthine-guanine phosphoribosyltransferase minigeneEcoRI cassette (39van der Lugt N. Maandag E.R. te Riele H. Laird P.W. Berns A. Gene. 1991; 105: 263-267Crossref PubMed Scopus (34) Google Scholar). The targeting vector was completed by the addition of an herpes simplex virus thymidine kinase minigene cloned into the XbaI site in the 3′ terminus of the short homology arm and the SalI site of the plasmid polylinker. HM-1 mouse embryonic stem cells (40Magin T.M. McWhir J. Melton D.W. Nucleic Acids Res. 1992; 20: 3795-3796Crossref PubMed Scopus (211) Google Scholar) were transfected with the targeting vector by electroporation and cultured in selective growth medium containing 0.1 mm hypoxanthine, 16 μmthymidine, 0.4 μm aminopterin (hypoxanthine/thymine/aminopterin supplement, Invitrogen), and 2 μm ganciclovir (Roche Molecular Biochemicals). Hypoxanthine/thymine/aminopterin-resistant clones were expanded and screened for the legitimate targeting event using a primer complementary to the hypoxanthine-guanine phosphoribosyltransferase minigene 5′ p01, 5′-ACC CTC TGG TAG ATT GTA GCT TAT C-3′, and a primer complimentary to sequences not included in the targeting vector 3′ p02, 5′-CCT TTC CCA ACA TTG TCA CTG C-3′. Cell clones containing the targeted allele were further characterized by Southern blot analysis. An exon 6-specific probe was hybridized toEcoRI-digested mouse genomic DNA. As a result of gene targeting, the endogenous EcoRI site present in the 5′ end of intron 4 was deleted and replaced by an EcoRI site at the 3′ end of the hypoxanthine-guanine phosphoribosyltransferase cassette. This results in a band of ∼6.5 kb in cells with the targeted construct and a band of ∼7.3 kb for the native allele (Fig. 1,A and B). To generate chimeric mice, targeted embryonic stem cells were injected into 72-h-old blastocysts from C57BL/6 mice and implanted into pseudopregnant B6D2 or C57BL/6 crossed with dilute brown agouti (NCI-Frederick, Frederick, MD). Offspring were mated to C57bl/6 wild type mice (NCI-Frederick, Frederick, MD) to generate heterozygous animals for the targeted gene. These were subsequently interbred to generate homozygous mutant progeny. Genotyping of animals was performed by PCR amplification of DNA obtained from tail biopsies with primers 5′ p03, 5′-CTG CGT CCC CAG ACT TTT GAT TT-3′, and 3′ p04, 5′-GCT TTT CAT GGC CAG AAT GCT CT-3′, to detect the targeted allele and primers 5′ p05, 5′-AAG TAG ACT GAA GTC AGG AGA GCC-3′, and 3′ p06, 5′-CTG TAG TGG TGA CCC TAG TCA TCT T-3′, to detect the wild type allele. Total RNA was prepared by flash-freezing tissue in liquid N2 followed by extraction in Trizol (Invitrogen). Twenty μg samples of total RNA were size-fractionated on formaldehyde-agarose gels, immobilized on nylon membranes, and hybridized to an exon 5-specific radiolabeled probe. First mandibular molars were extracted from 4.0–4.5-day-old pups, all non-mineralized tissues were removed, and proteins were extracted from mineralized tooth caps by placing them in gel loading buffer (Zymograms: 62.5 mmTris-HCl (pH 6.8), 1.0% SDS, 0.3% glycerol, and 0.005% bromphenol blue; in addition, protein gel loading buffers had 0.1% dithiothreitol). Casein zymography gels were prepared (12% acrylamide, 375 mm Tris-HCl (pH 8.8), 0.1% casein, 0.0005% TEMED, and 0.05% ammonium persulfate), and electrophoresis was performed at a constant current of 20 mA per gel for ∼2 h. After electrophoresis, protein gels were silver-stained (Amersham Biosciences), and zymography gels were washed twice for 10 min in 50 ml of 2.5% Triton X-100 solution (2.5% Triton X-100 in 100 mm Tris-HCl buffer (pH 8.0)). The gels were incubated for 1–2 days at 37 °C in 50 mm Tris-HCl buffer (pH 7.2) containing 10 mmCaCl2 and stained with Coomassie Brilliant Blue (CBB) R-250 solution (0.23% CBB R-250, 5.8% acetic acid, and 30% methanol) for 20 min and destained with 10% methanol and 10% acetic acid until clear bands of substrate lysis were observed (41Smith C.E. Issid M. Margolis H.C. Moreno E.C. Adv. Dent. Res. 1996; 10: 159-169Crossref PubMed Scopus (72) Google Scholar). Incisors obtained from three euthanized wild type, three heterozygous, and six enamelysin null mice were fixed in 5% neutral formalin/saline overnight, incubated in phosphate-buffered saline containing 0.1% Triton X-100 for 8 h, rinsed overnight with running water, and decalcified in 20% sodium citrate, 45% formic acid for 2 weeks. This and all subsequent incubations were performed at ambient temperature. The jaws were dehydrated in a graded series of ethanol washes and embedded in paraffin for sectioning. Deparaffinized and rehydrated sections were stained with hematoxylin/eosin. For scanning electron microscopy, erupted molar and incisor teeth were either examined whole or fractured transversely, air-dried, fastened to stubs, sputter-coated, and examined using a JEOL 6400 scanning electron microscope. The mouse enamelysin gene includes 10 exons and is located within the MMP cluster at the centromeric end of chromosome 9 (42Caterina J. Shi J. Krakora S. Bartlett J.D. Engler J.A. Kozak C.A. Birkedal-Hansen H. Genomics. 1999; 62: 308-311Crossref PubMed Scopus (14) Google Scholar). To disrupt the functional expression of the enamelysin gene, a 10.6-kb-segment-containing sequence starting at the 3′ end of intron 2 and extending through most of intron 5 was modified such that the majority of intron 4 and exon 5 was replaced by a phosphoglycerate kinase promoter-controlled hypoxanthine-guanine phosphoribosyltransferase minigene (Fig.1 A) (39van der Lugt N. Maandag E.R. te Riele H. Laird P.W. Berns A. Gene. 1991; 105: 263-267Crossref PubMed Scopus (34) Google Scholar, 43Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1112) Google Scholar). Exon 5 encodes the highly conserved zinc-binding site (HEXGHXXGXXH) present in the catalytic domains of the MMP family. This deletion renders any polypeptide expressed from this mutant gene catalytically inactive. The targeting construct was transfected by electroporation into HM-1 (hypoxanthine-guanine phosphoribosyltransferase-deficient mouse embryonic stem) cells (40Magin T.M. McWhir J. Melton D.W. Nucleic Acids Res. 1992; 20: 3795-3796Crossref PubMed Scopus (211) Google Scholar), and hypoxanthine/thymine/aminopterin resistant clones were selected for further characterization. Targeted alleles were identified by PCR and confirmed by Southern blot analysis. Chimeric offspring derived from two individual cell clones were mated to C57bl/6 mice, and germ line transmission was obtained with chimeras from both clones. Interbreeding of heterozygous mice yielded the expected Mendelian distribution of homozygous mutant (enamelysin−/−), heterozygous (enamelysin+/−), and wild type (enamelysin+/+) mice (Fig. 1 B). Total RNA prepared from enamelysin−/−-homogenized incisors probed with an exon 5-specific probe demonstrated the absence of transcripts containing this exon (Fig. 1 C). Zymography of proteins extracted from 4.0–4.5-day-old first molars verified the absence of enamelysin activity in the enamelysin-deficient mice (Fig.1 C). Note that two enamelysin bands are present on the zymogram. A study in which native enamelysin was purified from porcine enamel suggests that the two bands represent active intact enamelysin and active enamelysin with at least one cut site present within its hemopexin domain. 2Y. Yamada, Y. Yamakoshi, R. F. Gerlach, J. C-C. Hu, K. Matsumoto, M. Fukae, S. Oida, J. D. Bartlett, and J. P. Simmer, submitted for publication. Maxillas from wild type and enamelysin null mice were removed, the periradicular bone was dissected away, and the exposed molars were prepared for scanning electron microscopy. The first maxillary molars from a wild type (Fig.2 A) and enamelysin null mouse are shown (Fig. 2 B). The dashed lines in Fig. 2encompass the enamel-free areas of each molar. As shown in the wild type, enamel-free areas are normally present in the mouse molar at the marsal plateau of the cusps. These areas provide troughs that are necessary for the efficient side-to-side grinding of ingested food. In the enamelysin null mouse, however, the enamel that surrounds the cusps is virtually absent. Only the cervical margin of the tooth had an enamel covering that remained (Fig. 2). Thus, enamel from the null mouse delaminates from the dentin surface. To determine whether the characteristic decussating rod pattern was altered in enamel from enamelysin null mice, incisors were fractured and prepared for scanning electron microscopy. The fracture plane of the wild type and heterozygous tooth extended through the enamel and dentin (Fig. 3, A andB), whereas in the null mouse, fracture planes of enamel and dentin were separate (Fig. 3 C). This suggests that the null mouse enamel does not adhere properly to the dentin surface. Littermate wild type and heterozygous mice had an inner enamel layer (100 μm) consisting of alternating rows of enamel rods decussating at about 90°, an outer enamel layer of parallel rods (15 μm) slightly inclined to the tooth surface, and a surface layer without rods (6 μm). Inspection of the littermate null mouse inner enamel rods revealed the complete absence of the typical decussating rod pattern, and enamel rod diameters were notably uneven (Fig.3 C). Fractures in the sagittal plane of null mouse incisors (not shown) did reveal the three distinct enamel layers; that is, the inner enamel layer (50 μm) with parallel rods inclined at about 45° to the dentin surface, an outer enamel layer (15 μm) with rods nearly parallel to the tooth surface, and a surface layer without rods (5 μm). In addition to the abnormal rod pattern present in the null mice, a comparison of the enamel thickness from the dentin/enamel junction to the enamel surface revealed that the null mice had a significantly thinner (hypoplastic) layer of enamel (70 μm) than did the wild type (120 μm) mice (Fig. 3). Thus, the enamelysin null mouse incisor had enamel that fractured independently of the dentin, abnormal enamel rod pattern, uneven enamel rod diameter, and hypoplastic enamel. An advantage of observing tooth development in rodents is that rodent incisors are continuously erupting, and therefore, all the stages of tooth development are present along each forming incisor throughout adult life. A morphological comparison of enamel development present in a demineralized incisor from littermate wild type mice (Fig.4, A–C), heterozygous mice (Fig. 4, D–F), and enamelysin null mice (Fig. 4,G–I) is shown. Because the tissues were demineralized, only protein is observed. Thus, for the wild type and heterozygous mouse incisors, the staining pattern beneath the ameloblasts becomes lighter with each successive panel until, at the late maturation stage (Fig. 4,C and F), the most mature enamel is clear. This clear area represents demineralized mature enamel that is almost protein-free. Conversely, in the null mouse, with each successive panel (Fig. 4, G–I) the ameloblasts become progressively more disorganized, and the protein in the enamel matrix is not properly resorbed. A comparison of the enamel proteins in the secretory and early maturation panels between the wild type and null animals reveals that the normal protein pattern present in the wild type is consistent with proteins that had surrounded mineralized prism structures (Fig. 4,A and B). This organized enamel protein pattern is absent in the enamelysin null mouse (Fig. 4, G andH). Thus, in comparison to wild type and heterozygous animals, the morphology of the enamelysin null mouse incisor displays hypoplastic enamel, ineffective removal of proteins from the enamel matrix, a disorganized protein pattern, and an increasingly disorganized ameloblast morphology as development progresses. To directly demonstrate that enamelysin cleaves amelogenin in vivo, amelogenins were extracted from 4.0–4.5-day-old mouse molars and size-separated by SDS-PAGE. A clearly different pattern of amelogenin degradation was evident between the wild type and null mice (Fig. 5). Amelogenin proteins from the null mice displayed a prominent band at ∼27 kDa that was only faintly detectable in the amelogenins from the wild type controls. Only one amelogenin band of less than ∼23 kDa was present in the enamel from the null mice, whereas in the controls at least 5 bands were present below this molecular mass. Thus, enamelysin activity is responsible for generating at least four different amelogenin isoforms that are present in naturally maturing dental enamel. In summary, the enamelysin null mouse does not process amelogenin properly, possesses an altered enamel protein and associated rod pattern, has hypoplastic enamel, has enamel that delaminates from the dentin, and has a deteriorating tooth morphology as enamel development progresses. Previously, several studies show that recombinant enamelysin cleaves recombinant amelogenin (21Llano E. Pendas A.M. Knauper V. Sorsa T. Salo T. Salido E. Murphy G. Simmer J.P. Bartlett J.D. Lopez-Otin C. Biochemistry. 1997; 36: 15101-15108Crossref PubMed Scopus (186) Google Scholar, 24Bartlett J.D. Ryu O.H. Xue J. Simmer J.P. Margolis H.C. Connect. Tissue Res. 1998; 39: 101-109Crossref PubMed Scopus (42) Google Scholar, 26Fukae M. Tanabe T. Uchida T. Lee S.K. Ryu O.H. Murakami C. Wakida K. Simmer J.P. Yamada Y. Bartlett J.D. J. Dent. Res. 1998; 77: 1580-1588Crossref PubMed Scopus (90) Google Scholar, 44Li W. Machule D. Gao C. DenBesten P.K. Eur. J. Oral Sci. 1999; 107: 352-359Crossref PubMed Scopus (32) Google Scholar), including a study demonstrating that recombinant amelogenin was cleaved at virtually all of the precise cleavage sites that had previously been observed in vivo (28Ryu O.H. Fincham A.G. Hu C.C. Zhang C. Qian Q. Bartlett J.D. Simmer J.P. J. Dent. Res. 1999; 78: 743-750Crossref PubMed Scopus (171) Google Scholar). However, until now (Fig. 5), no study has presented direct evidence demonstrating that enamelysin is responsible for these cleavages in vivo. Because enamelysin is expressed primarily during the secretory stage of amelogenesis when the crystallites grow in length, it appears that enamelysin functions to initiate hydrolysis of the structural enamel matrix proteins so that the enamel crystals may grow in length. Prevention of this process by the elimination of enamelysin activity results in thin, brittle enamel that does not mature properly. Enamelysin activity is therefore essential for proper enamel development. In addition, we have observed (not shown) that the first molar of the null mouse possesses less enamel than the second molar, which in turn, has less enamel than the third molar. The mouse molars erupt in this very sequence, from first to third. The same phenomenon was observed in incisor teeth. Intact enamel covered the labial surface of the recently erupted incisor portion near the gingival margin, but at the incisal tip, the enamel was missing. This enamel wear pattern suggests that the teeth erupt with a complete covering of enamel but that over time the malformed enamel wears or chips away presumably due to normal stresses encountered during mastication. Amelogenin comprises ∼90% of the organic component of developing enamel. Thus, the lack of amelogenin processing in the enamelysin null mouse is likely an important aspect of the null mouse phenotype. Previously it was demonstrated that a solitary point mutation (proline to threonine) in exon 6 of the amelogenin gene caused amelogenesis imperfecta (AI) (45Collier P.M. Sauk J.J. Rosenbloom S.J. Yuan Z.A. Gibson C.W. Arch. Oral Biol. 1997; 42: 235-242Crossref PubMed Scopus (121) Google Scholar). This missense mutation was positioned at P5 relative to a Trp/Leu enamelysin cleavage site and was demonstrated to reduce the efficiency of hydrolysis by 25-fold compared with hydrolysis of the non-mutated peptide (46Li W. Gibson C.W. Abrams W.R. Andrews D.W. DenBesten P.K. Matrix Biol. 2001; 19: 755-760Crossref PubMed Scopus (45) Google Scholar). Therefore a small change in amelogenin structure can have a profound effect on enamel development. Also, the hydrolysis of enamel proteins can alter their functional properties. Proteolysis of amelogenin reduces both its crystal binding affinity and its solubility (47Aoba T. Tanabe T. Moreno E.C. Adv. Dent. Res. 1987; 1: 252-260Crossref PubMed Scopus (58) Google Scholar, 48Simmer J.P. Lau E.C. Hu C.C. Aoba T. Lacey M. Nelson D. Zeichner-David M. Snead M.L. Slavkin H.C. Fincham A.G. Calcif. Tissue Int. 1994; 54: 312-319Crossref PubMed Scopus (166) Google Scholar, 49Hu C.C. Ryu O.H. Qian Q. Zhang C.H. Simmer J.P. J. Dent. Res. 1997; 76: 641-647Crossref PubMed Scopus (40) Google Scholar, 50Ryu O.H. Hu C.C. Simmer J.P. Connect. Tissue Res. 1998; 38: 207-214Crossref PubMed Scopus (32) Google Scholar, 51Tan J. Leung W. Moradian-Oldak J. Zeichner-David M. Fincham A.G. J. Dent. Res. 1998; 77: 1388-1396Crossref PubMed Scopus (29) Google Scholar). Thus, the lack of amelogenin processing in the enamelysin null mouse likely eliminated necessary changes in the physical properties of amelogenin that are essential for proper enamel development. Interestingly, during the late maturation stage, the ameloblasts of the null mouse sometimes surrounded abnormal nodule structures. In addition, an abnormally thick layer of protein appeared to separate the ameloblasts from the enamel surface (Fig. 4 I). This result was difficult to interpret given that both enamelysin and amelogenin are not normally expressed during this late stage of enamel development. Perhaps, pre-processing of enamel proteins by enamelysin is necessary for their proper removal from the enamel matrix and/or their subsequent degradation by the ameloblasts. Because, the dental enamel disease amelogenesis imperfecta affects only dental enamel, the phenotype/genotype of the enamelysin null mice suggest that one form of human AI may be caused by the recessively inherited inactivation of the enamelysin locus. The human amelogenin gene in the p21.1-p22.3 region of the X chromosome and the human enamelin gene at 4q11-q21 are loci in known cases of AI (52Rajpar M.H. Harley K. Laing C. Davies R.M. Dixon M.J. Hum. Mol. Genet. 2001; 10: 1673-1677Crossref PubMed Scopus (181) Google Scholar, 53Lagerstrom M. Dahl N. Nakahori Y. Nakagome Y. Backman B. Landegren U. Pettersson U. Genomics. 1991; 10: 971-975Crossref PubMed Scopus (227) Google Scholar). The human enamelysin gene locates to 11q22.3, which has not yet been identified as an AI locus. However, in contrast to the phenotype observed for mutations in the amelogenin gene (X-linked) or the enamelin gene (autosomal-dominant), an enamelysin defect would likely be autosomal-recessive and, therefore, less prevalent within the population. Thus, the likelihood of identifying an enamelysin-deficient AI patient is greatly reduced compared with the known genes that cause AI. An intriguing aspect of the enamelysin null mouse is that because it displays a severe and profound phenotype and survives to breed, it may be useful for transgenic studies to assess the functional significance of MMP domain structure. MMPs are characterized by a domain structure that consists of a signal peptide of ∼20 amino acid residues that is removed after it has directed secretion of the enzyme from the cell, a propeptide composed of ∼80 amino acids that folds back to mask and inhibit the catalytic pocket, a catalytic domain composed of ∼160 amino acids, and except for matrilysin and matrilysin-2, a hemopexin-like domain comprised of ∼200 amino acids (for review, see Ref. 54Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2647) Google Scholar). In general, MMP hemopexin domain function is poorly characterized. We are therefore currently elucidating the function of the enamelysin hemopexin domain by inserting an enamelysin transgene that encodes all but the hemopexin domain into the null mouse background. Thus, the enamelysin null mouse may allow us an opportunity to identify functional aspects of specific MMP domains as the tooth develops. We thank Glenn Longenecker and Ashok Kulkarni for gene targeting expertise, Justine M. Dobeck, Nancy Marinos, and Victor Morgan, Jr. for histology expertise, Susan Yamada for technical assistance, Jeffrey A. Engler and the University of Alabama at Birmingham Cancer Center oligonucleotide core facility for intellectual input and oligonucleotides, Charles E. Smith for sharing his zymography expertise, David Melton for his generous gift of the HM-1 cells, and Conan Young and Daniel H. Lee for critical review of the manuscript.
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