Dentin Sialophosphoprotein Knockout Mouse Teeth Display Widened Predentin Zone and Develop Defective Dentin Mineralization Similar to Human Dentinogenesis Imperfecta Type III

Abstract

Dentin sialophosphoprotein (Dspp) is mainly expressed in teeth by the odontoblasts and preameloblasts. The Dspp mRNA is translated into a single protein, Dspp, and cleaved into two peptides, dentin sialoprotein and dentin phosphoprotein, that are localized within the dentin matrix. Recently, mutations in this gene were identified in human dentinogenesis imperfecta II (Online Mendelian Inheritance in Man (OMIM) accession number 125490) and in dentin dysplasia II (OMIM accession number 125420) syndromes. Herein, we report the generation of Dspp-null mice that develop tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure. Electron microscopy revealed an irregular mineralization front and a lack of calcospherites coalescence in the dentin. Interestingly, the levels of biglycan and decorin, small leucine-rich proteoglycans, were increased in the widened predentin zone and in void spaces among the calcospherites in the dentin of null teeth. These enhanced levels correlate well with the defective regions in mineralization and further indicate that these molecules may adversely affect the dentin mineralization process by interfering with coalescence of calcospherites. Overall, our results identify a crucial role for Dspp in orchestrating the events essential during dentin mineralization, including potential regulation of proteoglycan levels. Dentin sialophosphoprotein (Dspp) is mainly expressed in teeth by the odontoblasts and preameloblasts. The Dspp mRNA is translated into a single protein, Dspp, and cleaved into two peptides, dentin sialoprotein and dentin phosphoprotein, that are localized within the dentin matrix. Recently, mutations in this gene were identified in human dentinogenesis imperfecta II (Online Mendelian Inheritance in Man (OMIM) accession number 125490) and in dentin dysplasia II (OMIM accession number 125420) syndromes. Herein, we report the generation of Dspp-null mice that develop tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure. Electron microscopy revealed an irregular mineralization front and a lack of calcospherites coalescence in the dentin. Interestingly, the levels of biglycan and decorin, small leucine-rich proteoglycans, were increased in the widened predentin zone and in void spaces among the calcospherites in the dentin of null teeth. These enhanced levels correlate well with the defective regions in mineralization and further indicate that these molecules may adversely affect the dentin mineralization process by interfering with coalescence of calcospherites. Overall, our results identify a crucial role for Dspp in orchestrating the events essential during dentin mineralization, including potential regulation of proteoglycan levels. Tooth development depends on reciprocal interactions between oral epithelium and mesenchyme (1Lumsden A.G. Development. 1988; 103: 155-169Google Scholar). The mesenchymederived odontoblasts secrete several collagenous and non-collagenous proteins to form a unique extracellular matrix (2MacDougall M. Zeichner-David M. Slavkin H.C. Biochem. J. 1985; 232: 493-500Google Scholar). Among the non-collagenous proteins, dentin sialoprotein (Dsp) 1The abbreviations used are: Dsp, dentin sialoprotein; Dpp, dentin phosphoprotein; Dspp, dentin sialophosphoprotein; DGI, dentinogenesis imperfecta; ES, embryonic stem; GAG, glycosaminoglycans; RT, reverse transcription/transcriptase. and dentin phosphoprotein (Dpp) are highly tooth-specific and are believed to play a crucial role in converting predentin to form mineralized dentin. These two proteins are derived from the cleavage of a 940-amino acid polypeptide, dentin sialophosphoprotein (Dspp) (3MacDougall M. Simmons D. Luan X. Nydegger J. Feng J. Gu T.T. J. Biol. Chem. 1997; 272: 835-842Google Scholar). The Dspp gene consists of five exons spanning 16 kb and is transcribed as a 4.4-kb mRNA (4Feng J.Q. Luan X. Wallace J. Jing D. Ohshima T. Kulkarni A.B. D'Souza R.N. Kozak C.A. MacDougall M. J. Biol. Chem. 1998; 273: 9457-9464Google Scholar). mRNA for Dspp is expressed predominantly by odontoblasts and transiently by preameloblasts (5D'Souza R.N. Bronckers A.L. Happonen R.P. Doga D.A. Farach-Carson M.C. Butler W.T. J. Histochem. Cytochem. 1992; 40: 359-366Google Scholar, 6Begue-Kirn C. Krebsbach P.H. Bartlett J.D. Butler W.T. Eur. J. Oral Sci. 1998; 106: 963-970Google Scholar, 7D'Souza R.N. Cavender A. Sunavala G. Alvarez J. Ohshima T. Kulkarni A.B. MacDougall M. J. Bone Miner. Res. 1997; 12: 2040-2049Google Scholar). Recently, however, low levels of Dspp have been observed in the ear and in bone (8Xiao S. Yu C. Chou X. Yuan W. Wang Y. Bu L. Fu G. Qian M. Yang J. Shi Y. Hu L. Han B. Wang Z. Huang W. Liu J. Chen Z. Zhao G. Kong X. Nat. Genet. 2001; 27: 201-204Google Scholar, 9Qin C. Brunn J.C. Cadena E. Ridall A. Tsujigiwa H. Nagatsuka H. Nagai N. Butler W.T. J. Dent. Res. 2002; 81: 392-394Google Scholar). The Dsp, the amino-terminal part of Dspp, is a sialic acid-rich and glycosylated protein that shares similarities with the other sialoproteins, including bone sialoprotein, dentin matrix protein-1, and osteopontin. The Dpp, a highly phosphorylated protein with repeats of aspartic acid and phosphoserine, is thought to play a key role in the nucleation of hydroxyapatite formation during dentin calcification (10Veis A. J. Bone Miner. Res. 1993; 8: S493-S497Google Scholar, 11George A. Bannon L. Sabsay B. Dillon J.W. Malone J. Veis A. Jenkins N.A. Gilbert D.J. Copeland N.G. J. Biol. Chem. 1996; 271: 32869-32873Google Scholar, 12Butler W.T. Eur. J. Oral Sci. 1998; 106: 204-210Google Scholar). Because of its restricted expression and its physical localization on the human chromosome 4q within the dentinogenesis imperfecta (DGI)-II locus (13Aplin H.M. Hirst K.L. Crosby A.H. Dixon M.J. Genomics. 1995; 30: 347-349Google Scholar, 14MacDougall M. Eur. J. Oral Sci. 1998; 106: 227-233Google Scholar), Dspp was implicated as a potential candidate gene for this disorder. The other important genes involved in biomineralization mapped to this region of chromosome 4q include dentin matrix protein-1, bone sialoprotein, and osteopontin. DGI, an autosomal dominant disorder of the tooth that primarily affects dentin biomineralization, is classified into three subtypes, based on the clinical features; type I is the least severe and type III is the most severe (15Shields E.D. Bixler D. el-Kafrawy A.M. Arch. Oral Biol. 1973; 18: 543-553Google Scholar). DGI-I is associated with osteogenesis imperfecta, whereas the more severe forms (DGI-II and DGI-III) are restricted to the dentin. Opalescent dentin with obliterated pulp chambers are the characteristic features in DGI-II (16Rao S. Witkop Jr., C.J. Birth Defects Orig. Artic. Ser. 1971; 7: 153-184Google Scholar). The teeth of patients with DGI-III are referred to as ‘shell teeth,‘ in which the dentin mineralization does not occur after mantle dentin is formed. Radiographically, the pulp cavities in these teeth appear as enlarged pulp chambers along with high incidence of pulp exposures (17Witkop Jr., C.J. MacLean C.J. Schmidt P.J. Henry J.L. Ala. J. Med. Sci. 1966; 3: 382-403Google Scholar, 18Levin L.S. Leaf S.H. Jelmini R.J. Rose J.J. Rosenbaum K.N. Oral Surg. Oral Med. Oral Pathol. 1983; 56: 267-274Google Scholar). Recently, several mutations in the Dspp gene have been identified in families with DGI-II disorder. These mutations include a C to T transition at the end of the third exon (Gln45stop) resulting in a premature termination of Dspp protein (19Zhang X. Zhao J. Li C. Gao S. Qiu C. Liu P. Wu G. Qiang B. Lo W.H. Shen Y. Nat. Genet. 2001; 27: 151-152Google Scholar), a G to A transition mutation in intron 3 (splice donor site) causing exon skipping, and Pro17Thr and Val18Phe transitions (8Xiao S. Yu C. Chou X. Yuan W. Wang Y. Bu L. Fu G. Qian M. Yang J. Shi Y. Hu L. Han B. Wang Z. Huang W. Liu J. Chen Z. Zhao G. Kong X. Nat. Genet. 2001; 27: 201-204Google Scholar). Dentin dysplasia-II, another human disorder of dentin mineralization, is similar to DGI-II and is attributed to a Tyr6Asp protein transition mutation in the hydrophobic core sequence of the Dspp gene. This mutation disabled the entry of Dspp into the endoplasmic reticulum (20Rajpar M.H. Koch M.J. Davies R.M. Mellody K.T. Kielty C.M. Dixon M.J. Hum. Mol. Genet. 2002; 11: 2559-2565Google Scholar). All these mutations indicate a potential function for Dspp in tooth mineralization. To characterize the molecular events that control dentin mineralization during normal tooth development and disease, we have deleted the entire Dspp coding region in embryonic stem (ES) cells and generated Dspp–/– mice. These null mice displayed an enlarged pulp cavity, widened predentin zone, decreased dentin width, and high incidence of pulp exposures similar to that in DGI-III. In addition, these mice showed an increased accumulation of biglycan and decorin within the widened predentin and scalloped (void spaces) regions in the dentin, correlating well with defective mineralization. Generation of Dspp–/– Mice—Dspp genomic clones from a 129/SvJ mouse genomic library were cloned and characterized as reported previously (4Feng J.Q. Luan X. Wallace J. Jing D. Ohshima T. Kulkarni A.B. D'Souza R.N. Kozak C.A. MacDougall M. J. Biol. Chem. 1998; 273: 9457-9464Google Scholar). The targeting vector was constructed by standard recombinant DNA techniques; the Nsi/EcoRV region of exons 2–5 spanning the entire coding region of Dspp was replaced by a promoterless β-galactosidase gene with simian virus 40 polyadenylation sequences (21Sreenath T.L. Cho A. MacDougall M. Kulkarni A.B. Int. J. Dev. Biol. 1999; 43: 509-516Google Scholar) followed by a neomycin-resistance and thymidylate kinase cassettes. The targeting vector was linearized with NotI and electroporated into 129/SvJ R1 embryonic stem cells. ES-cell clones that survived G-418 and ganciclovir selection were evaluated for homologous recombination by Southern blot analysis. ES cells were injected into C57BL/6 blastocysts and the chimeric mice produced were mated to C57Bl/6 female mice to derive heterozygotes. Heterozygous mice were then interbred to obtain homozygous mice and maintained as homozygotes. The null mice were viable and able to reproduce normally. All mice were housed according to National Institutes of Health guidelines. In Situ Hybridization—Digoxigenin-11-dUTP-labeled sense and antisense riboprobes were generated from Dspp exons 3 and 4 (pSX1.7) and hybridized to the frozen tissue sections obtained from 6-day-old mouse teeth as described previously (22Thyagarajan T. Sreenath T. Cho A. Wright J.T. Kulkarni A.B. J. Biol. Chem. 2001; 276: 11016-11020Google Scholar). Microradiography—Wild-type and Dspp–/– mice were sacrificed, and the mandibles were separated and radiographed using a Faxitron MX20 Specimen Radiography System (Faxitron X-ray Corp., Wheeling, IL) at energy settings 120 s at 15 kV (22Thyagarajan T. Sreenath T. Cho A. Wright J.T. Kulkarni A.B. J. Biol. Chem. 2001; 276: 11016-11020Google Scholar). The images were captured on X-OMAT TL film (Eastman Kodak). Scanning and Transmission Electron Microscopy—The jaws were dissected from the control and mutant mice and immersed in a 2.5% glutaraldehyde and 2% paraformaldehyde buffered solution, pH 7.4, for 12 h. The jaws are then transferred to a 0.1 m cacodylate buffer solution, pH 7.4. The teeth were dissected from the jaws using a dissecting microscope and prepared for analysis using the following techniques. Teeth were fractured and the samples mounted with the fractured surface facing, coated with Au-Pd using a sputter coater, and examined in a JEOL 6300 scanning electron microscope. After fixation, whole mandibles were glued to a glass slide and polished to an optically flat surface (1/4 μm) using an automatic polisher. The jaws were removed from the glass slide using acetone and rinsed with ethanol and dH2O, air dried, mounted on a carbon stub, and carbon coated. The samples were examined using a JEOL 6300 scanning electron microscope fitted with a backscatter detector (25-mm working distance, 15 kV). Small pieces of the incisors and molars were dissected from the teeth and osmicated before serial dehydration in ethanol. After dehydration, the samples were embedded in and then with an were and with and The samples were examined with a electron microscope. and from and Dspp–/– mice were dissected and in The were embedded in for sections using an low and for by and for mandibles were in in 0.1 m buffer for 3 and embedded in sections μm) from and Dspp–/– mouse were and with of 0.1 for at to the and tooth sections were for the of with m at pH according to the of G. R.M. J. Oral Pathol. 1988; Scholar). and from and mutant mice were dissected and into two using a and pulp was at with in with The teeth were several with to the pulp cells. was by the odontoblasts that were in the dentin using a was using according to the using the following biglycan decorin and The were on a 2% of Dspp, and decorin was on The sections were in and in a ethanol biglycan and decorin the sections were with of 0.1 for at to the before The were with J.T. 1995; Scholar) at The were with The in the was by a according to the and Generation of Dspp–/– have previously reported and of the mouse Dspp gene (4Feng J.Q. Luan X. Wallace J. Jing D. Ohshima T. Kulkarni A.B. D'Souza R.N. Kozak C.A. MacDougall M. J. Biol. Chem. 1998; 273: 9457-9464Google Scholar). The targeting was to the entire coding region of Dspp with the promoterless β-galactosidase gene The of the Dspp gene was by Southern using a not The ES cells clones were injected into blastocysts to the The were mated with mice to obtain mice. of these mice were by Southern using a and the lack of gene expression was by and by in using a Dspp exon and Dspp protein expression was by and The null mice not tooth of the incisors in null mice Dspp–/– Tooth in between the control teeth and Dspp–/– molars and incisors were observed in mice. The pulp chambers in the molars from mutant mice showed a and defects in the Dspp–/– mouse incisors were characterized as dentin with The of dentin, a of mineralized dentin, was at the end of the Dspp–/– mice with the incisors of pulp were observed in the molars of Dspp–/– mice from of exposures to occur among the increased incidence of pulp was observed in Dspp–/– mice. Because of pulp and severe the tooth were to the of the and The molars from these mutant mice that are with pulp displayed tooth The of teeth observed in the mutant mice was by pulp followed by increased pulp of Dspp–/– mice not pulp of in the teeth and examined by displayed in the mutant mouse and The bone was the tooth in the Dspp–/– teeth that were by pulp as a of of dentin. been reported previously that bone as a of in pulp in P. Oral Surg. Oral Med. Oral Pathol. Oral 2002; Scholar). of and in Dspp–/– electron microscopy of fractured Dspp–/– teeth showed an increased predentin region a and with the scalloped and The irregular mineralization front observed in the null mice was by a lack of coalescence of calcospherites. spaces and scalloped dentin in the mineralized dentin were observed in Dspp–/– teeth The widened predentin in the teeth a collagenous the and the dentin was of of the by increased by proteins and I the most of the dentin extracellular normal in the Dspp–/– dentin and were in a similar to that in the tissue and The of the and the from electron microscopy were similar and normal in the Dspp–/– and control mice. in of the Dspp–/– analysis revealed a in the width of the mineralized dentin and an in the width of the The of and and dentin was similar to the widened predentin in human teeth with DGI-III (17Witkop Jr., C.J. MacLean C.J. Schmidt P.J. Henry J.L. Ala. J. Med. Sci. 1966; 3: 382-403Google Scholar, 18Levin L.S. Leaf S.H. Jelmini R.J. Rose J.J. Rosenbaum K.N. Oral Surg. Oral Med. Oral Pathol. 1983; 56: 267-274Google Scholar). dentinogenesis in the predentin was and to at the region of the mineralized dentin. are among the molecules that are identified in the dentin extracellular matrix and are and by the which proteoglycans, was observed in the widened predentin region in the null teeth and was increased levels and of in the predentin of the null mice to the control mice. of and in the of type I decorin, and was by in the from and Dspp–/– incisors and decorin, in dentin and in GAG, to a of small leucine-rich that are predominantly expressed in and teeth P. J.D. J. Histochem. Cytochem. Scholar, M. H. Y. A. J. Histochem. Cytochem. Scholar, N. M. H. Arch. Oral Biol. 1996; Scholar, G. D. M. Oral Biol. Med. 2001; 12: Scholar, D. G. M. Int. 2001; Scholar). Small leucine-rich have a well role in and A.M. T. D.J. Biol. 1998; Scholar). and decorin mRNA expression was increased in the Dspp–/– with the teeth. I is of the to play an essential role in the mineralization and of dentin. The expression of type I was not in the null teeth. is believed that the of from is to the the on the of matrix which is implicated in the of from in the predentin and the part of predentin to form the mineralization front D. G. M. Histochem. J. 1999; Scholar), was not in the Dspp–/– teeth. The expression of and was used as control in these and with Defects in Dspp–/– biglycan and decorin are involved in mineralization we levels in the Dspp–/– and mouse teeth. In 6-day-old Dspp–/– mouse for biglycan and decorin was observed in the predentin and with a and increased of biglycan and decorin was observed in and and and mouse teeth pulp exposure. The of the mineralization front observed in the teeth of Dspp–/– mice with the lack of coalescence of the calcospherites in the mineralization Interestingly, high levels of biglycan and decorin were in the void spaces in the dentin a and the calcospherites are not and These that increased localization of biglycan and decorin in the predentin and dentin may with the nucleation and coalescence of calcospherites in a mineralization of biglycan and decorin in the spaces of the Dspp–/– teeth. of mouse tooth sections with biglycan and decorin accumulation calcospherites. Scanning electron microscopy of the fractured teeth the void spaces in the dentin similar to the regions in and teeth of dentin extracellular matrix interactions among several collagenous and non-collagenous Dspp, an important in the dentin, is cleaved into and and localized predominantly in the dentin. on its gene expression and localization within the DGI-II locus on human chromosome Dspp is implicated as a in converting predentin to dentin, and observed mutations in the Dspp gene in patients further its potential role in dentin mineralization. The features in DGI-II and DGI-III are defective dentin that been attributed to defective levels of the Dspp and Y. S. Res. Scholar), to mutations in the Dspp gene (8Xiao S. Yu C. Chou X. Yuan W. Wang Y. Bu L. Fu G. Qian M. Yang J. Shi Y. Hu L. Han B. Wang Z. Huang W. Liu J. Chen Z. Zhao G. Kong X. Nat. Genet. 2001; 27: 201-204Google Scholar, X. Zhao J. Li C. Gao S. Qiu C. Liu P. Wu G. Qiang B. Lo W.H. Shen Y. Nat. Genet. 2001; 27: 151-152Google Scholar). These and of been that the DGI-III develop of the of the DGI-II mutation (17Witkop Jr., C.J. MacLean C.J. Schmidt P.J. Henry J.L. Ala. J. Med. Sci. 1966; 3: 382-403Google Scholar, M.H. Genet. 3: Scholar). To the of Dspp in tooth mineralization, we generated Dspp–/– which develop defective and dentin mineralization and pulp exposures similar to of DGI-III of dentin is at the and forms the mineralization characterized by the of These calcospherites and with the calcospherites to form a mineralization A lack of coalescence of calcospherites in the dentin, an irregular mineralization and scalloped dentin similar to of the DGI-III patients L.S. Leaf S.H. Jelmini R.J. Rose J.J. Rosenbaum K.N. Oral Surg. Oral Med. Oral Pathol. 1983; 56: 267-274Google Scholar) were observed in the Dspp–/– mice. I is the by the which form a the in the region and in the predentin a was not in the null mice of increased were in B. G. J. J. Dent. Res. Scholar). type I and in the dentin extracellular matrix normal in the Dspp-null mice. The mRNA levels were in these that the mineralization defects observed in the teeth of Dspp–/– mice were of dentin mineralization, sialoproteins, and with other to form which to form dentin. A number of and have the of and in including teeth M. H. Y. A. J. Histochem. Cytochem. Scholar). to a of and more to the protein The are to and with hydroxyapatite and are by G. R.M. J. Oral Pathol. 1988; Scholar, N. M. H. Arch. Oral Biol. 1996; Scholar, J. Scholar). of observed in the predentin region of Dspp–/– mice the of Among small leucine-rich proteoglycans, decorin and biglycan have been well characterized for in the In mouse decorin was localized in and dentin and biglycan in odontoblasts and predentin Y. H. P. Arch. Oral Biol. 1997; Scholar). In have that decorin and biglycan play a crucial role in and to of the hydroxyapatite nucleation a zone in which is in the of mineralization Biol. 1999; 12: Scholar, M.J. A. L. J. Biol. 1993; Scholar). as an of mineralization during of S. Y. N. H. J. Bone Miner. Res. 1999; Scholar). Interestingly, biglycan the of formation and the of A.L. L. L. Int. 1997; Scholar). that decorin and biglycan may have in the mineralization The teeth of and mice widened and dentin and in a crucial role for decorin and biglycan in the tooth mineralization. width of the in the biglycan null teeth further a role in mineralization M. D. M. L. J. Dent. Res. 2002; 81: Scholar). The increased levels and of biglycan and decorin in the predentin of Dspp–/– teeth indicate that the increased biglycan and decorin may play a crucial role in the formation of a mineralization The and in the are the hydroxyapatite nucleation in the mineralization S. W. Scholar, J. Mol. Res. Scholar). been that a of mineralization in dentin, is localized in the W. T. S. 1992; 12: Scholar). to the of may the mineralization and interactions between and the molecules that mineralization M. A. A. J. Biol. 1997; Scholar). is to that the Dspp and Dpp, implicated in the tooth mineralization, the levels of the A number of have been to the expression of biglycan and decorin in a of a the levels of biglycan in cells Y. T. E. Y. J. Bone Miner. Res. 1993; 8: Scholar). In an (22Thyagarajan T. Sreenath T. Cho A. Wright J.T. Kulkarni A.B. J. Biol. Chem. 2001; 276: 11016-11020Google Scholar), we of Dspp expression in mice to dentin. In these we observed levels of biglycan and decorin by The levels of in Dspp–/– mice and in the regions of teeth that the Dspp gene may control in the regions of the dentin, the mineralization process is In addition, type I levels in Dspp–/– teeth were we that the increased levels of in the mutant mice with the and the however, may to from the for dentin mineralization. This may a of a of of a of to the increased we that Dspp gene in to role in the nucleation of mineralization, may play a role in regulation of during Overall, our results indicate that the of Dspp and the regulation of may the that to the mineralization defects in this disorder. Ohshima and for of Dspp genomic clones and and for and on the