RESUMO
ClC-7, located in late endosomes and lysosomes, is critical for the function of osteoclasts. Secretion of Cl(-) by the ruffled border of osteoclasts enables H(+) secretion by v-H(+)-ATPases to dissolve bone mineral. Mice lacking ClC-7 show altered lysosomal function that leads to severe lysosomal storage. Maturation ameloblasts are epithelial cells with a ruffled border that secrete Cl(-) as well as endocytose and digest large quantities of enamel matrix proteins during formation of dental enamel. We tested the hypothesis that ClC-7 in maturation ameloblasts is required for intracellular digestion of matrix fragments to complete enamel mineralization. Craniofacial bones and developing teeth in Clcn7(-/-) mice were examined by micro-CT, immunohistochemistry, quantified histomorphometry and electron microscopy. Osteoclasts and ameloblasts in wild-type mice stained intensely with anti-ClC-7 antibody but not in Clcn7(-/-) mice. Craniofacial bones in Clcn7(-/-) mice were severely osteopetrotic and contained 1.4- to 1.6-fold more bone volume, which was less mineralized than the wild-type littermates. In Clcn7(-/-) mice maturation ameloblasts and osteoclasts highly expressed Ae2 as in wild-type mice. However, teeth failed to erupt, incisors were much shorter and roots were disfigured. Molars formed a normal dental crown. In compacted teeth, dentin was slightly less mineralized, enamel did not retain a matrix and mineralized fairly normal. We concluded that ClC-7 is essential for osteoclasts to resorb craniofacial bones to enable tooth eruption and root development. Disruption of Clcn7 reduces bone and dentin mineral density but does not affect enamel mineralization.
Assuntos
Calcificação Fisiológica , Canais de Cloreto/genética , Esmalte Dentário/metabolismo , Mutação/genética , Raiz Dentária/patologia , Ameloblastos/metabolismo , Animais , Densidade Óssea , Remodelação Óssea , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Canais de Cloreto/deficiência , Canais de Cloreto/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Imageamento Tridimensional , Camundongos Knockout , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoclastos/ultraestrutura , Microtomografia por Raio-XRESUMO
Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr-null mice by quantitative electron probe microanalysis. Maturation-stage enamel from Cftr-null mice contained less chloride and calcium than did wild-type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr-null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr-deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/metabolismo , Esmalte Dentário/química , Calcificação de Dente/fisiologia , Ameloblastos/metabolismo , Amelogênese/fisiologia , Animais , Bicarbonatos/análise , Soluções Tampão , Cálcio/análise , Cariostáticos/farmacologia , Cloretos/análise , Cloretos/metabolismo , Esmalte Dentário/efeitos dos fármacos , Microanálise por Sonda Eletrônica , Fluoresceínas , Corantes Fluorescentes , Fluoretos/análise , Fluoretos/sangue , Fluoretos/farmacologia , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos CFTR , Microtomografia por Raio-X/métodosRESUMO
Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irreversible enamel defects. Monofluorophosphate (MFP) was considered as less toxic than NaF but equally cariostatic. We compared the potency of MFP and NaF to induce pre-eruptive sub-ameloblastic cysts and post-eruptive white spots and pits in developing hamster enamel. Hamster pups were injected subcutaneously with either NaF or MFP in equimolar doses of either 9 mg or 18 mg F/kg body weight. At 9 mg F/kg, MFP induced more but smaller sub-ameloblastic cysts with a collective cyst volume twice as large as that induced by NaF. Eight days after F injection, all F-injected groups had formed 4-6 white spots per molar, with an additional 2 pits per molar in the low MFP group. Twenty-eight days after injection, most white spots had turned into pits (5-6 per molar) and only the high MFP group still contained 2 white spots per molar. We conclude that parenterally applied MFP is more potent in inducing enamel defects than NaF. Most white spots formed turn into pits by functional use of the dentition. The higher potency of parenteral MFP may be associated with sustained elevated F levels in the enamel organ by enzymatic hydrolysis of MFP by alkaline phosphatase activity.
Assuntos
Esmalte Dentário/efeitos dos fármacos , Fluoretos/administração & dosagem , Fluorose Dentária/etiologia , Fosfatos/administração & dosagem , Fluoreto de Sódio/administração & dosagem , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Cricetinae , Esmalte Dentário/enzimologia , Esmalte Dentário/patologia , Fluoretos/farmacologia , Fluorose Dentária/patologia , Infusões Parenterais , Fosfatos/farmacologia , Fluoreto de Sódio/farmacologiaRESUMO
Ameloblasts need to regulate pH during the formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine, and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear, and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues, including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts, and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as a pH regulator. SLC26A4 does not appear to be critical for ameloblast function and is probably compensated by other pH regulators.
Assuntos
Ameloblastos/metabolismo , Amelogênese/genética , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/fisiologia , Órgão do Esmalte/metabolismo , Animais , Proteínas de Transporte de Ânions/biossíntese , Especificidade de Anticorpos , Calcificação Fisiológica/genética , Linhagem Celular , Tecido Conjuntivo/metabolismo , Cricetinae , Cristalização , Concentração de Íons de Hidrogênio , Transporte de Íons , Camundongos , Camundongos Knockout , Ratos , Transportadores de SulfatoRESUMO
Intramembrane proteases are critically involved in signal transduction and membrane protein turnover. Signal-peptide-peptidase-like 2a (SPPL2A), a presenilin-homologue residing in lysosomes/late endosomes, cleaves type II-oriented transmembrane proteins. We recently identified SPPL2A as the enzyme controlling turnover and functions of the invariant chain (CD74) of the major histocompatibility complex II (MHCII) and demonstrated critical importance of this process for B cell development. Surprisingly, we found that SPPL2A is critical for formation of dental enamel. In Sppl2a knockout mice, enamel of the erupted incisors was chalky white and rapidly eroded after eruption. SPPL2A was found to be expressed in enamel epithelium during secretory and maturation stage amelogenesis. Mineral content of enamel in Sppl2aâ»/â» incisors was inhomogeneous and reduced by â¼20% compared to wild-type mice with the most pronounced reduction at the mesial side. Frequently, disruption of the enamel layer and localized detachment of the most superficial enamel layer was observed in the knockout incisors leading to an uneven enamel surface. In Sppl2a null mice, morphology and function of secretory stage ameloblasts were not noticeably different from that of wild-type mice. However, maturation stage ameloblasts showed reduced height and a characteristic undulation of the ameloblast layer with localized adherence of the cells to the outer enamel. This was reflected in a delayed and incomplete resorption of the proteinaceous enamel matrix. Thus, we conclude that intramembrane proteolysis by SPPL2A is essential for maintaining cellular homeostasis of ameloblasts. Because modulation of SPPL2A activity appears to be an attractive therapeutic target to deplete B cells and treat autoimmunity, interference with tooth enamel formation should be investigated as a possible adverse effect of pharmacological SPPL2A inhibitors in humans.
Assuntos
Ameloblastos/enzimologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Esmalte Dentário/enzimologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Incisivo/enzimologia , Proteínas de Membrana/metabolismo , Proteólise , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Ácido Aspártico Endopeptidases/genética , Esmalte Dentário/crescimento & desenvolvimento , Antígenos de Histocompatibilidade Classe II/genética , Incisivo/crescimento & desenvolvimento , Proteínas de Membrana/genética , Camundongos , Camundongos KnockoutRESUMO
Maturation stage ameloblasts of rodents express vacuolar type-H-ATPase in the ruffled border of their plasma membrane in contact with forming dental enamel, similar to osteoclasts that resorb bone. It has been proposed that in ameloblasts this v-H-ATPase acts as proton pump to acidify the enamel space, required to complete enamel mineralization. To examine whether this v-H-ATPase in mouse ameloblasts is a proton pump, we determined whether these cells express the lysosomal, T-cell, immune regulator 1 (Tcirg1, v-H-Atp6v(0)a(3)), which is an essential part of the plasma membrane proton pump that is present in osteoclasts. Mutation of this subunit in Tcirg1 null (or oc/oc) mice leads to severe osteopetrosis. No immunohistochemically detectable Tcirg1 was seen in mouse maturation stage ameloblasts. Strong positive staining in secretory and maturation stage ameloblasts however was found for another subunit of v-H-ATPase, subunit b, brain isoform (v-H-Atp6v(1)b(2)). Mouse osteoclasts and renal tubular epithelium stained strongly for both Tcirg1 and v-H-Atp6v(1)b(2). In Tcirg1 null mice osteoclasts and renal epithelium were negative for Tcirg1 but remained positive for v-H-Atp6v(1)b(2). The bone in these mutant mice was osteopetrotic, tooth eruption was inhibited or delayed, and teeth were often morphologically disfigured. However, enamel formation in these mutant mice was normal, ameloblasts structurally unaffected and the mineral content of enamel similar to that of wild type mice. We concluded that Tcirg1, which is essential for osteoclasts to pump protons into the bone, is not appreciably expressed in maturation stage mouse ameloblasts. Our data suggest that the reported v-H-ATPase in maturation stage ameloblasts is not the typical osteoclast-type plasma membrane associated proton pump which acidifies the extracellular space, but rather a v-H-ATPase potentially involved in intracellular acidification.
Assuntos
Ameloblastos/metabolismo , Membrana Celular/enzimologia , Osteoclastos/enzimologia , Subunidades Proteicas/metabolismo , Bombas de Próton/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Densidade Óssea , Imuno-Histoquímica , Incisivo/crescimento & desenvolvimento , Incisivo/metabolismo , Mandíbula/diagnóstico por imagem , Mandíbula/crescimento & desenvolvimento , Mandíbula/patologia , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Microtomografia por Raio-XRESUMO
Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl(-) channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts. Tissue sections of young mouse jaws and fetal human jaws were immunostained with various anti-Cftr antibodies. Specificity of the antibodies was validated in Cftr-deficient murine and human tissues. Immunostaining for Cftr was obtained in the apical plasma membranes of mouse maturation ameloblasts of both incisor and molar tooth germs. A granular intracellular immunostaining of variable intensity was also noted in bone cells and odontoblasts. In Cftr-deficient mice the incisors were chalky white and eroded much faster than in wild type mice. Histologically, only maturation ameloblasts of incisors were structurally affected in Cftr-deficient mice. Some antibody species gave also a positive cytosolic staining in Cftr-deficient cells. Transcripts of Cftr were found in maturation ameloblasts, odontoblasts and bone cells. Similar data were obtained in forming human dentin and bone. We conclude that Cftr protein locates in the apical plasma membranes of mouse maturation ameloblasts. In mouse incisors Cftr is critical for completion of enamel mineralization and conceivably functions as a regulator of pH during rapid crystal growth. Osteopenia found in CF patients as well as in Cftr-deficient mice is likely associated with defective Cftr operating in bone cells.