Perfluorooctanoic acid-induced cell death via the dual roles of ROS-MAPK/ERK signaling in ameloblast-lineage cells.

Perfluorooctanoic acid (PFOA) is an artificial fluorinated organic compound that has generated increased public attention due to its potential health hazards. Unsafe levels of PFOA exposure can affect reproduction, growth and development. During tooth enamel development (amelogenesis), environmental factors including fluoride can cause enamel hypoplasia. However, the effects of PFOA on ameloblasts and tooth enamel formation remain largely unknown. In the present study we demonstrate several PFOA-mediated cell death pathways (necrosis/necroptosis, and apoptosis) and assess the roles of ROS-MAPK/ERK signaling in PFOA-mediated cell death in mouse ameloblast-lineage cells (ALC). ALC cells were treated with PFOA. Cell proliferation and viability were analyzed by MTT assays and colony formation assays, respectively. PFOA suppressed cell proliferation and viability in a dose dependent manner. PFOA induced both necrosis (PI-positive cells) and apoptosis (cleaved-caspase-3, γH2AX and TUNEL-positive cells). PFOA significantly increased ROS production and up-regulated phosphor-(p)-ERK. Addition of ROS inhibitor N-acetyl cysteine (NAC) suppressed p-ERK and decreased necrosis, and increased cell viability compared to PFOA alone, whereas NAC did not change apoptosis. This suggests that PFOA-mediated necrosis was induced by ROS-MAPK/ERK signaling, but apoptosis was not associated with ROS. Addition of MAPK/ERK inhibitor PD98059 suppressed necrosis and increased cell viability compared to PFOA alone. Intriguingly, PD98059 augmented PFOA-mediated apoptosis. This suggests that p-ERK promoted necrosis but suppressed apoptosis. Addition of the necroptosis inhibitor Necrostatin-1 restored cell viability compared to PFOA alone, while pan-caspase inhibitor Z-VAD did not mitigate PFOA-mediated cell death. These results suggest that 1) PFOA-mediated cell death was mainly caused by necrosis/necroptosis by ROS-MAPK/ERK signaling rather than apoptosis, 2) MAPK/ERK signaling plays the dual roles (promoting necrosis and suppressing apoptosis) under PFOA treatment. This is the initial report to indicate that PFOA could be considered as a possible causative factor for cryptogenic enamel malformation. Further studies are required to elucidate the mechanisms of PFOA-mediated adverse effects on amelogenesis.

Interfrontal Bone Among Inbred Strains of Mice and QTL Mapping.

The interfrontal bone (IF) is a minor skeletal trait residing between the frontal bones. IF is considered a quasi-continuous trait. Genetic and environmental factors appear to play roles in its development. The mechanism(s) underlying IF bone development are poorly understood. We sought to survey inbred strains of mice for the prevalence of IF and to perform QTL mapping studies. Archived mouse skulls from a mouse phenome project (MPP) were available for this study. 27 inbred strains were investigated with 6-20 mice examined for each strain. Skulls were viewed dorsally and the IF measured using a zoom stereomicroscope equipped with a calibrated reticle. A two generation cross between C3H/HeJ and C57BL/6J mice was performed to generate a panel of 468 F2 mice. F2 mice were phenotyped for presence or absence of IF bone and among mice with the IF bone maximum widths and lengths were measured. F2 mice were genotyped for 573 SNP markers informative between the two strains and subjected to linkage map construction and interval QTL mapping. Results: Strain dependent differences in the prevalence of IF bones were observed. Overall, 77.8% or 21/27, of the inbred strains examined had IF bones. Six strains (C3H/HeJ, MOLF/EiJ, NZW/LacJ, SPRET/EiJ, SWR/J, and WSB/EiJ) lack IF bones. Among the strains with IF bones, the prevalence ranged from 100% for C57BL/6J, C57/LJ, CBA/J, and NZB/B1NJ and down to 5% for strains such as CAST/Ei. QTL mapping for IF bone length and widths identifies for each trait one strong QTL detected on chromosome 14 along with several other significant QTLs on chromosomes 3, 4, 7, and 11. Strain dependent differences in IF will facilitate investigation of genetic factors contributing to IF development. IF bone formation may be a model to understand intrasutural bone formation.

Proteomic Mapping of Dental Enamel Matrix from Inbred Mouse Strains: Unraveling Potential New Players in Enamel.

Enamel formation is a complex 2-step process by which proteins are secreted to form an extracellular matrix, followed by massive protein degradation and subsequent mineralization. Excessive systemic exposure to fluoride can disrupt this process and lead to a condition known as dental fluorosis. The genetic background influences the responses of mineralized tissues to fluoride, such as dental fluorosis, observed in A/J and 129P3/J mice. The aim of the present study was to map the protein profile of enamel matrix from A/J and 129P3/J strains. Enamel matrix samples were obtained from A/J and 129P3/J mice and analyzed by 2-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry. A total of 120 proteins were identified, and 7 of them were classified as putative uncharacterized proteins and analyzed in silico for structural and functional characterization. An interesting finding was the possibility of the uncharacterized sequence Q8BIS2 being an enzyme involved in the degradation of matrix proteins. Thus, the results provide a comprehensive view of the structure and function for putative uncharacterized proteins found in the enamel matrix that could help to elucidate the mechanisms involved in enamel biomineralization and genetic susceptibility to dental fluorosis.

4-phenylbutyrate Mitigates Fluoride-Induced Cytotoxicity in ALC Cells.

Chronic fluoride over-exposure during pre-eruptive enamel development can cause dental fluorosis. Severe dental fluorosis is characterized by porous, soft enamel that is vulnerable to erosion and decay. The prevalence of dental fluorosis among the population in the USA, India and China is increasing. Other than avoiding excessive intake, treatments to prevent dental fluorosis remain unknown. We previously reported that high-dose fluoride induces endoplasmic reticulum (ER) stress and oxidative stress in ameloblasts. Cell stress induces gene repression, mitochondrial damage and apoptosis. An aromatic fatty acid, 4-phenylbutyrate (4PBA) is a chemical chaperone that interacts with misfolded proteins to prevent ER stress. We hypothesized that 4PBA ameliorates fluoride-induced ER stress in ameloblasts. To determine whether 4PBA protects ameloblasts from fluoride toxicity, we analyzed gene expression of Tgf-ß1, Bcl2/Bax ratio and cytochrome-c release in vitro. In vivo, we measured fluorosis levels, enamel hardness and fluoride concentration. Fluoride treated Ameloblast-lineage cells (ALC) had decreased Tgf-ß1 expression and this was reversed by 4PBA treatment. The anti-apoptotic Blc2/Bax ratio was significantly increased in ALC cells treated with fluoride/4PBA compared to fluoride treatment alone. Fluoride treatment induced cytochrome-c release from mitochondria into the cytosol and this was inhibited by 4PBA treatment. These results suggest that 4PBA mitigates fluoride-induced gene suppression, apoptosis and mitochondrial damage in vitro. In vivo, C57BL/6J mice were provided fluoridated water for six weeks with either fluoride free control-chow or 4PBA-containing chow (7 g/kg 4PBA). With few exceptions, enamel microhardness, fluorosis levels, and fluoride concentrations of bone and urine did not differ significantly between fluoride treated animals fed with control-chow or 4PBA-chow. Although 4PBA mitigated high-dose fluoride toxicity in vitro, a diet rich in 4PBA did not attenuate dental fluorosis in rodents. Perhaps, not enough intact 4PBA reaches the rodent ameloblasts necessary to reverse the effects of fluoride toxicity. Further studies will be required to optimize protocols for 4PBA administration in vivo in order to evaluate the effect of 4PBA on dental fluorosis.

Proteomics of Secretory-Stage and Maturation-Stage Enamel of Genetically Distinct Mice.

The mechanisms by which excessive ingestion of fluoride (F) during amelogenesis leads to dental fluorosis (DF) are still not precisely known. Inbred strains of mice vary in their susceptibility to develop DF, and therefore permit the investigation of underlying molecular events influencing DF severity. We employed a proteomic approach to characterize and evaluate changes in protein expression from secretory-stage and maturation-stage enamel in 2 strains of mice with different susceptibilities to DF (A/J, i.e. 'susceptible' and 129P3/J, i.e. 'resistant'). Weanling male and female susceptible and resistant mice fed a low-F diet were divided into 2 F-water treatment groups. They received water containing 0 (control) or 50 mg F/l for 6 weeks. Plasma and incisor enamel was analyzed for F content. For proteomic analysis, the enamel proteins extracted for each group were separated by 2-dimensional electrophoresis and subsequently characterized by liquid-chromatography electrospray-ionization quadrupole time-of-flight mass spectrometry. F data were analyzed by 2-way ANOVA and Bonferroni's test (p < 0.05). Resistant mice had significantly higher plasma and enamel F concentrations when compared with susceptible mice in the F-treated groups. The proteomic results for mice treated with 0 mg F/l revealed that during the secretory stage, resistant mice had a higher abundance of proteins than their susceptible counterparts, but this was reversed during the maturation stage. Treatment with F greatly increased the number of protein spots detected in both stages. Many proteins not previously described in enamel (e.g. type 1 collagen) as well as some uncharacterized proteins were identified. Our findings reveal new insights regarding amelogenesis and how genetic background and F affect this process.

Ex vivo evaluation of new 2D and 3D dental radiographic technology for detecting caries.

OBJECTIVES: Proximal dental caries remains a prevalent disease with only modest detection rates by current diagnostic systems. Many new systems are available without controlled validation of diagnostic efficacy. The objective of this study was to evaluate the diagnostic efficacy of three potentially promising new imaging systems. METHODS: This study evaluated the caries detection efficacy of Schick 33 (Sirona Dental, Salzburg, Austria) intraoral digital detector images employing an advanced sharpening filter, Planmeca ProMax(®) (Planmeca Inc., Helsinki, Finland) extraoral "panoramic bitewing" images and Sirona Orthophos XG3D (Sirona Dental) CBCT images with advanced artefact reduction. Conventional photostimulable phosphor images served as the control modality. An ex vivo study design using extracted human teeth, ten expert observers and micro-CT ground truth was employed. RESULTS: Receiver operating characteristic analysis indicated similar diagnostic efficacy of all systems (ANOVA p > 0.05). The sensitivity of the Schick 33 images (0.48) was significantly lower than the other modalities (0.53-0.62). The specificity of the Planmeca images (0.86) was significantly lower than Schick 33 (0.96) and XG3D (0.97). The XG3D showed significantly better cavitation detection sensitivity (0.62) than the other modalities (0.48-0.57). CONCLUSIONS: The Schick 33 images demonstrated reduced caries sensitivity, whereas the Planmeca panoramic bitewing images demonstrated reduced specificity. XG3D with artefact reduction demonstrated elevated sensitivity and specificity for caries detection, improved depth accuracy and substantially improved cavitation detection. Care must be taken to recognize potential false-positive caries lesions with Planmeca panoramic bitewing images. Use of CBCT for caries detection must be carefully balanced with the presence of metal artefacts, time commitment, financial cost and radiation dose.

Fluoride Modulates Parathyroid Hormone Secretion in vivo and in vitro.

The study objective was to investigate the effects of fluoride on intact parathyroid hormone (iPTH) secretion. Thyro-parathyroid complexes (TPC) from C3H (n = 18) and B6 (n = 18) mice were cultured in Ca²âº-optimized medium. TPC were treated with 0, 250, or 500 µM NaF for 24 h and secreted iPTH assayed by ELISA. C3H (n = 78) and B6 (n = 78) mice were gavaged once with distilled or fluoride (0.001 mg [F⁻]/g of body weight) water. At serial time points (0.5-96 h) serum iPTH, fluoride, total calcium, phosphorus, and magnesium levels were determined. Expression of genes involved in mineral regulation via the bone-parathyroid-kidney (BPK) axis, such as parathyroid hormone (Pth), calcium-sensing receptor (Casr), vitamin D receptor (Vdr), parathyroid hormone-like hormone (Pthlh), fibroblast growth factor 23 (Fgf23), α-Klotho (αKlotho), fibroblast growth factor receptor 1c (Fgf1rc), tumor necrosis factor 11 (Tnfs11), parathyroid hormone receptor 1 (Pth1r), solute carrier family 34 member 1 (Slc34a1), solute carrier 9 member 3 regulator 1 (Slc9a3r1), chloride channel 5 (Clcn5), and PDZ domain-containing 1 (Pdzk1), was determined in TPC, humeri, and kidneys at 24 h. An in vitro decrease in iPTH was seen in C3H and B6 TPC at 500 µM (p < 0.001). In vivo levels of serum fluoride peaked at 0.5 h in both C3H (p = 0.002) and B6 (p = 0.01). In C3H, iPTH decreased at 24 h (p < 0.0001), returning to baseline at 48 h. In B6, iPTH increased at 12 h (p < 0.001), returning to baseline at 24 h. Serum total calcium, phosphorus, and magnesium levels did not change significantly. Pth, Casr,αKlotho,Fgf1rc,Vdr, and Pthlh were significantly upregulated in C3H TPC compared to B6. In conclusion, the effects of fluoride on TPC in vitro were equivalent between the 2 mouse strains. However, fluoride demonstrated an early strain-dependent effect on iPTH secretion in vivo. Both strains demonstrated differences in the expression of genes involved in the BPK axis, suggesting a possible role in the physiologic handling of fluoride.

Bone response to fluoride exposure is influenced by genetics.

Genetic factors influence the effects of fluoride (F) on amelogenesis and bone homeostasis but the underlying molecular mechanisms remain undefined. A label-free proteomics approach was employed to identify and evaluate changes in bone protein expression in two mouse strains having different susceptibilities to develop dental fluorosis and to alter bone quality. In vivo bone formation and histomorphometry after F intake were also evaluated and related to the proteome. Resistant 129P3/J and susceptible A/J mice were assigned to three groups given low-F food and water containing 0, 10 or 50 ppmF for 8 weeks. Plasma was evaluated for alkaline phosphatase activity. Femurs, tibiae and lumbar vertebrae were evaluated using micro-CT analysis and mineral apposition rate (MAR) was measured in cortical bone. For quantitative proteomic analysis, bone proteins were extracted and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free semi-quantitative differential expression analysis. Alterations in several bone proteins were found among the F treatment groups within each mouse strain and between the strains for each F treatment group (ratio ≥1.5 or ≤0.5; p<0.05). Although F treatment had no significant effects on BMD or bone histomorphometry in either strain, MAR was higher in the 50 ppmF 129P3/J mice than in the 50 ppmF A/J mice treated with 50 ppmF showing that F increased bone formation in a strain-specific manner. Also, F exposure was associated with dose-specific and strain-specific alterations in expression of proteins involved in osteogenesis and osteoclastogenesis. In conclusion, our findings confirm a genetic influence in bone response to F exposure and point to several proteins that may act as targets for the differential F responses in this tissue.

Uncoupling protein-2 is an antioxidant that is up-regulated in the enamel organ of fluoride-treated rats.

Dental fluorosis is characterized by subsurface hypomineralization and retention of enamel matrix proteins. Fluoride (F(-)) exposure generates reactive oxygen species (ROS) that can cause endoplasmic reticulum (ER)-stress. We therefore screened oxidative stress arrays to identify genes regulated by F(-) exposure. Vitamin E is an antioxidant so we asked if a diet high in vitamin E would attenuate dental fluorosis. Maturation stage incisor enamel organs (EO) were harvested from F(-)-treated rats and mice were assessed to determine if vitamin E ameliorates dental fluorosis. Uncoupling protein-2 (Ucp2) was significantly up-regulated by F(-) (∼1.5 & 2.0 fold for the 50 or 100 ppm F(-) treatment groups, respectively). Immunohistochemical results on maturation stage rat incisors demonstrated that UCP2 protein levels increased with F(-) treatment. UCP2 down-regulates mitochondrial production of ROS, which decreases ATP production. Thus, in addition to reduced protein translation caused by ER-stress, a reduction in ATP production by UCP2 may contribute to the inability of ameloblasts to remove protein from the hardening enamel. Fluoride-treated mouse enamel had significantly higher quantitative fluorescence (QF) than the untreated controls. No significant QF difference was observed between control and vitamin E-enriched diets within a given F(-) treatment group. Therefore, a diet rich in vitamin E did not attenuate dental fluorosis. We have identified a novel oxidative stress response gene that is up-regulated in vivo by F(-) and activation of this gene may adversely affect ameloblast function.

Ameloblasts require active RhoA to generate normal dental enamel.

RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up-regulated. Transgenic mice were generated that express a dominant-negative RhoA transgene in ameloblasts using amelogenin gene-regulatory sequences. Transgenic and wild-type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F-actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho-associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E-cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E-cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited.

Renal proteome in mice with different susceptibilities to fluorosis.

A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis due to their genetic backgrounds. They also differ with respect to several features of fluoride (F) metabolism and metabolic handling of water. This study was done to determine whether differences in F metabolism could be explained by diversities in the profile of protein expression in kidneys. Weanling, male A/J mice (susceptible to dental fluorosis, n = 18) and 129P3/J mice (resistant, n = 18) were housed in pairs and assigned to three groups given low-F food and drinking water containing 0, 10 or 50 ppm [F] for 7 weeks. Renal proteome profiles were examined using 2D-PAGE and LC-MS/MS. Quantitative intensity analysis detected between A/J and 129P3/J strains 122, 126 and 134 spots differentially expressed in the groups receiving 0, 10 and 50 ppmF, respectively. From these, 25, 30 and 32, respectively, were successfully identified. Most of the proteins were related to metabolic and cellular processes, followed by response to stimuli, development and regulation of cellular processes. In F-treated groups, PDZK-1, a protein involved in the regulation of renal tubular reabsorption capacity was down-modulated in the kidney of 129P3/J mice. A/J and 129P3/J mice exhibited 11 and 3 exclusive proteins, respectively, regardless of F exposure. In conclusion, proteomic analysis was able to identify proteins potentially involved in metabolic handling of F and water that are differentially expressed or even not expressed in the strains evaluated. This can contribute to understanding the molecular mechanisms underlying genetic susceptibility to dental fluorosis, by indicating key-proteins that should be better addressed in future studies.

Phenotypic variation of fluoride responses between inbred strains of mice.

Excessive systemic exposure to fluoride (F) can lead to disturbances in bone homeostasis and dental enamel development. We have previously shown strain-specific responses to F in the development of dental fluorosis (DF) and in bone formation/mineralization. The current study was undertaken to further investigate F responsive variations in bone metabolism and to determine possible relationships with DF susceptibility. Seven-week-old male mice from FVB/NJ, C57BL/6J, C3H/HeJ, A/J, 129S1/SvImJ, AKR/J, DBA/2J, and BALB/cByJ inbred strains were exposed to NaF (0 or 50 ppm as F(-)) in drinking water for 60 days. Sera were collected for F, Ca, Mg, PO(4), iPTH, sRANKL, and ALP levels. Bone marrow cells were subjected to ex vivo cell culture for osteoclast potential and CFU colony assays (CFU-fibroblast, CFU-osteoblast, CFU-erythrocyte/granulocyte/macrophage/megakaryocyte, CFU-granulocyte/macrophage, CFU-macrophage, and CFU-granulocyte). Femurs and vertebrae were subjected to micro-CT analyses, biomechanical testing, and F, Mg, and Ca content assays. DF was evaluated using quantitative fluorescence and clinical criteria. Strain-specific responses to F were observed for DF, serum studies, ex vivo cell culture studies, and bone quality. Among the strains, there were no patterns or significant correlations between DF severity and the actions of F on bone homeostasis (serum studies, ex vivo assays, or bone quality parameters). The genetic background continues to play a role in the actions of F on tooth enamel development and bone homeostasis. F exposure led to variable phenotypic responses between strains involving dental enamel development and bone metabolism.

Fine mapping of dental fluorosis quantitative trait loci in mice.

Genetic factors underlie the susceptibility and the resistance to dental fluorosis (DF). The A/J (DF susceptible) and 129P3/J (DF resistant) mouse strains have previously been used to detect quantitative trait loci (QTLs) associated with DF on chromosome (Chr) 2 and Chr 11. In the present study, increased marker density genotyping followed by interval mapping was performed to narrow the QTL intervals and improve the logarithm of the odds (to the base 10) (LOD) scores. Narrower intervals were obtained on Chr 2 where LOD ≥ 6.0 (57-84 cM or ≈ 51 Mb), LOD ≥ 7.0 (62-79 cM or ≈ 32 Mb), and LOD ≥ 8.0 (65-74 cM or ≈ 17 Mb); and on Chr 11 where LOD ≥ 6.0 (18-51 cM or ≈ 53 Mb), LOD ≥ 7.0 (28-48 cM or ≈ 34 Mb), and LOD ≥ 8.0 (31-45 cM or ≈ 22 Mb). Haplotype analysis between A/J and 129P3/J mice further reduced the QTL intervals. Accn1 was selected as a candidate gene based upon its location near the peak LOD score on Chr 11 and distant homology with the Caenorhabditis elegans fluoride-resistance gene, flr1. The severity of DF between Accn1(-/-) and wild-type mice was not significantly different. Hence, the loss of ACCN1 function does not modify DF severity in mice. Narrowing the DF QTL intervals will facilitate additional candidate gene selections and interrogation.

Disruption of the murine Ap2ß1 gene causes nonsyndromic cleft palate.

Development of the secondary palate in mammals is a complex process that can be easily perturbed, leading to the common and distressing birth defect cleft palate. Animal models are particularly useful tools for dissecting underlying genetic components of cleft palate. We describe a new cleft palate model resulting from a transgene insertion mutation. Transgene insertional mutagenesis disrupts the genomic organization and expression of the Ap2ß1 gene located on chromosome 11. This gene encodes the ß2-adaptin subunit of the heterotetrameric adaptor protein 2 complex involved in clathrin-dependent endocytosis. Homozygous cleft palate mutant mice express no Ap2ß1 messenger RNA or ß2-adaptin protein and die during the perinatal period. Heterozygous mice are phenotypically normal despite expressing diminished ß2-adaptin messenger RNA and protein compared with wildtype. Remarkably, the paralogous ß1-adaptin subunit of the adaptor protein 1 complex partially substitutes for the missing ß2-adaptin in embryonic fibroblasts from homozygous mutant mice, resulting in assembly of reduced levels of an adaptor protein 2 complex bearing ß1-adaptin. This variant adaptor protein 2 complex is, therefore, apparently capable of maintaining viability of the homozygous mutant embryos until birth but insufficient to support palatogenesis. Nonsyndromic cleft palate in an animal model is associated with disruption of the Ap2ß1 gene.

Putative stem cells in human dental pulp with irreversible pulpitis: an exploratory study.

INTRODUCTION: Although human dental pulp stem cells isolated from healthy teeth have been extensively characterized, it is unknown whether stem cells also exist in clinically compromised teeth with irreversible pulpitis. Here we explored whether cells retrieved from clinically compromised dental pulp have stem cell-like properties. METHODS: Pulp cells were isolated from healthy teeth (control group) and from teeth with clinically diagnosed irreversible pulpitis (diseased group). Cell proliferation, stem cell marker STRO-1 expression, and cell odonto-osteogenic differentiation competence were compared. RESULTS: Cells from the diseased group demonstrated decreased colony formation capacity and a slightly decreased cell proliferation rate, but they had similar STRO-1 expression and exhibited a similar percentage of positive ex vivo osteogenic induction and dentin sialophosphoprotein expression from STRO-1-enriched pulp cells. CONCLUSIONS: Our study provides preliminary evidence that clinically compromised dental pulp might contain putative cells with certain stem cell properties. Further characterization of these cells will provide insight regarding whether they could serve as a source of endogenous multipotent cells in tissue regeneration-based dental pulp therapy.

Detection of dental fluorosis-associated quantitative trait Loci on mouse chromosomes 2 and 11.

Systemic exposure to greater than optimal fluoride (F) can lead to dental fluorosis (DF). Parental A/J (DF-susceptible) and 129P3/J (DF-resistant) inbred mice were used for histological studies and to generate F2 progeny. Mice were treated with 0 or 50 ppm F in their drinking water for 60 days. A clinical criterion (modified Thylstrup and Fejerskov categorical scale) was used to assess the severity of DF for each individual F2 animal. Parental strains were subjected to histological examination of maturing enamel. F treatment resulted in accumulation of amelogenins in the maturing enamel of A/J mice. Quantitative trait loci (QTL) detection was performed using phenotypic extreme F2 animals genotyped for 354 single nucleotide polymorphism-based markers distributed throughout the mouse genome followed by chi(2) analysis. Significant evidence of association was observed on chromosomes 2 and 11 for a series of consecutive markers (p < 0.0001). Further analyses were performed to examine whether the phenotypic effects were found in both male and female F2 mice or whether there was evidence for gender-specific effects. Analyses performed using the markers on chromosomes 2 and 11 which were significant in the mixed-gender mice were also significant when analyses were limited to only the male or female mice. The QTL detected on chromosomes 2 and 11 which influence the variation in response to fluorosis have their effect in mice of both genders. Finally, the QTL in both chromosomes appear to have an additive effect.

Inheritance of susceptibility to root resorption associated with orthodontic force in mice.

INTRODUCTION: External apical root resorption (EARR) is an unwanted sequelae of orthodontic treatment. Genetic factors account for approximately 64% of the EARR variation in humans. Inbred mice offer a model to control the environmental factors and genetic heterogeneity that complicate human genetic studies. Genetically distinct inbred mice and their offspring (F1s) were analyzed to examine the mode of inheritance and the influence of parental sex on the susceptibility to root resorption associated with orthodontic force (RRAOF). METHODS: RRAOF was determined histologically for male and female mice of the A/J, DBA/2J, and BALB/cJ strains, and the A/JxDBA/2J and A/JxBALB/cJ crosses (10 males and 10 females/reciprocal cross). RRAOF was induced by tipping the maxillary first molar mesially for 9 days. RESULTS: Sex differences were observed only among the mice of the BALB/cJ strain. Two patterns of inheritance were observed; F1s from the A/JxBALB/cJ cross were resistant, suggesting that the A/J have dominant resistance alleles. On the other hand, F1s from the A/JxDBA/2J cross showed RRAOF intermediate between their parental mice, suggesting a polygenic trait. CONCLUSIONS: These results provide evidence of a traceable and polygenetic component affecting RRAOF in mice.

The AP-2 adaptor beta2 appendage scaffolds alternate cargo endocytosis.

The independently folded appendages of the large alpha and beta2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The beta2 subunit appendage contains a common binding site for beta-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing beta2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 alpha subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the beta2 chain with the closely related endogenous beta1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both beta1 and beta2 subunit transcripts recapitulates the strong alpha subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive beta2-yellow fluorescent protein (YFP) expressed in the beta1 + beta2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the beta appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a beta2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a beta-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with beta2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and beta-arrestin binding to a site upon the beta2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and beta-arrestin depend on a privileged beta2 appendage site for proper cargo recruitment to clathrin bud sites.

Genetic background influences fluoride's effects on osteoclastogenesis.

Excessive fluoride (F) can lead to abnormal bone biology. Numerous studies have focused on the anabolic action of F yet little is known regarding any action on osteoclastogenesis. Little is known regarding the influence of an individual's genetic background on the responses of bone cells to F. Four-week old C57BL/6J (B6) and C3H/HeJ (C3H) female mice were treated with NaF in the drinking water (0 ppm, 50 ppm and 100 ppm F ion) for 3 weeks. Bone marrow cells were harvested for osteoclastogenesis and hematopoietic colony-forming cell assays. Sera were analyzed for biochemical and bone markers. Femurs, tibiae, and lumbar vertebrae were subjected to microCT analysis. Tibiae and femurs were subjected to histology and biomechanical testing, respectively. The results demonstrated new actions of F on osteoclastogenesis and hematopoietic cell differentiation. Strain-specific responses were observed. The anabolic action of F was favored in B6 mice exhibiting dose-dependent increases in serum ALP activity (p<0.001); in proximal tibia trabecular and vertebral BMD (tibia at 50&100 ppm, p=0.001; vertebrae at 50 and 100 ppm, p=0.023&0.019, respectively); and decrease in intact PTH and sRANKL (p=0.045 and p<0.001, respectively). F treatment in B6 mice also resulted in increased numbers of CFU-GEMM colonies (p=0.025). Strain-specific accumulations in bone [F] were observed. For C3H mice, dose-dependent increases were observed in osteoclast potential (p<0.001), in situ trabecular osteoclast number (p=0.007), hematopoietic colony forming units (CFU-GEMM: p<0.001, CFU-GM: p=0.006, CFU-M: p<0.001), and serum markers for osteoclastogenesis (intact PTH: p=0.004, RANKL: p=0.022, TRAP5b: p<0.001). A concordant decrease in serum OPG (p=0.005) was also observed. Fluoride treatment had no significant effects on bone morphology, BMD, and serum PYD cross-links in C3H suggesting a lack of significant bone resorption. Mechanical properties were also unaltered in C3H. In conclusion, short term F treatment at physiological levels has strain-specific effects in mice. The expected anabolic effects were observed in B6 and novel actions hallmarked by enhanced osteoclastogenesis shifts in hematopoietic cell differentiation in the C3H strain.