NaF Activates MAPKs NaF Activates MAPKs and Induces Apoptosis and Induces Apoptosis

in Odontoblast-like Cellsin Odontoblast-like Cells

H. Karube, G. Nishitai, K. Inageda, H. Karube, G. Nishitai, K. Inageda, H.Kurosu and M. MatsuokaH.Kurosu and M. Matsuoka

Grace Ellen S. Dey, DMDGrace Ellen S. Dey, DMD

AIMAIM

The cytotoxic effects of fluoride on The cytotoxic effects of fluoride on odontoblasts are not clear. In this study, the odontoblasts are not clear. In this study, the researchers wanted to examine whether NaF researchers wanted to examine whether NaF induces apoptosis in MDPC-23 odontoblast-like induces apoptosis in MDPC-23 odontoblast-like cells and determine its involvement in mitogen-cells and determine its involvement in mitogen-activated protein kinase (MAPK) signaling activated protein kinase (MAPK) signaling pathways in NaF-induced apoptosis.pathways in NaF-induced apoptosis.

IntroductionIntroduction

Fluoride plays a key role in the prevention Fluoride plays a key role in the prevention and control of dental caries. However, excessive and control of dental caries. However, excessive exposure of teeth to fluoride produces adverse exposure of teeth to fluoride produces adverse effects such as fluorosis.effects such as fluorosis.

Odontoblasts, which are responsible for Odontoblasts, which are responsible for dentin matrix formation, are derived from the dentin matrix formation, are derived from the ectomesenchymal cells of the dental papilla and ectomesenchymal cells of the dental papilla and are located at the periphery of the dental pulp.are located at the periphery of the dental pulp.

Review of Related LiteratureReview of Related Literature

It has been reported that administration of It has been reported that administration of sodium fluoride (NaF) causes ultrastructural sodium fluoride (NaF) causes ultrastructural changes in rat incisor odontoblasts and dentin changes in rat incisor odontoblasts and dentin (Araki,1989).(Araki,1989).

Chen and Huang in 1994 observed in the Chen and Huang in 1994 observed in the

pulp of rabbits administered with NaF, had pulp of rabbits administered with NaF, had shown histopathologic alterations such as the shown histopathologic alterations such as the loss of palisading odontoblasts. loss of palisading odontoblasts.

In another study done by Moseley et al. in 2003, In another study done by Moseley et al. in 2003, they found no clear morphological changes detected in they found no clear morphological changes detected in the dentin-pulp complex of cultured tooth sections the dentin-pulp complex of cultured tooth sections from rat incisors instead there was collagen synthesis from rat incisors instead there was collagen synthesis suppression. suppression.

The above findings suggest that NaF might cause The above findings suggest that NaF might cause damage to odontoblast as well as ameloblasts.damage to odontoblast as well as ameloblasts.

Treatment with NaF has been reported to induce Treatment with NaF has been reported to induce apoptotic cell death in zebrafish inner enamel apoptotic cell death in zebrafish inner enamel epithelium cells (Bartlett et al., 2005), mouse epithelium cells (Bartlett et al., 2005), mouse ameloblast-derived cells (Kubota et al., 2005) and ameloblast-derived cells (Kubota et al., 2005) and ameloblast-lineage cells isolated from the human ameloblast-lineage cells isolated from the human enamel organ (Yan et al., 2007).enamel organ (Yan et al., 2007).

To our knowledge, however, the potential of NaF To our knowledge, however, the potential of NaF

to induce apoptosis in odontoblasts has not been to induce apoptosis in odontoblasts has not been demonstrated.demonstrated.

Definition of terms 1Definition of terms 1

MAPK (mitogen-activated protein kinase)MAPK (mitogen-activated protein kinase)

is serine/threonine-specific protein kinase is serine/threonine-specific protein kinase

that respond to extracellular stimuli that respond to extracellular stimuli

(mitogens) and regulates various cellular(mitogens) and regulates various cellular

activities, such as gene expression, mitosis, activities, such as gene expression, mitosis,

differentiation, proliferation, and cell differentiation, proliferation, and cell

survival/apoptosis.survival/apoptosis.

KinaseKinase is alternatively known as a phosphotransferase. In is alternatively known as a phosphotransferase. In chemistry and biochemistry, it is a type of enzyme that chemistry and biochemistry, it is a type of enzyme that transfers phosphate groups from high-energy donor transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates. The molecules, such as ATP, to specific substrates. The process is referred to as phosphorylation. process is referred to as phosphorylation.

Caspase 3Caspase 3 is a gene that encodes a protein that is a gene that encodes a protein that is a member of the cysteine-aspartic acid is a member of the cysteine-aspartic acid protease (caspase) family. Sequential protease (caspase) family. Sequential activation of caspases plays a central role in activation of caspases plays a central role in the execution-phase of cell apoptosis. the execution-phase of cell apoptosis.

Poly (ADP-ribose) polymerasePoly (ADP-ribose) polymerase [ [PARP]PARP] is a protein involved in a number of cellular is a protein involved in a number of cellular processes involving mainly DNA repair and processes involving mainly DNA repair and programmed cell death. programmed cell death. Role: PARP can deplete the ATP of a cell in Role: PARP can deplete the ATP of a cell in an attempt to repair the damaged DNA. an attempt to repair the damaged DNA. ATP depletion in a cell leads to lysis and cell ATP depletion in a cell leads to lysis and cell death.death.

DNA FragmentationDNA Fragmentation is a key feature in is a key feature in programmed cell death and also occurs in certain programmed cell death and also occurs in certain stages of necrosis. Apoptosis is characterized by stages of necrosis. Apoptosis is characterized by the activation of endogenous endonucleases with the activation of endogenous endonucleases with subsequent cleavage of chromatin DNA into subsequent cleavage of chromatin DNA into internucleosomal fragments of 180 BP and internucleosomal fragments of 180 BP and multiples thereof.multiples thereof.

Cytoplasmic NucleosomesCytoplasmic Nucleosomes are complex structures are complex structures

comprised of DNA wound around a multi- comprised of DNA wound around a multi-

subunit core and associated proteins, which subunit core and associated proteins, which

forms the primary packing unit of DNA in the forms the primary packing unit of DNA in the

cytoplasm into higher order structures. An early cytoplasm into higher order structures. An early

event in apoptosis, DNA fragmentation occurs event in apoptosis, DNA fragmentation occurs

followed by the release of nucleosomes in the followed by the release of nucleosomes in the

cytoplasm. cytoplasm.

In a study conducted by Hanks et al. in 1998, In a study conducted by Hanks et al. in 1998,

they assessed the MDPC-23 mouse they assessed the MDPC-23 mouse odontoblast-like cells, which are derived from odontoblast-like cells, which are derived from dental papillae of CD-1 mice after 18-19 days dental papillae of CD-1 mice after 18-19 days of fetal life were treated with NaF and of fetal life were treated with NaF and apoptosis using various apoptotic indices, apoptosis using various apoptotic indices, namely: caspase-3 activity, cleavage of poly namely: caspase-3 activity, cleavage of poly (ADP-ribose) polymerase [PARP], DNA (ADP-ribose) polymerase [PARP], DNA fragmentation and cytoplasmic nucleosomes. fragmentation and cytoplasmic nucleosomes.

MAPKs are known to play important roles in cellular MAPKs are known to play important roles in cellular responses, including apoptosis, to various extracellular responses, including apoptosis, to various extracellular stimuli. stimuli.

So, to clarify the signaling pathways leading to So, to clarify the signaling pathways leading to apoptosis, the researchers examined the phosphorylation apoptosis, the researchers examined the phosphorylation status of MAPKs, c-Jun NH2-terminal kinase (JNK), p38 status of MAPKs, c-Jun NH2-terminal kinase (JNK), p38 and extracellular signal-regulated protein kinase (ERK) in and extracellular signal-regulated protein kinase (ERK) in MDPC-23 cells treated with NaF. MDPC-23 cells treated with NaF.

In addition, functional evidence for the possible In addition, functional evidence for the possible involvement of these pathways in NaF-induced apoptosis involvement of these pathways in NaF-induced apoptosis was examined with inhibitors of each member of the was examined with inhibitors of each member of the MAPK family.MAPK family.

Materials and MethodsMaterials and Methods

Chemicals used in the study were as follows:Chemicals used in the study were as follows:2.2. Sodium FluorideSodium Fluoride3.3. SP 600125, SB203580 and U0126SP 600125, SB203580 and U0126

4.4. Antibodies against PARP, phospho- SAPK/JNK, Antibodies against PARP, phospho- SAPK/JNK, phospho-p38, p38, phospho-MK-2, phospho-p44/42 phospho-p38, p38, phospho-MK-2, phospho-p44/42 MAPK (ERK) and ERKMAPK (ERK) and ERK

5.5. Actin antibodyActin antibody6.6. Cell Count Reagent SFCell Count Reagent SF

7.7. Acetyl-Asp-Glu-Val-AspAcetyl-Asp-Glu-Val-Asp

The Methods used to determine apoptosis, The Methods used to determine apoptosis, phosphorylation of MAPKs by NaF exposure and phosphorylation of MAPKs by NaF exposure and the involvement of MAPKs in NaF-induced the involvement of MAPKs in NaF-induced apoptosis were WST-8 assay, DNA fragmentation apoptosis were WST-8 assay, DNA fragmentation assay, nucleosome assay and Western assay, nucleosome assay and Western Immunoblotting. Only Immunoblotting. Only in vitroin vitro experiments were experiments were done with the MDPC-23 cell line.done with the MDPC-23 cell line.

Cell Culture and TreatmentsCell Culture and Treatments

MDPC-23 cells were grown in MDPC-23 cells were grown in ααMEM medium MEM medium supplemented with 10% heat-inactivated fetal supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin bovine serum (FBS), 100 units/mL penicillin and 100ug/ mL streptomycin in a humidified and 100ug/ mL streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37˚C.atmosphere of 5% CO2 and 95% air at 37˚C.

Cells were incubated with serum-free medium Cells were incubated with serum-free medium containing the indicated concentration of NaF containing the indicated concentration of NaF for 0.5 -24 hrs at 37˚C. SP 600125, SB203580 for 0.5 -24 hrs at 37˚C. SP 600125, SB203580 and UO126 were dissolved in dimethyl and UO126 were dissolved in dimethyl sulfoxide (DMSO). Unless otherwise indicated, sulfoxide (DMSO). Unless otherwise indicated, cells were pre-incubated with serum-free cells were pre-incubated with serum-free medium containing DMSO (0.2%) or each medium containing DMSO (0.2%) or each compound for 1 hour and then treated with compound for 1 hour and then treated with 5mM NaF for 24 hours.5mM NaF for 24 hours.

RESULTSRESULTS

Induction of Apoptosis by NaF Induction of Apoptosis by NaF exposure in MDPC-23 cellsexposure in MDPC-23 cells

Effects of NaF exposure on Effects of NaF exposure on

MDPC-23 cell viabilityMDPC-23 cell viability

Cells were incubated with 0, Cells were incubated with 0, 2, 5 or 10 mM NaF for 4, 2, 5 or 10 mM NaF for 4, 8,16 or 24 hours and cell was 8,16 or 24 hours and cell was determined by WST-8 assays. determined by WST-8 assays. Incubation of MDPC-23 Incubation of MDPC-23 cells with NaF for 4, 8, 16 or cells with NaF for 4, 8, 16 or 24 hrs induced a dose-24 hrs induced a dose-dependent decrease in cell dependent decrease in cell viability. Following exposure viability. Following exposure to 5mM NaF for 24 hrs, cell to 5mM NaF for 24 hrs, cell viability was reduced at viability was reduced at approximately 50%. approximately 50%.

Induction of Apoptosis by NaF Induction of Apoptosis by NaF exposure in MDPC-23 cellsexposure in MDPC-23 cells

NaF- induced apoptosis in MDPC-23 NaF- induced apoptosis in MDPC-23 cells showing caspase-3 activitycells showing caspase-3 activity

Cells were incubated with 0, Cells were incubated with 0, 2 or 5 mM NaF for 24 2 or 5 mM NaF for 24 hrs and caspase-3 hrs and caspase-3 activity. Incubation for activity. Incubation for 24 hrs with 2 and 5 mM 24 hrs with 2 and 5 mM NaF increased caspase-3 NaF increased caspase-3 activity by 1.5 and 2.7 activity by 1.5 and 2.7 fold respectively.fold respectively.

Induction of Apoptosis by NaF Induction of Apoptosis by NaF exposure in MDPC-23 cellsexposure in MDPC-23 cells

NaF- induced apoptosis in NaF- induced apoptosis in MDPC-23 cells showing MDPC-23 cells showing PARP cleavage.PARP cleavage.

Western blot analysis Western blot analysis showed that PARP, one of showed that PARP, one of the substrates of caspase-3, the substrates of caspase-3, was cleaved into 89-kDa was cleaved into 89-kDa fragments in cells exposed fragments in cells exposed to 5mM NaF. The to 5mM NaF. The abundance of actin served abundance of actin served as a loading control. as a loading control.

Induction of Apoptosis by NaF Induction of Apoptosis by NaF exposure in MDPC-23 cellsexposure in MDPC-23 cells

Agarose gel Agarose gel electrophoresis showed electrophoresis showed that incubation with that incubation with 5mM NaF-induced 5mM NaF-induced internucleosomal DNA internucleosomal DNA fragmentation.fragmentation.

Definition of terms 2Definition of terms 2

AgaroseAgarose is a polysaccharide extracted from seaweed. It is is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. higher the agarose concentration the "stiffer" the gel.

ElectrophoresisElectrophoresis is a technique used to separate and is a technique used to separate and sometimes purify macromolecules - especially proteins sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology. used techniques in biochemistry and molecular biology.

Induction of Apoptosis by NaF Induction of Apoptosis by NaF exposure in MDPC-23 cellsexposure in MDPC-23 cells

Fold-increase of cytoplasmic nucleosomes Fold-increase of cytoplasmic nucleosomes

relative to untreated controlrelative to untreated control

The level of cytoplasmic The level of cytoplasmic nucleosomes increased nucleosomes increased by 12.7-fold in cells by 12.7-fold in cells exposed to 5mM NaF.exposed to 5mM NaF.

Phosphorylation of MAPKs by NaF Phosphorylation of MAPKs by NaF exposureexposure

Time-course of NaF-induced accumulation Time-course of NaF-induced accumulation

of phosphorylated JNKof phosphorylated JNK

In MDPC-23 cells In MDPC-23 cells treated with 5mM NaF, treated with 5mM NaF, the level of the level of phosphorylated JNK phosphorylated JNK (p46 and p54) increased (p46 and p54) increased after 1 hr, peaked at 2 after 1 hr, peaked at 2 hrs, and then declined hrs, and then declined slightly.slightly.

Definition of Terms 3Definition of Terms 3

PhosphorylationPhosphorylation is the addition of a phosphate (PO4) is the addition of a phosphate (PO4) group to a protein or other organic molecule. Protein group to a protein or other organic molecule. Protein phosphorylation in particular plays a significant role in a phosphorylation in particular plays a significant role in a wide range of cellular processes. wide range of cellular processes.

c-Jun N-terminal kinasesc-Jun N-terminal kinases (JNKs) (JNKs) are mitogen-activated are mitogen-activated protein kinases which are responsive to stress stimuli, protein kinases which are responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and are involved in T cell and osmotic shock, and are involved in T cell differentiation and apoptosis.differentiation and apoptosis.

Phosphorylation of MAPKs by NaF Phosphorylation of MAPKs by NaF exposureexposure

Time-course of NaF-induced accumulation Time-course of NaF-induced accumulation

of phosphorylated p38of phosphorylated p38

The level of The level of phosphorylated p38 phosphorylated p38 increased after 1 hr, increased after 1 hr, peaked at 4 or 6 hrs, and peaked at 4 or 6 hrs, and remained elevated at 24 remained elevated at 24 hrs.hrs.

Phosphorylation of MAPKs by NaF Phosphorylation of MAPKs by NaF exposureexposure

Time-course of NaF-induced Time-course of NaF-induced

accumulation of accumulation of

phosphorylated ERKphosphorylated ERK

The level of phosphorylated ERK The level of phosphorylated ERK increased after 30 min and returned to increased after 30 min and returned to control level at 2-6 hrs (initial peak). control level at 2-6 hrs (initial peak). Eight hours after NaF addition, the Eight hours after NaF addition, the levels of phosphorylated ERK levels of phosphorylated ERK increased again, becoming more increased again, becoming more markedly elevated as the duration of markedly elevated as the duration of incubation increased (secondary peak). incubation increased (secondary peak). During the 24 –hour incubation period During the 24 –hour incubation period without NaF exposure, less marked without NaF exposure, less marked levels of phosphorylated ERK were levels of phosphorylated ERK were found after 8 hrs.found after 8 hrs.

Effects of MAPK inhibitors on NaF-Effects of MAPK inhibitors on NaF-induced apoptosis in MDPC-23 cellsinduced apoptosis in MDPC-23 cells

In MDPC-23 cells exposed In MDPC-23 cells exposed to 5mM NaF for 24 hrs, to 5mM NaF for 24 hrs, treatment with JNK treatment with JNK inhibitor, SP600125 (5 and inhibitor, SP600125 (5 and 10 uM), suppressed JNK 10 uM), suppressed JNK phosphorylation and phosphorylation and reduced cytoplasmic reduced cytoplasmic nucleosome levels.nucleosome levels.

Effects of MAPK inhibitors on NaF-Effects of MAPK inhibitors on NaF-induced apoptosis in MPDC-23 cellsinduced apoptosis in MPDC-23 cells

While treatment with the While treatment with the p38 inhibitor, SB203580 p38 inhibitor, SB203580 (10 and 20uM), (10 and 20uM), suppressed the suppressed the phosphorylation of phosphorylation of MAPK-activated protein MAPK-activated protein kinase-2 (MK-2), a direct kinase-2 (MK-2), a direct target of p38, but failed target of p38, but failed to change nucleosome to change nucleosome levels significantly.levels significantly.

Effects of MAPK inhibitors on NaF-Effects of MAPK inhibitors on NaF-induced apoptosis in MPDC-23 cellsinduced apoptosis in MPDC-23 cells

In contrast, treatment In contrast, treatment with U0126 (10 and with U0126 (10 and 20uM), an inhibitor of 20uM), an inhibitor of both activated and non-both activated and non-activated forms of activated forms of MAPK/ERK kinase MAPK/ERK kinase (MEK1/2), suppressed (MEK1/2), suppressed ERK phosphorylation ERK phosphorylation and reduced nucleosome and reduced nucleosome levels.levels.

Effects of MAPK inhibitors on NaF-Effects of MAPK inhibitors on NaF-induced apoptosis in MDPC-23 cellsinduced apoptosis in MDPC-23 cells

MDPC-23 cells were incubated for MDPC-23 cells were incubated for 6-24 hrs with 20uM U0126 in the 6-24 hrs with 20uM U0126 in the presence of 5mM NaF for 24 hrs presence of 5mM NaF for 24 hrs causing suppression of secondary causing suppression of secondary peak in ERK phosphorylation. peak in ERK phosphorylation. Treatment with U0126 for 6-24 hrs Treatment with U0126 for 6-24 hrs completely abolished ERK completely abolished ERK phosphorylation at 24 hrs, but did phosphorylation at 24 hrs, but did not change NaF-induced increase not change NaF-induced increase in the level of the nucleosomes.in the level of the nucleosomes.

ConclusionsConclusions

The responses to MAPK inhibitors observed in The responses to MAPK inhibitors observed in the present experiments are consistent with the the present experiments are consistent with the involvement of MAPK phosphorylation in NaF-involvement of MAPK phosphorylation in NaF-induced apoptosis.induced apoptosis.

NaF-induced apoptosis was markedly NaF-induced apoptosis was markedly suppressed by treatment with the JNK inhibitor, suppressed by treatment with the JNK inhibitor, SP600125 and mildly suppressed by the SP600125 and mildly suppressed by the MEK1/2 inhibitor, U0126.MEK1/2 inhibitor, U0126.

In contrast, inhibition of p38 activity with In contrast, inhibition of p38 activity with SB203580 did not protect cells from apoptosis.SB203580 did not protect cells from apoptosis.

While persistent suppression of ERK While persistent suppression of ERK phosphorylation reduced NaF-induced phosphorylation reduced NaF-induced apoptosis in MDPC-23 cells, suppression of the apoptosis in MDPC-23 cells, suppression of the second phase of ERK phosphorylation did not second phase of ERK phosphorylation did not protect the cells from apoptosis.protect the cells from apoptosis.

Therefore, NaF-induced apoptosis in MDPC-23 Therefore, NaF-induced apoptosis in MDPC-23 cells depends primarily on JNK, with ERK cells depends primarily on JNK, with ERK pathways apparently making a lesser pathways apparently making a lesser contribution.contribution.

Further investigations concerning the precise Further investigations concerning the precise role of the initial and secondary ERK role of the initial and secondary ERK phosphorylation are needed.phosphorylation are needed.

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