MAP kinase phosphatase MKP-1 regulates p-ERK1/2 signaling pathway with uoride treatment

Dental uorosis is characterized by hypomineralization of tooth enamel caused by ingestion of excessive uoride during enamel formation. Excess uoride could have effects on the ERK signaling, which is essential for the ameloblasts differentiation and tooth development. MAP kinase phosphatase-1 (MKP-1) plays a critical role in regulating ERK related kinases. However, the role of MKP-1 in ameloblast and the mechanisms of MKP-1/ERK signaling in the pathogenesis of dental uorosis are incompletely understood. Here, we adopted an in vitro uorosis cell model using murine ameloblasts-like LS8 cells by employing sodium uoride (NaF) as inducer. Using this system, we demonstrated that uoride exposure led to an inhibition of p- MEK and p-ERK1/2 with a subsequent increase in MKP-1 expression in a dose-dependent manner. We further identied, under high dose uoride, MKP-1 acted as a negative regulator of the uoride-induced p-ERK1/2 signaling, leading to downregulation of CREB, c-myc, and Elk-1. Our results identify a novel MKP-1/ERK signaling mechanism that regulates dental uorosis and provide a framework for studying the molecular mechanisms of intervention and uorosis remodeling under normal and pathological conditions. MKP-1 inhibitors may prove to be a benet therapeutic strategy for dental uorosis treatment.


Results
Here, we adopted an in vitro uorosis cell model using murine ameloblasts-like LS8 cells by employing sodium uoride (NaF) as inducer. Using this system, we demonstrated that uoride exposure led to an inhibition of p-MEK and p-ERK1/2 with a subsequent increase in MKP-1 expression in a dose-dependent manner. We further identi ed, under high dose uoride, MKP-1 acted as a negative regulator of the uoride-induced p-ERK1/2 signaling, leading to downregulation of CREB, c-myc, and Elk-1.

Conclusion
Our results identify a novel MKP-1/ERK signaling mechanism that regulates dental uorosis and provide a framework for studying the molecular mechanisms of intervention and uorosis remodeling under normal and pathological conditions. MKP-1 inhibitors may prove to be a bene t therapeutic strategy for dental uorosis treatment.

Background
Fluoride plays a dual role in tooth development. Fluoride at low concentrations can strengthen enamel and prevent tooth decay. When permanent teeth are under development, high exposure to uoride leads to dental uorosis, as referred to a condition characterized by staining and pitting of the teeth that affects millions of people worldwide. Although much research has been conducted, the mechanisms underlying its onset and progression remains unknown.
The signaling networks responsible for properly building the dentition have been heavily investigated and the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway. This molecular cascade is initiated by binding of a growth factor to a receptor tyrosine kinase (RTK), leading to the increased phosphorylation of successive kinases, activated effector kinases and the transcription of target genes [1]. Previous research has reported a probable link between uoride exposure and ERK-MAPK pathway [2]. Investigation of the effects of uoride on enamel-forming cells isolated from rats (primary enamel cells) revealed ERK pathway as an important regulator during tooth development [3]. Moreover, phosphorylated ERK1/2 are highly expressed in ameloblasts and odontoblasts in mandibular molars and incisors [3]. The nding from a mouse-derived enamel cell line known as LS8 provided an in vitro model for understanding the molecular basis of dental uorosis due to its relative ease of handling in the lab compared with primary enamel cells. Through our previous study of uoride-treated LS8 cell, we identi ed a uoride-induced downregulation of p-ERK1/2 which is further involved in apoptosis [4] [1].
MAPK phosphatases (MKPs) is a negative regulator for Mitogen-activated protein kinase (MAPK) activity via dual-speci city phosphatases (DUSPs). Lately, MAPK phosphatase 1 (MKP-1) has emerged as the main counter-regulator of MAPK signaling [2]. MKP-1 locates in the nuclear region and controls gene expression by inactivating the subcellular group of MAPKs [3]. MKP-1 is the original member of a family of dual-speci city phosphatases that can remove phosphates from tyrosine and threonine in ERK and related kinases [4]. MKP-1 activity can manifest positively or negatively the signaling outcomes through a particular pathway, which varies in different cell types either as a function of the relative activities of the various MAPKs and/or abundance of the MAPK substrate [5]. However, the effect of MKP-1 in ameloblast and how MKP-1 regulates ERK signaling together with their downstream regulation of transcription factors in dental uorosis are unclear.
In the present study, we apply an established in vitro uorosis system by using murine ameloblasts-like LS8 cells and employed NaF as an inducer for dental uorosis. We show that uoride exposure inactivates both MEK and ERK1/2 pathways with a subsequent active in MKP-1, which negatively mediates the downstream regulation of transcription factors cAMP-response element-binding protein (CREB), c-myelocytomatosis oncogene cellular homolog (c-myc) and Elk-1. Moreover, blocking or enhancing either the ERK pathway attenuates the changes of MKP-1 in response to high dose uoride exposure. Together, these data provide evidence MKP-1/ERK mediated pathways contribute to dental uorosis pathogenesis and, importantly, indicating that MKP-1 inhibitors may prove to be bene t therapeutic strategy for dental uorosis treatment.

Results
Fluoride exposure inhibits phosphorylation of MEK and ERK1/2 with a subsequent increase in MKP-1 expression via a dose-dependent manner.
Fluoride is an environmental toxicant and induces dental uorosis. NaF is one of the most common inorganic uorides, which is frequently used in the research of uoride toxicity. The ERK-MAPK pathway plays a vital role in the developmental processes of the dental epithelium and tooth growth [3]. Previously, our group has established the in vitro dental uorisis model by treating NaF in murine ameloblasts-like LS8 cells [5]. Brie y, LS8 cells were incubated with NaF at a serial concentration of 0 mmol/L, 1 mmol/L and 2 mmol/L for 48 hours. We observed that NaF treatment induced a signi cant decrease on cell number with a dosage-depended manner ( Figure A1). However, the cell morphology was not changed after the NaF treatment ( Figure A1). By using this model, we examined whether uoride treatment in our cell system leads to MEK and ERK activation. LS8 cells were treated with NaF for 48 hours with various concentrations (0 mmol/L, 1 mmol/L, 2 mmol/L), and total cell lysates were subjected to western blot analysis using an antibody against the phosphorylated form of MEK (p-MEK) and antibodies against phosphorylated forms of ERK1/2 (p-ERK1/2). As shown in Fig. 1, NaF treatment led to a signi cant downregulated expression level of p-MEK ( Fig. 2A, a&b) and p-ERK1/2 (Fig. 2B, a&b) in NaF-treated cells compared to un-treated group in a dose-dependent manner. MAPKs are deactivated by members of the MAPK phosphatases such as MKP-1, which is vital in ERK and related kinases activation and plays a critical role in regulation of MAPK signaling in various peripheral tissues. Therefore, we investigated the MKP-1 expression in the cells in the present of NaF. In contrast, we showed that the phosphorylation of MKP-1 (p-MKP-1, Fig. 2C, a&b) was strongly activated in uoride-treated cells than those in control via a dose-dependent manner, suggesting the negative feedback regulation of the MAPK/ ERK pathway. These results indicate that uoride exposure inhibits phosphorylation of MEK and ERK1/2 with a subsequent increase in MKP-1 expression via a dose-dependent manner.
High dose uoride exposure activates MKP-1 gene transcription and induces downregulation of downstream transcription factors.
MKP-1 is an important kinase as the downstream regulators of both p38 and ERK1/2 [6]. Activation of MKP-1 is regulated through phosphorylation of several transcription factors, including cAMP-response element-binding protein (CREB), c-myc and Elk-1. CREB acts as an indirect mediated of ERK [7]. The Cmyelocytomatosis oncogene cellular homolog (c-Myc) family is one transcription factors that modulates the genes responsible for cellular homeostasis [8] and its gene transcription and protein stabilization and accumulation [9] are sustained by the hyper-activated RAS/MEK/ERK pathway. Transcription factor Elk-1 is a downstream substrate, as a feedback control mechanism to activate ERK l/2. The severity of dental uorosis is dependent upon uoride dose and the timing and duration of uoride exposure. Therefore, we used the LS8 cells with uoride treatment under a high dose (2 mmol/L) to mimic the in vivo dental uorosis and investigated whether this condition activates MKP-1 gene expression and their downstream transcription factors c-myc, CREB and Elk-1. QRT-PCR measurement demonstrated a signi cantly increased mRNA expression of MKP-1 (Fig. 3A), con rming the positive feedback control mechanism induced by uoride treatment as demonstrated above in Fig. 2C. Quanti cation of mRNA expression of the downstream molecule CREB displayed a downregulation of CREB gene level in uoride treated LS8 cells versus control cells, with similar change in both concentration (Fig. 3B). Furthermore, a signi cantly decreased level of c-myc (Fig. 3C) and Elk-1 (Fig. 3D) transcription expression was identi ed in both NaFtreated groups compared to un-treated cells with a dosage-dependent pattern. These data suggest a negative feedback between MKP-1 signaling and downstream regulation of transcription factors CREB, cmyc and Elk-1.
PD98059 and curcumin treatment attenuate the changes of MKP-1 protein expression in response to high dose uoride exposure.
PD98059 is a potent and selective inhibitor of MAPK kinases via binding to the inactive form of MAPK and prevents activation by upstream activators, such as c-Raf [10]. Curcumin, one of the main bioactive components extracted from a traditional Chinese medicinal herb, induces ERK/MARK activation with upregulating phospholortated ERK1/2 signaling [11]. To further con rm the linkage between MARK and ERK signaling in dental uorosis, the LS8 cells were treated with 50 µM PD98059 for 1 hour and then incubated with 2 mM NaF for 48 hours. Western blot assessment was then applied to reveal the protein expression changes of p-ERK1/2 and MKP-1 for the cells under different combinations of the treatment. Our results showed, compared to the control and NaF group, a decreased expression of p-ERK1/2 in PD98059, PD98059 and NaF treated groups, suggesting the treatment of PD98059 were able to inhibit p-ERK1/2. In addition, we demonstrated that PD98059 and NaF treated groups showed signi cantly lower levels of p-ERK1/2 than that with PD98059 alone (Fig. 4A). In contrast, the protein expression level of MKP-1 increased gradually from the cells treated with PD98059 alone, PD98059 and NaF or NaF alone (Fig. 4B), where PD98059 and NaF exposed cells demonstrated a signi cantly increased level of MKP-1 than that exposed in NaF alone (Fig. 4B). To validate this observation, we used 1 µM curcumin to treat the LS8 cells for 1 hours before NaF treatment and we found that, curcumin treated cells exhibited signi cantly higher p-ERK1/2 expression when normalized with total expression of ERK1/2 than untreated cells (Fig. 4C), suggesting treatment of curcumin actives the ERK1/2 signaling in LS8 cells. When the cells treated with curcumin alone, MKP-1 expression elevated in the all three treated groups ( Fig. 4D) with a gradual decrease from the cells treated with curcumin alone, curcumin and NaF or NaF alone. In addition, curcumin and NaF treated groups showed signi cantly higher level of MKP-1 than that with NaF alone (Fig. 4D). These nding further con rm the effect of the MKP-1 on negative regulation phosphorylation ERK signaling pathway in the cells with uoride exposure, as described above (Fig. 2).
D98059 and curcumin treatment convert the gene expression changes of transcript factors in response to high dose uoride exposure.
Our ndings in Fig. 2 suggested a negative feedback between MKP-1 signaling and downstream regulation of transcription factors CREB, c-myc and Elk-1. To further validate this observation, qRT-PCR measurement was applied to measure the of mRNA expression of c-myc, CREB, Elk-1 in LS8 cells preincubated with PD98059 (50 µM) or curcumin (1 µM) for 1 hour, followed by incubation with 2.0 mM NaF for 24 hours. Our data showed that mRNA expression of c-myc and Elk-1 decreased in the cells treated with PD98059, PD98059 and NaF than NaF group and control group, whereas the mRNA expression of CREB dropped in the group in the present of PD98059, PD98059 and NaF, and NaF than control group (Fig. 5A). In contrast, curcumin treated cells showed a signi cantly highest transcriptional expression level for c-myc, CREB and Elk-1 among the cells treated with control, curcumin and NaF, and NaF (Fig. 5B). To further con rm whether lack of MKP-1 protein expression in response to uoride treatment is regulated through a transcriptional mechanism via modulating downstream transcription factors c-myc, CREB and Elk-1, we then transfected the LS8 cells with siRNA targeting MKP-1 to know-out the MKP-1 expression before the qRT-PCR was performed. MOCK siRNAs with the sequences that do not target any gene product was used for negative control. After transfection of GFP siRNA-MKP-1 for 6 hours, we detected a bright green uorescent signal, indicating the cells expressed MKP-1 after transfection (Fig. 5C). We demonstrated that mRNA expression of c-myc, CREB and Elk-1 increased in LS8 cells treated with siRNA-MKP-1 than all the other groups (Fig. 5C). Similarly, a signi cantly upregulated mRNA expression of c-myc, CREB and Elk-1 was identi ed in LS8 cells transfected with siRNA-MKP-1 compared to that in MOCK control group (Fig. 5C). More importantly, curcumin and NaF treated cells showed a signi cant increase of c-myc, CREB and Elk-1 mRNA expression than NaF treat cells (Fig. 5E). These evidences suggest that MKP-1 can facilitate gene transcription through modulating downstream transcription factors c-myc, CREB and Elk-1 in uoride treated cells. Therefore, the previous observations in the qRT-PCR have been con rmed on this point.

Discussion
Our previous evidence showed the activation of ERK1/2 and other MAP Kinase in dental pulp cells [12] and excess uoride have effects on the ERK signaling [1]. MKP-1, as an inhibitor of MAPKs, plays an essential role in regulating ERK related kinases. However, it remains unclear on how MKP-1 is regulated in dental uorosis. Here, we present compelling evidence that treatment with uoride in vitro at the millimolar concentrations markedly activated MKP-1 expression and decreased MEK and ERK1/2 phosphorylation level in a dose-dependent manner in mouse ameloblasts-like LS8 cells [1], indicating a negative feedback between MKP-1 and MEK/ERK signaling. We further use high dose uoride to induce the dental uorosis and demonstrated a negative regulatorily role of MKP-1 in the uoride-induced p-ERK1/2 signaling. We also found that MKP-1 downregulated ERK/MAPK-mediated CREB, c-myc, and Elk-1 transcriptions. Our study clearly demonstrates the effect of MKP-1 on dental uorosis and its therapeutic potential for the treatment of dental uorosis.
In our previous work, we reported a positive expression of p-ERK, p-JNK, p-p38 and uoride-induced apoptosis led to the deceased phosphorylation and deactivation of ERK and JNK signaling cascade in a concentration-and time-dependent manner in ameloblasts cells, indicating a linkage of MAPK signaling on dental uorosis [1]. In present study, we con rm the MEK and ERK1/2 phosphorylation in ameloblasts and their downregulation induced by uoride exposure.
MAPK is a serine/threonine protein kinase that is widely present in eukaryotic cells. Previous studies found four different MAPKs, including ERK, c-JNK, ERK5 and p38 MAPK (p38) [13]. ERK cascade reaction can be activated by various stimuli, such as RTK and G protein-coupled receptors. After activation, it can regulate the proliferation, differentiation and apoptosis. Ras/Raf/MEK/ERK cascade reaction is an important signaling pathway in MAPKs. Various stimuli can activate the corresponding cell surface receptors that, in turn, activates the signal transduction pathway and produce an appropriate biological response. The Ras/Raf/MEK/ERK cascade reaction is the key factor in integrating the signal transduction pathway. Previous observation of individuals with orofacial and craniofacial disorders identi ed an association of Ras/Raf/MEK/ERK pathway and dental malformations [14]. MKP-1 has the capacity to bind and dephosphorylate ERK, and subsequently p38 and JNK kinases. MKP-1 exerts dual effects under different physiological conditions. Previous studies have suggested that MKP-1 at relatively low level has a weak binding a nity for ERK kinases and exerts little effect on their function, whereas MKP-1 is activated and shows a higher binding a nity for p38 and JNK kinases than ERK kinases under other physiological conditions [10,11]. When MKP-1 levels are high, it also inhibits ERK kinases, although to a lesser extent than MARK/p38 and JNK, and thus provides a negative feedback loop on many cellular processes. Consistent with these ndings, here, we report a gradual upregulation of MKP-1 and inhibition of MEK and ERK expression from low to high uoride exposure. Furthermore, we show that the relative high phosphorylation level of MKP-1 is associated with the negative feedback regulation of the MAPK/ERK pathway, which may contribute to the pathogenesis in dental uorosis.
ERK-induced Elk-1 phosphorylation leads to enhanced DNA-binding and TCF-mediated transcriptional activation. Several early genes, such as c-jun, c-fos, and c-myc, are regulated upstream by Ras/ERK and p38. They were found to be involved in controlling the cell growth and metabolism [15]. Elk-1, after phosphorylation by ERK, binds to the SRE cis-acting element in the promoter region of c-fos and induces its transcription. Transcription factor Elk-1 is a part of the ternary complex which can be combined with serum effect factors (SRE) to regulate gene activity in order to reply to the serum and growth factors [16]. The activation of ERKl/2 is transferred from the cytoplasmic to nuclear and activates its downstream substrate EIk-1, and the phosphorylation of EIk-1 will promote the function of cell differentiation, proliferation and apoptosis [17][18][19]. In addition, CREB and c-myc are also recognized as putative active speci c transcriptional factor substrates which mediate indirectly by extracellular signal-regulated kinase such as ERK [7]. Activated phosphorylation of CREB is reported in human molar odontoblasts and cementoblasts in vivo [20]. Study in osteoblast cells reports that ERK/CREB signaling can inhibit cellular oxidative stress [21]. However, some studies reported that MEK/ERK/CREB signaling pathway may not be the solely mediator in the signal transduction pathways in dental pulp cells [22]. The myelocytomatosis oncogene cellular homolog c-myc family consists of transcription factors which modulates genes responsible for cellular homeostasis [8]. C-myc gene transcription and protein stabilization and accumulation [9] are sustained by the hyper-activated RAS/MEK/ERK pathway. In this study, we demonstrate that MKP-1 reduction results in the deactivation of ERK-induced Elk-1 phosphorylation in cytoplasm, which consequently leads to downregulate the downstream substrates CREB and c-myc.
In this study, we used PD98059 and curcumin to inhibit and agitate the phosphorylation of MEK/ERK pathway, respectively, and then detected the trend of the expression of MKP-1 protein and expression of downstream transcription factors of ERK, and found that there was a interaction between the phosphorylation level of ERK1/2 and MKP-1. Silencing MKP-1 at the gene expression level and detecting the expression of ERK downstream transcription factors con rmed this interaction. As an exogenous stimulus, NaF reduced the phosphorylation of ERK in LS8 cells, which was caused by the decrease of MKP-1. The negative feedback regulation mechanism of the formation of MEK/ERK-MKP-1 was involved in the of dental uorosis.

Cell culture and treatments
The mouse ameloblast-like cell line (LS8) was kindly donated by Malcolm L. Snead (Department of Biomedical Sciences, University of Southern California) cultured in DMEM supplemented with 10% FBS and 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, CA, USA). The incubator atmosphere was humidi ed and adjusted at 5% CO 2 and 95% air at 37°C. When reached 70-80% con uence, the cells were incubated with serum-free medium containing the indicated concentrations (0~2 mM) of NaF. After the treatment, the cells were incubated for 24 hours or 48 hours at 37°C.

Transient transfection siRNA
SiRNA-MKP-1 purchased form Shanghai jima pharmaceutical technology co. LTD. At 40% con uence, LS8 cells were transfected with silencing RNA (siRNA) against MKP-1, using Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA) according to the manufacturer's instruction. Brie y, MKP-1-siRNA was diluted in serum-free culture medium with the transfection reagent, mixed by vertexing and incubated for 20 minutes at room temperature to allow the formation of the transfection complex. Then the MKP-1-siRNA was added to the cells for 24 hours. The effectiveness of gene silencing was monitored by measuring the MKP-1 levels in relation to GAPDH, as analyzed by qRT-PCR for mRNA expression level. Cells transfected with MKP-1-siRNA, were further exposed to NaF for 24 hours. The mRNA expression level of c-myc, CREB, Elk-1 was determined by using qRT-PCR.

Protein extraction
Cells were washed with chilled PBS and lysed in ice-cold RIPA buffer as previously described [5], consisting of 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 10 mM EGTA, 2 mM sodium pyrophosphate, 4 mM paranitrophenyl phosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl uoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin and 2 µg/ml pepstatin. Cell lysate was collected using a cells scraper (Corning, Acton, MA) and the homogenates sonicated on ice for 30 min. The lysate was collected by centrifugation at 12,000×g for 15 min at 4°C. The protein content was determined using a BCA protein assay kit (Pierce, Rockford, IL) by extrapolation to dye binding for a standard series of known protein concentration using spectrophotometry.

Western blotting
A volume of supernatant corresponding to an equal mass of protein for each experimental condition was mixed with loading buffer (5-sodium dodecyl sulfate, 5% v/v) and denatured by heating the samples at 95°C for 5 min. Lysate proteins were resolved to size by electrophoresis using 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22 µm polyvinylidene uoride (PVDF) membrane (Millipore, Bedford, MA, USA) using a semi-dry blotting system (Bio-Rad, Hercules, CA, USA). Non-speci c absorption by the membranes was blocked by incubation with 5g% (w/v) skin milk in Tris-buffered saline (TBS, 500 mM NaCl, 20 mM Tris-HCl pH 7.5) with 0.05% (v/v) Tween-20 for 2 hours. Samples were incubated overnight with one of the following primary antibodies at 4°C: Anti-phospho-ERK and total ERK (1:1000), Anti-phospho-MEK (1:1000), Anti-MKP-1 (1:500), GAPDH (1:1000), each diluted in TBS with 5% (v/v) bovine serum containing 0.1% Tween-20 for 24 hours at room temperature with gentle shaking. The membranes were washed using 0.1% Tween-20 TBS three times for 10 min each and incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse secondary antibody (1:10000), as appropriate for each primary antibody, for 1 hour at room temperature. After washing in TBS-0.1% Tween-20 three times, an enhanced chemiluminescence kit (Millipore, MA, USA) was used to detect immunoreactive protein bands. Blots were immuno-detected with an anti-GAPDH antibody (1:1000) to con rm equal mass of protein loaded among samples. The intensity for each immunoreactive protein band was quanti ed using a Quantity One densitometer (BioRad, Hercules, USA).

RNA Extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA of the cells was extracted using Trizol reagent (Carlsbad, CA, USA) according to the manufacturer's instruction. The quality and quantity of the isolated RNA was examined using a GTTCACACCCATCACAAAC3; were used as the internal control. A melting curve analysis was performed on each amplicon to ensure ampli cation of a single PCR product. The relative expression levels were calculated using the comparative threshold cycle (△△CT) method.

Statistical analysis
Statistical analyses were performed using SPSS software, Version 18.0 (SPSS Inc; Chicago, IL). All data were expressed as mean ± standard deviation (SD) with each experiment performed in triplicates. Differences among groups were tested by one-way ANOVA or two-way ANOVA, and the T test was used for two individual comparisons. For all analyses, two-tailed p values of less than 0.05 were considered signi cant.

Conclusions
In summary, we determine that upregulation of MKP-1 induced by uoride treatment via negatively regulating cellular p-MEK1/2 levels following feedback-regulated p-ERK1/2 signaling, which consequently facilitates gene transcription through modulating speci c transcription factor substrates c-myc, CREB and Elk-1 in uoride treated cells (Fig. 6). These ndings provide novel insights into the role of MKP-1 in the pathogenesis of dental uorosis and its potential as a new target for dental uorosis therapy. These ndings together with our previous evidence on uoride induced apoptosis through p-ERK and p-JNK [5], suggest the MEK-ERK-JNK signaling cascade controlling the mechanisms involved in ERK1/2-mediated cell apoptosis in dental uorosis.

Consent for publication
All authors have read and approved the nal transcript This research was funded by special talent project of Ningxia Medical University [No. XT2017020]. The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Competing interests
The authors declare no competing interests.

Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request The study design and reprehensive images of the NaF treated LC8 cells. (A). The study design demonstrates that LS8 cells are treated with NaF under at a serial nal concentration to model the dental uorosis. Western blotting analysis is used to measure p-MEK, p-ERK1/2 and MKP-1 expression. QRT-PCR is performed to demonstrate the mRNA expression for transcription factors CREB, c-myc and Elk-1 after the treatment. Inhibitor PD98059 and activator Curcumin for ERK1/2 signaling were added for further validation. (B). Bright-eld images for the cells treated with NaF at different concentration (0 mmol/L, 1 mmol/L and 2 mmol/L) for 48 hours. Magni cation is 100X.     Summary of the role of MKP-1 in ameloblast and the mechanisms of MKP-1/ERK signaling in the pathogenesis of dental uorosis. The present study we show the upregulation of MKP-1 induced by uoride treatment via negatively regulating cellular p-MEK1/2 levels following feedback-regulated p-ERK1/2 signaling (Black arrows). In addition to receiving P-ERK signals, p-ERK1/2 signaling interacted with P-JNK signal and lead to the apoptosis via several caspases signaling, which has been reported in our previous publication (Blue arrows).

Supplementary Files
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