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

https://doi.org/10.1016/j.bbrc.2020.12.100Get rights and content

Highlights

  • Fluoride exposure led to an inhibition of p-MEK and ERK1/2 and an increase in MKP-1 expression in a dose-dependent manner.

  • MKP-1 acted as a negative regulator of the fluoride-induced p-ERK1/2 signaling.

  • MKP-1 negatively mediates the downstream regulation of transcription factors CREB, c-myc and Elk-1.

  • PD98059 and curcumin attenuates the gene level changes of transcript factors in response to high dose fluoride exposure.

  • MKP-1 inhibitors may prove to be a benefit therapeutic strategy for dental fluorosis treatment.

Abstract

Dental fluorosis is characterized by hypomineralization of tooth enamel caused by ingestion of excessive fluoride during enamel formation. Excess fluoride 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 fluorosis are incompletely understood. Here, we adopted an in vitro fluorosis cell model using murine ameloblasts-like LS8 cells by employing sodium fluoride (NaF) as inducer. Using this system, we demonstrated that fluoride 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 identified, under high dose fluoride, MKP-1 acted as a negative regulator of the fluoride-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 fluorosis and provide a framework for studying the molecular mechanisms of intervention and fluorosis remodeling under normal and pathological conditions. MKP-1 inhibitors may prove to be a benefit therapeutic strategy for dental fluorosis treatment.

Introduction

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 fluoride leads to dental fluorosis, 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 fluoride exposure and ERK-MAPK pathway [2]. Investigation of the effects of fluoride 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 finding from a mouse-derived enamel cell line known as LS8 provided an in vitro model for understanding the molecular basis of dental fluorosis due to its relative ease of handling in the lab compared with primary enamel cells. Through our previous study of fluoride-treated LS8 cell, we identified a fluoride-induced downregulation of p-ERK1/2 which is further involved in apoptosis [1,4].

MAPK phosphatases (MKPs) is a negative regulator for mitogen-activated protein kinase (MAPK) activity via dual-specificity 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-specificity 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 fluorosis are unclear.

In the present study, we apply an established in vitro fluorosis system by using murine ameloblasts-like LS8 cells and employed sodium fluoride (NaF) as an inducer for dental fluorosis (Fig. 1A). We show that fluoride 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 the ERK pathway attenuates the changes of MKP-1 in response to high dose fluoride exposure. Together, these data provide evidence MKP-1/ERK mediated pathways contribute to dental fluorosis pathogenesis and, importantly, indicating that MKP-1 inhibitors may prove to be benefit therapeutic strategy for dental fluorosis treatment.

Section snippets

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, USA) cultured in DMEM supplemented with 10% FBS and 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, CA, USA). The incubator atmosphere was humidified and adjusted at 5% CO2 and 95% air at 37 °C. When reached 70–80% confluence, the cells were incubated with serum-free medium containing the indicated concentrations (0–2 mM) of

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 fluorosis. NaF is one of the most common inorganic fluorides, which is frequently used in the research of fluoride 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 fluorosis model by treating NaF in murine ameloblasts-like LS8 cells [5]. Briefly, LS8 cells were incubated with NaF at a serial

Discussion

The previous evidence showed the activation of ERK1/2 and other MAP kinase in dental pulp cells [12] and our previsou study reported the excess fluoride had 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 fluorosis. Here, we present compelling evidence that treatment with fluoride in vitro at the millimolar concentrations markedly activate MKP-1

Author’s contributions

L.Z, J.S, S.L, Y.L and T.X contribute to the experimental investigation; L.Z and K.L contribute to the writing original draft; all the authors contribute to review and edit; R.H and L.Z contribute the resources; R.H and K.L contribute to supervision and funding acquisition. All authors read and approved the final manuscript.

Funding

This research was funded by special talent project of Ningxia Medical University [No. XT2017020], China. The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgements

We thank Professor Malcolm L. Snead (Department of Biomedical Sciences, University of Southern California, USA) for donation of LS8 cells.

References (22)

  • T. Otani

    Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    Cell Death Dis.

    (2018)
  • Cited by (12)

    • Interaction of fluoride exposure and CREB1 gene polymorphisms on thyroid function in school-age children

      2022, Chemosphere
      Citation Excerpt :

      It is well known that thyroid hormones are involved in the process of brain development (Schiera et al., 2021; Weiss et al., 1993). Previous toxicological studies have provided evidence that fluoride exposure could decrease the CREB gene mRNA and protein expression levels in murine ameloblasts-like LS8 cells and mouse hippocampal neuronal cell lines (Wang et al., 2019; Zhao et al., 2021). Notably, CREB was involved in thyroid hormone levels, thyroid cell proliferation and differentiation, and thyroid development (Ayroldi et al., 2018; Nguyen et al., 2000), and single nucleotide polymorphisms (SNPs) of CREB1 have been reported to be associated with neurological disorders (Xiao et al., 2018).

    • Sodium fluoride suppresses spleen development through MAPK/ERK signaling pathway in mice

      2022, Ecotoxicology and Environmental Safety
      Citation Excerpt :

      Chen et al. (2014) pointed out that fluoride inhibits the expression of p-ERK protein through the ERK pathway, resulting in kidney injury in carp (Chen et al., 2014). Zhao et al. reported that NaF treatment resulted in the inhibition of phosphorylation of MEK and ERK1/2 (p-MEK and p-ERK1/2) in mouse ameloblasts (LS8 cells), suggesting that high-dose fluoride may affect ERK signal transduction, thus affecting the differentiation of ameloblasts (Zhao et al., 2021). Additionally, numerous researchers also reported that sodium fluoride (NaF) influenced organ development, cell cycle progression, promoted cell apoptosis, caused damage and dysfunction via ERK signaling pathway (Chen et al., 2014; Geng et al., 2014; Ma et al., 2017; Zhu et al., 2017).

    • NaF reduces KLK4 expression by decreasing Foxo1/Runx2 expression in LS8 cells

      2022, Archives of Oral Biology
      Citation Excerpt :

      However, no significant differences in runx2 mRNA levels were found between siCtrl- and siFoxo1-transfected cells, even under NaF conditions (Fig. 4C and D). Growing evidence has demonstrated that excess fluoride intake during enamel development could cause cell stress, that autophagy alleviates excess fluoride-mediated cell stress, and that unresolved cell stress impairs the function of ameloblasts, leading to the downregulation of KLK4 and the retention of enamel matrix proteins and eventually resulting in dental fluorosis (Fujiwara, Whitford, Bartlett, & Suzuki, 2021; Suzuki, Bandoski, & Bartlett, 2015; Suzuki, Ikeda, & Bartlett, 2018; Wei, Pang, & Sun, 2019; Zhao et al., 2021). As one of the critical factors causing dental fluorosis, high fluoride-mediated KLK4 downregulation could affect the cleavage and degradation of enamel hypomineralization.

    View all citing articles on Scopus
    1

    These first authors contributed equally to this work.

    2

    These corresponding authors contributed equally.

    View full text