Melatonin Rescues Enamel Defects Induced by Maternal Circadian Disruption via Targeting the BMAL1-JNK3 Axis

Abstract

Introduction and aims

Developmental defects of enamel (DDE) are highly prevalent, yet effective preventive measures remain elusive. The process of amelogenesis exhibits intrinsic circadian rhythmicity. While the photoperiod-regulated hormone melatonin is a recognized modulator of bone formation, its potential role in coordinating the circadian aspects of enamel biomineralization remains incompletely understood. This study aimed to decipher the impact of aberrant photoperiods on enamel development and to identify melatonin’s regulatory targets.

Methods

We assessed the impact of maternal circadian disruption on offspring enamel mineralization using 3D X-ray microscopy (3DXRM), ATR-FTIR, SEM, Western blot, and qRT-PCR. Plasma melatonin concentration was measured by ELISA. Melatonin was administered to pregnant mice under circadian disruption to investigate its effects on enamel mineralization in their offspring. To investigate the molecular mechanisms downstream of BMAL1, we performed Bmal1 knockdown in ameloblast-lineage cells (ALCs) and used RNA sequencing and siRNA transfection.

Results

Circadian disruption impaired enamel mineralization (reduced thickness/density, increased CO₃²⁻/PO₄³⁻ ratio, rough surface with disorganized prisms), which was partially rescued by melatonin. Bmal1 knockdown in ALCs disrupted mitochondrial respiration, increased JNK3 phosphorylation, and reduced mineralization; melatonin restored mitochondrial function and suppressed JNK3 overactivation.

Conclusion

Mechanistically, melatonin inhibits JNK3 phosphorylation to restore enamel mineralization. Bmal1 knockdown impairs the mitochondrial respiratory electron transport chain, while melatonin restores its function, thereby suppressing JNK3 overactivation and ultimately promoting mineralization.

Clinical relevance

The BMAL1-JNK3 axis is a therapeutic target for enamel mineralization, providing a novel theoretical basis for perinatal circadian regulation.

Introduction

Enamel is the body’s hardest mineralized tissue. Its matrix proteins are produced by ameloblasts, including amelogenins, non-amelogenins, and proteases.¹ Enamel formation (amelogenesis) occurs in 2 main stages: the secretory stage, during which ameloblasts secrete enamel matrix proteins (eg, AMELX, AMBN, ENAM), and the maturation stage, during which proteases (eg, KLK4, MMP-20) degrade the matrix to allow crystal expansion. Any disruption to these highly coordinated processes, whether genetic or environmental, can result in developmental defects of enamel (DDE).^2,3 DDE often results from abnormal ameloblast structure or function.⁴ DDE is associated with numerous clinical issues, such as the aesthetics of incisors, dentin hypersensitivity, early childhood caries, premature tooth loss, and malocclusion.⁵

Mammalian enamel formation follows a strict circadian rhythm,⁶ reflected in growth lines.⁷ This biological process is driven by the cyclic expression of core clock genes.^8,9 Core clock genes, including Bmal1, drive this process and regulate mitochondrial genes and metabolism,⁹ indicating a bidirectional feedback between circadian clocks and energy metabolism.¹⁰ Clock genes oscillate in ameloblasts in vitro, and circadian disruption reduces incisor eruption rates in vivo, confirming circadian involvement in amelogenesis.^11,12

The circadian rhythm in mammals is precisely regulated by the retinohypothalamic-pineal melatonin axis.⁸ Melatonin, which is secreted by the pineal gland, exhibits high concentrations at night and low levels during the day. Its secretion is initiated in darkness and suppressed by light exposure.^13,14 However, factors such as shift work, nighttime entertainment, and sleep deprivation lead to increased exposure to artificial light at night, which inhibits melatonin synthesis.^15,16 As epidemiological studies have confirmed, sleep disturbances and shift work are highly associated with the development of obesity, dyslipidaemia, and type 2 diabetes in humans.^15,17 This misalignment between endogenous rhythms, which is disrupted by artificial nighttime lighting, and the natural cycle triggers a cascade of health issues.16, 17, 18, 19 Particularly important is the role of the maternal organism as the nearly exclusive source of melatonin for the foetus; thus, the stability of the maternal circadian rhythm directly determines the stability of foetal rhythm.20, 21, 22

In the field of oral health, melatonin is recognized as an important local hormone that exerts paracrine effects on nearby cells.^23,24 It can promote osseointegration of implants, exert anti-inflammatory effects within the periodontal ligament, and regulate odontogenic functions.25, 26, 27 Currently, research on melatonin in oral contexts predominantly focuses on its effects on the periodontium and dentin.28, 29, 30 In contrast, its role and mechanisms in enamel development and mineralization have been scarcely studied. Melatonin plays distinct roles during the secretory and maturation stages of amelogenesis. During the secretory stage, ameloblasts produce enamel matrix proteins (eg, amelogenin, ameloblastin, enamelin), and melatonin has been shown to promote cell proliferation and matrix protein synthesis. During the maturation stage, ameloblasts modulate pH homeostasis to facilitate crystal growth and matrix degradation. Melatonin contributes to this phase by regulating mitochondrial function and oxidative stress, thereby supporting proper mineral deposition. Understanding these stage-specific functions is critical for interpreting the effects of maternal circadian disruption on enamel development. The stress-activated serine/threonine protein kinase JNK3, belongs to the MAPK family, represents a potential key node connecting the aforementioned pathways.³¹ Its expression is influenced by energy metabolism, and regulates various cellular activities during embryonic development especially enamel development, including cell proliferation, apoptosis, and differentiation.^32,33 Previous studies have shown that JNK3 is expressed in ameloblasts and is involved in mineralization processes.²⁹ Adding melatonin to ameloblast-like cells can promote the nuclear translocation of JNK3 to enhance mineralization capacity.³³ However, the network involving the circadian, core clock genes, melatonin, and the JNK3 in enamel development remains unclear. This study investigates the development and mineralization of dental enamel in neonatal mice under different circadian conditions and identifies the regulatory targets of melatonin in this process. Given that maternal circadian disruption significantly downregulates BMAL1 expression in offspring tooth germs, we performed in vitro Bmal1 knockdown experiments to simulate this key molecular event. This approach allowed us to dissect the downstream mechanisms involving mitochondrial dysfunction and JNK3 hyperactivation, and to test whether melatonin acts through targeting this pathway.

Materials and methods

Chemicals and reagents

Melatonin, ascorbic acid, dexamethasone, and β-glycerophosphate were purchased from SigmaAldrich. RIPA lysis buffer was obtained from Yamay, China. Bmal1 siRNA and negative control siRNA (siControl) were synthesized by GenePharma, China, and Lipofectamine 2000 was from Invitrogen. RNA extraction, reverse transcription, and qPCR were performed respectively using the EZ-press RNA Purification Kit (EZbio Science), PrimeScript™ RT Master Mix (TaKaRa Bio), and SYBR Green I Mastermix (Roche). Primary antibodies against beta-actin (ab32099), AMELX (ab163915), GAPDH (ab181602), JNK3 (ab314190), and p-JNK (ab124956) were acquired from Abcam. The ODAM antibody (DF13204) was from Affinity, and the HRP-conjugated Goat anti-Rabbit IgG secondary antibody (AB_1185567) was from Thermo Fisher Scientific. The ALP assay kit was from Beyotime Biotechnology, China. The JC-10 Mitochondrial Membrane Potential Assay Kit was from Yeasen Biotechnology, China. Mouse Melatonin (MT) ELISA Kit was purchased from FineTest, China.

Cell culture

ALCs were provided by the Shanghai Key Laboratory of Stomatology and were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal bovine serum at 37 °C. Odontogenic induction medium was DMEM containing 10 mM β-glycerophosphate, 50 mM ascorbic acid, and 100 nM dexamethasone.

Animals

All animal experiments were approved by the Animal Welfare Committee of Shanghai Ninth People's Hospital (SH9H-2021-A436-1). Eight- to ten-week-old BALB/c mice (female: 18-22 g body weight; male: 20-25 g body weight) were from the Shanghai Animal Experiment Center and timed-mated. Timed mating was performed by housing 1 female with 1 male overnight. The day a vaginal plug was observed was designated as embryonic day 0.5 (E0.5). From E0.5 to E16.5, all pregnant mice were housed under a standard 12L:12D cycle. At E16.5, pregnant mice were randomly assigned to experimental groups, and their light conditions were changed accordingly (12L:12D, 16L:8D, or 24L:0D), with daily intraperitoneal injections of melatonin (10 mg/kg) or vehicle initiated on the same day. Treatments continued until delivery (E18.5-E19.5). After birth, offspring were kept with their dams under the same conditions.

Two offspring cohorts were collected: (1) At postnatal day 3.5 (P3.5), mandibular first molar tooth germs and plasma were collected for molecular analyses, a time point when enamel matrix secretion is highly active. (2) A separate cohort was raised to 3 weeks of age (P21) for 3D XRM analysis of enamel thickness and density. P21 was chosen because mandibular first molars have completed crown mineralization with minimal occlusal wear, allowing assessment of intrinsic developmental phenotypes without the confounding wear that occurs by 4 weeks of age.³¹

The experimental groups were as follows:

Control group (C): Housed under a 12-hour light/12-hour dark cycle (12L:12D).

Continuous-light group (CL): Housed under constant 24-hour light (35W white light at 30 cm distance).

High-light group (HL): Housed under a 16-hour light/8-hour dark cycle (16L:8D).

Normal light + Melatonin group (12L:12D + MEL, NM): Housed under 12L:12D and received daily intraperitoneal injections of melatonin (10 mg/kg) dissolved in vehicle (0.5% ethanol in saline).

High-light + Melatonin group (16L:8D + MEL, HM): Housed under 16L:8D and received daily injections of melatonin (10 mg/kg).

Normal light + Vehicle group (12L:12D + Veh, NV): Housed under 12L:12D and received daily injections of vehicle only.

High-light + Vehicle group (16L:8D + Veh, HV): Housed under 16L:8D and received daily injections of vehicle only. For simplicity, these groups are hereafter referred to as C, CL, HL, NM, HM, NV, and HV, respectively.

This dosage was selected based on our previously established protocol, which has been demonstrated to effectively elevate plasma melatonin to levels that elicit physiological and protective effects in mouse models of developmental disruption.³¹

Odontogenic induction, alkaline phosphatase (ALP) staining and activity assay

Cells were cultured in 12-well plates with odontogenic induction medium, supplemented with 10 μM melatonin based on previous concentration screening,²⁹ for 1 week with daily medium changed. Following manufacturer's instructions, cells were fixed in 4% paraformaldehyde for ALP staining using a commercial kit. Mineralized nodules were documented using both digital camera and inverted phase contrast microscopy. For quantitative analysis, cells were lysed on day 7 and ALP activity was measured using phosphate (pNPP) substrate incubated at 37 °C for 30 minutes. Following the measurement of optical density at 405 nm, the activity was normalized to the total protein concentration.

Gene expression analysis by qRT-PCR

Total mRNA was extracted using EZ-press RNA Purification Kit, and its concentrations were quantified by Nanodrop-800 spectrophotometer. The cDNA was synthesized from the extracted RNA by reverse transcription using the PrimeScript™ RT Master Mix. Quantitative Real Time PCR (RT-qPCR) utilized the SYBR Green I Mastermix. The reaction conditions were set according to manufacturer's protocol. Gene expression was assessed using the 2-ΔΔCT method with relative levels normalized to β-actin as the reference gene. The corresponding primer sequences can be found in the supplementary table.

Protein extraction and western blotting

Cells were lysed in ice-cold RIPA buffer containing protease and phosphatase inhibitors. After quantifying protein concentration via BCA assay, equal amounts of protein (40 μg for tissue, 20 μg for lysates) were separated on 8% to 16% ExpressPlus PAGE gels and transferred to PVDF membranes. After being blocked with either 10% skim milk for 2 hours or 5% BSA for 1 hour at room temperature, the membranes were incubated with specific primary antibodies overnight at 4 °C. To ensure equal protein loading, blots were probed with either β-actin or GAPDH antibody as an internal control. After washing, the membranes were incubated with an HRP-conjugated anti-rabbit secondary antibody for 1 hour at room temperature. Protein bands were visualized using an ECL detection system, and their intensities were quantified with ImageJ software.

ELISA

From postnatal day 3 (P3) to day 4 (P4), pups were euthanized by decapitation under deep isoflurane anaesthesia at 6-hour intervals. Blood samples were collected into heparinized microcentrifuge tubes and centrifuged to obtain plasma. The plasma concentration of melatonin was then quantified using a commercial Mouse Melatonin (MT) ELISA Kit (FineTest,), according to the manufacturer's instructions, with the final optical density measured at 450 nm.

3D x-ray microscope (3D XRM) analysis

Mouse mandibles and maxillae were dissected following euthanasia under deep anaesthesia. The samples were scanned using a 3D X-ray microscope. The acquired data were imported into Dragonfly software (Comet Technologies, Canada) for processing and analysis. A thickness mesh algorithm was applied to generate a 3D enamel thickness heatmap. Mineral density values were obtained from the scanning data, which were calibrated using 2 standard phantoms with known densities.

Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR)

Mandibular first molars were dissected, and the lingual surface of each tooth was gently cleaned with deionized water. Enamel samples were dried at 60 °C for 2 hours before analysis. ATR-FTIR spectra were collected using a Thermo Fisher Scientific Nicolet iS 5 FT-IR spectrometer equipped with a diamond ATR crystal. Spectra were recorded in absorbance mode over the range of 600 to 4000 cm⁻¹ at a resolution of 4 cm⁻¹, with 32 scans per sample. For each tooth, 3 measurement points were taken from the mid-lingual surface and averaged. After baseline correction and normalization to the phosphate band, the carbonate-to-phosphate peak area ratio CO₃²⁻/PO₄³⁻ was calculated to assess relative mineral maturity. The integrated area of the carbonate band (1410-1460 cm⁻¹) and the phosphate band (1000-1100 cm⁻¹) were determined using Origin software (OriginLab Corporation).

Scanning electron microscopy (SEM)

Enamel surface morphology was examined using Hitachi SU8600 field-emission scanning electron microscope. Mandibular first molars were fixed in 2.5% glutaraldehyde at 4 °C for 4 hours, then dehydrated through a graded ethanol series: 30%, 50%, 70%, 85%, and 90% for 15 minutes each, followed by 100% ethanol twice for 30 minutes each, and finally subjected to critical point drying. The dried samples were mounted on aluminium stubs with conductive carbon tape and sputter-coated with gold (10-15 nm thickness) to ensure conductivity. Images were taken from the lingual surface at an accelerating voltage of 5 kV and a working distance of 10 to 15 mm, at magnifications of × 1000 (scale bar = 50 µm), × 5000 (scale bar = 10 µm), and × 20,000 (scale bar = 2 µm). SEM analysis was performed on 3 teeth per group, and representative images are shown.

Assessment of mitochondrial membrane potential

The mitochondrial membrane potential in ALCs was assessed with the JC-10 fluorescent probe (Yeasen Biotechnology, China). Cells were cultured in 6-well plates until reaching appropriate density, followed by incubation with JC-10 working solution in culture medium at 37 °C for 20 minutes in the dark. Cells were washed twice with ice-cold JC-10 staining buffer. For fluorescence microscopy imaging, dual-channel observations were performed with excitation/emission wavelengths set at 490/530 nm for JC-10 monomers (green fluorescence) and 525/590 nm for JC-10 aggregates (red fluorescence). For flow cytometry, at least 10,000 events per sample were acquired. JC-10 monomers (green) and aggregates (red) were detected in the FITC and PE channels, respectively.

RNA-seq and computational analysis

To recapitulate the BMAL1 downregulation observed in vivo (Figure 1A-D) and dissect downstream mechanisms, we knocked down Bmal1 in ALCs with or without melatonin treatment. Total RNA was then extracted for transcriptome sequencing. The purified RNA was then subjected to ribosomal RNA depletion, library preparation, and sequencing on an Illumina platform. Clean reads were aligned to the reference genome, and gene expression levels were quantified. Differential expression analysis was performed using DESeq2 (|fold change| > 2, FDR < 0.05). Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) were performed on the differentially expressed genes.

Fig. 1.

Maternal circadian rhythm disruption impairs enamel mineralization in offspring. (A) Relative mRNA expression levels of Bmal1 and Amelx in mandibular first molar tooth germs at P3.5, measured by qRT-PCR (n = 3). (B) Western blot analysis showing protein expression levels of BMAL1, p-JNK, total JNK3, AMELX, and ODAM across different Zeitgeber Time (ZT) points in the control and HL groups. (C) Fluctuations in serum melatonin concentration and expression patterns of Bmal1 and Amelx mRNA across a 24‑hour period in the control group (C) (n = 3). D. Fluctuations in serum melatonin concentration and expression patterns of Bmal1 and Amelx mRNA across a 24‑hour period in the HL group (D) (n = 3). (E) 3D XRM images of mandibular first molar enamel thickness in the control, CL, and HL groups. Colour scale from purple to red indicates low to high tissue thickness. (F) 3D reconstruction images of enamel thickness corresponding to the samples in (E). (G) Frequency distribution analysis of enamel thickness across the 3 groups. (H) Quantitative analysis of enamel thickness and mineral density in mandibular first molars (n = 6). (I) ATR-FTIR analysis showing the carbonate-to-phosphate (CO₃²⁻/PO₄³⁻) peak area ratio in the control, CL, and HL groups. Data are presented as mean ± SD. Statistical significance was determined by 1-way ANOVA with Bonferroni's post-hoc test: ns (not significant), *P < .05, **P < .01, ***P < .001.*Abbreviations: 3D XRM, 3D X-ray microscope; Amelx, amelogenin; Bmal1, brain and muscle ARNT-Like 1. Odam, odontogenic ameloblast-associated protein; Ambn, ameloblastin; JNK3, c-Jun N-terminal kinase 3.

Accession number

The sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1356859.

Statistical analysis

All data were analysed with GraphPad Prism 9.5.1 and are expressed as mean ± SD. Student's t-test was used for 2-group comparisons. For multi-group data, normally distributed datasets were analysed by 1-way ANOVA with Bonferroni's post-hoc test, while non-normally distributed datasets were analysed by the Kruskal-Wallis H test, followed by Dunn's test when significance was reached. A P-value below .05 was considered statistically significant, and all experiments were repeated independently at least 3 times.

Ethics approval and consent to participate

All animal procedures in this study were approved by the Animal Welfare Committee of Shanghai Ninth People's Hospital, under protocol number [SH9H-2021-A436-1]. All methods were carried out in accordance with relevant guidelines and regulations.

Results

Gene expression oscillates in synchrony with serum melatonin

Expression of Bmal1 and Amelx was suppressed, with the most pronounced decrease in the CL group (Figure 1A).Western blot analysis revealed that the diurnal expression rhythm of BMAL1 in the tooth germs of the high-light group offspring was disrupted compared to the control group. Concurrently, JNK3 phosphorylation was elevated during the light phase and reduced during the dark phase. In contrast, a converse pattern was observed for Bmal1, Amelx, and Odam, whose expression levels increased during the dark phase (Figure 1B).In the control group and high-light group, melatonin concentration progressively decreased with extended light exposure and increased during the dark phase. We observed that the expression trends of clock genes Bmal1 and mineralization-related genes Amelx closely paralleled the melatonin concentration patterns in both groups: expression levels decreased with prolonged light exposure and increased during darkness (Figure 1C, and D).

Circadian rhythm disruption leads to hypomineralisation during enamel development

To further assess the impact of maternal circadian disruption on offspring enamel structure, we performed 3D XRM, FTIR, and SEM analyses. Heatmap and 3D reconstruction images revealed that both the continuous-light (CL) and high-light (HL) groups exhibited a marked reduction in enamel thickness compared to the control group (Figure 1E, and F). Thickness distribution analysis revealed a progressive decrease in maximum enamel thickness from 160 μm (control) to 140 μm (HL) and 130 μm (CL), with the predominant thickness range shifting from 60-80 μm to 20-30 μm (Figure 1G). Circadian rhythm disruption caused enamel hypomineralisation in offspring, with the continuous-light (CL) group showing more severe reductions in mandibular molar enamel thickness and density than the high-light (HL) group (Figure 1H). ATR-FTIR spectroscopy showed that the CO₃²⁻/PO₄³⁻ peak area ratio was significantly increased in the CL and HL groups compared to the control group (Figure 1I). SEM analysis further revealed that the control group displayed a smooth enamel surface with regularly arranged enamel prisms, whereas the CL and HL groups showed rough surfaces with enlarged interprismatic spaces and increased porosity (Supplementary Figure 1).

Exogenous melatonin supplementation can partially rescue enamel hypoplasia

We next examined whether exogenous melatonin could rescue the enamel defects. Offspring from the CV, CM, HV, and HM groups were analysed. qRT-PCR analysis revealed that the mRNA expression levels of Bmal1, Amelx, and Odam were significantly decreased in the HV group compared to the CV group, whereas maternal melatonin supplementation (HM group) partially restored their expression (Figure 2A). Western blot analysis further showed that p-JNK levels were elevated in the HV group, indicating activation of stress signalling, while melatonin treatment reduced p-JNK expression in the HM group (Figure 2B). 3D XRM analysis of mandibular first molars demonstrated that maternal melatonin supplementation enhanced offspring enamel thickness and density compared to the vehicle groups (Figure 2C, and D). Specifically, melatonin increased the average maximum enamel thickness from 110 μm (NV) to 130 μm (NM) and partially restored it to 120 μm in the HM group (Figure 2E). Similarly, the reduced enamel density in the HV group was mitigated by melatonin (Figure 2F). ATR-FTIR spectroscopy revealed that the CO₃²⁻/PO₄³⁻ peak area ratio was significantly increased in the HV group compared to the CV group, and reduced in the HM group (Figure 2G). SEM analysis further supported these findings. The CV group displayed a smooth enamel surface with regularly arranged, compact enamel prisms and minimal porosity. In contrast, the HV group exhibited a rough, irregular surface with disorganized enamel prisms, enlarged interprismatic spaces, and increased porosity. Notably, the HM group showed partial structural rescue, with a smoother surface and more regular prism arrangement compared to the HV group (Supplementary Figure 2).Collectively, these data confirm that exogenous melatonin ameliorates the enamel hypomineralisation induced by maternal circadian disruption.

Fig. 2.

Melatonin partially rescues the impaired enamel mineralization caused by maternal circadian disruption. (A) qRT-PCR analysis of Bmal1, Amelx, and Odam mRNA expression in the CV, CM, HV, and HM groups. (B) Western blot analysis of BMAL1 and p-JNK protein levels in the different treatment groups and quantitative analysis. (C) 3D XRM heatmap showing enamel thickness distribution. (D) 3D reconstruction images showing enamel thickness distribution. (E) Frequency distribution analysis of enamel thickness. (F) Quantitative analysis of enamel thickness and mineral density. (G) ATR-FTIR analysis showing the carbonate-to-phosphate (CO₃²⁻/PO₄³⁻) peak area ratio. Data are presented as mean ± SD. Statistical significance is denoted as *P < .05, **P < .01, ***P < .001.

Melatonin modulates the expression of Bmal1 and its associated mineralization-related genes in ALCs

ALCs were cultured in mineralization induction medium at 37 °C, following supplementation with either 10 μM melatonin or 10 μM luzindole. RT- qPCR analysis revealed that melatonin enhanced the expression of both the Bmal1 gene and mineralization-related genes in ALCs, whereas luzindole suppressed the expression of these genes (Figure 3A). ALP activity assays demonstrated that melatonin-treated ALCs exhibited a higher degree of mineralization compared to untreated controls in vitro, while luzindole treatment inhibited mineralization (Figure 3B).

Fig. 3.

Melatonin promotes the mineralization of ALCs, Bmal1 knockdown impairs ameloblast mineralization. (A) Relative mRNA expression levels in ALCs (n = 3). (B) Alkaline phosphatase (ALP) staining (upper panel) and corresponding quantitative analysis (lower panel) of ALCs after 7 days of odontogenic induction under the different culture conditions. (C) Knockdown efficiency of Bmal1 siRNA confirmed by qRT-PCR. (D) Knockdown efficiency of Bmal1 siRNA confirmed by Western blot. (E) Relative mRNA expression levels control (NC) and Bmal1-knockdown (si-Bmal1) ALCs, with or without melatonin treatment (n = 4). F. ALP staining (left panel) and quantitative analysis (right panel) of ALCs after 7 days of odontogenic induction. Data are presented as mean ± SD. Statistical significance is denoted as *P < .05, **P < .01, ***P < .001. Abbreviations: Ambn, ameloblastin; Enam, Enamelin.

Knockdown of Bmal1 downregulates the expression of genes associated with cellular mineralization and impairs the oxidative respiratory chain

We used small interfering RNA (siRNA) to knock down Bmal1 expression in ameloblast-lineage cells (ALCs), with the knockdown efficiency confirmed by qRT‑PCR and Western blot (Figure 3C, and D). The NC (negative control) and Bmal1‑knockdown ALCs were allocated to 2 treatment conditions (with or without melatonin in mineralization induction medium), with 3 independent biological replicates (n=3) per condition for each cell type. At 48 hours post‑knockdown, qPCR analysis showed that Bmal1 knockdown significantly suppressed the expression of mineralization‑related genes (Figure 3E). The ALP activity assay indicated that in vitro mineralization was significantly reduced in Bmal1‑knockdown ALCs, and only marginally recovered after melatonin addition (Figure 3F).

Melatonin partially rescued Bmal1 knockdown-induced hypomineralisation by inhibiting JNK phosphorylation

To dissect the transcriptional changes downstream of Bmal1, we performed RNA-seq on ALCs after Bmal1 knockdown with or without melatonin treatment. Using |log2FoldChange| > 1 and P-adjust < 0.05 as screening thresholds, a total of 308 differentially expressed genes (DEGs) were identified between the si-Bmal1 group and the NC group, including 103 upregulated genes and 205 downregulated genes. Between the si-Bmal1+MEL group and the si-Bmal1 group, 114 DEGs were identified, comprising 61 upregulated and 53 downregulated genes (Figure 4A). Hierarchical clustering analysis of these 114 DEGs was performed based on FPKM values, and a gene expression heatmap was generated (Figure 4B). The results revealed marked differences in gene expression patterns between the 2 groups, indicating that melatonin treatment reversed the transcriptional dysregulation induced by Bmal1 knockdown. KEGG pathway analysis further indicated that Bmal1 knockdown mainly affected pathways related to energy metabolism, metabolism of cofactors and vitamins, immune system, and signal transduction, while melatonin treatment exhibited regulatory effects on pathways including amino acid metabolism, signalling molecules and interaction, cell motility, and the immune system (Figure 4C). GO enrichment analysis of the overlapping genes revealed that the differentially expressed genes were primarily enriched in biological processes such as cellular processes, biological regulation, response to stimulus, developmental regulation, and immune system processes (Figure 4D). These findings suggest that BMAL1 deficiency disrupts core cellular and metabolic functions, whereas melatonin partially restores homeostasis by modulating stress-responsive and immune-related pathways. Western blot analysis confirmed that Bmal1 knockdown significantly increased the phosphorylation level of JNK in ameloblast-lineage cells (ALCs), accompanied by downregulated expression of mineralization-related genes and impaired in vitro mineralization capacity. Notably, exogenous melatonin supplementation significantly reduced p-JNK levels regardless of whether Bmal1 was knocked down (Figure 4E). This indicates that melatonin mitigates BMAL1 deficiency-induced cellular stress, at least in part, by inhibiting JNK overactivation.

Fig. 4.

Analysis of overlapping DEGs identifies core pathways affected by NC and si-Bmal1. (A) Venn diagram illustrating the overlap of DEGs between the si-Bmal1 vs si-NC and si-Bmal1+MEL vs si-Bmal1 comparisons. (B) Hierarchical clustering heatmap of the 38 overlapping DEGs. Red indicates upregulation, blue indicates downregulation. (C) Gene Ontology (GO) enrichment analysis of the overlapping DEGs. Terms are categorized into biological processes (BP), cellular components (CC), and molecular functions (MF). (D) KEGG pathway enrichment analysis of the overlapping DEGs, showing the top significantly enriched pathways. (E) Western blot analysis validating the protein levels of BMAL1, p-JNK, total JNK3, and AMELX in ALCs under the indicated conditions and quantitative analysis of Western blot. Data are presented as mean ± SD. Statistical significance is denoted as *P < .05, **P < .01, ***P < .001.

Melatonin restores mitochondrial function and suppresses JNK overactivation

GSEA analysis revealed that, in the comparison between the si-Bmal1 group and the NC group, mitochondrial function-related gene sets such as RESPIRATORY ELECTRON TRANSPORT were significantly enriched and negatively regulated, indicating that Bmal1 knockdown led to a global downregulation of genes associated with the respiratory electron transport chain (Figure 5A). In the comparison between the si-Bmal1+MEL group and the si-Bmal1 group, these suppressed metabolic pathways showed significant positive enrichment (Figure 5B).Based on the transcriptomic and GSEA findings, we further performed functional experiments to validate the impact of Bmal1 knockdown on mitochondrial function in ameloblasts and the regulatory effect of melatonin. Mitochondrial membrane potential (ΔΨm) in ALCs from each group was assessed using the JC-10 fluorescent probe. Under fluorescence microscopy, NC group cells exhibited bright red fluorescence with weak green fluorescence, indicating normal mitochondrial function. In the si-Bmal1 group, red fluorescence was markedly reduced, green fluorescence was significantly increased, and the red/green fluorescence ratio was decreased, indicating that Bmal1 knockdown led to a significant loss of mitochondrial membrane potential. In the si-Bmal1+MEL group, red fluorescence intensity was partially restored compared to the si-Bmal1 group, suggesting that melatonin treatment could partially rescue the impaired mitochondrial membrane potential (Figure 5C).Flow cytometry analysis of JC-10 fluorescence intensity further confirmed these findings. NC group cells predominantly displayed red fluorescence with a high red/green fluorescence ratio. In the si-Bmal1 group, red fluorescence intensity was decreased, green fluorescence was increased, and the red/green fluorescence ratio was reduced by 25% compared to the NC group, indicating a significant loss of mitochondrial membrane potential following Bmal1 knockdown. In the si-Bmal1+MEL group, red fluorescence intensity was increased and green fluorescence was decreased compared to the si-Bmal1 group, with a slight recovery in the red/green fluorescence ratio, although it remained lower than that of the NC group (Figure 5D). These results quantitatively confirm that melatonin partially ameliorates Bmal1 knockdown-induced mitochondrial dysfunction. To further validate the association between mitochondrial dysfunction and JNK activation, we examined the phosphorylation levels of JNK in ALCs from each group by Western blot. The results showed that, compared with the NC group, p-JNK protein levels were significantly elevated in the si-Bmal1 group, while total JNK3 protein levels remained unchanged, indicating that Bmal1 knockdown specifically activated the JNK signalling pathway. In the si-Bmal1+MEL group, p-JNK levels were significantly decreased compared to the si-Bmal1 group (P < .05), but remained higher than those in the NC group (Figure 5E).

Fig. 5.

Bmal1 knockdown significantly disrupts metabolic processes, which is restored by melatonin. (A, B) Gene Set Enrichment Analysis (GSEA) enrichment plots. (C) Representative fluorescent images (left) and quantitative analysis (right) of mitochondrial membrane potential (ΔΨm) in ALCs using JC-1 staining. A decrease in the red/green fluorescence ratio indicates mitochondrial depolarization. (D) Flow cytometric analysis of ΔΨm using JC-10 staining (left) and its quantification (right). The ratio of red (PE, ∼590 nm) to green (FITC, ∼530 nm) fluorescence intensity was used for quantification. Statistical significance is denoted as *P < .05, **P < .01, ***P < .001.

Discussion

Enamel formation involves secretory and maturation stages as detailed in the Introduction, and disruption to either stage can lead to Developmental defects of enamel (DDE). DDE represent a clinical challenge in dentistry due to the lack of regenerative capacity.^4,5 Although previous studies have largely focused on hereditary causes, dental fluorosis, and inflammatory insults to enamel defects,³⁴ the impact of systemic circadian rhythm disruption on enamel biomineralization still unclear.^6,7,12 Our research found that maternal circadian disruption reduced enamel thickness and density in offspring mice (Figure 1A), a phenotype partially rescued by maternal melatonin supplementation (Figure 2). We further observed synchronized fluctuations between serum melatonin levels and Bmal1 expression in ameloblasts across normal and disrupted cycles, with key mineralization proteins upregulated during the dark phase (Figure 1F). ATR‑FTIR spectroscopy revealed that maternal circadian disruption significantly increased the carbonate‑to‑phosphate (CO₃²⁻/PO₄³⁻) peak area ratio in offspring enamel (Figures 1I, 2G), indicating a higher degree of carbonate substitution in the hydroxyapatite lattice. This biochemical abnormality is a hallmark of enamel hypomineralisation, as increased carbonate content reduces crystallinity and compromises mechanical properties. SEM analysis further showed that the enamel surface in circadian‑disrupted groups was rough and irregular, with disorganized enamel prisms, enlarged interprismatic spaces, and markedly increased porosity (Supplementary Figs. 1, 2). These structural defects are consistent with the impaired mineral quality detected by FTIR. Notably, melatonin treatment reduced the CO₃²⁻/PO₄³⁻ ratio and partially restored normal enamel architecture, supporting its role in improving both mineral maturity and structural integrity. These results suggest that melatonin acts as a critical messenger linking maternal light-dark cycles to offspring enamel formation, potentially facilitating circadian control over mineralization through BMAL1.

BMAL1, a core circadian transcription factor, is fundamental to cellular metabolism.^9,35 When we knocked down Bmal1 in ameloblast-lineage cells, it triggered broad transcriptomic changes and impaired mitochondrial electron transport (Figure 5), leading to cellular energy deficit. As central to energy production and stress sensing, mitochondria respond to dysfunction by releasing signals like ATP depletion and reactive oxygen species. These in turn activate the JNK pathway.^29,35 JNK acts as a master stress regulator, curbing differentiation and promoting apoptosis. Our data indicate that loss of Bmal1 elevates JNK3 phosphorylation (Figure 4), shifting cell effort from energy-intensive differentiation (enamel matrix secretion) toward stress adaption.^32,33 The evidence confirmed BMAL1′s established role in metabolism,^30,31 and defines a coherent signalling route that begins with BMAL1, involves mitochondrial energy metabolism, activates JNK, and ultimately resets cell fate. The in vitro Bmal1 knockdown model was designed to recapitulate the molecular consequences of maternal circadian disruption observed in vivo, specifically the downregulation of Bmal1 in ameloblast-lineage cells. This approach enabled us to isolate the cell-autonomous effects of BMAL1 deficiency on mitochondrial function and JNK3 signalling, independent of systemic circadian cues.

Melatonin, an endogenous hormone regulated by light-dark cycle, supports mineralized tissues.25, 26, 27, 28 We confirmed its direct action by showing that it improves enamel mineralization in vivo (Figure 2) and enhancing mineralization in ALCs in vitro (Figure 3). Emerging evidence suggests that metabolic disturbances in oral tissues can have systemic consequences, including regulation of autophagy and neuroinflammation.^36,37 These findings support our hypothesis that circadian disruption-induced metabolic impairment in ameloblasts may impact enamel mineralization. However, melatonin’s effect almost vanished upon Bmal1 knockdown. Without BMAL1, melatonin still suppressed JNK phosphorylation (Figure 4E), indicating a BMAL1-independent mechanism for stress mitigation. Yet, the execution of the energy-intensive mineralization program requires more than just removing the stress brake (JNK); it necessitates activating the pro-anabolic machinery. We propose that BMAL1 functions as a driver. Under physiological conditions, melatonin and BMAL1 act synergistically—the former alleviates stress, while the latter powers differentiation. In the absence of BMAL1, melatonin's action is reduced to a primarily defensive role, resulting in only partial phenotypic rescue.

Our study has several limitations. Although the strong trend of melatonin enhancing ALP activity suggests a biologically meaningful effect, it lacked statistical significance, potentially due to sample size (Figure 3). X‑ray diffraction (XRD) analysis was not performed in this study. A single XRD measurement typically requires approximately 50 mg of powdered enamel sample, yet each mouse mandible yields only about 1 to 2 mg of enamel. Thus, obtaining sufficient material would require approximately 25 -50 mice per experimental group, totalling more than 200 animals, which exceeds ethical limits. Moreover, XRD studies usually employ whole human tooth crowns ground into powder (including dentin),³⁸ an approach not feasible with the limited amount of mouse enamel available. Therefore, XRD analysis was not pursued. Furthermore, although light-based disruption effectively models shift work or nighttime light exposure, we cannot rule out contributions from other rhythm-sensitive hormones like glucocorticoids. The detailed signalling steps linking mitochondria to JNK3 remain unclear. Finally, clinical translation of melatonin supplementation will necessitate careful pharmacokinetic and dosing studies during pregnancy.

Conclusions

We trace a pathological pathway: BMAL1 loss leading to mitochondrial failure, activating JNK stress signalling and impairing mineralization. Melatonin intervenes by suppressing JNK, offering context-aware protection that directly enhances mineralization under stability and provides stress relief during circadian disruption. These results nominate the BMAL1-JNK3 axis as a potential therapeutic target and reinforce the importance of circadian health during the perinatal period.

Author contributions

Xuanyu Wang: Contributed to conception and design, contributed to acquisition, analysis and interpretation, drafted the manuscript, critically revised it, gave final approval, and agrees to be accountable for all aspects of the work ensuring its integrity and accuracy. Jing Liu: Contributed to analysis, critically revised the manuscript, gave final approval, and agrees to be accountable for all aspects of the work ensuring its integrity and accuracy. Mengning Bi: Contributed to conception, contributed to acquisition and analysis, drafted the manuscript, gave final approval, and agrees to be accountable for all aspects of the work ensuring its integrity and accuracy. Xue Li: Contributed to interpretation, critically revised the manuscript, gave final approval, and agrees to be accountable for all aspects of the work ensuring its integrity and accuracy. Xuejiao Yang: Contributed to interpretation, drafted the manuscript, critically revised it, gave final approval, and agrees to be accountable for all aspects of the work ensuring its integrity and accuracy. Wenke Xu: Contributed to analysis, drafted the manuscript, gave final approval, and agrees to be accountable for all aspects of the work ensuring its integrity and accuracy. Zhihua Chen: Contributed to analysis, drafted the manuscript, gave final approval, and agrees to be accountable for all aspects of the work ensuring its integrity and accuracy. Yueying Wang: Contributed to acquisition, analysis and interpretation, drafted the manuscript, critically revised it, gave final approval, and agrees to be accountable for all aspects of the work ensuring its integrity and accuracy. Fang Ji: Contributed to conception and design, contributed to acquisition, analysis and interpretation, drafted the manuscript, critically revised it, gave final approval, and agrees to be accountable for all aspects of the work ensuring its integrity and accuracy. All authors read and approved the final manuscript.

Funding

This work was supported by the Cross-disciplinary Research Fund of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong university School of Medicine [JYJC202311].

Availability of data and materials

The RNA-seq datasets generated during the current study are available in the NCBI Sequence Read Archive (SRA) repository, under BioProject accession number [PRJNA1356859]. All other data generated or analysed during this study are included in this published article and its supplementary information files.

Ethics approval

All animal procedures in this study were approved by the Animal Welfare Committee of Shanghai Ninth People's Hospital, under protocol number SH9H-2021-A436-1. All methods were carried out in accordance with relevant guidelines and regulations.

Conflict of interest

None disclosed.

Appendix. Supplementary materials

Supplementary Fig. 1. Representative scanning electron microscopy (SEM) images of enamel surface morphology. Mandibular first molars from offspring mice in the control group, continuous-light group and high-light group were examined. Images were taken at an accelerating voltage of 5 kV. Scale bars: 50 µm ( × 1000), 10 µm ( × 5000) and 2 µm ( × 20,000). The continuous-light group and high-light group showed increased porosity and surface irregularity compared to the control group.

Supplementary Fig. 2. Representative scanning electron microscopy (SEM) images of enamel surface morphology. Mandibular first molars from offspring mice in the normal light (NV), high light (HV), and high light plus melatonin (HM) groups were examined. Images were taken at an accelerating voltage of 5 kV. Scale bars: 10 µm ( × 5000) and 2 µm ( × 20,000). The HV group showed increased porosity and surface irregularity compared to the NV group, while the HM group exhibited partial structural rescue.