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. 2020 Nov 24;18(2):265–277. doi: 10.1007/s13770-020-00310-3

Autophagy-Related Protein MAP1LC3C Plays a Crucial Role in Odontogenic Differentiation of Human Dental Pulp Cells

Heui Seung Cho 1,#, Sam Young Park 1,#, Sun Mi Kim 2, Won Jae Kim 1,✉, Ji Yeon Jung 1,✉
PMCID: PMC8012421  PMID: 33230801

Abstract

Background:

Autophagy plays important roles in odontogenic differentiation of dental pulp cells (DPCs) in the developmental stage of tooth bud. Few studies have reported the role of autophagy during reparative dentin formation process. The objective of this study was to discover gene expression pattern correlated to autophagy and their role during odontogenic differentiation process in DPCs.

Methods:

After tooth cavities were prepared on the mesial surface of lower first molar crown of rats. Odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining.

Results:

After tooth cavities were prepared on the mesial surface of lower first molar crown of rats, odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining and dentin sialophosphoprotein (DSPP), whereas autophagy inhibitor 3-methyladenine (3MA) reversed. Results of quantitative polymerized chain reaction array of autophagosome formation related genes revealed that GABARAPL2 was prominently upregulated while expression of other ATG8 family members were moderately increased after tooth cavity preparation. In addition, human DPCs incubated in differentiation medium predominantly upregulated MAP1LC3C, which selectively decreased by 3MA but not by autophagy enhancer trehalose. Knock-down of MAP1LC3C using shRNA resulted in strong downregulation of dentin matrix protein 1 and DSPP as well-known odontogenic marker compared to knock-down of MAP1LC3B during odontogenic differentiation process of human DPCs.

Conclusion:

Our results suggest that MAP1LC3C plays a crucial role in odontogenic differentiation of human DPCs via regulating autophagic flux.

Electronic supplementary material

The online version of this article (10.1007/s13770-020-00310-3) contains supplementary material, which is available to authorized users.

Keywords: Cell differentiation, Odontogenesis, Pulp biology, Stem cells

Introduction

During tooth eruption, dental papilla as part of the tooth germ contains mesenchymal cells that have potential to differentiate into odontoblast. In the presence of exogenous stimuli, such as tooth eruption, expansion of caries lesions and pulpal exposure by trauma or cavity preparation, dental pulp cells (DPCs) reside in dental pulp tissue can proliferate and differentiate into odontoblast-like cells to form secondary and reactionary dentin [1, 2]. It has been suggested that reparative dentin formation is a natural dental recovery process due to odontogenic differentiation of DPCs upon external stimuli [3]. The mechanisms underlying odontogenic differentiation process have not been completely elucidated, although it has been reported that complicate signaling pathways are involved in the differentiation process of DPCs [4–6].

Autophagy is a process of self-degradation that is crucial for balancing energy sources at important times during development or in the response to stress due to insufficient nutrient. Autophagy also plays a housekeeping role by eliminating misfolded proteins, damaged organelles, and intracellular pathogens. It has a peculiar cargo named autophagosome which has a double-membrane structure containing a set of proteins called autophagy-related (ATG) proteins [7]. ATG proteins are known to control initiation and expansion of autophagosomal membrane to encapsulate cytosolic contents for lysosomal degradation. Autophagy is initiated by unc-51-like autophagy activating kinase 1/2 complex (ULK1/2 complex) and ATG9A is a transmembrane protein of autophagy. These two components are both derived from the site of autophagosome formation. Class III phosphatidylinositol 3-kinase complex is known to subsequently induce the generation of phospholipid phosphatidylinositol-3-phosphate (PI3P), which gathers downstream factor WIPI1/2 along with other autophagy proteins (including ATG1A/B and ATG8 family proteins). It then expands and closes the autophagosomal membrane to complete autophagosome formation [8]. In the past few years, genetic analysis of ATG products has advanced markedly and multiple mammalian homologues of ATG have been characterized. After deleting ATG7, glycogen metabolism is altered, causing hepatomegaly in mice [9]. ATG13, one of components of ULK1/2 complex, was revealed to be essential for cardiac development in mice via autophagy [10].

The roles of autophagy have implicated in various cellular functions, including cell proliferation, differentiation, aging, and immunity [11]. Focusing on autophagic function in adult stem cell differentiation, recent studies have proven that autophagy is required for differentiation of human adult stem cells, including epidermal, dermal, hematopoietic, and cardiac stem cells [12]. Particularly, odontoblasts are involved in the transmission of sensory stimuli from the dentin-pulp complex and in the cellular defense against pathogens. Their homeostasis and longevity is maintained by an elaborate autophagic-lysosomal system that renews necessary organelles and proteins [13].

Previous studies have also shown that autophagy is essential to the differentiation process of human DPCs [14, 15]. However, research on gene expression pattern correlated with autophagy activated during odontogenic differentiation and reparative dentin formation of DPCs are little. Therefore, the objective of this study was to verify autophagy-related gene expression and the roles in odontogenic differentiation of DPCs.

Materials and methods

Animals and chemicals

Male Sprague–Dawley (SD) rats at 8 weeks old were purchased from Daehan Biolink (Chungbuk, Korea). These animals were kept under standard conditions (22 °C, humidity of 55%, and 12-hour light/12-hour dark cycle). They were provided free access to food and water. Rats were divided into the following three groups: non-treated group (control), phosphate buffered saline (PBS) intraperitoneal (IP) injection group, and 3-methyladenine (3MA, diluted in PBS) (Sigma-Aldrich, Saint Louis, MO, USA) IP injection group (5 rats/each group). 3MA (10 mg/kg weight) in 0.5 ml of PBS or PBS only was injected on right lower abdomen of rats. The injection was performed on alternate days from 4 days before preparation and continued until the day of sacrifice. For tooth cavity preparation experiments, rats were lightly anesthetized with diethyl ether (Junsei, Tokyo, Japan) for 1 min followed by immediate IP injection of solution (1.5 mL/kg weight) with Zoletil 50 (Virbac, Carros, France), Rompun (Bayer, Leverkusen, Germany), and PBS in ratio of 9:5:8. All experiments were approved by the Animal Care and Use Committee of Chonnam National University. All chemicals used in experiments were purchased from Sigma-Aldrich.

Tooth cavity preparation on rat molars

Under deep anesthesia, limbs of rat were fixed and mouth was opened using a mouth holder. For tooth cavity preparation, autoclaved low-speed handpiece with medium grit diamond bur (Dentsply Sirona, York, PA, USA) was used under water spray cooling. Tooth cavities were made on the mesial surface of tooth crown of lower left first molars. Teeth were not perforated into dental pulp. They were drilled only into the dentin layer. Lower right first molars were preserved and used as controls of each day. Rats were sacrificed at 0, 3, 8, and 10 days after tooth cavity preparation without or with autophagy inhibitor 3MA (10 mg/kg weight).

Preparation of rat dental tissue for hematoxylin and eosin (H&E) staining

Rats were perfused transcardially with 0.1 M PBS followed by fresh cold 4% paraformaldehyde (PFA) in 0.1 M PBS. Rat mandible and molars were extracted together and fixed in 4% PFA in 0.1 M PBS at 4 °C overnight. They were then decalcified at 4 °C for 3–4 months in RapidCal Immuno (BBC Biochemical, Mt Vernon, WA, USA) with one-week exchange period. After decalcification, tissues were fixed again in 4% PFA in 0.1 M PBS at 4 °C overnight and washed with 0.1 M PBS for 3 h twice. They were then stored in 0.1 M PBS at 4 °C overnight. On the next day, tissues were dehydrated gradually using 50, 60, 70, 80, 90, and 100% ethyl alcohol (Duksan, Ansan, Korea) each for 1 h. They were then stored in ACS grade ethyl alcohol (Honeywell, Morris Plains, NJ, USA) at 4 °C overnight. Before paraffin embedding, tissues were immersed in xylene (Duksan) for 2 h at room temperature twice. Tissues were then embedded in 50, 75, and 100% solution of paraffin wax (CellPath, Wales, UK) in xylene for 1 h each in a vacuum dry oven (65 °C). They were then embedded in new paraffin wax overnight. Paraffin embedded mandible and molars were cut sagittally at thickness of 4 µm using a microtome (RM2235, Leica Microsystems, Wetzlar, Germany). Sections were mounted on charged slide glasses for IHC.

H&E staining for paraffin sections

Paraffin sections were washed with xylene for 5 min twice and hydrated with 100, 90, 80, 70, and 60% ethanol in distilled water for 5 s each. After washing in running water for 2–3 min, sections were dipped in hematoxylin for 1.5 min, washed with running water for 2–3 min, and then immersed in eosin for 30 s. Sections were then dehydrated in 60, 70, 80, 90, and 100% ethanol in distilled water for 3 s each. After natural drying, Canada balsam was used as mount medium. Every procedure was done at room temperature. H&E staining images were acquired using a microscope (DFC450C, Leica Microsystems).

Tooth crown-pulp lysate preparation

Rats were anesthetized by the same method mentioned in ‘Animals and chemicals’ section. Rat mandible was fixed with Halsted mosquito hemostatic forceps. Lower first molars were extracted with Allis tissue forceps by pulling up and down several times smoothly. Roots and attached soft tissues of teeth were removed before homogenization for protein or RNA preparation. Such removal was done using autoclaved micro forceps and micro scissors with observation under a microscope (SZ51; Olympus, Tokyo, Japan). To compensate insufficient quantity of protein or RNA for preparation, four rats were pooled as one and homogenized.

Cell culture and chemicals

Human dental pulp tissues were obtained from cut teeth aseptically, rinsed with Hanks’ buffed saline solution, and placed on 60-mm Petri dishes. These tissues were then chopped finely with a blade into small fragments and incubated in α-Minimum Essential Medium (α-MEM) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) along with 1% penicillin and streptomycin (Thermo Fisher Scientific). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. For odontogenic differentiation and dentin formation experiments, cells were cultured in differentiation media (DM) containing 50 μM ascorbic acid and 5 mM β-glycerophosphate. Trehalose (50 mM; Sigma-Aldrich) and 3MA (1 mM; Sigma-Aldrich) were dissolved in distilled water and sterilized using 0.2 μm filters.

Total RNA preparation and cDNA synthesis

For tissues, tooth crown-pulp pools were mixed with Trizol reagent (RiboEx; GeneAll, Seoul, Korea) and crushed using RNase free mortar and pestle first. They were then homogenized on ice using a homogenizer (T10B; IKA). For cultured cells, harvested human DPC were directly homogenized on ice with pellet pestle motor (Kontes). After total RNA extraction, remaining genomic DNA was eliminated using DNase kit (DNA-Eraser; Intron Biotechnology, Seongnam, Korea). DNA-eliminated total RNA (1.5 μg) was then subjected to cDNA synthesis using Prime RT-premix kit (Genet Bio, Daejeon, Korea). After cDNA synthesis, 180 μl of RNase-free water was added to minimize enzymatic effect.

Quantitative polymerase chain reaction (qPCR) arrays

RT2 Profiler PCR Arrays (PARN-084Z; Qiagen, Hilden, Germany) were performed as qPCR arrays for 84 autophagy-related genes using cDNAs prepared from tooth lysates of rat tooth cavity preparation models on a real-time thermal cycler (Step One Plus; Thermo Fisher Scientific). Data normalization was based on correcting every ct values for the average ct value of ACTB, B2M, and HPRT1 gene present on the array. Three independent experiments were performed. To analyze PCR-array results, web-based data analysis program (http://www.qiagen.com/kr/shop/genes-and-pathways/data-analysis-center-verview-page/) was used.

qPCR of ATG8 family and odontogenic marker genes

qPCR mixtures (10 μL) contained 1 μL of synthesized cDNA, 1 μL of 10 pM qPCR primer pairs, and 5 μL of SYBR green mix (Quanti Speed; PhileKorea, Daejeon, Korea). The following qPCR primers were used: for human MAP1LC3A, 5′-CAGCATGGTGAGTGTGT CCA-3′ (forward), 5′-TCAGAAGCCGAAGGTTTCCT-3′ (reverse), for human MAP1LC3B, 5′-AGGCCTTCTTCCTGTTGGTG-3′ (forward), 5′-ATTTCATCCCGAACGTCTCC-3′ (reverse), for human MAP1LC3C, 5′-ACAAGAGGAAGTTGCTGGAATCCG-3′ (forward), 5′-GATGATGCTGAGGAACTGGGTCAT-3′ (reverse), for human GABARAP, 5′-CTCTG AGGGCGAGAAGATCC-3′ (forward), 5′-TGAGATCAGAAGGCACCAGG-3′ (reverse), for human GABARAPL1, 5′-TTTGGTGCCCCTTATCTCAC-3′ (forward), 5′-GGCCATCA TGTAGCATTCCTT-3′ (reverse), for human GABARAPL2, 5′-CGAAATATCCCGACAG GGTT-3′ (forward), 5′-TCCACAAACAGGAAGATCGC-3′ (reverse), for human DMP1, 5′-TGGTCCCAGCAGTGAGTCCA-3′ (forward), 5′-TGTGTGCGAGCTGTCCTCCT-3′ (reverse), for human DSPP, 5′-GGGAATATTGAGGGCTGGAA-3′ (forward), 5′-TCATTG TGACCTGCATCGCC-3′ (reverse), for human GAPDH, 5′-AAGGGTCATCATCTCTGCC C-3′ (forward), 5′-G TGATGGCATGGACTGTGGT-3′ (reverse). Product accumulation was monitored by SYBR green fluorescence using a real-time thermal cycler (Rotor Gene 3000; Qiagen). Relative expression was determined from a standard curve of serial dilutions of cDNA samples. ATG8 family and odontogenic marker mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level. To quantitatively compare gene expressions, 2−ΔΔCt (Ct: cycle threshold) method was used.

RNA interference with shRNA

To silence specific genes, shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA) of MAP1LC3B or MAP1LC3C were seeded in incubated human DPC following the manufacturer’s instructions. Gene knockdown was confirmed by comparing gene levels after treatment with specific shRNA lentiviral particles with those after treatment with sh-control (mock) lentiviral particles (Santa Cruz Biotechnology) in cultured human DPCs by reverse transcription-qPCR.

Autophagosome detection by acridine orange

Acidic autophagosomes were visualized by acridine orange staining. Human DPCs were cultivated on confocal dish (SPL, Pocheon, Korea) in α-MEM containing 10% FBS, 50 μM ascorbic acid, and 5 mM β-glycerophosphate without or with RNA interference of MAP1LC3B or MAP1LC3C for 0 or 11 days. At the end of each time period of incubation, cells were treated with 1 μg/mL acridine orange (Sigma-Aldrich) in FBS-free medium for 15 min at 37 °C. Next, cells were washed three times with PBS. After removing acridine orange solution, cells were examined with a confocal microscope (Zeiss). Depending on acidity, autophagosomes appeared as bright orange fluorescent in cytoplasm while nuclei was green.

Immunofluorescence stain

Human DPCs were cultured on confocal dish (SPL) in α-MEM containing 10% FBS, 50 μM ascorbic acid, and 5 mM β-glycerophosphate with knock-down of MAP1LC3B, MAP1LC3C, or mock knock-down for 0 or 11 days. At the end of the incubation period, cells were fixed with 4% paraformaldehyde first, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 10 min, and incubated with 1% bovine serum albumin in PBS for 1 h at room temperature. After washing with PBS, cells were incubated with rabbit anti-DSPP (1:200) primary antibody in PBS at 4 °C overnight followed by incubation with FITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) secondary antibody. DAPI hard set mount medium (Vector Laboratories, Burlingame, CA, USA) was dropped into the center of the confocal dish to stain nuclei with blue fluorescence.

Data analyses

H&E images were loaded with Leica LAS AF (Version 2.3.5 build 5379, Leica Microsystems). Fluorescence IHC images were acquired with Zeiss LSM Image Browser (Version 4.2.0.121, Carl Zeiss). Adobe Photoshop (Version CS6, Adobe Systems, San Jose, CA, USA) was used to make composites. For analysis of qPCR arrays, Data Analysis Center (Qiagen) web-based program was used. Data are presented as mean ± SEM of three independent experiments. Statistical difference between means was analyzed with Student t test with analysis of variance (ANOVA). Difference was considered statistically significant at p-value < 0.05.

Results

Induction of odontogenic differentiation and reparative dentin formation near tooth cavity preparation site is regulated by autophagy

To examine the induction of odontogenic differentiation and reparative dentin formation in rat DPCs, tooth cavity models were prepared on lower first molars of male SD rats. By using water cooling spray with low-speed dental handpiece, cavity was prepared into dentin layer without perforation into dental pulp on mesial surfaces of rat lower first molars (Fig. 1A). The sagittal sections of rat lower first molar tissues were visualized by H&E staining to verify reparative dentin appeared under the cavity preparation site. At 3 days after tooth cavity preparation, DPCs subjacent to cavity preparation site showed balloon shaped morphology, indicating that they were in the process of odontogenic differentiation. However, non-cavity prepared control group displayed spindle shaped morphology. At 8 and 10 days after tooth cavity preparation, newly-formed reparative dentin layer appeared on the odontoblast layer close to cavity preparation site (Fig. 1B). These data demonstrated that reparative dentin layer was newly formed with odontogenic differentiation of DPCs upon external stimulation.

Fig. 1.

Fig. 1

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Induction of odontogenic differentiation and reparative dentin formation in rat DPCs near cavity preparation site. A Tooth cavities were prepared on the mesial surface (M) of lower first molar of SD rats while distal surface (D) was conserved. Cavity was prepared into dentin layer with water cooling spray of low-speed dental handpiece on mesial surfaces of rat lower first molars. The sagittal sections of rat lower first molar tissues were visualized by hematoxylin and eosin (H&E) staining. B Sagittal rat lower first molar sections at 0, 3, 8, and 10 days after tooth cavity preparation (Prep) were stained with H&E. Non-cavity prepared teeth were used as the control group (Ctrl). Black arrowed curve: balloon shaped cells; Black arrowed lines: newly formed reparative dentin. C Immunoreactivity of DSPP in sagittal lower first molar sections from rats sacrificed at 0, 3, 8, and 10 days after tooth cavity preparation with or without 3-methyladenine (3MA) injection was visualized by fluorescence immunohistochemistry. Scale bars: 500 μm in 50X and 50 μm in 400X. D Densitometry analysis for the protein expression of DSPP was presented as relative ratio compared with β-Actin. Data are expressed as mean ± SEM. Student’s t-test was used to compare means of independent or paired groups (n = 3; *p < 0.05, **p < 0.01 vs. Day 0 control or preparation group)

To verify general expression level of odontoblastic differentiation marker associated with autophagy modulation in DPCs, fluorescence IHC was performed in rat lower first molar at 3, 8, and 10 days after tooth cavity preparation with or without autophagy inhibitor 3-methyladenine (3MA) injection. Expression level of DSPP was increased in the cavity prepared group in a time-dependent manner, whereas 3MA injection showed reversed effect compared to preparation without 3MA (Fig. 1C, D). Additionally, upregulation of autophagy indicator p62 and proliferation marker Ki67 in the cavity prepared group were blocked by 3MA (Supplementary Figs. S1, S2). These results demonstrated that autophagy is involved in odontoblast differentiation during reparative dentin formation process.

Autophagy-related gene shows significant change of expression level during odontogenic differentiation and reparative dentin formation process in rat DPCs

To investigate the signaling molecules regulating autophagy during odontogenic differentiation and reparative dentin formation process in DPCs, qPCR arrays were used to determine expression levels of 84 genes, which are associated with autophagosome formation, autophagosome targeting, protein transport, ubiquitination, proteases, co-regulators of autophagy & apoptosis, co-regulators of autophagy and the cell cycle, autophagy induction by intracellular pathogens, autophagy in response to other intracellular signals, and chaperone-mediated autophagy. Total RNA was extracted from rat lower first molar crown-pulp lysates at 3 and 8 days after cavity preparation and reversely transcribed into cDNA for use in qPCR array. Results showed no significant changes in expression levels of these genes in the non-cavity prepared control group at different time points. However, there were minor (less than two-fold) changes in mRNA expression levels at 3 days after tooth cavity preparation (Fig. 2A). In addition, two-fold or greater mRNA changes were shown at 8 days after tooth cavity preparation (Fig. 2B). Pronounced changes of GABARAPL2, ATG9A, and PARK7 expression were found. GABARAPL2 level was increased over ten-fold while ATG9A level was upregulated over four-fold at 8 days after tooth cavity preparation (Fig. 2B).

Fig. 2.

Fig. 2

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Analysis of qPCR arrays for autophagy-related genes in odontogenic differentiation and reparative dentin formation process of rat DPCs. A Rat qPCR arrays for autophagy-related genes were performed to validate mRNA expression levels in DPCs from rat lower first molar at 3 and 8 days after tooth cavity preparation (Day 3, 8). No significant changes were found in the non-cavity prepared control group at all time points (Ctrl). However, in tooth cavity prepared experimental group, changes of less than two-fold were detected at 3 days after tooth cavity preparation while changes of two-fold or greater were found at 8 days after cavity preparation (Prep). B Rat qPCR arrays for autophagy-related genes in DPCs at 8 days after tooth cavity preparation confirmed that some genes showed two-fold or greater changes. Pronounced changes in expression level were found for GABARAPL2, ATG9A, and PARK7. Black solid line means no change while black dotted line indicates two-fold change. Normalization of data was based on average ct values of ACTB, B2M, and HPRT1 in the array. Scatter plot analysis was performed for each gene. Three independent biological replicates were used

Mammalian ATG8 family genes are particularly expressed in odontogenic differentiation and reparative dentin formation process of rat DPCs

Since qPCR array data implied evident expression of GABARAPL2 and ATG9A known to be important components of autophagosome formation process, qPCR array data at 8 days after tooth cavity preparation were reconstituted for selected genes associated with autophagosome formation process. These selected genes were classified based on their sub-functions (initiation, nucleation, elongation & closure, and fusion & degradation) and their changed expression levels were plotted. Results showed prominent mRNA increment (over ten-fold) of GABARAPL2, one homologue of mammalian ATG8 family genes. However, other mammalian ATG8 family genes (GABARAP, MAP1LC3A, and MAP1LC3B) only showed moderate upregulation (about two-fold) at 8 days after tooth cavity preparation (Fig. 3). These data suggested that ATG8 family genes might be particularly expressed in DPCs during odontogenic differentiation after tooth cavity preparation.

Fig. 3.

Fig. 3

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Fold change of genes related to autophagosome formation in odontogenic differentiation and reparative dentin formation process of rat DPCs. Rat qPCR arrays for autophagy-related genes at 8 days after tooth cavity preparation were rearranged based on their association with autophagosome formation process. Selected genes were categorized into four groups according to stages of autophagosome formation (Initiation, Nucleation, Elongation & Closure, and Fusion & Closure). Changes in mRNA expression of selected genes in tooth cavity prepared group at 8 days after cavity preparation (Day 8 Prep) relative to expression level in non-cavity prepared control group at day 0 (Day 0 Ctrl) were plotted. Normalization of data was based on average ct value of ACTB, B2M, and HPRT1 gene. Data are expressed as mean ± SEM. Data were statistically analyzed using ANOVA (n = 3; *p < 0.05, **p < 0.01 vs. Day 0 control group)

MAP1LC3C plays a crucial role in odontogenic differentiation process of human DPCs

To examine expression levels of mammalian ATG8 family genes in the process of odontogenic differentiation in vitro culture, human DPCs were cultivated for 7 and 11 days (Day 7 and 11) in non-differentiation media (NM) or differentiation media (DM). Expression levels of six ATG8 family genes (MAP1LC3A, MAP1LC3B, MAP1LC3C, GABARAP, GABARAPL1, and GABARAPL2) and odontogenic marker genes (DMP1 and DSPP) in human DPCs were then assessed by qPCR. Expression levels of all ATG8 family genes in DPCs after incubating with DM showed two-fold or greater increment after 11 days of incubation compared to those in control group of DPCs incubated with NM. Expression level of MAP1LC3C was increased significantly higher (near seven-fold in 11 days) than those of other ATG8 family genes (MAP1LC3A, MAP1LC3B, GABARAP, GABARAPL1, and GABARAPL2). Significant increment in expression level of DMP1 and DSPP in DPCs incubated with DM was also found in a time-dependent manner compared to those of culture cells in NM (Fig. 4A).

Fig. 4.

Fig. 4

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Effect of autophagy regulators on expression of mammalian ATG8 family genes in odontogenic differentiation process of human DPCs. A Human DPCs were cultivated in non-differentiation normal media (NM) or differentiation media (DM) for 0, 7, and 11 days. They were then harvested at indicated time. Expression was assessed by qPCR. Expression levels of six ATG8 family genes and odontogenic marker genes (DMP1 and DSPP) of human DPCs incubated in NM or DM were plotted. B Human DPCs were incubated in DM only (DM), DM with 1 mM 3MA (3MA), or DM with 50 mM trehalose (TRE) for 0, 7, and 11 days. Human DPCs incubated with DM, DM with 3MA, or DM with trehalose were harvested at indicated days. Total cell lysates were used for RNA extraction. Expression levels were determined by qPCR. Expression levels of six ATG8 family genes and odontogenic marker genes DMP1 and DSPP in human DPCs incubated with DM, DM with 3MA, or DM with trehalose were plotted. GAPDH expression was used as internal control. Data are expressed as mean ± SEM. Student’s t-test was used to compare means of independent or paired groups (n = 3; *p < 0.05, **p < 0.01 vs. Day 0 control group;#p < 0.05, ##p < 0.01 vs. Day 11 control group)

To determine whether MAP1LC3C among ATG8 family genes had selective role in odontogenic differentiation process, expression levels of six ATG8 family genes and odontogenic marker genes DMP1 and DSPP in human DPCs incubated for 7 and 11 days (Day 7 and 11) in DM only, DM with 1 mM 3MA (an autophagy inhibitor), or DM with 50 mM trehalose (an autophagy enhancer) were examined by qPCR. Expression levels of GABARAPL2 and MAP1LC3C in DPCs incubated in DM with 3MA were significantly downregulated compared to those in DPCs incubated in DM only after 11 days incubation. Expression level of DSPP was also significantly downregulated in DPCs incubated in DM with 3MA compared to that in DPCs incubated in DM only after 11 days. However, expression levels of MAP1LC3C and DSPP were significantly upregulated in DPCs incubated in DM with trehalose compared to those in DPCs incubated in DM only after 11 days of culture (Fig. 4B). These results indicate that MAP1LC3C is prominently expressed in human DPCs during odontogenic differentiation.

MAP1LC3C regulates expression of DSPP in odontogenic differentiation process of human DPCs

To determine the role of MAP1LC3C in odontogenic differentiation process of human DPCs, cells infected with lentivirus expressing MAP1LC3B or MAP1LC3C specific shRNA or control shRNA were incubated in DM and mRNA expression levels of mammalian ATG8 family genes and DMP1 and DSPP as odontogenic marker genes were determined by qPCR. Results verified successful knockdown of each targeted gene. Expression level of DSPP in MAP1LC3B or MAP1LC3C knockdown group was decreased compared to that in mock-infected control group. Knock-down of MAP1LC3C resulted in prominent and significant downregulation of DSPP expression compared to knock-down of MAP1LC3B at 7 and 11 days after incubation in DM. However, expression level of DMP1 was only decreased in MAP1LC3C knock-down group, not in MAP1LC3B knock-down group, compared to that in mock-infected control group at 7 and 11 days after incubation in DM (Fig. 5).

Fig. 5.

Fig. 5

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Effect of MAP1LC3C knockdown in odontogenic differentiation process of human DPCs. Human DPCs were infected with lentivirus suppressing MAP1LC3B (sh-LC3B), MAP1LC3C (sh-LC3C), or mocking (sh-Cont) specific shRNA and incubated in DM for 0, 7, and 11 days. Cells harvested in indicated days were used for qPCR analysis. Successful knock-down of each target gene was confirmed. Expression levels of six ATG8 family genes and odontogenic marker genes DMP1 and DSPP in human DPCs with indicated gene knockdown cultivated in DM were plotted. GAPDH expression level was used as internal control. Data are expressed as mean ± SEM. Student’s t-test was used to compare means of independent or paired groups (n = 3; *p < 0.05, **p < 0.01 vs. Day 0 control group;#p < 0.05,##p < 0.01 vs. Day 11 control group)

To confirm that knockdown of MAP1LC3C could reduce autophagosome formation during odontogenic differentiation, lentivirus expressing MAP1LC3B or MAP1LC3C specific shRNA or control shRNA was used to infect human DPCs incubated in DM for 0 or 11 days and visualized by confocal fluorescence microscope after acridine orange staining. Cells in MAP1LC3C knockdown group displayed predominantly green fluorescence with minimal orange fluorescence. However, moderate orange fluorescence was observed in MAP1LC3B knockdown group and considerable orange fluorescence was found in mock-infected control group at 11 days after incubation with DM (Fig. 6A).

Fig. 6.

Fig. 6

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Effect of MAP1LC3C knockdown on formation of autophagosome and expression of DSPP in odontogenic differentiation process of human DPCs. A Human DPCs plated in confocal dishes were preliminarily infected with lentivirus down-regulating MAP1LC3B (sh-LC3B), MAP1LC3C (sh-LC3C), or mocking (sh-Cont) specific shRNA and cultured in DM for 0 or 11 days. These cells were then fixed with 4% paraformaldehyde, stained with acridine orange, and visualized by confocal fluorescence microscope. Autophagosome formation was shown in puncta fluorescence (orange). Scale bars: 50 μm. B Human DPCs plated in confocal dishes were preliminarily infected with lentivirus suppressing MAP1LC3B (sh-LC3B), MAP1LC3C (sh-LC3C), or mocking (sh-Cont) specific shRNA and cultured in DM for 0 or 11 days. They were then analyzed by immunofluorescence using anti-DSPP antibody. Blue fluorescence indicates DAPI. Scale bars: 50 μm

To determine whether knockdown of MAP1LC3C could inhibit protein expression of DSPP during odontogenic differentiation, DSPP in cytoplasm of human DPCs stained with immunofluorescence was observed with a confocal microscope. Expression level of DSPP was markedly decreased in MAP1LC3C knockdown group. However, its level was maintained in MAP1LC3B knockdown group and increased in mock-infected control group at 11 days after incubation with DM compared to that in control (0-day) (Fig. 6B). These data indicate that MAP1LC3C plays a crucial role in odontogenic differentiation of human DPCs.

Discussion

The reparative mechanism that restores pulp-dentin complex following carious or dental damage such as cavity preparation with dental handpiece is crucial to dental pulp regeneration. It consists of a series of highly conserved processes, including newly derived proliferation and differentiation of DPCs by external stimuli. Reparative dentin formation is one important process in the recovery of pulp-dentin complex [16–18]. Following intensive injuries of dentin, subjacent DPCs are attracted to the damaged site. They will undergo odontogenic differentiation to build up new dentin to cover lost enamel-dentin layer thickness [19, 20]. In the present study, tooth cavity models were prepared on the mesial surface of rat lower first molar crown for convenient cavity preparation and accurate observation of morphology change in odontoblast and dentin layer near the cavity preparation site. Results of H&E staining confirmed that reparative dentin formation with thickness of 100 μm occurred at 8 and 10 days after tooth cavity preparation. In supplementary Figs. S1 and S2, fluorescence IHC revealed that upregulation of autophagy marker p62 or proliferation indicator Ki67 near the prepared cavity was diminished by 3MA autophagy inhibitor, which blocks autophagosome formation via the inhibition of type III Phosphatidylinositol 3-kinases (PI-3K) [21]. In addition, autophagy inhibitor 3MA injection restored their morphological changes and odontogenic differentiation marker DSPP expression suppressed after cavity preparation, suggested that autophagy is involved in odontogenic differentiation as well as proliferation of DPCs during reparative dentin formation process.

Recent studies have proven that autophagy is required for differentiation of human adult stem cells, including epidermal, dermal, hematopoietic, and cardiac stem cells [12]. It has been demonstrated that defect in autophagy causes abnormal cell differentiation and development in various organisms, including Caenorhabditis elegans, drosophila, and mouse [22–24]. The previous studies have also suggested that autophagy is essential for the odontoblastic differentiation process of incubated human DPCs [14, 15]. Despite these advances, the mechanisms underlying the relation between autophagy and differentiation of mammalian stem cells including DPCs remain unclear. The present study verified the role of autophagy in odontogenic differentiation and reparative dentin formation process by altering expression of autophagy-related genes.

Following the first discovery of ATG1, more than forty ATGs involved in different steps of autophagy pathway have been identified in yeast [25, 26]. In a recent review, ATGs in core machinery of autophagosome formation are classified under five heads: ATG1/ULK complex, ATG9 and its cycling system, PtdIns3K complex, ATG8 Ubl conjugation system, and ATG12 Ubl conjugation system. ATG1/ULK complex regulates the initiation of autophagosome. ATG9 and its cycling system play a role in delivery of membrane. PtdIns3K complex has roles in nucleation of vesicles. ATG8 and ATG12 conjugation systems act in expansion of vesicles [26]. To determine expression level changes of genes related to autophagy in odontogenic differentiation and reparative dentin formation process, qPCR gene array of 84 autophagy-related genes, including components for autophagosome formation and autophagy regulation, was done for tooth crown-pulp after 0 or 8 days of cavity preparation. Expression levels of GABARAPL2, ATG9A, PARK7, and MAPT showed more than four-fold increments in cavity prepared group at 8 days after preparation compared to those in non-cavity prepared group. Focusing on increased expression of GABARAPL2 and ATG9A as components related to autophagosome formation, genes associated with autophagosome formation were selected and their fold changes were plotted. Interestingly, expression of GABARAPL2, one of ATG8 family genes was predominantly increased more than ten-fold, although expression levels of others were increased about two-fold. These findings imply that predominant ATG8 family members could regulate autophagosome formation in odontogenic differentiation.

ATG8 family is one of highly conserved eukaryotic gene families and the key molecular components involved in autophagy. Although yeast only has a single ATG8 gene, mammalian has three different ATG8 subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3), γ-amino-butyric acid receptor-associated protein (GABARAP), and Golgi-associated ATPase enhancer of 16 kDa (GATE-16) [27, 28]. In humans, MAP1LC3 subfamily has four genes (MAP1LC3A, MAP1LC3B, MAP1LC3B2, and MAP1LC3C) while GABARAP subfamily is comprised of two genes (GABARAP, GABARAPL1). However, GATE-16 subfamily is composed of only one gene (GABARAPL2 or GATE-16). A recent research has revealed that MAP1LC3, GABARAP, and GATE-16 subfamilies are all essential during formation of autophagosome in human epithelioid cervical cell line, although they act differently. MAP1LC3 subfamily contributes to the elongation of autophagosome membrane while GABARAP and GATE-16 subfamilies might mediate sealing and maturation of autophagosome [29]. MAP1LC3A and MAP1LC3B are considered as the most important markers of autophagosome formation, whereas MAP1LC3C is not treated as a novel marker of autophagy based on belief that MAP1LC3C is weakly or not expressed in normal tissues mostly [30–32]. However, recent studies have suggested that MAP1LC3C also plays a crucial role in selective autophagy of human cells by autophagy receptor CALCOCO2/NDP52 [33, 34].

There are some differences in distribution of ATG8 family genes. Some ATG8 family members have different physiological roles and tissue or cell-specific function [28]. Predominant expression of MAP1LC3C has been verified in the lung, although it may not have a primary role generally compared to other members of ATG8 subfamilies [30, 35]. In GABARAP and GATE-16 subfamilies, GABARAP is markedly expressed in endocrine glands while GABARAPL1 is strongly expressed in the brain and the central nervous system [36, 37]. In addition, LC3 expression level is increased whereas GABARAP expression is decreased in human dementia with Lewy bodies [38]. In the present study, expression levels of six human ATG8 family genes and odontogenic marker genes DMP1 and DSPP during odontogenic differentiation process of human DPCs were assessed by qPCR and fold changes compared to their expression in control group with NM were determined. Human DPCs incubated in DM for 11 days showed prominent increment of MAP1LC3C expression (about six-fold) while other ATG8 family members displayed moderate increment (lower than four-fold) (Fig. 4A). Additionally, autophagy inhibition with 3MA treatment resulted in selective down-regulation of MAP1LC3C expression. However, autophagy enhancement with trehalose augmented the expression of MAP1LC3C in human DPCs incubated with DM for 7 or 11 days (Fig. 4B). DSPP showed similar expression and features associated with regulation of MAP1LC3C (Fig. 5). These data confirmed that MAP1LC3C was predominantly expressed and regulated during odontogenic differentiation process of human DPCs.

It has been recently reported that MAP1LC3C plays a role in autophagosome formation and coat protein complex II (COPII) dependent ER export through binding to tectonin beta-propeller repeat-containing protein 2 (TECPR2), indicating that secretory pathways could be associated with autophagy [39]. Calcium-binding and coiled-coil domain-containing protein 2 (CALCOCO2; NDP52), a selective autophagy receptor, also shows preferable binding with LC3C (translational product of MAP1LC3C) while MAP1LC3C has selective role in antibacterial effect of autophagy [33, 34]. However, no study has reported the role of MAP1LC3C (or LC3C) in odontogenic differentiation process of human DPCs. In the present study, MAP1LC3B and MAP1LC3C were selectively knocked-down by infecting human DPCs with lentivirus expressing specific sh-RNA and incubated in DM for 0, 7 or 11 days. MAP1LC3C knock-down lead to down-regulated DMP1 or DSPP mRNA level the most whereas MAP1LC3B knock-down only exhibited moderate decrement of odontogenic markers. However, mock knock-down displayed no decrement in these genes (Fig. 5).

In addition, fluorescence of autophagosomal puncta in MAP1LC3B and MAP1LC3C knock-down groups cultivated in DM for 11 days were decreased by acridine orange staining, although acidic autophagosomes in mock knock-down control group were increased. Immunofluorescence results also showed that MAP1LC3C knock-down displayed higher efficiency in inhibiting DSPP expression after 11 days incubation in DM odontogenic differentiation of human DPCs compared to MAP1LC3B or mock knock-down. These results confirmed that MAP1LC3C had important role during odontogenic differentiation process of human DPCs. High sequence similarity among these members lessens antibody specificity, making it difficult to exploit specific epitopes to construct novel antibodies. Overexpression study is not optimal either because of their tendency to form aggregates when they are expressed transiently [40]. Therefore, further studies are needed to determine the underlying mechanisms involved in the regulation of odontogenic differentiation process via MAP1LC3C by overcoming these limitations.

This study demonstrated that MAP1LC3C, one of ATG8 family, plays a crucial role in odontogenic differentiation process of human DPCs via regulating autophagy flux. Further functional studies of MAP1LC3C using in vivo model are necessary for understanding the molecular mechanism of odontogenic differentiation. These findings may provide new candidate for the developing therapy and regeneration of damaged pulp tissue.

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Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MSIT) (No. 2019R1A5A2027521 and No. 2018R1D1A1B07049876). It was also financially supported by Chonnam National University (Grant Number: 2018-3404).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no financial or commercial conflicts of interest that are directly related to the content of this article.

Ethical statement

This study was approved by the Institutional Review Board of Chonnam National University Dental Hospital, Gwangju, Korea (CNUDH-2013-002).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Heui Seung Cho and Sam-Young Park contributed equally to this study.

Contributor Information

Won Jae Kim, Email: wjkim@jnu.ac.kr.

Ji Yeon Jung, Email: jjy@jnu.ac.kr.

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