The effects of 4-Phenylbutyric acid on ER stress during mouse tooth development

Introduction: During tooth development, proper protein folding and trafficking are significant processes as newly synthesized proteins proceed to form designated tissues. Endoplasmic reticulum (ER) stress occurs inevitably in tooth development as unfolded and misfolded proteins accumulate in ER. 4-Phenylbutyric acid (4PBA) is a FDA approved drug and known as a chemical chaperone which alleviates the ER stress. Recently, several studies showed that 4PBA performs therapeutic effects in some genetic diseases due to misfolding of proteins, metabolic related-diseases and apoptosis due to ER stress. However, the roles of 4PBA during odontogenesis are not elucidated. This study revealed the effects of 4PBA during molar development in mice. Methods: We employed in vitro organ cultivation and renal transplantation methods which would mimic the permanent tooth development in an infant period of human. The in vitro cultivated tooth germs and renal calcified teeth were examined by histology and immunohistochemical analysis. Results and Discussion: Our results revealed that treatment of 4PBA altered expression patterns of enamel knot related signaling molecules, and consequently affected cellular secretion and patterned formation of dental hard tissues including dentin and enamel during tooth morphogenesis. The alteration of ER stress by 4PBA treatment during organogenesis would suggest that proper ER stress is important for pattern formation during tooth development and morphogenesis, and 4PBA as a chemical chaperone would be one of the candidate molecules for dental and hard tissue regeneration.

Introduction: During tooth development, proper protein folding and trafficking are significant processes as newly synthesized proteins proceed to form designated tissues. Endoplasmic reticulum (ER) stress occurs inevitably in tooth development as unfolded and misfolded proteins accumulate in ER. 4-Phenylbutyric acid (4PBA) is a FDA approved drug and known as a chemical chaperone which alleviates the ER stress. Recently, several studies showed that 4PBA performs therapeutic effects in some genetic diseases due to misfolding of proteins, metabolic related-diseases and apoptosis due to ER stress. However, the roles of 4PBA during odontogenesis are not elucidated. This study revealed the effects of 4PBA during molar development in mice.
Methods: We employed in vitro organ cultivation and renal transplantation methods which would mimic the permanent tooth development in an infant period of human. The in vitro cultivated tooth germs and renal calcified teeth were examined by histology and immunohistochemical analysis.
Results and Discussion: Our results revealed that treatment of 4PBA altered expression patterns of enamel knot related signaling molecules, and consequently affected cellular secretion and patterned formation of dental hard tissues including dentin and enamel during tooth morphogenesis. The alteration of ER stress by 4PBA treatment during organogenesis would suggest that proper ER stress is important for pattern formation during tooth development and morphogenesis, and 4PBA as a chemical chaperone would be one of the candidate molecules for dental and hard tissue regeneration.
KEYWORDS chemical chaperones, dentinogenesis, morphogenesis, secretion, signaling OPEN ACCESS EDITED BY Introduction Tooth development starts from thickening of oral epithelium followed by the migration of neural crest-derived mesenchyme below the epithelium (Jussila and Thesleff, 2012;Balic, 2018). Interactions between epithelium and the underlying mesenchyme play important roles for development of tooth from bud, cap, bell to secretory stages (Santosh and Jones, 2014;Balic, 2018). At cap stage, enamel knot (EK), a non-proliferating signaling center of tooth development closely linked with epithelial-mesenchymal interactions, expresses important signaling molecules including autocrine and paracrine factors, and transcription factors for proper tooth morphogenesis (Yu and Klein, 2020;Abramyan et al., 2021). At the secretory stage, inner enamel epithelium (IEE) begins differentiate into ameloblasts and the underlying mesenchymal dental papilla (DP) differentiate into odontoblasts for the secretion of enamel and dentin respectively (Otsu et al., 2011;Kawashima and Okiji, 2016). However, when there is disturbance in the secretion of extracellular matrix (ECM) proteins, it may lead to Amelogenesis Imperfecta (AI) or Dentinogenesis Imperfecta (DI) (Smith et al., 2017). It is suggested that AI would be one of the results of ER stress which arises when secreting cells such as ameloblasts have inadvertent stress (Brookes et al., 2014Aryal et al., 2019).
The regulation of ER stress is important for proper modulation of cell signaling and secretion during tooth development (Kim J. W. et al., 2014;Brookes et al., 2014Brookes et al., , 2017Aryal et al., 2020). ER stress occurs when the capacity of protein-folding of ER is exceeded (Tabas and Ron, 2011). As ER stress occurs, it induces the phenomenon called unfolded protein response (UPR) in order to maintain protein homeostasis through collection of intracellular signal transduction reactions (Sano and Reed, 2013). During ER stress regulation, the binding immunoglobulin protein (BiP) binds the unfolded proteins in the ER lumen for proper folding of unfolded and misfolded proteins (Pobre et al., 2019). ER stress triggers accumulation of unfolded proteins which leads to nutrient deprivation, hypoxia and point mutation (Zhang et al., 2010;Oakes and Papa, 2015). Recent studies in ER stress revealed that ER stress modulation plays an important role in hard tissue formation, especially dentin formation (Brookes et al., 2014Aryal et al., 2019).
The chemical chaperones correct the folding and assemblance of proteins, thus controlling protein trafficking by preventing their degradation or aggregation for proper protein function to alleviate ER stress (Kim et al., 2013;Cortez and Sim 2014). Because of their proteostatic function, it draws increasing attention for many therapeutic agents. Phenylbutric acid (4PBA) is a chemical chaperone in the ER which enhances the capacity of protein folding and also facilitates the proper folding of mutant proteins (Iannitti and Palmieri, 2011;Kim et al., 2013). The clinical use of 4PBA as an ammonia scavenger has been approved (Uppala et al., 2017). In addition, some studies proved the efficacy of 4PBA in metabolic diseases such as obesity and diabetes and in inflammatory diseases (Luo et al., 2010;Kim et al., 2013;Chen et al., 2016;Wu et al., 2020).
However, the effects of 4PBA during dodntogenesis has not yet elucidated. Based on our previous study, which revealed the expression patterns of ER stress related signaling molecules during various stages of tooth development (Aryal et al., 2020), thus, we designed to examine the role of 4PBA in tooth development using in vitro organ cultivation system.

Materials and methods
The animal experiments performed in this study were ethically approved (KNU 2020-0107). We followed the guidelines from Intramural Animal Use and Care Committee, Kyungpook National University, School of Dentistry.

Animals
The healthy time-mated pregnant ICR mice at embryonic stage 14 (E14) were obtained for embryo collection. Similarly, adult ICR 7-week old male mice were used for renal transplantation experiment. Mice were housed in optimum conditions, including room temperature (22°C ± 2°C), 55 ± 5% humidity and artificial illumination with lights on from 05:00 to 17:00 h, with access to food and water ad libitum.

In vitro organ cultivation and drug treatment
Tooth germs at E14 stage were micro-dissected in PBS and then cultivated with either 100 μM 4PBA (Sigma Aldrich, St. Louis, Missouri, United States) or 0.07% DMSO with DMEM (HyClone, Logan, UT, United States; cat. no.-SH30243.01) and 10% fetal bovine serum (Hyclone, Logan, UT, United States) for 48 h using a modified Trowell's culture method as previously described (Kim et al., 2009). The cultured tooth germs were then transplanted into the kidney capsule of 7 week old male mice for one and 3 weeks to obtain the calcified teeth as described previously (Neupane et al., 2020). The harvested renal calcified teeth were photographed using a Leica MZ16FA.

Histology and immunohistochemistry
The in vitro cultivated tooth germs and renal calcified teeth were sectioned for histology and immunohistochemical analysis. Before sectioning, the one and 3 week calcified teeth were decalcified using 0.5 M EDTA. The histology and immunohistochemistry (IHC) were performed as described previously (Neupane et al., 2020) Similarly, the biotinylated anti-rabbit or anti-rat IgG were used as secondary antibodies. The antibody binding was visualized using the diaminobenzidine tetrahydrochloride reagent kit (GBI Labs, Bothell, WA, United States, cat no. C09-12).

Quantitative PCR (qPCR)
RNA extraction from in vitro cultivated tooth germs and cDNA synthesis for qPCR analysis were performed as described previously (Aryal et al., 2020). The data of real time PCR were normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene and are expressed as mean ± standard deviation. The primers used during qPCR experiment are presented inSupplementary Table S1.

In situ hybridization
To examine the expression patterns of genes, whole mount and section in situ hybridizations were performed in the in vitro cultivated tooth germs as described previously (Sohn et al., 2012). For hybridization, digoxigenin-labeled mRNA probes were hybridized overnight at 65°C. The images were photographed using MZ16FA and DM2500 microscope (Leica, Wetzlar, Germany).

Three-dimensional (3D) reconstruction
For 3D reconstruction, the serial sections of cultivated tooth with thickness of 7 μm were photographed. The tooth germs were then reconstructed for each specimen using "Voloom 2.3" software (Micro Dimensions, Germany). The images were then aligned automatically and manually.

Statistical analysis
ImageJ software (http://imagej.net/) was used to count the immunostaining and TUNEL positive cells as described previously (Neupane et al., 2020). The number of Ki67 and TUNEL positive cells in the DAB-stained sections were counted in the defined area of 50 × 50 μm 2 . The number of Ki67 positive cells were counted from the epithelium and adjacent mesenchyme, whereas the TUNEL positive cells were counted from the enamel knot area. Similarly, the cusp, crown, root and mesiodistal length of renal calcified teeth were measured using ImageJ (http://imagej.net/) as described previously (Neupane et al., 2020). Data were represented as ± standard deviations and the mean was determined by comparing control and 4PBA treated groups using Student's t-test. p < .05 indicates significance.

ER stress in the developing tooth
The normal developing tooth at E14 and E16 represent cap and bell stage respectively ( Figures 1A, B). Similar to previous report (Aryal et al.,2 020), the localizations of ER stress related markers: 74-kDa glucose-regulated protein (GRP78) and HMG-CoA reductase degradation protein 1 (HRD1) were observed in the cap and bell stage of tooth development (Figures 1C-F). GRP78, a major ER chaperone protein and a master regulator for ER stress, localized at DP, EK and IEE at cap stage ( Figure 1C). However, at bell stage, the localization of GRP78 was restricted only to enamel forming area ( Figure 1D). Similarly, HMG-CoA, a key enzyme for ER-associated degradation (ERAD) of misfolded proteins, was localized along the entire tooth germ during cap stage ( Figure 1E), whereas, strong localization was observed along the enamel forming area during bell stage ( Figure 1F).

Altered cellular physiology after 4PBA treatment
To examine the effect of 4PBA in the developing tooth germ, we performed in vitro organ cultivation of E14 tooth germs for 2 days. Cellular proliferation were examined by immunostaining against Ki67, and the apoptosis were examined by TUNEL assay (Figures 2A-F). The number of proliferative cells were decreased in the 4PBA treated groups when compared to control ( Figures  2C, D, G). Particularly, the EK area showed decreasing number of Frontiers in Physiology frontiersin.org proliferative cells in the 4PBA treated group (Figures 2A-D). Whereas, apoptotic event of 4PBA treated group showed more apoptotic cells, especially at the EK area when compared to control ( Figures 2E, F, H). To compare the exact size of EK, the expression pattern of Fgf4 was examined using whole mount in situ hybridization, however, there was no obvious differences between control and 4PBA treated specimens (Supplementary Figure S1), To examine the activity of ER stress during in vitro cultivation, we performed immunostainings against GRP78 and HRD1 ( Figures 3A-D). The localization of GRP78 in the 4PBA treated group was more intense than those of controls throughout the entire tooth germ (Figures 3 A, B, Supplementary Figure S2). Similarly, the localization of HRD1 was stronger along the IEE and dental papilla in the 4 PBA treated tooth germs when compared to control (Figures 3 C, D, Supplementary Figure S2). We also measured the relative expression levels of signaling molecules related to amelogenesis, odontogenesis and ER stress after 24 h of in vitro organ cultivation at E14 with treatment of 4PBA. After 12 h of cultivation, all the molecules were up-regulated with treatment of 4PBA, however, their expressions levels did not altered significantly after 24 h (data not shown).

Tooth phenotype after 4PBA treatment
To examine the long term effect of 4PBA in tooth phenotype, we performed kidney capsule transplantation for one and 3 week after in vitro cultivation of tooth germs at E14 for 2 days. One  (Figures 5A-D). Particularly, thicker predentin was observed along the cuspal area in the 4PBA treated teeth when compared to control ( Figures 5A-D). To compare the control and 4PBA treated teeth precisely, we randomly selected 5 teeth from both groups and measured the cusp, crown, root and mesio-distal length. Interestingly, 4PBA treated group showed shorter cusp and crown length, however, the root and mesio-distal lengths were longer when compared to control ( Figure 5E). Especially, root length of 4PBA treated tooth was almost double than that of control group ( Figure 5E).

Discussion
ER stress pathway is involved during development and differentiation of hard tissue formation (Cameron et al., 2015;Li et al., 2018;Chen et al., 2020). Especially in tooth development, Frontiers in Physiology frontiersin.org ER stress plays a key role in the ameloblast (Brookes et al., 2014;Suzuki et al., 2017;Aryal et al., 2019Aryal et al., , 2020 and odontoblast (Kim Y.-G. et al., 2014) differentiation. In addition, ER stress molecules play a vital role in maintaining cellular homeostasis during odontogenesis because their expression patterns coincide with the novel transcription-regulating signaling molecules (Uchibe et al., 2012;Aryal et al., 2020). Particularly, the role of UPR in cap stage is not only alleviating protein overload in the ER, but also interacting with signaling molecules for cellular homeostasis during cap to bell stage transition (Aryal et al., 2020). In addition, the expressions of ER stress regulator genes: Ire1 and Xbp1 at IEE and dental papilla during bell stage suggest that Ire1/Xbp1 ER stress pathway initiates the ameloblast precursor cells to secrete ECM and dental papilla cells to differentiate into odontoblasts beforehand (Tsuchiya et al., 2008;Brookes et al., 2014;Kawashima and Okiji, 2016;Aryal et al., 2020), thereby emphasizing the importance of ER stress molecules and chemical chaperones during extracellular matrix secretion and tooth morphogenesis. Therefore, this study examined the effects of chemical chaperone, 4PBA, during tooth development using in vitro organ cultivation system. The clinical trials of 4PBA in both in vitro and in vivo as one of the potential therapeutic agent for metabolic diseases such as obesity and diabetes, inflammatory disorders and cancers has been studied (Luo et al., 2010;Iannitti and Palmieri, 2011;Kim et al., 2013;Chen et al., 2016). Especially, 4PBA rescued malformed tooth enamel, mirroring human AI and improved the gross appearance of incisor teeth of (Barron et al., 2010;Brookes et al., 2014). According to Brookes et al., 4PBA does not directly interact with secretory load of ER to restore normal  Frontiers in Physiology frontiersin.org 06 secretory function, but it appears to inhibit ameloblast apoptosis itself (Brookes et al., 2014). In our study, after treatment of 4PBA, localization of ER stress markers: GRP78 and HRD1, as well as NESTIN and AMELOGENIN were increased with denser alignment of odontoblasts and thicker dentin/enamel space (Figures 3, 4). These results suggest that chemical chaperone (4PBA) facilitates matrix formation in normal tooth development, and as a result of extensive secretion of matrices, ER stress was increased than in normal developing condition. Moreover, alteration in tooth phenotype was due to alteration in cellular events such as proliferation, apoptosis and ER stress in the 4PBA treated teeth (Figures 2, 3) because the alteration in enamel knot signaling play a vital role for future cusp, crown and root morphogenesis (Cho et al., 2007;Aryal et al., 2020;Neupane et al., 2020). The longer root length and thicker matrices of predentin in the 4PBA treated specimen suggest that this chemical chaperone facilitates the transcription of genes during development of tooth root and secretion of matrices because 4PBA as a histone deacetylase inhibitor can possibly facilitate transcription of multiple genes which regulate apoptosis and promote survival in the formation of matrices (Duncan et al., 2013;Brookes et al., 2014;Zhang and Zhong, 2014;Chotikatum et al., 2018).
The application of 4PBA as a drug repositioning method in dental procedures has been practiced for dentin regeneration (Lee et al., 2022). Based on these results, we speculated that proper amount of concentrated drug, 4PBA would affect tooth development in fetal period. The in vitro organ cultivation using embryonic stage tooth may imitate human oral environment of tooth transition period because permanent teeth in human initiate to develop in the jaws at birth and continue after a child is born. Our results suggest that direct absorption of chemical chaperone in oral environment during normal primary tooth development may affect matrix formation of enamel and dentin. Additionally, we speculated Frontiers in Physiology frontiersin.org that disorders like dentin dysplasia type I which is characterized by short or absence of tooth roots, obliterated pulp chambers (Malik et al., 2015) would be improved through exposure of chemical chaperone, such as 4PBA during permanent tooth development period.
In summary, ER stress occurs inevitably during secretory stage of tooth development. When protein folding capacity is overloaded, the UPR is activated which functions as a significant factor for normal tooth development. Our results showed the alterations in cellular physiology and morphology of developing tooth when treated with chemical chaperone, 4PBA. These results imply that when 4PBA is treated during tooth development period, or specifically during human tooth transition period, there would be both positive and negative effects of chemical chaperone. Further studies about these effects need to be proceeded in the near future.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by KNU 2020-0107. Author contributions E-SL, YA, J-HH and J-YK contributed to conception, design, and data interpretation, critically revised the manuscript. T-YK, EP, J-YK, HY, C-HA, S-YA, YL, W-JS and J-KJ contributed to data analysis, interpretation, and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Funding
This study is supported by the National Research Foundation of Korea (NRF) NRF-2017R1A5A2015391, 2021R1F1A1062736 and 2022R1I1A2063745.