Yuqiao Zhou, University of Pittsburgh, United States Claudio Cantù, University of Zurich, Switzerland Jan Hu, University of Michigan, United States

> \*Correspondence: Claire Bardet<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology

> Received: 27 March 2017 Accepted: 05 May 2017 Published: 24 May 2017

#### Citation:

Bardet C, Ribes S, Wu Y, Diallo MT, Salmon B, Breiderhoff T, Houillier P, Müller D and Chaussain C (2017) Claudin Loss-of-Function Disrupts Tight Junctions and Impairs Amelogenesis. Front. Physiol. 8:326. doi: 10.3389/fphys.2017.00326 Claudins are a family of proteins that forms paracellular barriers and pores determining tight junctions (TJ) permeability. Claudin-16 and -19 are pore forming TJ proteins allowing calcium and magnesium reabsorption in the thick ascending limb of Henle's loop (TAL). Loss-of-function mutations in the encoding genes, initially identified to cause Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC), were recently shown to be also involved in Amelogenesis Imperfecta (AI). In addition, both claudins were expressed in the murine tooth germ and Claudin-16 knockout (KO) mice displayed abnormal enamel formation. Claudin-3, an ubiquitous claudin expressed in epithelia including kidney, acts as a barrier-forming tight junction protein. We determined that, similarly to claudin-16 and claudin-19, claudin-3 was expressed in the tooth germ, more precisely in the TJ located at the apical end of secretory ameloblasts. The observation of Claudin-3 KO teeth revealed enamel defects associated to impaired TJ structure at the secretory ends of ameloblasts and accumulation of matrix proteins in the forming enamel. Thus, claudin-3 protein loss-of-function disturbs amelogenesis similarly to claudin-16 loss-of-function, highlighting the importance of claudin proteins for the TJ structure. These findings unravel that loss-of-function of either pore or barrier-forming TJ proteins leads to enamel defects. Hence, the major structural function of claudin proteins appears essential for amelogenesis.

Keywords: Amelogenesis Imperfecta, enamel, barrier-forming tight junction protein, pore-forming tight junction protein, claudins

Epithelial cells are attached to each other at their lateral membranes by a complex of intercellular junctions (Farquhar and Palade, 1963). The most apical complex of the intercellular junctions is the zona occludens also named tight junctions (TJ). TJ represent the principal component of the paracellular diffusion barrier by determining epithelial permeability of small molecules and water, and participate in the control of the diffusion of membrane components between the basolateral and apical regions. Abrogation of such a barrier function in epithelia that interfaces with the environment is associated with a variety of gastrointestinal (Barmeyer et al., 2017), renal (Hou, 2014), and cutaneous disorders (Basler and Brandner, 2017). TJ are composed of several

(Continued)

#### FIGURE 1 | Continued

mouse in post-natal day 3 tooth germ, liver and heart. Identification of claudin-3 in the continuously growing incisor was performed by immunofluorescence using claudin-3 antibody (#34-1700, Invitrogen) at 1/100 dilution and a Goat anti-Rabbit antibody (#R-6394, Invitrogen). Claudin-3 was localized at the distal end of secretory ameloblasts (am). Protein expression was also observed at the basal end of the cells (n = 3 per group). (p) pulp. (B) Recapitulative schema of claudin TJ proteins expression at the apical end of secretory ameloblasts. Claudin-3, -16, and -19 were shown to be expressed at TJ level of ameloblast secretory ends during enamel formation in the mouse. (C) 3D volume rendering from Micro-CT data showed severe enamel loss on lingual molar cusps in Claudin-3 KO mice. Quantitative analysis confirmed a significant lower enamel volume in Claudin-3 KO mice when compared to WT (0.022 vs. 0.120 mm3 respectively) (n = 8 per group). In WT mice, incisor mineralization is observed under the third molar (M3) whereas in Claudin-3 KO mice it is detected under the second molar (M2). \*\*P < 0.00001 (M1) first molar. (D) Scanning electron microscopy (SEM) analysis showed enamel prisms normally constituted in Claudin-3 KO incisor when compared to WT (n = 3 per group). Transmission electron microscopy (TEM) analysis of WT tooth germs showed TJ as adjoining ameloblast membranes converging to form a thin intermediate line at the distal end of the cells. In contrast, the TJ structure was altered in Claudin-3 KO tooth germs, with thicker and packed structures (red arrows). (am) ameloblast. (E) Enamel matrix protein expression by secretory ameloblasts of Claudin-3 KO mice (n = 3 per group). Western blot analysis of the protein extracts of the soft part of the growing incisor showed slightly higher levels of amelogenin (amel). No difference was observed by immunostaining regarding amelogenin expression in the forming enamel matrix (e) between Claudin-3 KO and WT incisors.

transmembrane and membrane-associated proteins including the claudin proteins. Claudins form either paracellular barrier or pores that determine TJ properties. They interact with each other and with additional membrane and non-membrane proteins, such as the intracellular zonula occludens ZO-1 and ZO-2 and other PDZ domain-containing proteins. Claudins are considered as core components of TJ strands and determine epithelial permeability of small molecules and water (Gunzel and Yu, 2013). Only few claudins are unequivocally qualified as pore-forming proteins, including claudin-2, -10b, and -15 as cation pores and claudin-10a and -17 as anion pores. Other claudins have been reported to form pores only when specifically interacting with another claudin (Gunzel and Yu, 2013). Such is the case of claudin-16 and claudin-19, which form a cationselective TJ complex in the thick ascending limb of Henle's loop (Hou et al., 2008).

Mineral transport involves the epithelial permeability which is tightly related to the type and properties of TJ. During amelogenesis, secretory ameloblast TJ are responsible for restricted paracellular access to the enamel compartment. The paracellular permeability (tightness) of the ameloblast layer depends on the composition of TJ relying on claudin proteins. Hence, a combination of different claudins may either allow some paracellular ion passage or restrict this passage (Denker and Sabath, 2011; Bronckers, 2017). To date, 11 claudins have been identified in the tooth germ at various developmental stages (Ohazama and Sharpe, 2007; Bardet et al., 2016; Yamaguti et al., 2017). We recently associated the abrogation of a pore function and a dental disorder. Indeed, we demonstrated that loss-offunction mutations in Claudin-16 and -19 (CLDN16 and 19) genes, initially identified to cause Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC), also resulted in Amelogenesis Imperfecta (AI) (Bardet et al., 2016; Yamaguti et al., 2017). At the time of this discovery, it was acknowledged that claudin-16 and -19 were mainly expressed in the thick ascending limb of Henle loop in the kidney, suggesting that the AI diagnosed in patients with FHHNC was a consequence of the disturbed mineral homeostasis. However, we were able to show that claudin-16 and claudin-19 were also expressed in the ameloblast TJ, indicating that the AI diagnosed in patients with FHHNC was an intrinsic consequence of the Claudin mutation. Furthermore, studying Claudin-16 Knockout (KO) mice, we showed that the structure of ameloblast TJ was altered.

Claudin-3, which is expressed in epithelia of a wide variety of organs such as intestine, kidney, liver, skin, and lung, acts as a barrier-forming TJ protein (Milatz et al., 2010; Gunzel and Yu, 2013). To analyze the substantial role of a barrierforming TJ protein in amelogenesis, we explored the dental phenotype of Claudin-3 KO mice using the methods previously described in Bardet et al. (2016) (ethical agreement D92-049- 01). We first determined that, similar to claudin-16 and claudin-19 (Bardet et al., 2016; Yamaguti et al., 2017), claudin-3 was expressed in the tooth germ and located in the TJ at the apical end of secretory ameloblasts (**Figures 1A,B**). Dental examination of adult Claudin-3 KO mice revealed a lower enamel volume in molars when compared to WT mice and a mineralization delay in the continuously growing incisor (**Figure 1C**). At the structural level, when examining the forming enamel in the intrabony part of the incisor, we observed normally formed prisms by scanning electron microscopy in Claudin-3 KO mice (**Figure 1D**). However, transmission electron microscopy (TEM) analysis of tooth germs showed altered TJ structure in Claudin-3 KO ameloblasts displaying thicker and packed structures compared to the thin intermediate line observed in the WT mice (**Figure 1D**). At the molecular level, enamel matrix formation was disturbed by claudin-3 deficiency, manifesting by an accumulation of enamel proteins further confirmed by Western blot analysis of the protein extracts of the soft part of the growing incisor (**Figure 1E**). Thus, claudin-3 protein lossof-function disturbs amelogenesis similar to claudin-16 loss-offunction, where accumulation of enamel matrix protein led to Amelogenesis Imperfecta (Bardet et al., 2016).

During amelogenesis, TJ are composed of pore- and barrierforming claudin proteins. Deficiency of both types of claudin proteins in murine models as well as in human disorders such as FHHNC led to enamel defects. To date, no human disease was associated with CLDN3 mutation. Interestingly, the hemizygous contiguous gene microdeletion at 7q11.23 in Williams-Beuren syndrome (WBS; OMIM 194050) includes the CLDN3 gene (Dutra et al., 2011) and several dental manifestations have been reported in patients with WBS, including hypodontia, abnormal tooth shape (microdontia), but also hypoplastic enamel defects and higher caries susceptibility (Hertzberg et al., 1994; Axelsson et al., 2003).

Our observations highlight the importance of claudin proteins for TJ structure. Indeed, either barrier or a pore forming claudins link to adaptor proteins such as ZO proteins and other PDZ protein family of the cytosolic TJ plaque. This interaction allows direct or indirect bonding to actin, anchoring the TJ within the underlying cytoskeleton. This scaffold facilitates the assembly of highly ordered structures, such as junctional complexes, regulating epithelial cell polarity, proliferation and differentiation (Sluysmans et al., 2017). This is consistent with our data showing a disturbed actin filament network at apical end of ameloblasts and a more diffused ZO-1 labeling in Claudin-16 KO mice (Bardet et al., 2016). Claudins are necessary for the TJ assembly process and their loss impairs TJ organization in ameloblasts and consequently disturbs enamel formation.

Overall, although TJ insure suitable microenvironments for enamel deposition and concomitant early maturation by determining the paracellular permeability and selectivity of solutes, the ion transport is tightly regulated during amelogenesis, involving barrier or channel properties of a certain claudin. Impaired TJ structure resulting from claudin loss-of function disturbs enamel formation. Hence, the major structural function of claudin proteins appears essential for amelogenesis.

## AUTHOR CONTRIBUTIONS

CB and CC wrote the manuscript with contributions from all authors. CB, SR, YW, and CC contributed to the design of

## REFERENCES

the experiments. CB, SR, YW, MD, BS, TB, PH, DM, and CC performed and analyzed experiments. CB and CC supervised the project. All authors reviewed and approved the final version of the manuscript.

## FUNDING

This work was supported by grants from Paris Descartes University, from Foundation pour la Recherche Médicale for EA2496 and Plateforme d'Imagerie du Vivant Paris Descartes (FRM DGE20111123012). DM was supported by a CRG grant of the Berlin Institute of Health. YW (post-doctoral position) was supported by the French Chinese Foundation for Science and Applications (FFSCA), Académie des Sciences, and the China Scholars Council (CSC).

## ACKNOWLEDGMENTS

Authors thank Stephane Le Goff (EA 4462, Paris Descartes University, France) for his help producing SEM data, and Jean-Marc Massé and Alain Schmitt (Cochin Institute, INSERM U1016, CNRS UMR8104, Paris Descartes University, France) for their help producing TEM data.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer YZ and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2017 Bardet, Ribes, Wu, Diallo, Salmon, Breiderhoff, Houillier, Müller and Chaussain. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells

Sarah Y. T. Robertson1 , Xin Wen1 , Kaifeng Yin1 , Junjun Chen1, 2, 3, Charles E. Smith4 and Michael L. Paine1 \*

1 Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, United States, 2 Department of Oral Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 3 Shanghai Key Laboratory of Tumor Microenvironment and Inflammation, Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 4 Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University, Montreal, QC, Canada

#### Edited by:

Steven Joseph Brookes, Leeds Dental Institute, United Kingdom

#### Reviewed by:

Petros Papagerakis, University of Michigan, United States Felicitas B. Bidlack, Forsyth Institute, United States Javier Catón, CEU San Pablo University, Spain

> \*Correspondence: Michael L. Paine<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology

> Received: 13 March 2017 Accepted: 08 May 2017 Published: 23 May 2017

#### Citation:

Robertson SYT, Wen X, Yin K, Chen J, Smith CE and Paine ML (2017) Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells. Front. Physiol. 8:336. doi: 10.3389/fphys.2017.00336 Calcium export is a key function for the enamel organ during all stages of amelogenesis. Expression of a number of ATPase calcium transporting, plasma membrane genes (ATP2B1-4/PMCA1-4), solute carrier SLC8A genes (sodium/calcium exchanger or NCX1-3), and SLC24A gene family members (sodium/potassium/calcium exchanger or NCKX1-6) have been investigated in the developing enamel organ in earlier studies. This paper reviews the calcium export pathways that have been described and adds novel insights to the spatiotemporal expression patterns of PMCA1, PMCA4, and NCKX3 during amelogenesis. New data are presented to show the mRNA expression profiles for the four Atp2b1-4 gene family members (PMCA1-4) in secretory-stage and maturation-stage rat enamel organs. These data are compared to expression profiles for all Slc8a and Slc24a gene family members. PMCA1, PMCA4, and NCKX3 immunolocalization data is also presented. Gene expression profiles quantitated by real time PCR show that: (1) PMCA1, 3, and 4, and NCKX3 are most highly expressed during secretory-stage amelogenesis; (2) NCX1 and 3, and NCKX6 are expressed during secretory and maturation stages; (3) NCKX4 is most highly expressed during maturation-stage amelogenesis; and (4) expression levels of PMCA2, NCX2, NCKX1, NCKX2, and NCKX5 are negligible throughout amelogenesis. In the enamel organ PMCA1 localizes to the basolateral membrane of both secretory and maturation ameloblasts; PMCA4 expression is seen in the basolateral membrane of secretory and maturation ameloblasts, and also cells of the stratum intermedium and papillary layer; while NCKX3 expression is limited to Tomes' processes, and the apical membrane of maturation-stage ameloblasts. These new findings are discussed in the perspective of data already present in the literature, and highlight the multiplicity of calcium export systems in the enamel organ needed to regulate biomineralization.

Keywords: amelogenesis, biomineralization, calcium channels, calcium exchangers, calcium pumps

**45**

## INTRODUCTION

Enamel is the hardest and most calcified tissue in mammals, and understanding enamel formation is crucial for developing strategies to repair or regenerate it (Smith, 1998; Hubbard, 2000; Lacruz et al., 2013). Amelogenesis, the process of enamel development, can be divided into the secretory and maturation stages with a brief pre-secretory stage before the secretory stage and a transition stage between the secretory and maturation stages. Epithelial-derived enamel-forming cells (ameloblasts) differentiate from the inner enamel epithelium (IEE) during the pre-secretory stage (Orrenius et al., 2015).  During the secretory stage, ameloblasts migrate away from the dentin while synthesizing and secreting enamel matrix proteins (EMPs) such as amelogenin, ameloblastin, and enamelin into the enamel area from Tomes' processes at their apical ends. These EMPs serve as a scaffold for the orientation and elongation of enamel hydroxyapatite (Hap) crystals (Smith, 1998). Each enamel rod follows a single ameloblast's Tomes' process with the interrod following the border of the cell, giving enamel its characteristic rod-interrod pattern (Skobe, 2006; Hu et al., 2007). There is a massive shift in gene expression during the transition stage, when approximately 25% of ameloblasts undergo apoptosis, after which another 25% undergo apoptosis throughout the following stages of amelogenesis (Tsuchiya et al., 2009). During the maturation stage, the ameloblasts undergo cyclical changes between ruffle-ended (RA) and smooth-ended (SA) morphology (Smith, 1998; Lacruz et al., 2013). Maturation-stage ameloblasts become specialized for ion transport and resorptive activities, which includes the secretion of the protease KLK4 to aid in the degradation of EMPs that are subsequently removed through endocytosis (Smith, 1998; Lacruz et al., 2012a, 2013). The continuously growing incisor of mice makes it a good model for studying the chronological progression of amelogenesis. While general concepts of ion transport throughout amelogenesis have been well-studied and discussed elsewhere (Arquitt et al., 2002; Paine et al., 2007; Lyaruu et al., 2008; Bronckers et al., 2010, 2015; Josephsen et al., 2010; Yin et al., 2015), and in particular the transcellular calcium ion (Ca2+) transport (reviewed in Nurbaeva et al., 2015b), in this paper we focus primarily on Ca2+ export.

## Overview—Calcium Transport

In general high intracellular concentrations of calcium (Ca2+) catalyze cell death signaling cascades, so cells maintain a gradient of ∼10−3 M Ca2+ concentration outside the cell, in the mitochondria, and in the endoplasmic reticulum (ER) where Ca2+ is stored; while in the cytoplasm, the concentration is ∼10−7 M (Brini and Carafoli, 2011). The plasma membrane contains a variety of Ca2+ channels that transiently open to allow Ca2+ influx in response to plasma membrane voltage changes, ligand-receptor interaction, or emptying Ca2+ stores of the ER and mitochondria (Brini and Carafoli, 2011). Calcium is removed from the cytoplasm through a number of mechanisms including the SERCA pump that replenishes ER stores, the mitochondrial Ca2+ uniporter, that replenishes mitochondrial stores, the plasma membrane low-affinity high capacity Na+/Ca2+ exchanger proteins (NCX), the Na+/Ca2+ K + exchanger proteins (NCKX), and the high-affinity low-capacity plasma membrane Ca2+- ATPase (PMCA) pump proteins (Berridge et al., 2003; Brini and Carafoli, 2011; Hu et al., 2012; Bronckers et al., 2015).

Calcium (Ca2+) transport is crucial to understand the process of amelogenesis because not only is unbound Ca2+ a major component of hydroxyapatite (Hap), Ca2+ can also act as a major signaling molecule capable of regulating cell processes in eukaryotic cells such as cell division, cell attachment, motility, survival, differentiation as well as gene expression (Hubbard, 1996; Blair et al., 2011).

## Calcium Extrusion

The SLC8A (sodium/calcium exchangers or NCX), SLC24A (potassium-dependent sodium/calcium exchangers or NCKX), and ATP2B (ATPase plasma membrane Ca2+ transporting pumps or PMCA pumps) gene families of Ca2+ transporters mediate Ca2+ extrusion in most cell types (Brini and Carafoli, 2011), and proteins from all three of these families have been reported in enamel organ cells (Sasaki and Garant, 1986c; Borke et al., 1995; Zaki et al., 1996; Okumura et al., 2010; Hu et al., 2012; Wang et al., 2014). The SLC8A gene family has 3 members (NCX1-3) and all have a generally accepted stoichiometry of the extrusion of 1 Ca2+ in exchange for the intrusion of 3 Na+ (Brini and Carafoli, 2011), while the SLC24A gene family has 5 members (NCKX1-5) and extrudes 1 Ca2+ and 1 K+ in exchange for 4 Na+, typically against the Ca2+ gradient; however, the directionality both NCX and NCKX exchangers can be reversed depending on the Na+ and Ca2+ gradients (Jalloul et al., 2016b; Zhekova et al., 2016). SLC8A and SLC24A gene families are electrogenic because there is a translocation of net charge across the plasma membrane, have a low Ca2+ affinity, and are capable of transporting Ca2+ in bulk rapidly across the plasma membrane (Brini, 2009). They are reversible but in ameloblasts they likely operate in extruding Ca2+ from the cytoplasm facilitated by transport of Na+ and K+ down their gradients (Brini and Carafoli, 2011; Hu et al., 2012). PMCA pumps/proteins have 4 members (PMCA1-4, coded by genes ATP2B1-4) and are part of a larger family of genes, called P-type primary ion transport ATPases, that catalyze the auto-phosphorylation of a conserved aspartyl residue within the pump from ATP (Palmgren and Nissen, 2011).

## Calcium Extrusion—SLC8A and SLC24A Gene Products

The SLC8A and SLC24A families are primarily expressed in excitable tissues such as muscle and heart, as their rapid bulk transport of Ca2+ is important in, for example, muscle and heart contraction (Brini and Carafoli, 2011). The SLC8A/NCX and SLC24A/NCKX transporters are Na+/Ca+ exchangers and can be either K+-dependent (NCKX) or K+-independent (NCX) (Shumilina et al., 2010).

NCX1 is expressed in heart, brain, bladder, kidney, and cells of the enamel organ; NCX2 is expressed in brain and skeletal muscle; and NCX3 is expressed in brain, skeletal muscle and cells of the enamel organ (Lytton, 2007; Okumura et al., 2010; Lacruz et al., 2012b; Sharma and O'halloran, 2014). Okumura et al. demonstrated NCX1 and NCX3 expression at the apical pole of both secretory and maturation ameloblasts, and expression of NCX1 was also observed in cells of the stratum intermedium and papillary layer (Okumura et al., 2010). In addition, protein levels of NCX1 and NCX3 throughout amelogenesis remained relatively unchanged (Okumura et al., 2010). Using real-time PCR, Lacruz et al. confirmed that the mRNA levels of both NCX1 and NCX3 did not significantly change from secretory- to maturation-stage enamel organ cells (Lacruz et al., 2012b).

NCKX1 is expressed primarily in retinal rod photoreceptors and platelets (Schnetkamp, 2004; Lytton, 2007). NCKX2 is expressed in cone photoreceptors and is involved in mouse motor learning and memory (Schnetkamp, 2004; Lee et al., 2009, 2013), and NCKX3 is expressed in the brain and the kidneys (Schnetkamp, 2004; Lee et al., 2009) though it is expressed in the kidneys at higher levels in female mice than in male mice (Lee et al., 2009). NCKX3 is also highly expressed in the human endometrium during the menstrual cycle, where its expression is partially regulated by the steroid hormone 17β-estradiol (Yang et al., 2011). NCKX4 is expressed in olfactory neurons (Stephan et al., 2011), and also in the maturation-stage ameloblasts (Hu et al., 2012).  NCKX6/NCLX was originally considered a member of the NCKX family but is now considered part of the Ca2+ cation (CCX) exchanger branch (Cai and Lytton, 2004; Sharma and O'halloran, 2014) as a mitochondrial membrane Ca2+, Li+/Na + exchanger with a wide tissue distribution (Schnetkamp, 2004; Lytton, 2007; Sharma and O'halloran, 2014).

## Calcium Extrusion—ATP2B Gene Products

The ATPase plasma membrane Ca2+ transporting (or PMCA) gene family is postulated to be involved in Ca2+ homeostasis, as it has a high affinity for Ca2+ but cannot transport Ca2+ as rapidly as either the NCX or NCKX transporters (Brini and Carafoli, 2011). The PMCA family is part of the superfamily of P-type ATPase pumps that form a stable phosphorylated intermediate as it hydrolyzes one molecule of ATP for each Ca2+ transported (Strehler and Zacharias, 2001; Cai and Lytton, 2004). The phosphorylated enzyme intermediate of the P-type ATPases, which include the SERCA family of transporters in the ER membrane (Giacomello et al., 2013), occurs between γ-phosphate of a hydrolyzed ATP with a D-residue in a highly conserved region of the ATPase pump (Brini and Carafoli, 2011).

PMCA1 is expressed in most tissues throughout development. Its expression is highest in the nervous system, heart, skeletal muscle, and intestine (Zacharias and Kappen, 1999), and regulated by growth factors such as glucocorticoids and Vitamin D (Zacharias and Kappen, 1999; Giacomello et al., 2013).  PMCA3 has the highest calmodulin affinity and is detected primarily in the brain and skeletal muscles (Krebs, 2009; Giacomello et al., 2013). PMCA4 is involved in the fertilization process and cardiac function, and has been found to associate with lipid rafts, which often function to aggregate protein complexes important in signaling pathways (Giacomello et al., 2013). It has been suggested that PMCA4 is more involved in cell-specific Ca2+ signaling than as a pump for bulk Ca2+ export (Strehler, 2013; Brini et al., 2017). PMCA4 interacts with nitric oxide synthase and with calcineurin, which regulates NFAT signaling (Brini, 2009; Kim et al., 2012; Strehler, 2013).

PMCA1 and PMCA4 are important in osteoclast differentiation, maturity, and survival, and Atp2b1+/− and Atp2b4−/− mice have decreased bone density due to an increased number of mature osteoclasts and increased osteoclast apoptosis (Kim et al., 2012)**.** The PMCA family members can also influence IP3-mediated calcium signaling by binding to phosphatidylinositol-4,5-bisphosphate (PIP2) on the plasma membrane as well as removing Ca2+ necessary for phospholipase C (PLC) activity, which prevents cleaving by PLC and thereby prevents Ca2+ release from the ER (Penniston et al., 2014). Altered PMCA expression is a characteristic of many cancers (Curry et al., 2011) and many other human diseases (Brini et al., 2013), but the diversity in isoforms and splicing and lack of specificity of small molecules to target PMCAs present challenges in therapeutic agent development (Strehler, 2013).

## Summary—Calcium Export Exchangers and Pumps in Amelogenesis

There now are a number of reports that show expression and localization data for NCX1 and NCX3 (Okumura et al., 2010; Lacruz et al., 2012b), and NCKX4 (Hu et al., 2012; Wang et al., 2014) in the enamel organ. Reports on PMCA expression and activities throughout amelogenesis are scant (Sasaki and Garant, 1986c; Borke et al., 1995; Zaki et al., 1996), and to the authors' knowledge, the only investigations into the role of PMCA proteins in amelogenesis date back decades. Data presented here better defines the mRNA profiles of all SLC8A, SLC24A, and ATP2B gene family members, and adds additional insight into the protein and spatiotemporal expression profiles of NCKX3, PMCA1, and PMCA4.

## Calcium Export Exchangers and Pumps and Disease

A number of the ATP2B, SLC8A, and SLC24A gene family members are linked to mammalian disease, but notably mutations to SLC24A4 are associated with non-syndromic amelogenesis imperfecta (AI) (Parry et al., 2013; Seymen et al., 2014; Wang et al., 2014; Herzog et al., 2015). A comprehensive list of the PMCA, NCX, and NCKX pumps and exchangers, their links to human pathologies, and mouse models of each gene is found in **Table 1**.

## MATERIALS AND METHODS

## Animals

All vertebrate animal manipulation was carried out in accordance with Institutional and Federal guidelines. The animal protocols were approved by the Institutional Animal Care and Use

TABLE 1 | Pathologies associated with genes ATP2B1-4, SLC8A1-3 and SLC24A1-6.

Note that SLC8B1 is found in the mitochrodria of mammalian skeletal and heart muscle, neurons and a few other cell types.

Committee at the University of Southern California (Protocol #20461).

## Quantitative PCR Analysis

Secretory-stage and maturation-stage enamel organ cells from mandibular incisors of 4-week old Wistar Hanover rats were collected as previously described (Lacruz et al., 2012b; Wen et al., 2014), and RNA extraction was performed using a QIAshredder, an RNeasy Protect Mini Kit, and DNase I solution from Qiagen (Valencia, CA, USA). Reverse transcription and real-time PCR were performed using the iScript cDNA Synthesis kit and SYBR Green Supermix from BioRad, respectively. Real-time PCR was performed on the CFX96 system (BioRad Laboratories, Hercules, CA, USA) in 10 µl volumes with a final primer concentration of 100 nm, for 40 cycles at 95◦C for 10 s and 58◦C for 45 s. Six independent real-time PCR analyses were conducted using samples from a total of 6 rats, 3 males, and 3 females, for each gene of interest (primers are listed in **Table 2**), and for both stages of amelogenesis. The male and female data were analyzed separately and no significant differences were noted between the sexes, so the data presented in the graph were generated from all 6 animals (n = 6). Rat enamel organ is preferred to mouse enamel organ for real-time PCR and western blot studies because separating secretory and maturation stage from adult mouse incisors is technically difficult and yields less RNA and protein per animal.

## Western Blot Analysis

Secretory (S) and maturation (M) enamel organ cells from mandibular incisors of 4-week old Wistar Hanover rats were collected. Brain (B) and heart (H) tissues were also collected as control tissues. Total protein extraction was performed with RIPA buffer (1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, 150 mm NaCl, 50 mm Tris, pH 8.0) and protease inhibitor cocktail, complete mini (Roche Applied Sciences, Indianapolis, IN, USA). Samples were homogenized manually with a pestle six times, then sonicated with a BRANSON digital sonifier Model 450 (All-Spec Industries, Wilmington, NC, USA; 10% intensity, 10 s on and 10 s off). Samples were then cleared by centrifugation (15,000 g, 15 min, 4◦C). Proteins were quantified using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA) and equal quantities were loaded (15 µg per lane) onto 4–12% SDS–PAGE resolving gels. Protein was transferred to a PVDF membrane, then blocked with 5% milk in TBST. Antibodies against PMCA1 (AbCam, Cambridge, MA, USA; catalog #ab190355), PMCA2 (ab3529), PMCA3 (ab3530), PMCA4 (ab2783), NCKX3 (St. John's Laboratory, London, UK, catalog #STJ94358), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA, catalog #sc-32233), amelogenin (ThermoFisher Scientific, catalog #PA5-31286), and cardiac muscle actin (ACTC1) (GeneTex Inc., Irvine, CA, catalog #GTX101876) were used at dilutions of 1:500, 1:2,000, 1:300, 1:5,000, 1:500, 1:500, 1:3,000, and 1:500, respectively in 5% milk in TBST. Secondary antibody for PMCA1-4 from Cell Signaling (Danvers, MA, USA; catalog #7074 and #7076) was applied at a dilution of 1:10,000. Secondary antibodies for NCKX3, amelogenin, and ACTC1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA; catalog #sc-2004 and sc-2418) were applied at a dilution of 1:7500. Pierce ECL Plus Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA; catalog #32132) was used as the detection system for all antibodies. TBST (tris-buffered saline with.1% Tween-20) was used as a wash buffer.

## Immunofluorescence

Mandibular incisors were dissected from 9-day-old wild type mice and placed in 4% paraformaldehyde in PBS overnight. Mouse incisors were preferred to rat incisors in the immunofluorescence studies because mouse incisors decalcify more rapidly and all stages of amelogenesis are visible in one sagittal section. The incisors were then washed in PBS and decalcified in 10% EDTA in PBS pH 7.4 for 4 weeks at 4◦C. The sample was embedded in paraffin and 4 µm sections were cut with a microtome. The sections were deparaffinized and rehydrated. The primary antibodies for PMCA1 and PMCA4 (AbCam, Cambridge, MA, USA; catalog #ab3528 and #ab2783, respectively) were used at dilutions of 1:40 and 1:200 in 1% BSA in PBS, respectively. The primary antibody for NCKX3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; catalog #sc-50129) was used at a dilution of 1:50. The secondary antibodies (Vector Laboratories, Burlingame, CA, catalog #DI-1088, DI-2488, DI-2594, DI-3094) were used at a dilution of 1:300 in 1% BSA in PBST. Sections were mounted with mounting medium with DAPI (Vector Laboratories, Burlingame, CA, catalog #H-1200) and imaged on a Leica TCS SP8 confocal microscope (Leica Biosystems). PBST (0.1% Tween-20) was used as a wash buffer for the experiments. Negative control sections, using secondary antibody only, under identical conditions, were included and showed negligible auto fluorescence—see Supplemental Figure 1.

## RESULTS

## Messenger RNA Expression Profiles

Quantitative PCR (qPCR) comparing mRNA expression levels in secretory and maturation enamel organ cells for all PMCA (Atp2b), NCX (Slc8) and NCKX (Slc24) gene family members indicate that: (1) PMCA1 (Atp2b1), 3 (Atp2b3), and 4 (Atp2b4), and NCKX3 (Slc24a3) expression is highest during secretory-stage amelogenesis; (2) NCX1 (Slc8a1) and 3 (Slc8a3), and NCKX6 (Slc24a6) were expressed during secretory and maturation stages; and (3) NCKX4 (Slc24a4) is most highly expressed during maturation-stage amelogenesis (**Figure 1**). The expression levels of PMCA2 (Atp2b2), NCX2 (Slc8a2), NCKX1

FIGURE 1 | Real-time PCR for rat Atp2b, Slc8a, and Slc24a gene family members. Atp2b1, Atp2b3, Atp2b4, Slc8a3, and Slc24a3 have significantly higher expression in secretory stage than maturation stage, while Slc8a1, Slc24a2, and Slc24a4 were significantly more highly expressed in maturation stage compared to secretory stage. β-actin (Actb) served as a normalizing control, and enamelin (Enam) and Odam as control transcripts that were significantly down-regulated and up-regulated, respectively (as expected), during maturation-stage amelogenesis. Slc24a4, Enam and Odam (arrows) have all been linked to non-syndromic cases or amelogenesis imperfecta. The x-axis is placed at the 0.001 expression level relative to Actb, and below this "cut-off" figure is arbitrarily considered non-significant. The Student's t-test (paired two-tail) was used to compare the expression of each gene between the secretory and maturation stages (\*p < 0.05, and \*\*p < 0.01). Standard deviations are also included.

(Slc24a1), NCKX2 (Slc24a2), and NCKX5 (Slc24a5) are negligible throughout amelogenesis (**Figure 1**). These data for NCX (Slc8) and NCKX (Slc24) gene family members are consistant with previously published gene expression data (Okumura et al., 2010; Hu et al., 2012), and add novel information suggesting that PMCA1, PMCA4, and to a lesser extent PMCA3 (which is expressed in secretory enamel organ cells at a level an order of magnitude lower than seen for PMCA1 and PMCA4), play an important role in secretory-stage amelogenesis.

## Western Blot Analysis Confirms Expression of PMCA Proteins in Enamel Organ Cells

Western blot data indicate that PMCA1 and PMCA4 are more highly expressed in secretory stage than in maturation stage, and PMCA2 is not expressed at any appreciable level in amelogenesis, consistent with the qPCR data (**Figures 2Ai,Aiv,Aii**respectively). Contrary to the qPCR data, PMCA3 and NCKX3 appear to be expressed at similar levels during both secretory stage and maturation stage (**Figures 2Aiii,B** respectively). Rat brain and heart protein samples were used as control tissues and analyzed with the secretory- and maturation-stage protein samples. The expected molecular weights for PMCA1-4 are ∼130, 133, 123, and 129 kDa respectively, and relate to the single bands seen at approximately the 150 kDa molecular weight mark (as indicated by an arrow, **Figure 2A**). The expected molecular weight of NCKX3 is ∼60 kDa (**Figure 2B**). Gapdh has been included as a loading control for all samples, and additional controls include Western analysis for both amelogenin (Amelx) and cardiac muscle alpha actin (Actc) (**Figure 2B**).

## PMCA1 and PMCA4 Localization by Immunofluorescence

In the enamel organ, PMCA1 expression is seen primarily on the basolateral membrane of both secretory- and maturationstage ameloblasts, with stronger signals seen in secretory ameloblasts (green; **Figures 3A–C**). These data complement both the qPCR (**Figure 1**) and Western blot data (**Figure 2**) on the spatiotemporal expression of PMCA1 in enamel organ cells. When compared to ameloblasts, a weaker signal of PMCA1 is seen in the stratum intermedium (**Figure 3A**) and papillary layer cells of the enamel organ (**Figure 3B**); as reflected by the orange color observed in the merged images (**Figures 3G–I**). In the enamel organ, PMCA4 expression is also seen on the basolateral membrane of secretory- and maturation-stage ameloblasts, and also cells of the stratum intermedium and papillary layer cells (red; **Figures 3D–F**). Similar to the PMCA1 data, these PMCA4 immunolocalization data complement the qPCR and Western blot data (**Figures 1**, **2**). The co-localization of both PMCA1 and PMCA4 in polarized ameloblasts can be appreciated in the merged image (yellow; **Figures 3G–I**), while PMCA4 (but not PMCA1) is also expressed in the stratum intermedium and papillary layer cells of the enamel organ (red; **Figures 3G–I**).

## NCKX3 Localization by Immunofluorescence

NCKX3 expression is highest in the Tomes' processes (**Figure 3M**) and the apical membrane of transition- and maturation-stage ameloblasts (**Figures 3N,O**), while some minor ameloblast-specific intracellular granular immune-reaction is also apparent (**Figures 3M–O**). We compared the expression

samples tested, with similar expression noted in both secretory-stage and maturation-stage enamel organ cells, and brain tissue. Relatively higher levels of NCKX3 expression can be appreciated in heart tissue. Amelx and Actc are used as controls as expression is highest in secretory-stage ameloblasts (Lacruz et al., 2012a,b) and heart tissue (Hamada et al., 1982) respectively. GAPDH is used here as a loading control.

to I; J,M merged to P; K,N merged to Q; and L and O merged to R). Am, Ameloblasts; ES, enamel space; Si, stratum intermediu (TP; secretory ameloblasts only), Tomes' processes and (PL, maturation ameloblasts only), papillary layer. The proximal/basal poles (p/b) and distal/apical poles (d/a) of ameloblast cells are identified, as are the lateral membranes of ameloblasts (broken while line in G). Scale for (A–I) shown in (I); and scale for (J–R) shown in R.

profile for NCKX3 (red; **Figures 3M–O**) to the expression profile of the control PMCA1 (green; **Figures 3J–L**). As can be appreciated from the images (**Figures 3M–R**), the expression profile for NCKX3 in the enamel organ is highest at the distal/apical pole, and this is distinct from the expression profiles seen for PMCA1 and PMCA4 where expression is seen on the lateral membranes of polarized ameloblasts (for both PMCA1 and PMCA4) and stratum intermedium and papillary layer cells (only PMCA4) (**Figures 3A–L**).

## DISCUSSION

From data presented here and prior studies, it is possible to make the following generalizations. First, of the four unique genes coding the PMCAs, PMCA1, and PMCA4 are highly expressed on the basolateral membranes of polarized ameloblasts; and both are expressed during secretory- and maturationstage amelogenesis. These data somewhat contradicts previously published data suggesting PMCA1 and PMCA4 are localized primarily to Tomes' processes of secretory ameloblasts (Sasaki and Garant, 1986c; Borke et al., 1995). These differences likely result from the different specificities of antibodies used to carry out these studies, and as noted previously, protein localization differences may also result from the different chemical and processing techniques used by the various laboratories (Takano, 1995). While expression of PMCA1 is primarily in the basolateral membrane of ameloblasts, there are also lower expression levels noted in the cells of stratum intermedium and papillary layer. Similarly, while expression of PMCA4 is seen in the basolateral membrane of ameloblasts, expression of PMCA4 is also recognized as a feature of the cells of the stratum intermedium and papillary layer cells. Of the three unique genes coding for the NCXs, NCX1, and NCX3 are highly expressed at the apical pole of both secretory- and maturation-stage ameloblasts (Okumura et al., 2010). Finally, of the six unique genes coding for NCKXs, NCKX3 (data reported here; **Figures 1**–**3**) and NCKX4 (Hu et al., 2012; Wang et al., 2014) are both highly expressed at the apical pole of polarized ameloblasts. A similar level of expression of NCKX3 is noted in both secretory- and maturationstage ameloblasts (**Figures 2**, **3**). While expression of NCKX4 is negligible in secretory-stage ameloblasts, it is highly expressed in maturation-stage ameloblasts (Hu et al., 2012; Wang et al., 2014). All six proteins expressed in ameloblasts (PMCA1, PMCA4, NCX1, NCX3, NCKX3, and NCKX4) export Ca2+ from the

cytoplasm to the extracellular space, thus ameloblasts may be one of the more complicated epithelial cell types when it comes to understanding ion movements related to Ca2+ transport as they relate to a mineralizing dental enamel.

The data suggest that there are likely redundancies amongst similarly functioning proteins from these gene families. For example, from this list of six Ca2+ export proteins expressed in ameloblasts, only mutations to SLC24A4/NCKX4 have been linked to enamel pathologies (Parry et al., 2013; Seymen et al., 2014; Wang et al., 2014; Herzog et al., 2015). NCKX4 exports Ca2+ from the apical pole of maturation-stage ameloblasts at the developmental stage where enamel mineralization is at its greatest; thus, NCKX4 may play a greater role in enamel formation than either NCX1 or NCX3, which have expression localized to the apical pole throughout the entire process of amelogenesis. It is conceivable that if the function of either NCX1 or NCX3 is less than optimal, the other may compensate such that no overt enamel phenotype results. Future studies may be able to address whether NCX1 and NCX3 are equivalent in enamel formation.

Similar to NCX1 and NCX3 in ameloblasts, PMCA1 and PMCA4 or other calcium handling proteins may be able to overcome the effects of Atp2b1 or Atp2b4 mutations, or gene silencing. No human pathologies have yet been linked to ATP2B1 mutations (as noted in the Online Mendelian Inheritance in Man; http://omim.org/entry/108731), however Atp2b1-null mice are embryonic lethal (Okunade et al., 2004). Only recently a case of familial spastic paraplegia has been linked to an ATP2B4 mutation (Ho et al., 2015; http://omim.org/entry/108732), and while Atp2b4-null mice have no overt phenotype, the male mice are infertile due to reduced sperm motility (Okunade et al., 2004; Schuh et al., 2004; Kim et al., 2012). The similar expression profiles of PMCA1 and PMCA4 in ameloblasts (that being to the basolateral membrane) may suggest that loss of PMCA4 function in ameloblasts may be compensated by PMCA1, while the loss of PMCA1 function remains embryonic lethal. If this is correct, studying PMCA1 activities in in vivo enamel formation would, in the future, be limited to conditional knockout or heterozygote animal models. The localization of PMCA1 and PMCA4 on the ameloblast basolateral membrane may suggest that these Ca2+ pumps are unlikely to have a critical role in enamel mineralization; i.e., Ca2+ removed by the PMCA pumps may not directly be incorporated into the Hap mineral phase. Instead, the PMCA pumps may be indirectly involved in amelogenesis by maintaining ameloblast Ca2+ homeostasis; or being a part of ameloblast cell signaling pathways. As calcium is transported through the stratum intermedium and papillary layer to the ameloblasts and the enamel organ, the PMCA

TABLE 3 | Major and most highly expressed calcium export pumps and exchangers in enamel organ cells.

family may additionally be valuable in shuttling the calcium from circulation to the ameloblasts. The PMCA family can be involved in IP3-mediated calcium signaling (Penniston et al., 2014), and PMCA1 and PMCA4 are involved in RANKL signaling and regulate osteoclast differentiation (Kim et al., 2012). In cultured osteoclasts, the knockdown and/or silencing of both PMCA1 and PMCA4 increased protein expression of SERCA2 and TRPV5 (Kim et al., 2012). Indeed, the PMCA transporters may have evolved to fine-tune intracellular calcium concentration as they have higher calcium affinity and lower capability for bulk Ca2+ transport than the NCX/NCKX exchangers (Brini and Carafoli, 2011), and are therefore more likely to play a housekeeping role by removal of intracelullar Ca2+ during maturation stage enamel mineralization which may also prevent calcium overload and possibly ameloblasts apoptosis. The PMCA transporters have multiple known expressed isoforms in other tissues but this has not yet been studied in the enamel organ.

Our novel data adds to the current understanding of Ca2+ transport in secretory and maturation stage enamel organ, described in **Figure 4**. Ca2+ import by the CRAC channel, ER Ca2+ export by IP3R, and ER Ca2+ import by the SERCA2 pump have been well-described elsewhere (Nurbaeva et al., 2015a,b, 2017). In summary, when Ca2+ is released from the ER through IP3R, STIM1 associates with ORAI1 and forms the CRAC channel that allows Ca2+ to flow into the cell. Ca2+ is then removed from the cytoplasm through PMCA1 and PMCA4 on the basal and lateral membranes, NCX1, NCX3, NCKX3, and NCKX4 on the apical membrane, and SERCA2 on the ER membrane. NCX1 and PMCA4 are also involved in Ca2+ export in the stratum intermedium and papillary layer. This process of Ca2+ cycling occurs more during the maturation stage, when large amounts of Ca2+ are necessary for enamel mineralization. During the secretory stage, PMCA1, PMCA4, NCX1, NCX3, and NCKX3 are the known Ca2+ exporters expressed, but further studies are necessary to understand Ca2+ import and other mechanisms of Ca2+ export during enamel secretion.

## CONCLUSION

Based on the available data, we have reviewed and summarized the expression profiles of the major Ca2+ export pumps and exchangers in enamel organ cells (**Table 3**; Okumura et al., 2010; Hu et al., 2012; Wang et al., 2014), and illustrated these Ca2+ export proteins along with the current model for Ca2+ import in enamel organ cells as proposed by Nurbaeva et al. (2015a,b, 2017) (**Figure 4**). As future studies continue to better define Ca2+ export (and also Ca2+ import—see Nurbaeva et al., 2015a,b, 2017) during amelogenesis, the information in **Table 3** will undoubtedly expand and become more precisely defined. Ultimately, elucidating the multitude of mechanisms involved in transcellular Ca2+ movements in the enamel-forming cells will result in a better understanding of the physiology and formation of enamel, the hardest and most calcified tissue in mammals.

## AUTHOR CONTRIBUTIONS

SR, XW, CS, and MP designed the experiments and wrote the manuscript; SR and XW performed the experiments; SR, XW, KY, JC, CS, and MP analyzed the data; SR, XW, and MP prepared the figures and tables; All listed authors critically read, edited, and approved the final manuscript. MP accepts full responsibility for the integrity of the data analysis.

## ACKNOWLEDGMENTS

The authors would like to thank Bridget Samuels for her help in the preparation of this manuscript, and Dr. Rodrigo S. Lacruz for his helpful comments and edits of the final version. This work was supported by grants DE019629 (MP), DE021982 (SR), and DE022528 (KY) from the National Institute of Dental and Craniofacial Research/National Institutes of Health. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphys. 2017.00336/full#supplementary-material

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Zaki, A. E., Hand, A. R., Mednieks, M. I., Eisenmann, D. R., and Borke, J. L. (1996). Quantitative immunocytochemistry of Ca2+-Mg2+ ATPase in ameloblasts associated with enamel secretion and maturation in the rat incisor. Adv. Dent. Res. 10, 245–251. doi: 10.1177/08959374960100022101

Zanni, G., Cali, T., Kalscheuer, V. M., Ottolini, D., Barresi, S., Lebrun, N., et al. (2012). Mutation of plasma membrane Ca2+ ATPase isoform 3 in a family with X-linked congenital cerebellar ataxia impairs Ca2+ homeostasis. Proc. Natl. Acad. Sci. U.S.A. 109, 14514–14519. doi: 10.1073/pnas.1207488109

Zhekova, H., Zhao, C., Schnetkamp, P. P., and Noskov, S. Y. (2016). Characterization of the cation binding sites in the NCKX2 Na+/Ca2+-K+ exchanger. Biochemistry 55, 6445–6455. doi: 10.1021/acs.biochem.6b00591

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Robertson, Wen, Yin, Chen, Smith and Paine. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Deletion of *Slc26a1* and *Slc26a7* Delays Enamel Mineralization in Mice

Kaifeng Yin1, 2, Jing Guo3 , Wenting Lin1 , Sarah Y. T. Robertson1 , Manoocher Soleimani 4 and Michael L. Paine1 \*

*1 Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry of University of Southern California, Los Angeles, CA, USA, 2 Department of Orthodontics, Herman Ostrow School of Dentistry of University of Southern California, Los Angeles, CA, USA, 3 Department of Endodontics, Herman Ostrow School of Dentistry of University of Southern California, Los Angeles, CA, USA, 4 Department of Medicine, University of Cincinnati, Research Services, Veterans Affairs Medical Center, Cincinnati, OH, USA*

Amelogenesis features two major developmental stages—secretory and maturation. During maturation stage, hydroxyapatite deposition and matrix turnover require delicate pH regulatory mechanisms mediated by multiple ion transporters. Several members of the Slc26 gene family (*Slc26a1, Slc26a3, Slc26a4, Slc26a6, and Slc26a7)*, which exhibit bicarbonate transport activities, have been suggested by previous studies to be involved in maturation-stage amelogenesis, especially the key process of pH regulation. However, details regarding the functional role of these genes in enamel formation are yet to be clarified, as none of the separate mutant animal lines demonstrates any discernible enamel defects. Continuing with our previous investigation of *Slc26a1*−/− and *Slc26a7*−/− animal models, we generated a double-mutant animal line with the absence of both *Slc26a1* and *Slc26a7*. We showed in the present study that the double-mutant enamel density was significantly lower in the regions that represent late maturation-, maturation- and secretory-stage enamel development in wild-type mandibular incisors. However, the "maturation" and "secretory" enamel microstructures in double-mutant animals resembled those observed in wild-type secretory and/or pre-secretory stages. Elemental composition analysis revealed a lack of mineral deposition and an accumulation of carbon and chloride in double-mutant enamel. Deletion of *Slc26a1* and *Slc26a7* did not affect the stage-specific morphology of the enamel organ. Finally, compensatory expression of pH regulator genes and ion transporters was detected in maturation-stage enamel organs of double-mutant animals when compared to wild-type. Combined with the findings from our previous study, these data indicate the involvement of SLC26A1and SLC26A7 as key ion transporters in the pH regulatory network during enamel maturation.

Keywords: amelogenesis, enamel maturation, pH regulation, bicarbonate transport, SLC26a1, SLC26A7

## INTRODUCTION

Acid-base balance is one of the major essential processes during amelogenesis (Simmer and Fincham, 1995; Smith et al., 1996; Smith and Nanci, 1996; Lacruz et al., 2010a, 2012b), and it has been suggested that fluctuations in extracellular pH level during maturation-stage enamel development are essential for mineral growth (Simmer and Fincham, 1995). Digestion of enamel

#### *Edited by:*

*Steven Joseph Brookes, Leeds Dental Institute, UK*

#### *Reviewed by:*

*Eric Everett, University of North Carolina at Chapel Hill, USA Pamela DenBesten, University of California, San Francisco, USA*

> *\*Correspondence: Michael L. Paine<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology*

> *Received: 16 March 2017 Accepted: 28 April 2017 Published: 16 May 2017*

#### *Citation:*

*Yin K, Guo J, Lin W, Robertson SYT, Soleimani M and Paine ML (2017) Deletion of Slc26a1 and Slc26a7 Delays Enamel Mineralization in Mice. Front. Physiol. 8:307. doi: 10.3389/fphys.2017.00307*

**57**

matrix proteins (EMPs) through the endosome/lysosome pathway, following trafficking from the enamel space, relies highly on the acidic intracellular luminal environment (Lloyd, 1996). Previous studies have identified the functional role of several groups of genes, including carbonic anhydrases (Lezot et al., 2008), cystic fibrosis transmembrane conductance regulator (CFTR), chloride channels (CLCNs), solute carrier gene family 4 (SLC4s) and solute carrier gene family 9 (SLC9s), in maintaining the ameloblast-mediated pH homeostasis within both extracellular space and intracellular lumens (Dogterom and Bronckers, 1983; Lin et al., 1994; Wright et al., 1996a,b; Arquitt et al., 2002; Lyaruu et al., 2008; Paine et al., 2008; Bronckers et al., 2009, 2010; Josephsen et al., 2010; Wang et al., 2010; Lacruz et al., 2010a,b, 2012c, 2013a; Chang et al., 2011; Duan et al., 2011; Duan, 2014; Jalali et al., 2014; Reibring et al., 2014; Wen et al., 2014).

The solute carrier (SLC) 26A gene family encodes multiple anion transporters with chloride/bicarbonate exchanger activities (Xie et al., 2002; Petrovic et al., 2003a,b, 2004; Alper and Sharma, 2013). Animal models with mutations of Slc26a1, Slc26a6, and Slc26a7 exhibit disorders featuring disruption of ion homeostasis, such as urolithiasis, hepatotoxicity, renal tubular acidosis and impaired gastric secretion (Freel et al., 2006; Jiang et al., 2006; Xu et al., 2009; Dawson et al., 2010). Based on our previous study and those of Bronckers et al., Slc26a1/Sat1, Slc26a3/Dra, Slc26a4/pendrin, Slc26a6/Pat1 and Slc26a7/Sut1 are immunolocalized in secretory- and maturationstage ameloblasts (Bronckers et al., 2011; Jalali et al., 2015; Yin et al., 2015). In particular, these genes mainly localize to the apical membrane/subapical vesicles of maturation ameloblast. In addition, the expression of Slc26a1, Slc26a6, and Slc26a7 is significantly upregulated at both RNA and protein levels during maturation stage compared to secretory stage (Yin et al., 2014, 2015). These are strong indications of the functional involvement of the Slc26 gene family in pH regulation during amelogenesis. However, the deletion of these genes individually fails to induce any abnormal enamel phenotypes, likely due to the compensatory expression of other pH regulatory genes and Slc26a isoforms, suggesting a yet-to-be-identified master pH response regulatory mechanism in amelogenesis (Bronckers et al., 2011; Jalali et al., 2015; Yin et al., 2015).

In this study, we generated an animal model with the absence of both Slc26a1 and Slc26a7 by breeding homozygous parents (Slc26a1−/− and Slc26a7−/−). We showed that the double-null enamel density was significantly lower in the regions that represent late maturation-, maturation-, and secretory-stage enamel development in wild-type mandibular incisors. However, the "maturation" and "secretory" enamel microstructures in double-mutant animals resembled those observed in wild-type secretory and/or pre-secretory stages. Elemental composition analysis revealed a lack of mineral deposition and an accumulation of carbon and chloride in double-mutant enamel, although absence of Slc26a1 and Slc26a7 did not affect the stage-specific morphology of the enamel organ including ameloblasts. Finally, compensatory expression of pH regulators and ion transporters at RNA level was detected in maturation-stage enamel organs of double-mutant animals. Taken together, the data obtained from double mutant animals (Slc26a1−/− and Slc26a7−/−) provide new evidence from a functional perspective to support the hypothesis that SLC26A1/SAT1 and SLC26A7/SUT1 are actively involved in ameloblast-mediated pH regulation during maturation-stage amelogenesis.

## MATERIALS AND METHODS

## Animals

All vertebrate animal manipulation was carried out in accordance with Institutional and Federal guidelines. The animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Southern California (Protocol # 11736). For immunofluorescence analysis, we dissected mandibles and kidneys from rats (Wistar Hannover, 4-week, 100–110 g). Slc26a1+/− mice were purchased from the Jackson Laboratory (stock # 012892) and Slc26a7+/− mice were a kind gift from Dr. Manoocher Soleimani (Xu et al., 2009; Dawson et al., 2010). Slc26a1−/− and Slc26a7−/− mice were generated by breeding heterozygous (Slc26a1+/− or Slc26a7+/−) parents. To generate double-mutant animals with the absence of Slc26a1 and Slc26a7, we crossed Slc26a1−/− and Slc26a7−/− mice. The double-mutant lines were genotyped by PCR using primers designed in earlier studies (Xu et al., 2009; Dawson et al., 2010).

## Immunofluorescence

The expression patterns of Slc26a1 and Slc26a7 in maturationstage ameloblasts were shown by co-localization using immunofluorescence (IF). Hemi-mandibles and kidneys obtained from Wistar Hannover rats (100–110 g body weight, 4 weeks old) were fixed in 4% paraformaldehyde (PFA) at 4◦C overnight. The hemi-mandibles were then decalcified in 10% EDTA (pH 7.4) for 2 months. Sagittal sections were prepared from paraffin-embedded tissue blocks with a thickness of 7 µm. After being dewaxed, rehydrated and blocked by 1% bovine serum albumin (BSA) in PBST (1X, pH 7.4), the tissue sections were incubated with primary antibodies against Slc26a1 (Santa Cruz Biotechnology, Catalog # sc-132090, dilution 1:400) and Slc26a7 (Abcam, Catalog # ab65367, dilution 1:300). All tissue sections were stained with DAPI (Vector Laboratories; Catalog # H-1200) before cover slides were applied.

## µCT Analysis

Mandibles were dissected from 4-week-old double-mutant animals and their age-matched wild-type controls. Samples from 12 animals in each group were prepared for µCT analysis (n = 12, SkyScan 1174) with the scanner setting to 50 kVp, 800 µA, and 6.7 µm resolution. The reconstruction and calculation of the enamel density of mandibular incisors and first molars were performed with Amira 3D Visualization and Analysis Software 5.4.3 (FEI Visualization Science Group, Burlington, MA, USA) (Wen et al., 2015). The potential statistical differences in the relative enamel density between double-mutant and wild-type groups were evaluated by a two-tailed Student's t-test using IBM SPSS Statistics 22.0 (significance level defined as P < 0.05).

## Scanning Electron Microscopy and Energy-dispersive X-ray Spectroscopy (EDS)

The hemi-mandibles prepared for µCT analysis (n = 12) were used for the subsequent SEM and EDS analyses. The samples were scanned and imaged by SEM and EDS according to previously published protocols (Lacruz et al., 2010b; Wen et al., 2014; Yin et al., 2015).

## Hematoxylin and Eosin (H & E) Staining

Mandibles were dissected from 4-week-old double-mutant animals and wild-type controls for H & E staining. The protocols followed those described in a previous study (Lacruz et al., 2012a).

## Realtime PCR Analysis

RNA samples of maturation-stage enamel organs were extracted from mandibles of double-mutant and wild-type animals (n = 6) using a method described previously (Yin et al., 2015). cDNA used for real-time PCR analysis was prepared using the miScript II RT Kit with miScript HiFlex Buffer (Qiagen). To detect the expression changes in the genes that have been identified to be involved in maturation-stage pH regulation (Dogterom and Bronckers, 1983; Wright et al., 1996a,b; Andrejewski et al., 1999; Arquitt et al., 2002; Lyaruu et al., 2008; Paine et al., 2008; Bertrand et al., 2009; Bronckers et al., 2009, 2010, 2011; Josephsen et al., 2010; Wang et al., 2010; Lacruz et al., 2010a,b, 2011, 2012b,c, 2013b; Chang et al., 2011; Duan, 2014; Jalali et al., 2014; Yin et al., 2014, 2015), real-time PCR reactions were performed on a CFX96 TouchTM Real-Time PCR Detection System (Biorad Life Sciences) with iQ SYBR R Green supermix (Bio-rad Life Science) and mouse-specific primers (**Table 1**). The Ct values were normalized to those of Actb (Beta-actin). The 11Ct method was used to calculate the fold changes in gene expression

(double-mutant relative to wild-type; Livak and Schmittgen, 2001; Schmittgen and Livak, 2008). Two-tailed Student's t-tests were used to detect the potential differences in the expression levels of gene transcripts between double-mutant and wildtype groups (significance level defined as P < 0.05). Data were analyzed using IBM SPSS Statistics 22.0 software.

## RESULTS

## SLC26A1 and SLC26A7 Do Not Colocalize in Maturation-Stage Ameloblasts

We revisited the expression patterns of SLC26A1 and SLC26A7 in rodent maturation-stage ameloblasts by conducting colocalization analysis using immunofluorescence. The expression of Slc26a1 was mainly immunolocalized to the apical membrane of maturation ameloblast (**Figure 1A**). In contrast, SLC26A7 showed more expression in the cytoplasmic area in addition to an apical/subapical distribution (**Figure 1A**). No apparent overlaps in fluorescence signals from SLC26A1 and SLC26A7 were observed (**Figure 1A**). Tissue sections prepared from rat kidneys were stained with the same antibodies as a reference (**Figure 1B**).

## Double-Mutant Mandibular Incisors Demonstrate Decreased Enamel Density

We dissected hemi-mandibles from 4-week-old double-mutant animals and their age-matched wild-type controls for µCT analysis. After 3D reconstruction from raw dicom files, we selected three regions to analyze the enamel density of incisors, which were indicated by the three reference planes along the long axis of the mandibular incisors (**Figure 2**). The first reference plane was placed at the region where bony support ends (**Figures 2A3,B3**). The second and the third reference planes sectioned though the first and the third mandibular

confocal microscopy at 63x magnification. SLC26A1 immunolocalized to the apical membrane of maturation-stage ameloblasts. SLC26A7 showed more expression in the cytoplasmic area in addition to an apical/subapical distribution. Apparent overlaps in fluorescence from SLC26A1 and SLC26A7 were not observed. (B) Tissue sections prepared from rat kidneys were stained with the same antibodies as a reference. All sections were counterstained with DAPI to highlight the nuclei (blue).

molars (**Figures 2A5,A7,B5,B7**). The three reference planes from anterior to posterior represent late-maturation, maturation and secretory stages, respectively (Nanci, 2008; Lacruz et al., 2011, 2012b; Yin et al., 2014, 2015). In a 4-week-old wildtype mouse, the enamel of the mandibular incisor is fully mature (maturation-stage) between the first and the second reference planes (**Figures 2A2–A6**) (Yin et al., 2015). In contrast, the double-mutant enamel at the first and second reference planes showed statistically significant decreases in relative density (**Figures 2B2–B6**, **3A,B**, P < 0.05). The double-mutant enamel density was approximately 14.3% lower than wild-type enamel density at the first reference plane (**Figure 3A**). At the second reference plane, the density gap between double-mutant and wild-type enamel was even higher—35.7% (**Figure 3B**). The enamel on mandibular incisors at the third reference plane is in secretory stage in wild-type animals (Yin et al., 2015), and the difference in relative enamel density of incisors was not statistically significant between wild-type and double-mutant groups (**Figures 2A8,B8**, **3C**).

Based on µCT analysis, we also quantified the relative density of mandibular first molars. For calculating the enamel density of each molar, we averaged the measurements obtained from mesial, middle and distal cusps (**Figure 4**). Although the doublemutant molars demonstrated lower enamel density than wildtype molars, the differences were not statistically significant (**Figure 4C**, P = 0.35).

## Absence of *Slc26a1* and *Slc26a7* Disrupts Development of Enamel Microstructure

For SEM analysis, we used the same reference planes as in the µCT analysis (**Figures 2**, **5**). We exposed the surface of interest by fracturing the mandibular incisors in the coronal direction, which was consistent with the orientation of the reference planes. At the first and the second reference planes, wild-type enamel showed typical microstructure of maturation-stage enamel with rods and interrods laid out in a decussating and orderly pattern (**Figures 5A1,A1',B1,B1'**). In wild-type enamel at secretory stage, which was marked by the third reference plane, enamel rods did not reach full thickness and the boundary between rod and interrod structure was not yet well defined (**Figures 5C1,C1**). In comparison, the structure of double-mutant enamel at the first and the second reference planes was similar to that observed at the second and the third reference planes in wild-type group, respectively (**Figures 5A2,A2',B2,B2'**), suggesting the double knockout animals showed a delay in maturation. Furthermore, there was a complete lack of decussating pattern in doublemutant enamel in the region labeled by the third reference plane, and aprismatic enamel/enamel-like structure dominated the whole vision field (**Figures 5C2,C2**′ ).

## Double Mutations Impact Mineral Deposition

Following SEM, we analyzed the elemental compositions using EDS on the same regions of mandibular incisor enamel marked by the three previously mentioned reference planes (**Figures 2**, **5**). At the first reference plane, there were no statistically significant differences between wild-type and double-mutant enamel in the atomic percentages (At%) of all the elements analyzed—Ca, P, O, C, Cl, Na, and Mg (**Figures 6A1–A3**). At the second reference plane, statistically significant changes were detected in the At% of Ca, P, C, and Cl (**Figures 6B1–B3**). The At% of Ca and P in double-mutant enamel decreased by ∼26.7 and ∼35.1%, respectively, compared to those in wild-type enamel (**Figures 6B1–B3**), while there were increases in the At% of C and Cl— ∼268.4 and ∼18.6%, respectively (double-mutant/wildtype, **Figures 6B1–B3**). Changes in the elemental compositions at the third reference plane showed similar trends to those detected at the second reference plane—double-mutant enamel showed significantly lower At% of Ca, P, O, Mg (∼92.8, ∼69.2, ∼29.6, and ∼57.9%, **Figures 6B1–C3**), and higher At% of C and Cl (∼20.0 and ∼35.6%, **Figures 6B1–C3**).

## Deletion of *Slc26a1* and *Slc26a7* Does Not Affect Morphology of Ameloblasts

We prepared tissue sections from 4-week-old mouse mandibles (wild-type and double-mutant) for H & E staining. At the regions marked by the three reference planes, wild-type enamel organs demonstrated typical morphology of ameloblasts in latematuration, maturation, and secretory stages (**Figures 7A1–A3**). Compared with the findings in the wild-type group, cell morphology in double-mutant enamel organs in the same regions was not significantly different (**Figures 7B1–B3**).

## pH Regulators Show Compensatory Expression in Double-Mutant Animals

The expression levels of 16 genes involved in pH regulation and ion transport during maturation-stage amelogenesis were quantified by realtime PCR using RNA samples isolated from wild-type and double-mutant maturation-stage enamel organs. Significant upregulation was detected for all the genes quantified (double-mutant/wild-type, **Figure 8**). Note that Ae4 and Slc26a9 showed the most striking fold changes—∼70.5 and ∼83.0%,

respectively (**Figure 8B**), and the fold changes for the remaining genes were all above 2, except for Lamp3, Rab21, and Nhe1 (**Figure 8B**).

## DISCUSSION

Enamel formation during maturation-stage amelogenesis involves mineral deposition, crystal growth, protease activities and the degradation of the internalized organic matrix, all of which are highly pH-dependent (Simmer and Fincham, 1995; Smith et al., 1996; Smith and Nanci, 1996; Lacruz et al., 2010a, 2012b).  Although details regarding the mechanism of pH control are yet to be clarified, the critical roles of many genes in maturation-stage pH regulation have been implicated by previous studies on transgenic animal models. For example, NBCe1 is a sodium-bicarbonate cotransporter expressed mainly on the basolateral membrane of maturation-stage ameloblasts (Lacruz et al., 2010b; Jalali et al., 2014). NBCe1−/− animals demonstrated hypomineralized and weak enamel with an abnormal prismatic architecture. Severe enamel phenotypes have also been documented from Cftr−/− and Ae2−/− animals (Arquitt et al., 2002; Lyaruu et al., 2008; Bronckers et al., 2010; Chang et al., 2011). Bronckers et al. started to investigate the role of Slc26 family genes in tooth enamel formation in 2011 (Bronckers et al., 2011). Since then, all the animal studies on Slc26 mutations have reach similar conclusions: mutation or silencing of individual Slc26 gene members (Slc26a1, Slc26a3, Slc26a4, Slc26a6, and Slc26a7) is not sufficient to generate abnormal enamel phenotypes, yet the deletion of a single Slc26 gene can induce strong compensatory expression of other pH regulatory genes and SLC26 family members (Bronckers et al., 2011; Jalali et al., 2015; Yin et al., 2015).

In the present study, we continued with our previous investigation of Slc26a1−/− and Slc26a7−/− animal models. We generated a double-null animal line with the absence of both Slc26a1 and Slc26a7 (Slc26a1−/−/Slc26a7−/−). The enamel density of double-null animals was significantly lower in the regions that represent late maturation-, maturation- and

reference plane, the double-mutant enamel density was approximately 14.3% lower than that of wild-type enamel (*P* = 0.015). (B) At the second reference plane, the density gap between double-mutant and wild-type enamel was even higher—35.7% (*P* = 0.010). (C) The difference in relative enamel density of mandibular incisors was not significant between the wild-type and double-mutant groups (*P* = 0.56). \**P* < 0.05.

1, 2, and 3. (C) We averaged the measurements obtained from mesial, middle and distal cusps (reference planes 1, 2, and 3). The double-mutant molars demonstrated lower enamel density than wild-type molars, but the difference was not statistically significant (*P* = 0.35).

secretory-stage enamel development in age-matched wild-type siblings (**Figures 2**, **3**). However, the difference in enamel density between double-mutant and wild-type mandibular first molars was not statistically significant, which suggests that incisor and molar maturation events differ to some extent (**Figure 4**). In addition, the "maturation" and "secretory"

enamel microstructures in double-mutant animals resembled those observed in wild-type secretory and/or pre-secretory stages (**Figure 5**). This indicates that deletion of Slc26a1 and Slc26a7 delayed enamel development in mandibular incisors, although such an impact was not observed after full eruption of the mandibular first molars in double-mutant animals (only data from 4-week-old animals are shown in **Figure 4**). Subsequent elemental composition analysis of double-mutant incisors revealed that decreased enamel density could be attributed to a lack of mineral deposition (Ca2+ and HPO3, **Figure 6**). The accumulation of Cl− in double-mutant enamel was consistent with our previous findings in Slc26a1−/− and Slc26a7−/− animals (Yin et al., 2015), while the increase of carbon in double-mutant enamel is a possible manifestation of disrupted EMP retrieval and hydrolysis (**Figure 6**). These findings indicate functional redundancy within the SLC26 gene family, and also in the scope of the pH regulatory network during enamel maturation (Yin et al., 2015). Such a redundancy is not uncommon in developmental processes and pathogenesis of diseases, e.g., matrix metalloproteinases (MMPs) in embryonic development and amyloid precursor protein (APP) genes in Alzheimer's disease (Heber et al., 2000; Page-McCaw et al., 2007).

Amelogenesis imperfecta (AI) is the most severe inherited disorder among all enamel pathologies. The genes responsible for AI in human patients include AMELX, AMBN, ENAM, MMP20, KLK4, WDR72, FAM83H, LAMB3, ITGB6, and SLC24A4, and current evidence tends to support a single-gene origin for many AI cases (Aldred et al., 1992; Lench et al., 1994; Lagerstrom-Fermer and Landegren, 1995; Lagerstrom-Fermer et al., 1995; Collier et al., 1997; MacDougall et al., 1997; Hart et al., 2000,

2003, 2004, 2009; Kindelan et al., 2000; Mardh et al., 2002; Kim et al., 2004, 2005, 2008, 2013; El-Sayed et al., 2009; Wright et al., 2009; Poulter et al., 2014a,b,c; Wang S. et al., 2014; Wang S. K. et al., 2014; Herzog et al., 2015). Our data from the double-mutant animals (Slc26a1−/−/Slc26a7−/−) suggest that polygenic etiologic factors might also be involved

in the pathogenesis of AI/AI-like symptoms, which increases the complexity of genetic diagnosis for enamel disorders. The statement is further corroborated by the findings from a recent study on BMPs, in which double deletion of Bmp2 and Bmp4 in the epithelium led to hypoplastic enamel in mice (Xie et al., 2016).

We proposed in our previous study that pH regulation during enamel maturation might be achieved by the coordination of functional protein complexes (Yin et al., 2015). This is based on our findings that physical protein-protein interactions exist between Cftr and Slc26 gene family members Slc26a1, Slc26a6, and Slc26a7 in maturationstage ameloblasts, as supported by colocalization analyses, including co-immunofluorescence and co-immunoprecipitation studies. Here we examined the co-distribution pattern of SLC26A1 and SLC26A7 in maturation-stage ameloblasts by conducting immunostaining. We did not observe any overlaps in fluorescence of SLC26A1 and SLC26A7, which indicates a possible lack of colocalization of these two anion exchangers on the apical membrane of ameloblasts (**Figure 1A**). Nevertheless, the interactions between other different pH regulators in enamel maturation still warrants further investigation.

In conclusion, the data obtained from Slc26a1−/−/Slc26a7−/− mutant mice provide new evidence in support of the hypothesis that SLC26A1 and SLC26A7 are actively involved in the ameloblast-mediated pH regulation process during maturationstage amelogenesis.

## AUTHOR CONTRIBUTIONS

KY, JG, MS and MP designed the experiments; KY and WL performed the experiments; MS developed the mutant animal model; KY, JG, SR and MP analyzed the data; KY and JG prepared the Figures and tables; and KY and MP wrote the manuscript. All listed authors critically read, edited, and approved the final manuscript. MP accepts full responsibility for the integrity of the data analysis.

## FUNDING

This work was supported by NIH/NIDCR [grants # R01 DE019629 and R21 DE024704 (MP), R90 DE021982 (KY), and from the Department of Veterans Affairs [grant - Merit Review 5 I01 BX001000-06 award (MS)].

## REFERENCES

## ACKNOWLEDGMENTS

We sincerely thank Thach-Vu Ho and Dr. Jingtan Su (University of Southern California) for their assistance in µCT scanning and EDS analysis. We also thank Bridget Samuels for her critical reading and editing of the manuscript.

Yin, K., Lei, Y., Wen, X., Lacruz, R. S., Soleimani, M., Kurtz, I., et al. (2015). SLC26A Gene family participate in pH regulation during enamel maturation. PLoS ONE 10:e0144703. doi: 10.1371/journal.pone.0144703

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Yin, Guo, Lin, Robertson, Soleimani and Paine. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo

#### Felicitas B. Bidlack 1, 2, Yan Xia1 and Megan K. Pugach1, 2 \*

*1 Forsyth Institute, Cambridge, MA, United States, 2 Department of Developmental Biology, Harvard School of Dental Medicine, Boston, MA, United States*

Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ∼40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin transgenes (*Tg*) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180*Tg*, CTRNC*Tg* and LRAP*Tg* mice to generate M180*Tg* and CTRNC*Tg* double transgene and M180*Tg*, CTRNC*Tg*, LRAP*Tg* triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (*p* < 0.05). The presence of the LRAP*Tg* did not improve the phenotype of M180*Tg*/CTRNC*Tg*/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ∼80% that of molar and ∼40% that of incisor wild-type enamel.

Keywords: enamel development, transgenic, knockout, amelogenin, mineralization

## INTRODUCTION

Tooth enamel forms through appositional growth in an organic matrix that is secreted in daily increments until the full thickness of the crown is reached. During this first, secretory, stage of enamel formation, enamel crystallites grow primarily in length. Once the final enamel thickness is attained, the mineral content increases as crystallites grow

#### Edited by:

*Alexandre Rezende Vieira, University of Pittsburgh, United States*

#### Reviewed by:

*Pierfrancesco Pagella, University of Zurich, Switzerland Yuqiao Zhou, University of Pittsburgh, United States Lucia Jimenez-Rojo, University of Zurich, Switzerland*

> \*Correspondence: *Megan K. Pugach<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology*

Received: *22 June 2017* Accepted: *02 November 2017* Published: *16 November 2017*

#### Citation:

*Bidlack FB, Xia Y and Pugach MK (2017) Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo. Front. Physiol. 8:932. doi: 10.3389/fphys.2017.00932*

**70**

in thickness, while the organic phase is removed in a highly controlled way. The enamel matrix facilitates mineralization and organization, and is transient in abundance as well as composition. Starting out with a ratio of 70 wt% organic matter and water, and 30 wt% mineral (Smith, 1998) the ratio of organic matrix to mineral changes over the course of enamel development and reaches 95% mineral content, with about 1–2% organic matter retained in completed enamel. During the secretory stage, the full-length enamel matrix molecules amelogenin, ameloblastin, and enamelin are cleaved upon their secretion by matrix metalloproteinase 20 (MMP20). After the secretory stage, a second enzyme, kallikrein 4 (KLK-4) is active to further cleave the matrix proteins and allow for the removal of matrix until only a very small amount remains, which is important for the mechanical properties through the control of crack propagation. The three structural enamel matrix proteins and the alternative splice product of amelogenin, leucine rich amelogenin protein (LRAP) have been described (Smith, 1998; Bartlett, 2013; Tarasevich et al., 2015; Lacruz et al., 2017). Yet, is not resolved what the in vivo function of these matrix components is, what role the full-length molecules, their alternative splice products as well as their cleavage products play for the control of mineral phase, crystallite shape and orientation, pH regulation, and potentially feed-back to ameloblasts (Lacruz et al., 2017).

The organic enamel matrix comprises the three structural matrix proteins amelogenin, enamelin, and ameloblastin, with amelogenin accounting for 90 wt% of the composition. However, the amelogenin primary RNA transcript is extensively alternatively spliced to produce 16 amelogenin isoforms reported (Bartlett et al., 2006). It is not clear, whether these isoforms are critical for enamel formation, or what their roles are in amelogenesis. The most abundant of these isoforms are the fulllength molecule of 180 amino acids and the 59-amino acid long LRAP, leucine-rich amelogenin protein, which consist of the 33 N-terminal and 26 C-terminal amino acids, but lacks the hydrophobic core of the full-length molecule.

Ameloblasts secrete the full-length 180 amino acid sized amelogenin, which shortly thereafter is cleaved by the metalloproteinase MMP20, beginning from the C-terminus. The most abundant cleavage product is 167 amino acids long, and referred here to as CTRNC. All three structural matrix proteins are required for proper enamel formation and the AmelxKO enamel is hypoplastic, with no prismatic architecture. In order to determine the roles of the most abundant amelogenin isoforms, transgenic mice have been developed that overexpress (a) the full-length amelogenin M180, (b) the major amelogenin cleavage product CTRNC, and (c) LRAP in both C57BL6/J wild-type and AmelxKO genetic backgrounds (see **Table 1** for abbreviations).

Addition of LRAPTg to M180Tg in the amelogenin KO model improves the thickness and structure of enamel, suggesting that transgenes have a complementary function (Gibson et al., 2011). Among amelogenin isoforms, M180 by itself is sufficient for the formation of normal mechanical properties and prism patterns in enamel. Yet, additional amelogenin splice products are required to restore enamel thickness (Gibson et al., 2011; Snead et al., 2011; Pugach et al., 2013). The overexpression of CTRNCTg and LRAPTg together improved significantly the enamel phenotype of LRAPTg/KO and CTRNCTg/KO mouse enamel, however enamel microhardness was recovered only when M180Tg was expressed, alone or with LRAPTg. Expression of LRAP and CTRNC together provides all three regions of the amelogenin protein N-terminus, C-terminus and hydrophobic core further improved the phenotype to reach normal WT enamel thickness and prism organization in the reported mouse model (Chen et al., 2003; Pugach et al., 2010; Xia et al., 2016). Enamel phenotypes in M180Tg/KO, CTRNCTg/KO, LRAPTg/KO, and double transgenic mice have been heterogeneous. This is due to varying transgene dosages and suggests a cumulative effect on improving the AmelxKO enamel phenotype (Xia et al., 2016).

The importance of the ratio between cleaved and uncleaved amelogenin has been previously reported (Shin et al., 2014). In order to determine the optimal ratio of amelogenin major cleavage products and splice variants required for normal enamel structure, thickness, and mechanical properties, we generated double and triple transgenic mice with two different transgene dosages in KO backgrounds.

# MATERIALS AND METHODS

## Generation of Transgenic Mice and Genotyping

To generate M180Tg/CTRNCTg/LRAPTg/KO and M180Tg/CTRNCTg/KO mice, animals overexpressing M180, CTRNC, and LRAP transgenes from the bovine amelogenin promoter were mated with male Amelx–/0 mice. After two generations of mating, both male and female mice were genotyped using PCR primers to detect transgenes as well as amelogenin WT and Amelx−/− (KO) DNA (Gibson et al., 2001, 2007; Chen et al., 2003; Pugach et al., 2010). Four different genotypes were generated with either 2 (M180Tg and CTRNCTg) or 3 (M180Tg, CTRNCTg, and LRAPTg) transgenes, and either homozygous (++) or hemizygous (+/−) for the transgenes (N = 3–5 mice per genotype). PCR primers for M180Tg, CTRNCTg, LRAPTg, and KO mice have been previously published (Gibson et al., 2001; Chen et al., 2003; Li et al., 2008; Pugach et al., 2010). Relative copy number determined transgene homo- (++ = on both alleles) vs. hemizygosity (+ = on one allelle), and was determined by qPCR analysis using M180, CTRNC, and LRAP Tg-specific probes designed by Transnetyx (Cordova, TN) (**Table 2**). WT, KO, and hemizygous (+/−) M180Tg/LRAPTg/KO mice, which have been previously reported were used as controls (Gibson et al., 2001, 2011).

## Western Blot Analysis

To analyze expression of endogenous and transgenic amelogenin in M180Tg/CTRNCTg/LRAPTg/KO and M180Tg/CTRNCTg/KO male and female mice and controls, first molars were harvested from 5-day-old mouse pups (with ameloblasts in the secretory stage) and protein was extracted. Equal amounts of protein were loaded in each lane and run on a 4–20% SDS-PAGE gel (BioRad). Membranes were immunoblotted with an antibody against full-length Amelx (FL-191, Santa Cruz Biotechnology, Santa Cruz, CA) and against TABLE 1 | Abbreviations for amelogenin protein, transgenes, and mouse models.

TABLE 2 | Mouse genotypes (KO and transgenic status) as determined by PCR for genotyping for endogenous amelogenin and qPCR for measuring relative copy number of the three transgenes, using transgene-specific probes against M180*Tg*, CTRNC*Tg*, and LRAP*Tg*.

β-actin (A2103, Sigma-Aldrich, St. Louis, MO), with a goat anti-rabbit secondary antibody (Santa Cruz Biotechnology). Relative transgene expression was confirmed by Western blot, and mice with the same genotypes but Mmp20KO background were used as controls (**Figure 1**). β-actin was used as the loading control to quantify relative transgene expression levels.

## Tooth Sample Preparation and Analyses of Enamel

Six 6-week-old male and female adult hemi-mandibles were dissected and fixed in Zinc-formalin for 24 h, then rinsed and transferred to 50% ethanol. Samples were first analyzed by µCT, then dehydrated in a graded ethanol series and embedded in LR-White (Electron Microscopy Sciences, Hatfield, PA, USA) for micro-hardness testing, and lastly SEM analyses.

### Enamel Mineral Density

Enamel mineral density was determined by µCT and compared between standardized regions of interest in incisor and molar enamel in mutant and control mice as described previously (Pugach et al., 2013). Hemimandibles with soft tissues removed were scanned in a µCT-40 (Scanco, Brüttisellen, Switzerland) at 70 kV, 114 mA, and 6µm resolution. Images were processed with µCT-40 evaluation software and FIJI (https://fiji.sc/) was used to orient the mandibles in a standardized way based on anatomical landmarks to clearly observe and compare enamel mineralization in two locations: (1) In the first molar, in a coronal plane through the distal root and, extending this plane, in the early maturation stage of the developing incisor and, (2) in the maturation stage incisor, in coronal plane through the mandible proximal to the point of tooth eruption, that is where the incisor is still completely enclosed by bone.

## Enamel Hardness

Enamel hardness was determined from first molars on LR White embedded samples that were polished on M3 polishing film (Precision Surfaces International, Houston Texas, USA) to 0.3µm grit size in parasagittal plane. The polished samples were tested for enamel microhardness on an M400 HI testing machine (Leco, St. Joseph, MI) with a load of 10 g for 5 s with a Vickers tip, applying 20 indentations per sample on at least four teeth per group, with data averaged per group.

## Enamel Thickness Analyses

Enamel thickness analyses were performed on samples subsequently to micro-hardness testing. The sample surface was etched with 0.1 M phosphoric acid for 15 s, gold coated, and imaged with a Zeiss Evo LS 10 SEM at 15 kV, 6–8 mm WD, and 120 pA probe current. Enamel thickness was measured in first molars and incisors on images taken at 1000X at the mesial side of the first molar using lateral enamel and in incisors in the area underneath the mesial first molar root tip, respectively, in at least six samples per genotype.

## Enamel Microstructure

Enamel microstructure was analyzed on both para-sagittal sections prepared for enamel thickness measurements as well as coronal sections through the mesial root of the first molar and visualized at 2000X and higher magnification.

## Toluidine Blue Staining

Toluidine Blue staining was applied to visualize organic matter in enamel. The same samples analyzed for microstructure by SEM were used for toluidine blue staining. The gold coating was polished off, the sample mounted on a glass slide, and a thin section prepared through polishing to a final thickness of

homozygous vs. hemizygous M180*Tg*, CTRNC*Tg*, and LRAP*Tg* as determined by qPCR analyses with transgene-specific probes designed and tested by Transnetyx (Cordova, TN) (\*\*indicates significant difference, *p* < 0.0001). (B–D) Western blot of *Amelx* protein expression using anti-Amelogenin FL-191 (Santa Cruz), with β-actin control (Sigma) in transgenic 5 day-old developing molars. (B) Relative transgene protein expression of homozygous vs. hemizygous M180*Tg*, CTRNC*Tg*, and LRAP*Tg* as determined by Western blot analyses as measured by ImageJ using β-actin to normalize. Since we could not differentiate between M180*Tg* and CTRNC*Tg* in lanes 1, 3, 4, and 8, M180*Tg* and CTRNC*Tg* expression intensities were pooled when quantifying. Due to the lack of Mmp20 in lanes 5, 6, and 7, we were able to differentiate between M180Tg and CTRNC*Tg*. (*n* = 3 blots, \*\*indicates significant difference, *p* < 0.005). (C) Relative transgene protein expression for different genotypes as determined by Western blot analyses as measured by ImageJ using β-actin to normalize. Lane 1, WT; Lane 2, *Amelx*KO; Lane 3, M180*Tg*/CTRNC*Tg*/LRAP*Tg*/KO++; Lane 4, M180*Tg*/CTRNC*Tg*/KO++; Lane 5, M180*Tg*/CTRNC*Tg*/KO+ (*Mmp20*KO background control); Lane 6, M180*Tg*/CTRNC*Tg*/LRAP*Tg*/KO++ (*Mmp20*KO background control); Lane 7, M180*Tg*/CTRNC*Tg*/LRAP*Tg*/KO+ (*Mmp20*KO background control); Lane 8, M180*Tg*/CTRNC*Tg*/KO++; Lane 9, M180*Tg*/LRAP*Tg*/KO+. (D) Western blot with primary antibodies anti-Amelogenin and anti-β-actin, with lane numbers corresponding to genotypes in (C). In lane 1, molars had a smear of amelogenin protein between 17 and 20 kD representing most of the WT splice variants and cleavage products expressed during the secretory stage, which are absent in the *Amelx*KO lane 2. The M180*Tg* band is visible at ∼25 kD in all lanes except lane 2. The CTRNC*Tg* is visible ∼22 kD in lanes 5, 6, and 7, since the absence of *Mmp20* prevented its proteolytic degradation. The LRAP*Tg* band is visible ∼7 kD in lanes 3, 6 7, and 9, but below the detection level in lane 1. The β-actin loading control bands is visible in all lanes ∼40 kD.

∼100µm. A 1% toluidine blue solution was used for 3 min, rinsed off and samples air-dried before viewing in a Leica upright microscope.

## Statistical Methods

We used ANOVA with the Tukey post-hoc test to detect differences (p < 0.05) in RNA expression, protein expression, enamel thickness, and microhardness between groups of teeth analyzed for enamel thickness and Vickers microhardness (GraphPad Software, San Diego, CA, USA).

## RESULTS

## Relative Amelogenin Transgene Protein Expression

qPCR and Western blot analyses confirmed that we generated four different genotypes with either two transgenes (M180 & CTRNC) or three transgenes (M180, CTRNC, LRAP) and homozygous (++) or hemizygous (+) expression in KO mice (**Table 2**). As expected, qPCR data confirm that the transgene copy numbers are higher in the homozygous than in hemizygous transgenic mice. However, the double transgene expression level of M180 with only CTRNC, without LRAP, is decreased in the homozygous transgenic compared to the triple homozygous transgenic containing LRAP (**Figure 1A**). Western Blot analyses show that in developing day-5 molars, that in the triple transgene on AmelxKO background most of the splice variants and cleavage products are expressed during the secretory stage and visible as bands between 17 and 20 kD for M180 and CTRNC, and the LRAP band around 7 kD (**Figure 1B**). The LRAPTg expression in triple transgenic/Amelx−/− mice is higher in the homozygous mouse compared to hemizygous, as expected, but also higher than that in WT molars.

## Relative Enamel Mineral Density

Results from µCT analyses of incisor and molar enamel show differences between the five different genotypes and compared to WT and AmelxKO (**Figure 2**). In no transgene combination was the enamel thickness of WT enamel achieved. In addition, mineral density was decreased in both molar and incisor enamel with homozygous (++) transgene expression (**Figures 2A–C, G–H**), compared to hemizygous (+) transgene expression (**Figures 2D–F, J–O**). The enamel layer seen in the homozygous triple transgene M180Tg/CTRNCTg/LRAPTg/KO++ (**Figures 2A–C**) is also thinner than in the hemizygous double transgene M180Tg/CTRNCTg/KO+ (**Figures 2J–L**) and M180Tg/LRAPTg/KO+ (**Figures 2M–O**) on both molar and incisor. We observed ectopic depositions in mice with excess CTRNCTg (++) without LRAPTg (**Figures 2A–C, G–I**).

## Enamel Thickness Analyses

Based on SEM data (**Table 3**), incisor enamel of triple transgenic homozygous (++) mice was statistically (p < 0.05) thicker than triple transgenic enamel with less transgene expression (+), indicating a dosage effect. This effect is not seen in the molars. However, a difference between double and triple homozygous transgenic mice was clear in molar enamel, which was thicker in the triple transgene (p = 0.038), relating molar enamel thickness to the presence of LRAP in the matrix (**Table 3**). Interestingly, enamel thickness is highest in hemizygous double transgenic mice, which is when the matrix lacks LRAP but does contain M180 and CTRNC.

## Enamel Microhardness

Vickers microhardness data of the four different genotypes are shown in **Table 3**. The enamel phenotypes are highly variable due to a mosaic appearance of properties including ectopic depositions and resulted in such high standard deviations of microhardness data that differences in hardness were not statistically significant between groups. However, molar double transgenic hemizygous enamel was harder than homozygous double transgenic molar enamel (p < 0.05).

## Enamel Structure Analyses by SEM and Organic Matter Content

The incisors of all transgene combinations analyzed here differ from WT enamel in both an excess of retained organic matter and a distinctly layered structural organization of inner and outer enamel. This layering is clearly seen in the homozygous triple transgene M180Tg/CTRNCTg/LRAPTg/KO ++ incisor and molar, with layers distinguished by the pattern and clarity of prism decussation and amount of organic matrix (**Figures 3A–C**, **4B**). In comparison, the hemizygous triple transgene expression appeared to result in fewer layers in the incisor (**Figure 3D**) and less organic matrix retention in molars (**Figures 3E,F**), while maintaining prism organization (**Figures 3D–F**). Toluidine blue staining shows more homogeneity within the molar compared to the incisor where we see organic matter close to the DEJ and near the enamel surface (**Figures 4C,D**). In contrast, homozygous double transgenic enamel had extensive disruption of structural organization, appeared to contain more organic matrix and showed only rudimentary prismatic organization (**Figures 3G–I**, **4E,F**). Ectopic depositions were observed in mice with homozygous expression of the CTRNC transgene (**Figure 3G**).

Retained organic matrix was observed in incisor and molar enamel from all four genotypes, especially those with homozygous transgene expression of CTRNCTg (**Figures 3B,C,G,H**, **4A–H**). The inclusion of the LRAPTg did not improve the structure of M180Tg/CTRNCTg enamel in homozygous nor hemizygous transgenes. Consistent with µCT and SEM derived thickness data, the enamel of hemizygous double transgenic mouse molars M180Tg/CTRNCTg/KO+ and M180Tg/LRAPTg/KO+ are most similar to WT (**Figures 3K,L,N,O,T,U**). In a given animal the incisor enamel contains more retained organic matrix in the inner enamel, compared to the molars and WT as seen in SEM (**Figures 3J,M,S**), and toluidine blue stained samples (**Figures 4G,K**).

## DISCUSSION

It has been shown previously that enamel prism decussation and 83% of thickness is recovered in the double hemizygous transgenic mouse M180Tg/LRAPTg/KO model (Gibson et al., 2011). To better understand the in vivo role of full-length amelogenin vs. cleavage and alternative splice products, we generated four genotypes that differ from each other in their relative abundance of M180, its most abundant cleavage product

through the distal root of the first molar, representing early maturation stage. Last column: Incisor enamel in coronal plane adjacent and mesial to the first molar, representing maturation stage enamel. White arrowheads point to mineralized enamel layers visible in molars and incisors. WT seen in last row (S–U); (P–R) *Amelx*KO with little enamel on molars and no enamel in the incisor enamel. (A–C) homozygous triple transgene M180*Tg*/CTRNC*Tg*/LRAP*Tg*/KO++, showing the thinnest enamel layer compared to all samples shown on both molars and incisors. (D–F) Hemizygous triple transgene M180*Tg*/CTRNC*Tg*/LRAP*Tg*/KO+ and (M–O) hemizygous double transgene M180*Tg*/LRAP*Tg*/KO+ with thicker enamel than all other transgene phenotypes shown, but thinner than WT. (G–I) double transgene M180*Tg*/CTRNC*Tg*/KO++ with a thin layer of enamel as is also seen in (J–L) M180*Tg*/CTRNC*Tg*/KO+. Ectopic depositions (orange arrowheads) were visible in incisors of homozygous transgenes M180*Tg*/CTRNC*Tg*/LRAP*Tg*/KO++ and M180*Tg*/CTRNC*Tg*/KO++ (A–C, G–I). Scale bars, 500µm.

*Significant differences (p* < *0.05) are indicated as follows: wfrom WT; x from Amelogenin KO; 3ofrom M180Tg/CTRNCTg/LRAPTg/KO (*++*); 3tfrom M180Tg/CTRNCTg/LRAPTg/KO (*+*); 2ofrom M180Tg/CTRNCTg/KO (*++*); 2tfrom M180Tg/CTRNCTg/KO (*+*).*

*aGibson et al. (2011); bXia et al. (2016), cLi et al. (2008), dPugach et al. (2013), ePugach et al. (2010), f Chen et al. (2003), gGibson et al. (2009).*

CTRNC that lacks the C-terminus, and the most abundant alternative splice product LRAP, which contains only the N- and C-terminus of the full-length molecule, but not the hydrophobic core region. However, in the present study, enamel thickness did not rescue by more than 58%, and prism organization was not fully achieved in incisors (**Figure 3**; **Table 3**).

In normal enamel development, M180 is cleaved shortly after secretion. Mutations in either the C-terminus or Nterminus compromise enamel formation. There are five reported mutations in the AMELX C-terminal region, and six mutations in the N-terminal region, leading to thin, discolored, and hypoplastic enamel (Lagerström et al., 1991; Lagerström-Fermer et al., 1995; Lench and Winter, 1995; Kindelan et al., 2000; Sekiguchi et al., 2001; Greene et al., 2002; Hart et al., 2002; Kim et al., 2004; Cho et al., 2014). Mutations in the N-terminus cause self-assembly defects in vitro (Buchko et al., 2013). Amelogenin lacking the N-terminus does not form nanospheres in vitro, and in vivo, the enamel is thin with short crystallites and irregular enamel prisms, indicating the key role of the N-terminus in amelogenin self-assembly and crystallite elongation (Zhu et al., 2006).

Both recent in vitro and in vivo evidence suggest that CTRNC plays an important role in enamel mineralization (Kwak et al., 2009, 2011; Martinez-Avila et al., 2011; Xia et al., 2016). In vitro studies have suggested a role of full-length amelogenin in the control of mineral phase, specifically the stabilization of amorphous mineral phases such as amorphous calcium phosphate (ACP), and alignment of forming crystallites (Kwak et al., 2011). With cleavage of the full-length molecule, one would expect the transformation of ACP to hydroxyapatite to proceed. In vivo, amelogenin transcription and MMP20 activity regulate the ratio of M180 to its cleavage product. The choreographed interplay between amelogenin secretion, MMP20 activity, and the resulting abundance and ratio of M180 to CTRNC can provide a mechanism to regulate mineralization rate.

Our data show that the dose of transgene expression has a major effect on enamel formation as is seen in the comparison between homozygous double transgene of M180Tg++/CTRNCTg++/KO and hemizygous transgene of M180 and CTRNC. This hemizygous transgene best rescues the structural organization and prism decussation of enamel. However, the produced enamel is softer than in any other transgene model used in this study. In contrast, the homozygous transgene provides excess protein that disrupts the process of both mineral phase regulation and crystallite alignment, as seen in the small platelets compared to the elongated crystals (**Figure 3I**) in the hemizygous enamel. The excess organic matrix in the homozygous M180Tg++/CTRNCTg++/KO transgene remains in the extracellular space, covers enamel prisms and seems to prevent proper decussation resulting in diminished hardness (**Figures 3G,H**; **Table 3**). In addition, matrix secretion is disrupted in the homozygous transgene, resulting in ectopic depositions (**Figures 2H,I**, **3G**) and decreased enamel thickness. This finding highlights the importance of sheer abundance of matrix components at a given time, and in relation to MMP20 activity, and is paralleled in the results for the homozygous transgene M180Tg++/CTRNCTg++/LRAPTg++, where also ectopic depositions are observed, excess organic material covering prisms and diminished enamel thickness (**Figures 2**, **3**). The rate of enamel apposition and crown extension is higher in incisors compared to molars, which could contribute to the observed differences in structural organization, matrix deposition, and matrix removal between molars and incisors (Smith and Warshawsky, 1977).

Interestingly, enamel organization and prism decussation, as well as hardness is better rescued than in the homozygous transgene without LRAP, M180Tg++/CTRNCTg++. The capacity of LRAP to regulate mineral phase in vitro, specifically stabilize ACP, has been shown by Le Norcy et al. (2011). In contrast to the homozygous transgene model, a comparison between the hemizygous transgenic mice M180Tg+/CTRNCTg+/LRAPTg+ and M180Tg+/CTRNCTg+ shows that the structural organization is much better without LRAP than when it is expressed uniformly with M180 and CTRNC. Our data further suggest that LRAPTg overexpressed with M180Tg and CTRNCTg does not affect enamel thickness

Incisors, scale bars 10µm. Second column: Molars, scale bars 10µm. Third column: Molars, scale bars 10µm. Yellow triangles: LR White resin; turquoise dashed lines: DEJ; magenta colored circles: organic matrix in forming enamel.

M180Tg/CTRNCTg/KO+ enamel, (I,J) AmelX null, (K,L) WT enamel. Coronal sections through incisors (first column) and molars (second column). Nuclei and organic matter stained blue and marked by arrows, DEJ highlighted in yellow. Scale bars, 10µm. Magnification 400X. (**Table 3**). These findings underscore the importance of other

factors besides absolute abundance of matrix molecules, namely, the timing of transgene expression and the relative abundance of cleavage product and alternative splice products. While LRAP uses the same promoter in vivo as M180, it is not known how LRAP expression varies between stages of enamel development. In addition, the pH buffering effect of matrix components is relevant for mineralization rate and produced mineral phase.

A possible role of the central core domain of amelogenin is the buffering of pH during enamel mineralization and it has been suggested previously that amelogenins may act as a buffer to neutralize protons generated during enamel crystal formation in the secretory stage (Smith, 1998; Smith et al., 2005). Although the role of the central hydrophobic core remains elusive, seven amelogenesis imperfecta-causing mutations in this region are published to date (Aldred et al., 1992; Lench et al., 1994; Lench and Winter, 1995; Collier et al., 1997; Hart et al., 2000, 2002; Ravassipour et al., 2000; Sekiguchi et al., 2001; Greene et al., 2002). In all the transgene models presented here, enamel thickness is decreased, indicating abridged matrix secretion compared to normal enamel development in the WT, where the ending of amelogenin secretion coincides with both the attainment of the full enamel thickness and the enamel matrix becoming acidic (Smith, 1998). Amelogenins including the central hydrophobic core contain 14 histidine residues, which can bind protons such that a single amelogenin molecule can bind up to 15 protons in vitro (Ryu et al., 1998). Further support for a suggested role of amelogenin in pH regulation comes from data comparing ion channel expression in ameloblasts during secretory and maturation stage between WT and AmelxKO mice. In WT, secretory ameloblasts do not express the anion exchanger Ae2 basolaterally (Lyaruu et al., 2008; Bronckers et al., 2009). However, the expression of Ae2 in secretory ameloblasts of amelogenin KO mice indicate a mechanism to compensate for the lack of buffering in the absence of amelogenin through upregulation of Ae2 expression to secrete bicarbonate (Guo et al., 2015).

Taken together, our data suggest that all three domains of amelogenin play key roles in enamel formation and that the relative abundance over time is critical. The N- and C-termini of amelogenin, which are present in both the most abundant amelogenin (M180) and LRAP, are highly conserved and believed to have different but critical roles in enamel formation (Delgado et al., 2007). Our results support the hypothesis that the core domain affects enamel formation, in particular the aspects of enamel thickness in vivo through crystal elongation. This study highlights the need to appreciate the relative abundance of enamel matrix molecules and their role for pH regulation as a key factor of enamel formation. In conclusion, we have shown that excess amelogenin transgenes disrupted the process of enamel formation, likely through the disproportionate presence of amelogenin splice products and disruption of matrix removal. The presence of excess retained organic matrix, layering within enamel, and ectopic depositions in the mouse models studied, suggest that an optimal ratio between M180, CTRNC, and LRAP is critical for normal enamel structure, thickness, and hardness.

## ETHICS STATEMENT

This study was carried out in accordance with the recommendations of Institute, Federal and Institutional Animal Care and Use Committee (IACUC) guidelines. The protocol was approved by the IACUC at Forsyth.

## AUTHOR CONTRIBUTIONS

MP and FB wrote the manuscript with contributions from all authors. MP, FB, and YX contributed to the design of the experiments. MP, FB, and YX performed and analyzed experiments. MP and FB supervised the project. All authors reviewed and approved the final version of the manuscript.

## FUNDING

Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research of the

## REFERENCES

National Institutes of Health grant R00DE022624 (MP) and R01DE025865 (FB).

## ACKNOWLEDGMENTS

We gratefully acknowledge Bhumi Patel for assistance with Western blot experiments, Carolyn Gibson and Ashok Kulkarni for developing the AmelxKO mouse model, and John Bartlett for contributing the Mmp20KO mice used as the Western blot control lanes.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer YZ and handling Editor declared their shared affiliation.

Copyright © 2017 Bidlack, Xia and Pugach. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development

Mirali Pandya1 , Tiffani Lin2, 3, Leo Li 3, 4, Michael J. Allen5 , Tianquan Jin3, 6 , Xianghong Luan3 and Thomas G. H. Diekwisch1, 3 \*

1 Texas A&M Center for Craniofacial Research and Diagnosis, Dallas, TX, United States, 2 UCLA School of Dentistry, Los Angeles, CA, United States, 3 Brodie Laboratory for Craniofacial Genetics, University of Illinois at Chicago, Chicago, IL, United States, 4 University of Michigan Medical School, Ann Arbor, MI, United States, 5 Biometrology Inc., Chicago, IL, United States, 6 Biocytogen, Worcester, MA, United States

#### Edited by:

Steven Joseph Brookes, Leeds Dental Institute, United Kingdom

### Reviewed by:

Harald Osmundsen, University of Oslo, Norway Michel Goldberg, Institut National de la Santé et de la Recherche Médicale, France Claudio Cantù, University of Zurich, Switzerland

\*Correspondence:

Thomas G. H. Diekwisch<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology

Received: 22 June 2017 Accepted: 26 September 2017 Published: 17 October 2017

#### Citation:

Pandya M, Lin T, Li L, Allen MJ, Jin T, Luan X and Diekwisch TGH (2017) Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development. Front. Physiol. 8:790. doi: 10.3389/fphys.2017.00790 The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8–16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle

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compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.

Keywords: amelogenin, extracellular matrix, self-assembly, stippled materials, apatite crystal growth

## INTRODUCTION

Tooth enamel is a remarkable bioceramic characterized by extraordinary hardness, resilience and fracture resistance. The formation of this extremely hard biomineral within the soft and gel-like extracellular enamel matrix remains an enigma in biomedical research to this day. In-depth understanding and visualization of the biological processes and mechanisms involved in amelogenesis are hampered by the limitations of conventional imaging techniques and artifacts introduced because of sample preparation. Specifically, optical in vitro and in vivo imaging are limited by the resolution of conventional light microscopy; scanning and transmission electron microscopy are limited by sample preparation for near-vacuum electron beam imaging conditions, contrasting procedures for organic matrix visualization, and beam damage to the mineral phase; atomic force microscopy provides high resolution but is restricted to surface topographies; and nuclear magnetic resonance spectroscopy lacks the topographical information provided by electro-optical imaging techniques. Yet, disregarding the weaknesses of individual strategies, the combination of information derived from complimentary approaches has yielded much progress toward a comprehensive understanding of the many aspects contributing to early enamel biomineralization. The combination of imaging and analytical techniques together with a plethora of individual approaches has resulted in a number of model systems that explain various aspects of enamel biomineralization.

Early enamel researchers thought of the enamel matrix as a "concentrated amorphous gel structure, rather than a more highly oriented assembly of fibers"(Fearnhead, 1963)and believed that the concept of the enamel matrix as a thixotropic gel would explain its "potential mobility as the protein would flow from regions where rapid growth of apatite crystallites caused a local increase in pressure to adjacent, relatively unmineralized regions, where it could initiate further crystals" (Eastoe, 1963). John E. Eastoe fathomed that the enamel proteins should be considered "as the true matrix of enamel in which the apatite crystallites are laid down, by a process which is not yet explored by which may be analogous to the epitactic mechanism believed to occur in collagenous mineralized tissues"(Glimcher, 1959; Eastoe, 1963). Thus, Eastoe imagined the enamel matrix as a homogeneous gel in which the enamel proteins freely float between further crystallized regions close to the dentin-enamel junction to the sites of early apatite crystallization at the ameloblast cell membrane, readily aiding each crystal to be initiated and grown "until they come into contact with their neighbors" (Eastoe, 1963). Notwithstanding this insightful speculation related to the function of the enamel matrix, Eastoe deserves much credit for the discovery of amelogenins as tissue-specific enamel proteins rich in proline, glutamic acid, and histidine (Eastoe, 1960).

Eastoe's contemporaries, the electron microscopists Dorothy F. Travis and Marie U. Nylen, pioneered an ultrastructural perspective of the developing enamel matrix by recognizing the stippled or finely granular materials located at the mineralization front as morphological building blocks of the enamel matrix (Frank et al., 1964; Travis and Glimcher, 1964; Reith, 1967; Nylen, 1979). They and others reported the presence of 5–7 nm granules in sectioned material and in suspensions of developing enamel (Fearnhead, 1965; Nylen, 1979). The existence of stippled materials was briefly called into question when the effect of fixative temperature on matrix structure was discovered (Lyaruu et al., 1982, 1984). Changes in enamel suprastructure at 4◦C temperature had been described earlier and attributed to the high proline content of the enamel matrix (Nikiforuk and Simmons, 1965). Needless to say, faithful ultrastructural examination of the enamel matrix requires fixation at 37◦C or room temperature as the mammalian body temperature does not drop to 4◦C (Diekwisch et al., 1993, 1995). The functional significance of the enamel matrix stippled materials as supramolecular subunit compartments responsible for the control of enamel crystal growth became evident in study in which the translation of the key enamel matrix protein amelogenin was inhibited using an antisense strategy (Diekwisch et al., 1993). The concept of enamel matrix supramolecular assemblies as the basis for enamel crystal spacing and growth was thereafter confirmed using organ culture data (Diekwisch et al., 1995) and atomic force microcroscopy of unfixed freshly prepared enamel matrix (Diekwisch et al., 1993, 1995; Diekwisch, 1998).

Inversely interpreted transmission electron micrographs together with atomic force microscopy images and dynamic light scattering data helped to advance the nanosphere theory of enamel crystal growth (Fincham et al., 1994, 1995; Paine et al., 2001). A simplified sketch (Fincham et al., 1999) illustrates beaded rows of amelogenin nanospheres surrounding growing enamel crystals, and begs for the question as to how the needle-shaped thin enamel crystals would possibly grow while surrounded by densely packed globular structures. Indeed, the task of reconciling the rounded globes of amelogenin nanospheres with the sharp-edged hexagonal cross-sections of enamel apatite crystals resembles the fitting of a square peg in a round hole. Today, none of the three pillars of the nanosphere theory clearly provides evidence for the presence of spherical subunits in the enamel matrix: (i) light scattering data simply reference radii and not necessarily imply the presence of spherical assemblies, (ii) atomic force micrographs visualize the surface topographies of enamel proteins assembled on mica sheets and not in three dimensions, and (iii) the electron micrographs

initially recruited to support the nanosphere theory incorrectly refer to the circular spaces in between nanospheres as protein assemblies instead of the electron dense protein coats associated with the growing enamel crystals (Diekwisch, 1998), a reversal of stained and unstained matrix compartments analogous to Edgar Rubin's young girl/old woman optical illusion. Moreover, the concept of self-assembly of amelogenins into spherical subunits is not universally accepted, as some investigators have argued that the organic enamel matrix organizes into fibrillar (Frank et al., 1960), lamellar (Ronnholm, 1962), or helical structures (Smales, 1975), or filaments and ribbons (Martinez-Avila et al., 2012; Carneiro et al., 2016). Recent small angle X-ray scattering (SAXS) studies propose that amelogenins self-assemble as nanooblates with a 1:2 aspect ratio (Aichmayer et al., 2005; Margolis et al., 2006). Nevertheless, the nanosphere theory has established a model for the role of globular enamel protein assemblies as structural entities involved in enamel hydroxyapatite crystal growth.

The ubiquitous presence of the amelogenin-rich extracellular enamel matrix throughout all stages of enamel crystal formation infers an involvement in multiple aspects of matrix-mediated enamel crystal growth, including (i) matrix assembly, (ii) enamel crystal nucleation, (iii) initial crystal fusion of apatite precursors into apatite ribbons, and (iv) eventual crystal elongation and growth of true apatite crystals. Three models have been established to explain amelogenin nanosphere assembly and interaction among nanospheres. A first model based on SAXS data postulates that amelogenin nanospheres assemble into nanospheres with a dense hydrophobic core and a shell of hydrophilic and negatively charged chain segments (Aichmayer et al., 2005; Margolis et al., 2006). A second model, the amelogenin micelle model, focuses on the distribution of hydrophilic and hydrophobic regions within the amelogenin molecule and hypothesize that amelogenins aggregate into micelles through the ionic interactions between positively and negatively charged mini-domains and the complementary domain of another amelogenin molecule in reverse orientation (Fukae et al., 2007). A third model based on heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) spectra and analytical ultracentrifugation proposes that amelogenins self-assemble as donut-shaped entities through ipsilateral interactions at the α-helical N-terminus of the molecule, while the hydrophilic C-termini point toward the outside of the assembly (Zhang et al., 2011). Together, these studies provide a good understanding of the in vitro self-assembly capacity of amelogenin into nanoscale subunits. However, a universally accepted model explaining the in vivo structural entities of protein-mediated enamel crystal growth and their transformation throughout development is still lacking and in need of further investigation (Ruan and Moradian-Oldak, 2015).