biomolecules

Article Plasma Membrane Ca2+–ATPase in Rat and Human Odontoblasts Mediates Dentin Mineralization

Maki Kimura 1,†, Hiroyuki Mochizuki 1,†, Ryouichi Satou 2, Miyu Iwasaki 2, Eitoyo Kokubu 3, Kyosuke Kono 1, Sachie Nomura 1, Takeshi Sakurai 1, Hidetaka Kuroda 1,4,† and Yoshiyuki Shibukawa 1,*,†

1 Department of Physiology, Tokyo Dental College, 2-9-18, Kanda-Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan; [email protected] (M.K.); [email protected] (H.M.); [email protected] (K.K.); [email protected] (S.N.); [email protected] (T.S.); [email protected] (H.K.) 2 Department of Epidemiology and Public Health, Tokyo Dental College, Chiyodaku, Tokyo 101-0061, Japan; [email protected] (R.S.); [email protected] (M.I.) 3 Department of Microbiology, Tokyo Dental College, Chiyodaku, Tokyo 101-0061, Japan; [email protected] 4 Department of Dental Anesthesiology, Kanagawa Dental University, 1-23, Ogawacho, Kanagawa, Yokosuka-shi 238-8570, Japan * Correspondence: [email protected] † These authors contributed equally to this study.

Abstract: Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts   is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+–Ca2+ exchanger (NCX) and plasma membrane Ca2+–ATPase (PMCA) to Citation: Kimura, M.; Mochizuki, H.; maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ Satou, R.; Iwasaki, M.; Kokubu, E.; extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Kono, K.; Nomura, S.; Sakurai, T.; Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of Kuroda, H.; Shibukawa, Y. Plasma Membrane Ca2+–ATPase in Rat and mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain Human Odontoblasts Mediates unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under 2+ Dentin Mineralization. Biomolecules physiological conditions. We also examined the role of PMCA in the Ca extrusion pathway during 2+ 2+ 2021, 11, 1010. https://doi.org/ hypotonic and alkaline stimulation-induced increases in intracellular free Ca concentration ([Ca ]i). 2+ 10.3390/biom11071010 We performed RT-PCR and mineralization assays in human odontoblasts. [Ca ]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor Academic Editors: teeth and human odontoblasts. We detected mRNA encoding PMCA1–4 in human odontoblasts. The Taneaki Nakagawa and 2+ application of hypotonic or alkaline solutions transiently increased [Ca ]i in odontoblasts in both Vladimir N. Uversky rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution- 2+ induced [Ca ]i increase was decreased by PMCA inhibitors in both types. Alizarin red and Received: 15 April 2021 von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline Accepted: 5 July 2021 Published: 10 July 2021 stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did

Publisher’s Note: MDPI stays neutral not affect DMP-1 or DSPP mRNA levels at pH 7.4–8.8 and under isotonic and hypotonic conditions, with regard to jurisdictional claims in respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These 2+ published maps and institutional affil- findings indicate that PMCA participates in maintaining [Ca ]i homeostasis in odontoblasts by 2+ 2+ iations. Ca extrusion following [Ca ]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of Copyright: © 2021 by the authors. high-pH dental materials. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: dentinogenesis; odontoblasts; plasma membrane -transporting ; cal- distributed under the terms and cium signaling conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Biomolecules 2021, 11, 1010. https://doi.org/10.3390/biom11071010 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 1010 2 of 17

1. Introduction Odontoblasts play critical roles in the generation of dentinal sensitivity (known as the odontoblast hydrodynamic receptor model [1]) and dentin formation (dentinogenesis) in both physiological and pathological settings. We previously reported that odontoblasts are sensory receptor cells capable of detecting multiple stimuli applied to the dentin sur- face [1–8]. These stimuli at the surface are transformed into dentinal fluid movements, which elicit intracellular Ca2+ signaling by increasing the concentration of intracellular free 2+ 2+ 2+ Ca ([Ca ]i) through Ca influx activated by mechanosensitive ion channels, transient re- 2+ ceptor potential (TRP) channel subtypes, and Piezo1 channels [1,5]. This increase in [Ca ]i results in intercellular odontoblast–odontoblast and odontoblast–neuron signal communi- cation mediated by ATP and glutamate, which are released from mechanically stimulated odontoblasts [1,4,8]. ATP release from odontoblast pannexin-1 channels induced by the 2+ [Ca ]i increase activates ionotropic adenosine triphosphate receptors (P2X3 receptors) in intradental Aδ neurons, which induces an action potential in Aδ neurons, generating dentinal sensitivity [5]. In addition, the increased intracellular Ca2+ is extruded extracellu- larly to the mineralizing front by the Na+–Ca2+ exchanger (NCX) in odontoblasts [1–3,9] to 2+ 2+ maintain [Ca ]i homeostasis. The data show that intracellular Ca signaling participates in sensory signal transduction sequences that produce dentinal pain or promote dentino- 2+ genesis. Therefore, in odontoblasts, the regulation of [Ca ]i is important for regulating cellular function. Intracellular Ca2+ concentrations are tightly controlled by multiple plasma mem- brane/intracellular Ca2+ transport proteins. Transport mechanisms are mediated by trans- membrane Ca2+ influx and intracellular Ca2+ mobilization, as well as Ca2+ extrusion mech- 2+ 2+ 2+ anisms. The [Ca ]i increase induced by Ca influx from the or Ca 2+ 2+ release from Ca stores subsides and returns to resting [Ca ]i via the sarco/ Ca2+–ATPase (SERCA)-mediated uptake of Ca2+ into Ca2+ stores or Ca2+ ex- 2+ trusion to the extracellular space [10,11]. The extrusion of increased [Ca ]i is catalyzed by NCX or plasma membrane Ca2+–ATPase (PMCA) [12]. Thus, NCX and PMCA play key roles in the maintenance of cellular Ca2+ homeostasis. In a previous study, we found that NCX1 and NCX3 were highly expressed on the distal membrane of odontoblasts [9]. However, in non-excitable cells, PMCA is the major driver of Ca2+ efflux from the cy- tosol [13]. PMCA belongs to the family of P-type (subclass P2B) ATPases, which use the high energy produced by ATP hydrolysis to carry Ca2+ against membrane electrochemical gradients [13–15]. Odontoblasts have been shown to express PMCA in the distal and its processes [16,17]. Lundgren and Linde (1988 and 1995) previously demonstrated ATP- dependent Ca2+ extrusion, most likely by Ca2+–ATPase, across the plasma membrane in odontoblasts [18,19]. In addition, PMCA epitopes have been reported to be present in odontoblasts [17]. These results suggest the involvement of the PMCA in odontoblasts in regulating the delivery of Ca2+ to the mineralizing front and dentinogenesis. However, the levels of mRNA-encoding PMCA subtypes and the pharmacological properties and cellular roles of PMCA in odontoblasts have yet to be determined. In the present study, to elucidate the role of PMCA in the maintenance of intracellular calcium levels in odontoblasts and mineralization during dentinogenesis, we assessed PMCA1–4 mRNA levels in human odontoblasts (HOB cells). In addition, changes in the mRNA levels of non-collagenous extracellular matrix proteins in cells exposed to various external conditions, with or without a PMCA inhibitor, were evaluated. We also examined the role of PMCA during dentinogenesis using mineralizing assays, and investigated PMCA1 immunoreactivity using immunofluorescence analysis in HOB cells. Finally, to clarify the involvement of PMCA during Ca2+ extrusion, we measured Ca2+ extrusion efficiencies during hypotonic or high-pH stimulation-induced Ca2+ mobilization in acutely isolated rat and human odontoblasts. Biomolecules 2021, 11, 1010 3 of 17

2. Materials and Methods 2.1. Ethical Approval All animals (N = 25 in total) were treated in accordance with the Guiding Principles for the Care and Use of Animals in the field of physiological sciences, which were approved by the Council of the Physiological Society of Japan and the American Physiological Society. All animal experiments were carried out in accordance with the guidelines established by the National Institutes of Health regarding the care and use of animals for experimental procedures. The experiments followed the United Kingdom Animal (Scientific Procedures) Act, 1986. All experimental protocols were approved by the Ethics Committee of Tokyo Dental College (numbers 300301, 190301, and 200301).

2.2. Odontoblast Cell Culture The human odontoblast cell line (HOB cell) was obtained from a healthy third molar and immortalized by transfection with the human telomerase transcriptase [6,20,21]. The resulting cells showed mRNA expression of dentin sialophosphoprotein (DSPP), type 1 collagen, alkaline phosphatase, and bone sialoprotein, and exhibited nodule formation by Alizarin red staining in the mineralizing medium [20]. HOB cells were cultured in basal medium containing alpha-minimum essential medium, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (Life Technologies Japan, Tokyo, Japan), and 1% amphotericin B ◦ (Sigma-Aldrich, St Louis, MO, USA) at 37 C in a 5% CO2 incubator.

2.3. Acute Isolation of Rat Odontoblasts from Dental Pulp Slices Dental pulp slices were obtained from newborn Wistar rats (5−8 days old) using a previously described method [1,2,6,22–24]. The animals were housed with food and water available ad libitum. Briefly, the mandible was dissected under isoflurane (3%) and pentobarbital sodium (25 mg/kg, i.p.) anesthesia. The hemimandible was embedded in alginate impression material and sliced transversely through the incisor at a 500 µm thickness using a standard vibrating slicer (ZERO 1; Dosaka EM, Kyoto, Japan). A section of the mandible was sliced to the level at which the dentin and enamel were directly visible between the bone tissue and the dental pulp. Mandible sections in which the dentin layer was thin and the enamel and dentin were clearly distinguishable by microscopy were selected to avoid cellular damage to odontoblasts. The surrounding impression material, enamel, bone tissue, and dentin were removed from the mandible section under a stereomicroscope. The remaining dental pulp slices were used in further experiments. Pulp slices were treated with standard Krebs solution supplemented with 0.17% collagenase ◦ 2+ and 0.03% trypsin (30 min at 37 C). For [Ca ]i measurement, enzymatically treated dental pulp slices were plated onto a culture dish, soaked in alpha-minimum essential medium containing 5% horse serum and 10% fetal bovine serum (Life Technologies Japan), and ◦ 2+ maintained at 37 C in a 5% CO2 incubator. [Ca ]i in odontoblasts located on the periphery of the primary cultured dental pulp slices was measured within 24 h of isolation. In a previous study, cells isolated using the same protocol were validated as odontoblasts with positive immunofluorescence for odontoblast marker proteins, dentin matrix protein-1 (DMP-1), dentin sialoprotein, and nestin within 24 h of isolation [2].

2.4. RT-PCR For real-time RT-PCR analysis, HOB cells were cultured under physiological or high- pH conditions, as well as isotonic or hypotonic conditions, for 3 days with or without 5(6)-carboxyeosin (CE) (10 µM). To obtain HOB cells under high-pH conditions, culture ◦ plates with basal medium were maintained in an incubator (37 C) without CO2 for 12 h per day (21:00–09:00) for 3 days. We also examined the effects of isotonic or hypotonic conditions on the changes in levels of the mRNA of interest. For the isotonic medium, we prepared modified basal medium by reducing NaCl to 28.7 mM and adding 150 mM mannitol (300 mOsm/L) (Functional Peptides. Co., Higashine, Japan). To prepare the hypotonic medium, we modified the medium by reducing NaCl to 28.7 mM and adding Biomolecules 2021, 11, 1010 4 of 17

50 mM mannitol (200 mOsm/L) (Functional Peptides. Co.). HOB cells were also cultured in each medium for 3 days, with or without CE (10 µM). Total RNA from HOB cells was extracted using the modified acid guanidium–phenol– chloroform method. The purity and concentration of total RNA was determined us- ing a NanoDrop ND-2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA), and 50 ng/µL of total RNA was used for the RT-PCR analysis. In addition, RNA in- tegrity and the RNA integrity number (RIN) were determined using Agilent 4200 TapeS- tation (Agilent Technologies, Inc., Santa Clara, CA, USA). The 28S:18S rRNA ratio was 1.80 ± 0.24, and the RIN was 9.66 ± 0.25 (47 samples). Reverse transcription, complemen- tary DNA amplification, and PCR were performed using a One-Step SYBR Primescript RT-PCR Kit with Thermal Cycler Dice for semi-quantitative real-time RT-PCR (TaKaRa-Bio, Shiga, Japan). Primer sets and PCR conditions are listed in Table1. Real-time RT-PCR data were quantified using the comparative threshold (2−∆∆Ct) method [25,26]. Levels of the mRNA of interest were normalized to β-actin levels. Mean Ct values were obtained for each primer set. Changes in Ct were calculated as the difference between the average Ct for the target gene and β-actin as the control for the total starting RNA quantity. Fold changes in mRNA levels relative to β-actin mRNA levels were assessed using the ∆∆Ct method.

Table 1. Primer sets for real-time RT-PCR: plasma membrane Ca2+ ATPase (PMCA) and odontoblast marker proteins.

Name 50-Sequence-30 GenBank Number Forward TGGCACCCAGCACAATGAA β-actin NM_001101.5 Reverse CTAAGTCATAGTCCGCCTAGAAGCA Forward ACCATATGCTAGAATGCCCACCTC PMCA1 NM_001001323.2 Reverse CTGGTGAAATCTGGGCCCTAAC Forward AGAGCTTCCGCATGTACAGCAA PMCA2 NM_001001331.4 Reverse CAAGCCATGGGCTCAATCAC Forward CGTAACGTCTATGACAGCATCTCCA PMCA3 NM_001001344.2 Reverse TCCATGATCAAGTTCACCCACAA Forward TGGCATGGTTAAATCTGAATGG PMCA4 NM_001001396.2 Reverse CTGCTTCAATTGTAAGGCAAAGG Forward TCCAGTCTCACAGCAGCTCA DMP-1 NM_004407.4 Reverse TCTCCGTGGAGTTGCTATCTTC Forward TGATAGCAGTGACAGCACATCTGAC DSPP NM_014208.3 Reverse GTTGTTACCGTTACCAGACTTGCTC The conditions for real-time RT-PCR were as follows: 1 cycle at 42 ◦C for 5 min, followed by 1 cycle at 95 ◦C for 10 s, 40 cycles at 95 ◦C for 5 s, and then 60 ◦C for 30 s. The conditions for dissociation curve analysis were as follows: 1 cycle at 95 ◦C for 15 s, 1 cycle at 60 ◦C for 30 s, and 1 cycle at 95 ◦C for 15 s.

2+ 2.5. Fluorescence Measurement of [Ca ]i Odontoblasts in dental pulp slices and HOB cells were loaded with 10 µM fura-2- acetoxymethyl ester (Dojindo Laboratories, Kumamoto, Japan) [27] and 0.1% (w/v) pluronic acid F-127 (Life Technologies, Japan) in Krebs solution at 37 ◦C. After 30 min of loading fura- 2, they were rinsed with fresh Krebs solution. Fura-2-loaded odontoblasts were imaged by fluorescence microscopy (IX73; Olympus, Tokyo, Japan) with an excitation wavelength selector, HCImage software, and an intensified charge-coupled device camera system (Hamamatsu Photonics, Shizuoka, Japan). Fura-2 fluorescence was recorded at 510 nm in 2+ response to excitation wavelengths of 380 nm (F380) and 340 nm (F340). The [Ca ]i was determined using the fluorescence ratio (RF340/F380) of F340 to F380, which is represented as F/F0 units; the RF340/F380 value (F) was normalized to the resting value (F0). The F/F0 baseline was arbitrarily set at 1.0. All experiments were carried out at room temperature (25 ± 1.0 ◦C). Hypotonic, isotonic, high-pH, and Krebs solutions and those containing PMCA inhibitors were applied by superfusion using a rapid gravity-fed perfusion system (ValveLink8.2 Controller; AutoMate Scientific, Berkeley, CA, USA). It is worth noting that Biomolecules 2021, 11, 1010 5 of 17

these solutions included 2.5 mM extracellular Ca2+. At the start of the experiment using caloxin 1b1, the odontoblasts in the recording chamber were perfused with a standard extracellular solution or isotonic solution. By changing the standard extracellular solution or isotonic solution to high-pH or hypotonic solution, respectively, with caloxin 1b1 (see Figure legends for details), followed by changing the solution into a standard or isotonic 2+ solution, we analyzed hypotonic- or high-pH-mediated increases and extrusion of [Ca ]i. For the experiments using CE, the odontoblasts were perfused by standard extracellular or isotonic solution with CE, and the solution was changed to high-pH or hypotonic solution with CE, followed by changing the solution to a standard extracellular or isotonic solution with CE (see Figure legends for details). For the control, the cells were perfused by standard extracellular or isotonic solution, and the solution was changed to high-pH or hypotonic solution, followed by changing the solution to a standard extracellular or isotonic solution.

2.6. Mineralization Assay HOB cells were grown to full confluency in basal medium and then transferred to mineralization medium (10 mM β-glycerophosphate and 100 µg/mL ascorbic acid in basal ◦ medium) for growth at 37 C in 5% CO2. To determine the effects of PMCA activity on mineralization, HOB cells were cultured in mineralization medium without (as control) or with PMCA inhibitors CE (10 µM) or caloxin 1b1 (100 µM) for 28 days. During the 28-day culture period, mineralization media with or without PMCA inhibitors was changed twice a week [6]. To detect the deposition of calcium and calcium phosphate [6,28], cells were subjected to Alizarin red and von Kossa staining, and the mineralizing efficiencies were measured using ImageJ software (NIH, Maryland, USA). Images were obtained with a digital camera (Sony, Tokyo, Japan), converted to 8-bit, and converted to reversed grayscale. Regions of interest (ROIs) were then determined for each whole well to measure the mean luminance intensities of the total pixel numbers (I) of the ROI. Mineralizing efficiencies were normalized and represented as I/I0 units; the intensities (I) of Alizarin red and von Kossa staining were normalized to the mean intensity values of areas without cells (I0).

2.7. Solutions and Reagents

Krebs solution containing 136 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, 10 mM glucose, and 12 mM NaHCO3 (pH 7.4, Tris) was used as a standard extracellular solution. The isotonic solution was comprised of 36 mM NaCl, 200 mM mannitol, 5 mM KCl, 2.5 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, 10 mM glucose, and 12 mM NaHCO3 (pH 7.4, Tris). To induce membrane stretching [3,29], a hypotonic solution (200 mOsm/L) was prepared by decreasing the concentration of mannitol to 64 mM. For the high-pH (pH 8) extracellular solution, 12 mM NaHCO3 in Krebs solution was changed to 8 mM (pH 8) NaOH [6,7]; this change had no effect on the extracellular free Ca2+ concentration in the test solution. Caloxin 1b1 was obtained from Karebay Biochem, Inc. (Monmouth Junction, NJ, USA). CE was obtained from Abcam (Cambridge, UK). All other reagents were purchased from Sigma-Aldrich. A stock solution of caloxin 1b1 was prepared in 10% ethanol. A CE stock solution was prepared in dimethyl sulfoxide. Stock solutions were diluted to the appropriate concentrations using the Krebs, isotonic, hypotonic, high-pH solution, or medium before use. The concentrations of the PMCA inhibitors used in this study were determined by Groten et al. (2016) and Chen et al. (2014) for CE, while those for caloxin 1b1 were determined according to Pande et al. (2006 and 2008) [11,30–32].

2.8. Immunostaining HOB cells were cultured in 8-well glass chambers (AGC Techno Glass Co.,Ltd., Shizuoka, Japan) for 1 day. Cells were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) and washed with 1× PBS (Thermo Fisher Scientific K.K., Tokyo, Japan). After 10 min of incubation with blocking buffer (Nacalai Tesque, Kyoto, Biomolecules 2021, 11, x 6 of 16

2.8. Immunostaining HOB cells were cultured in 8-well glass chambers (AGC Techno Glass Co.,Ltd., Shi- zuoka, Japan) for 1 day. Cells were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) and washed with 1× PBS (Thermo Fisher Scientific K.K., Tokyo, Japan). After 10 min of incubation with blocking buffer (Nacalai Tesque, Kyoto, Japan) at room temperature, mouse monoclonal anti-PMCA1 (Santa Cruz Biotechnology, Dallas, Texas, USA; sc-398413, F-10, 1:200) was applied for 6 h to detect human PMCA1. Secondary antibody (Alexa Fluor® 555 donkey anti-mouse; Thermo Fisher Scientific K.K.) Biomolecules 2021, 11, 1010 6 of 17 was then applied for 1 h. Stained samples were mounted in mounting medium containing 4,6-diamidino- 2-phenylindole (Abcam, Cambridge, UK). Immunostained samples were

Japan)analyzed at room and temperature, observed mouseusing monoclonala fluorescence anti-PMCA1 microscope (Santa Cruz(BZ9000; Biotechnology, KEYENCE Co., Osaka, Dallas,Japan). Texas, For USA;negative sc-398413, control, F-10, the 1:200) cells was were applied incubated for 6 h to with detect nonimmune human PMCA1. antibody diluted Secondaryto equivalent antibody concentrations (Alexa Fluor® 555 to that donkey of anti-mouse;the primary Thermo antibody Fisher (data Scientific not K.K.) shown). was then applied for 1 h. Stained samples were mounted in mounting medium containing 4,6-diamidino-2.9. Statistics 2-phenylindole and Offline Analysis (Abcam, Cambridge, UK). Immunostained samples were analyzed and observed using a fluorescence microscope (BZ9000; KEYENCE Co., Osaka, Japan).Data For negative are presented control, the as cells means were incubated± standard with errors nonimmune (SE) antibodyof the mean diluted of to N observations, equivalentwhere Nconcentrations represents the to thatnumber of the of primary independent antibody (dataexperiments. not shown). Parametric statistical signif-

2.9.icance Statistics was and determined Offline Analysis using one-way ANOVA with Tukey’s post-hoc test to analyze de- tectedData PMCA are presented mRNA as meanslevels ±instandard HOB cells. errors Non- (SE)parametric of the mean of statisticalN observations, significance was de- whereterminedN represents using the the numberMann–Whitney of independent test or experiments. Kruskal–Wallis Parametric test statistical with Dunn’s sig- post-hoc test. nificanceStatistical was significance determined using was one-way set at ANOVAP < 0.05. with Statistical Tukey’s post-hoc analyses test to were analyze conducted using detectedGraphPad PMCA Prism mRNA 7.0 levels (GraphPad in HOB cells.Software, Non-parametric La Jolla, statisticalCA, USA). significance was determined using the Mann–Whitney test or Kruskal–Wallis test with Dunn’s post-hoc test. Statistical significance was set at P < 0.05. Statistical analyses were conducted using GraphPad3. Results Prism 7.0 (GraphPad Software, La Jolla, CA, USA). 3.1. Measurement of PMCA1–4 mRNA Levels in Human Odontoblasts 3. Results 3.1. MeasurementUsing real-time of PMCA1–4 RT-PCR mRNA Levelsanalysis, in Human we robustly Odontoblasts detected mRNAs encoding PMCA1, PMCA3,Using real-timeand PMCA4 RT-PCR in analysis,HOB cells, we robustly but the detected detected mRNAs level encodingof PMCA2 PMCA1, mRNA was signifi- PMCA3,cantly lower and PMCA4 than inthat HOB of cells,its paralogs but the detected (Figure level 1). of PMCA2 mRNA was signifi- cantly lower than that of its paralogs (Figure1).

FigureFigure 1. mRNA1. mRNA levels levels of plasma of plasma membrane membrane Ca2+–ATPase Ca2+ (PMCA)1–4–ATPase (PMCA)1–4 in human odontoblast in human cell odontoblast cell lineslines (HBO (HBO cells). cells). Real-time Real-time RT-PCR RT-PCR was used towas quantify used theto detectionquantify level the ofdetection PCMA1–4 level mRNAs of byPCMA1–4 mRNAs measuringby measuring the increase the increase in fluorescence in fluorescence elicited by the elicited binding ofby SYBR the binding green dye of to double-strandedSYBR green dye to double- −[∆∆Ct] DNA.stranded Data wereDNA. analyzed Data were by the analyzed 2 method, by the with 2−[ΔΔβCt]-actin method, as an internal with β control-actin as (which an internal was control positive(which in was all samples). positive Each in all bar samples). denotes the Each mean bar± SE deno of 7tes experiments. the mean Expression ± SE of 7 levels experiments. were Expression β normalizedlevels were to normalized-actin mRNA levels.to β-actin Statistically mRNA significant levels. differencesStatistically between significant columns differences (shown by between col- solid lines) are marked with asterisks. * P < 0.05. umns (shown by solid lines) are marked with asterisks. * P < 0.05. 3.2. Extrusion of Ca2+ by PMCA Following Hypotonic or High-pH Stimulation in Acutely Isolated Rat Odontoblasts and HOB Cells

To demonstrate the contribution of PMCA to Ca2+ extrusion following mechanosensitive- 2+ 2+ or high-pH sensitive-[Ca ]i increases, we measured [Ca ]i and analyzed the extru- sion efficiencies following the application of hypotonic or high-pH (pH 8) solutions Biomolecules 2021, 11, 1010 7 of 17

with or without non-selective PMCA inhibitors (10 µM CE [11,31,33] or 100 µM caloxin 1b1 [15,30,32]) in acutely isolated rat odontoblasts (Figures 2 and 4) and HOB cells (Figures 3 and 5). The Ca2+ extrusion efficiency was determined by the extrusion rate, calculated as:

Extrusion rate (%) = (F/F0peak − F/F0 at termination of stimulation)/(F/F0peak − F/F0) (1)

where F/F0 is the baseline, F/F0peak is the peak value of F/F0 upon stimulation, and F/F0 at termination of stimulation is the value at the termination of stimulation. The F/F0 baseline ratio was normalized to 1. In the presence of extracellular Ca2+, hypotonic stimulation transiently increased 2+ [Ca ]i in both the absence (Figures2A and3A) and the presence of caloxin 1b1 (Figures2B and3B) and CE (Figures2C and3C) in acutely isolated rat odontoblasts (Figure2) and HOB cells (Figure3). In the presence of 10 µM CE (Figures2C and3C), 2+ the peak values of hypotonic stimulation-induced [Ca ]i increases were significantly lower than those in the absence of CE and caloxin 1b1 in both cultured and isolated cells 2+ (Figures2D and3D). These [Ca ]i increases decayed during the administration of hypo- tonic solution in both cells (Figure2A–C and Figure3A–C). In the presence of 100 µM 2+ caloxin 1b1 or 10 µM CE, the extrusion rate in [Ca ]i during hypotonic stimulation de- creased significantly relative to that in the absence of PMCA inhibitors in both cultured and isolated cells (Figures2E and3E). In the presence of extracellular Ca2+, the application of Krebs solution (pH 8) to acutely 2+ isolated rat odontoblasts (Figure4) and HOB cells (Figure5) transiently increased [Ca ]i in the presence of caloxin 1b1 (Figures4B and5B) and CE (Figures4C and5C), as well 2+ as in the control cells in their absence (Figures4A and5A). The [Ca ]i increase with the addition of Krebs solution (pH 8) in the presence of 10 µM CE (Figures4C and5C) was significantly lower than that in the absence of CE and caloxin 1b1 in both cultured 2+ and isolated cells (Figures4D and5D). These [Ca ]i increases showed a decay in the increase during high-pH stimulation in both cells. The application of 100 µM caloxin 1b1 (Figures4B and5B) or 10 µM CE (Figures4C and5C) significantly decreased the extrusion 2+ rates of the high-pH-induced [Ca ]i increase in both cell types (Figures4E and5E).

3.3. PMCA Mediates Mineralization by Odontoblasts We investigated the effects of PMCA activity on mineralization induced by human odontoblasts. Alizarin red (left) and von Kossa (right) staining (Figure6A,C) were de- termined to be indicative of the mineralization levels based on the staining intensity (see Materials and Methods) represented as I/I0 units; the intensities (I) of both stains were normalized to the mean intensities of adjacent areas without cells (I0). We compared the mineralization levels in HOB cells cultured in mineralization medium for 28 days in the presence or absence of PMCA inhibitors. The application of 10 µM CE (Figure6A,B) or 100 µM caloxin 1b1 (Figure6C,D) to mineralization media resulted in decreased mineral- ization levels compared to those without CE or caloxin 1b1 (controls). Biomolecules 2021, 11, x 7 of 16

3.2. Extrusion of Ca2+ by PMCA Following Hypotonic or High-pH Stimulation in Acutely Isolated Rat Odontoblasts and HOB Cells To demonstrate the contribution of PMCA to Ca2+ extrusion following mechanosen- sitive- or high-pH sensitive-[Ca2+]i increases, we measured [Ca2+]i and analyzed the extru- sion efficiencies following the application of hypotonic or high-pH (pH 8) solutions with or without non-selective PMCA inhibitors (10 µM CE [11, 31,33] or 100 µM caloxin 1b1 [15,30,32]) in acutely isolated rat odontoblasts (Figure 2 and 4) and HOB cells (Figure 3 and 5). The Ca2+ extrusion efficiency was determined by the extrusion rate, calculated as:

Extrusion rate (%) = (F/F0peak − F/F0 at termination of stimulation)/(F/F0peak − F/F0) (1)

where F/F0 is the baseline, F/F0peak is the peak value of F/F0 upon stimulation, and F/F0 Biomoleculesat termination2021, 11, 1010 of stimulation is the value at the termination of stimulation. The F/F0 baseline8 of 17ratio was normalized to 1.

FigureFigure 2. PMCA 2. PMCA mediates mediates Ca2+ extrusion Ca2+ following extrusion hypotonic following stimuli inhypotonic acutely isolated stimuli rat odontoblasts. in acutely (A –isolatedC) Rep- rat 2+ 2+ resentative traces of intracellular free Ca concentration ([Ca ]i) following hypotonic stimuli (black boxes at tops of graphs) in odontoblasts. (A–C) Representative traces of intracellular free Ca2+ concentration ([Ca2+]i) the absence of 100 µM caloxin 1b1 and 10 µM 5(6)-carboxyeosin (CE) (A), with 100 µM caloxin 1b1 (white box at the top) (B), orfollowing with 10 µM CEhypotonic (white box atstimuli the top) (C(black), in the boxes presence at of extracellulartops of graphs) Ca2+ (white in boxes the at absence bottom in Aof to 100 C). ( DµM) Peak caloxin 1b1 2+ valuesand of10 [Ca µM]i during5(6)-carboxyeosin hypotonic stimuli without (CE) 100(A),µM with caloxin 100 1b1 µM and 10 caloxinµM CE(open 1b1 column;(white 1.88 box± 0.08at the F/F0 top)units), (B), or with with10 µM 100 µCEM caloxin(white 1b1 box (upper at the gray top) column; (C), 2.08 in ±the0.05 presence F/F0 units), of andextracellular with 10 µM CECa (lower2+ (white gray boxes column; at bottom in 1.37 ± 0.03 F/F0 units). Each column denotes the mean ± SE from 10, 10, and 14 independent experiments, respectively. A to C). (D) Peak values of [Ca2+]i during hypotonic stimuli without 100 µM caloxin 1b1 and 10 (E) Ca2+ extrusion rates during hypotonic stimulation in the absence of 100 µM caloxin 1b1 and 10 µM CE (open col- umn;µM 68.2CE± (open2.8%), withcolumn; 100 µM 1.88 caloxin ± 1b10.08 (upper F/F0 gray units), column; with 55.8 100± 3.2%), µM and caloxin with 10 µ1b1M CE (upper (lower gray gray column; column; 2.08 ± 46.30.05± 2.4%).F/F0 units), Each column and denotes with the 10 mean µM± CESE from(lower 10, 10, gray and 14 column; independent 1.37 experiments, ± 0.03 F/F respectively.0 units). Statistically Each column significantdenotes differences the mean (in (±D ,SEE)) betweenfrom 10, columns 10, and (shown 14 by independent solid lines) are indicated experiments, by asterisks. respectively. * P < 0.05. (E) Ca2+ extrusion rates during hypotonic stimulation in the absence of 100 µM caloxin 1b1 and 10 µM CE (open column; 68.2 ± 2.8%), with 100 µM caloxin 1b1 (upper gray column; 55.8 ± 3.2%), and with 10 µM CE (lower gray column; 46.3 ± 2.4%). Each column denotes the mean ± SE from 10, 10, and 14 independent experiments, respectively. Statistically significant differences (in (D,E)) between columns (shown by solid lines) are indicated by asterisks. * P < 0.05. Biomolecules 2021, 11, x 8 of 16 Biomolecules 2021, 11, 1010 9 of 17

2+ 2+ FigureFigure 3. PMCA 3. PMCA mediates Camediatesextrusion followingCa2+ extrusionhypotonic stimuli following in HOB cells. hypotonic (A–C) Representative stimuli traces in ofHOB [Ca ] i cells. (A–C) following hypotonic stimuli (black boxes at tops of graphs) in the absence of 100 µM caloxin 1b1 and 10 µM CE (A), with 2+ i 100RepresentativeµM caloxin 1b1 (white traces box at of the top)[Ca (B),] or following with 10 µM CE hypotonic (white box at thestimuli top) (C), (black in the presence boxes of extracellularat tops of Ca graphs)2+ in the 2+ (whiteabsence boxes of at bottom 100 µM in A tocaloxin C). (D) Peak 1b1 values and of [Ca10 µM]i during CE hypotonic(A), with stimuli 100 without µM 100caloxinµM caloxin 1b1 1b1 (white and 10 µ boxM at the top) 2+ CE(B (open), or column;with 10 1.19 µM± 0.02 CE F/F (white0 units), withbox 100 atµ theM caloxin top) 1b1 (C (upper), in the gray presence column; 1.17 of± 0.03 extracellular F/F0 units), and Ca with (white boxes 10 µM CE (lower gray column; 1.06 ± 0.00 F/F units). Each column denotes the mean ± SE from 11, 4, and 7 independent at bottom in A to C). (D) Peak values0 of [Ca2+]i during hypotonic stimuli without 100 µM caloxin experiments, respectively. (E) Ca2+ extrusion rates during hypotonic stimulation in the absence of 100 µM caloxin 1b1 0 and1b1 10 andµM CE 10 (open µM column; CE (open 53.4 ± 2.2%), column; with 100 1.19µM caloxin± 0.02 1b1 F/F (upper units), gray column; with 32.4100± 7.3%),µM caloxin and with 101b1µM (upper gray CEcolumn; (lower gray 1.17 column; ± 0.03 29.7 F/F± 3.0%).0 units), Each and column with denotes 10 theµM mean CE± (lowerSE from gray 11, 4, andcolumn; 7 independent 1.06 ± experiments, 0.00 F/F0 units). Each respectively.column denotes Statistically the significant mean differences ± SE from (in (D,E 11,)) between 4, and columns 7 independent (shown by solid lines)experiments, are indicated byrespectively. asterisks. (E) Ca2+ *extrusionP < 0.05. rates during hypotonic stimulation in the absence of 100 µM caloxin 1b1 and 10 µM CE (open column; 53.4 ± 2.2%), with 100 µM caloxin 1b1 (upper gray column; 32.4 ± 7.3%), and with 10 µM CE (lower gray column; 29.7 ± 3.0%). Each column denotes the mean ± SE from 11, 4, and 7 independent experiments, respectively. Statistically significant differences (in (D,E)) between columns (shown by solid lines) are indicated by asterisks. * P < 0.05. Biomolecules 2021, 11, x 9 of 16 Biomolecules 2021, 11, 1010 10 of 17

2+ FigureFigure 4. PMCA4. PMCA mediates mediates Ca extrusion Ca2+ following extrusion high-pH following stimuli in acutely high-pH isolated ratstimuli odontoblasts. in acutely (A–C) Representative isolated rat traces for pH 8 (black boxes)-induced [Ca2+] increases, in the absence of 100 µM caloxin 1b1 and 10 µM CE (A), with 100 µM i 2+ odontoblasts.caloxin 1b1 (white box(A at–C the) top)Representative (B), or with 10 µM traces CE (white for box pH at the 8 top)(black (C), in boxes)-induced the presence of extracellular [Ca Ca]i 2+increases,(white in the 2+ absenceboxes at bottom of 100 in A µM to C). caloxin (D) Peak values 1b1 and of [Ca 10]i duringµM CE high-pH (A), stimulationwith 100 in µM the absence caloxin of 100 1b1µM (white caloxin 1b1 box and at the top) (10B),µM or CE with (open 10 column; µM 1.35CE± (white0.02 F/F box0 units), at withthe 100top)µM (C caloxin), in 1b1the (upper presence gray column; of extracellular 1.23 ± 0.02 F/F Ca0 units),2+ (white boxes and with 10 µM CE (lower gray column; 1.11 ± 0.01 F/F units). Each column denotes the mean ± SE from 9, 12, and at bottom in A to C). (D) Peak values of [Ca0 2+]i during high-pH stimulation in the absence of 100 10 independent experiments, respectively. (E) Ca2+ extrusion rates during high-pH stimulation in the absence of 100 µM µMcaloxin caloxin 1b1 and 1b1 10 µM and CE (open10 µM column; CE 74.2(open± 2.5%), column; with 100 1.35µM caloxin± 0.02 1b1 F/F (upper0 units), gray column;with 100 58.0 µM± 4.2%), caloxin and 1b1 (upperwith 10 µ Mgray CE (lower column; gray column;1.23 ± 44.00.02± 3.9%).F/F0 units), Each column and denotes with the10 meanµM CE± SE (lower from 9, 12, gray and 10column; independent 1.11 ± 0.01 F/Fexperiments,0 units). respectively. Each column Statistically denotes significant the differences mean (in± SE (D,E from)) between 9, 12, columns and (shown 10 independent by solid lines) are experiments, indicated respectively.by asterisks. * P < ( 0.05.E) Ca2+ extrusion rates during high-pH stimulation in the absence of 100 µM caloxin 1b1 and 10 µM CE (open column; 74.2 ± 2.5%), with 100 µM caloxin 1b1 (upper gray column; 58.0 ± 4.2%), and with 10 µM CE (lower gray column; 44.0 ± 3.9%). Each column denotes the mean ± SE from 9, 12, and 10 independent experiments, respectively. Statistically significant differences (in (D,E)) between columns (shown by solid lines) are indicated by asterisks. * P < 0.05. Biomolecules 2021, 11, x 10 of 16 Biomolecules 2021, 11, 1010 11 of 17

2+ Figure 5. PMCA mediates Ca extrusion2+ following high-pH stimuli in HOB cells. (A–C) Representative traces for pH 8 Figure 5. PMCA mediates2+ Ca extrusion following high-pH stimuli in HOB cells. (A–C) (black boxes)-induced [Ca ]i increases, in the absence of 100 µM caloxin 1b1 and 10 µM CE (A), with 100 µM caloxin 1b1 2+ i Representative(white box at the top)traces (B), or for with pH 10 µ M8 CE(black (white boxboxes)-induced at the top) (C), in the[Ca presence] increases, of extracellular in Cathe2+ (white absence boxes atof 100 µM 2+ caloxinbottom 1b1 in A toand C). (D10) PeakµM values CE ( ofA [Ca), with]i during 100 high-pH µM caloxin stimulation 1b1 in the(white absence box of 100 atµ Mthe caloxin top) 1b1 (B and), or 10 µwithM 10 µM 2+ CECE (white (open column; box at 1.15 the± top)0.01 F/F (C0 ),units), in the with presence 100 µM caloxin of extracellular 1b1 (upper gray column; Ca (white 1.15 ± 0.02 boxes F/F0 units), at bottom and with in A to C). 10 µM CE (lower gray column; 1.07 ± 0.01 F/F units). Each column denotes the mean ± SE from 6, 3, and 4 independent (D) Peak values of [Ca2+]i during high-pH0 stimulation in the absence of 100 µM caloxin 1b1 and 10 experiments, respectively. (E) Ca2+ extrusion rates during high-pH stimulation in the absence of 100 µM caloxin 1b1 and µM10 CEµM CE(open (open column; column; 55.6 1.15± 3.0%),± 0.01 with F/F 1000 units),µM caloxin with 1b1 (upper100 µM gray caloxin column; 32.41b1± (upper8.5%), and gray with column; 10 µM 1.15 ± 0.02CE F/F (lower0 units), gray column; and with 29.0 ± 104.4%). µM Each CE column(lower denotes gray thecolumn; mean ± 1.07SE from ± 0.01 6, 3, and F/F 40 independentunits). Each experiments, column denotes therespectively. mean ± SE Statistically from 6, significant 3, and 4 differences independent (in (D,E)) experiments, between columns (shownrespectively. by solid lines) (E) are Ca indicated2+ extrusion by asterisks. rates during high-pH* P < 0.05. stimulation in the absence of 100 µM caloxin 1b1 and 10 µM CE (open column; 55.6 ± 3.0%), with 100 µM caloxin 1b1 (upper gray column; 32.4 ± 8.5%), and with 10 µM CE (lower gray column; 29.0 ± 4.4%). Each column denotes the mean ± SE from 6, 3, and 4 independent experiments, respectively. Statistically significant differences (in (D,E)) between columns (shown by solid lines) are indicated by asterisks. * P < 0.05.

In the presence of extracellular Ca2+, hypotonic stimulation transiently increased [Ca2+]i in both the absence (Figure 2A and 3A) and the presence of caloxin 1b1 (Figure 2B and 3B) and CE (Figure 2C and 3C) in acutely isolated rat odontoblasts (Figure 2) and HOB cells (Figure 3). In the presence of 10 µM CE (Figure 2C and 3C), the peak values of hypotonic stimulation-induced [Ca2+]i increases were significantly lower than those in the absence of CE and caloxin 1b1 in both cultured and isolated cells (Figure 2D and 3D). These [Ca2+]i increases decayed during the administration of hypotonic solution in both cells (Figure 2A–2C and 3A–3C). In the presence of 100 µM caloxin 1b1 or 10 µM CE, the extrusion rate in [Ca2+]i during hypotonic stimulation decreased significantly relative to that in the absence of PMCA inhibitors in both cultured and isolated cells (Figure 2E and 3E). In the presence of extracellular Ca2+, the application of Krebs solution (pH 8) to acutely isolated rat odontoblasts (Figure 4) and HOB cells (Figure 5) transiently increased [Ca2+]i in the presence of caloxin 1b1 (Figures 4B and 5B) and CE (Figures 4C and 5C), as well as in the control cells in their absence (Figures 4A and 5A). The [Ca2+]i increase with the addition of Krebs solution (pH 8) in the presence of 10 µM CE (Figures 4C and 5C) was significantly lower than that in the absence of CE and caloxin 1b1 in both cultured Biomolecules 2021, 11, x 11 of 16

and isolated cells (Figures 4D and 5D). These [Ca2+]i increases showed a decay in the in- crease during high-pH stimulation in both cells. The application of 100 µM caloxin 1b1 (Figures 4B and 5B) or 10 µM CE (Figures 4C and 5C) significantly decreased the extrusion rates of the high-pH-induced [Ca2+]i increase in both cell types (Figures 4E and 5E).

3.3. PMCA Mediates Mineralization by Odontoblasts We investigated the effects of PMCA activity on mineralization induced by human odontoblasts. Alizarin red (left) and von Kossa (right) staining (Figure 6A,C) were deter- mined to be indicative of the mineralization levels based on the staining intensity (see Materials and Methods) represented as I/I0 units; the intensities (I) of both stains were normalized to the mean intensities of adjacent areas without cells (I0). We compared the mineralization levels in HOB cells cultured in mineralization medium for 28 days in the presence or absence of PMCA inhibitors. The application of 10 µM CE (Figure 6A,B) or Biomolecules 2021, 11, 1010 100 µM caloxin 1b1 (Figure 6C,D) to mineralization media resulted in decreased12 of 17 mineral- ization levels compared to those without CE or caloxin 1b1 (controls).

Figure 6.6.PMCA PMCA inhibitors inhibitors decrease decrease mineralization mineralization levels. levels. (A,C) ( HOBA,C) cells HOB were cells cultured were cultured for 28 days for 28 days in mineralizationmineralization medium medium with with PMCA PMCA inhibitors inhibitors (each (each lower lower column), column), 10 µM 10 CE µM (A), CE or 100 (A),µ Mor 100 µM caloxin 1b11b1 ( C(C),), or or without without them them (each (each upper upper column column in (A ,inC)) (A at,C pH)) 7.4.at pH Alizarin 7.4. Alizarin red (left red columns; (left columns; red indicativeindicative of of calcium calcium deposition) deposition) and and von von Kossa Kossa (right (right columns; columns; black indicative black indicative of phosphate of phosphate and calciumcalcium deposition) deposition) staining. staining. (B,D) ( MineralizationB,D) Mineralization levels with levels (gray with columns) (gray and columns) without (open and without columns)(open columns) PCMA inhibitorsPCMA inhibitors 10 µM CE 10 (B )µM or 100 CEµ (MB) caloxin or 100 1b1µM ( Dcaloxin), assessed 1b1 by(D Alizarin), assessed red (upperby Alizarin red columns),(upper columns), and von and Kossa von (lower Kossa columns) (lower staining.columns) Each staining. column Each in (B column,D) denotes in (B the,D mean) denotes±SE the mean from± SE 8from and 128 and experiments, 12 experiments, respectively. respectively. The mineralization The mineralization levels in the absence levels ofin CEthe (as absence controls of CE (as 0 0 incontrols (B)) were in 1.15(B)) ±were0.03 1.15 I/I0 with± 0.03 Alizarin I/I with red Alizarin staining andred 1.08staining± 0.02 and I/I 01.08units ± with0.02 vonI/I units Kossa with von 0 staining.Kossa staining. The mineralization The mineralization levels with levels CE (gray with bars CE in ((grayB)) were bars 1.00 in± (B0.01)) were I/I0 units 1.00with ± 0.01 Alizarin I/I units with 0 redAlizarin staining red and staining 1.01 ± and0.01 I/I1.010 units ± 0.01 with I/I von units Kossa with staining. von Kossa The mineralization staining. The levels mineralization without levels without caloxin 1b1 (as controls in (D)) were 1.34 ± 0.02 I/I0 units with Alizarin red staining and 1.11 caloxin 1b1 (as controls in (D)) were 1.34 ± 0.02 I/I0 units with Alizarin red staining and 1.11 ± 0.01 ± 0.01 I/I0 units with von Kossa staining. The mineralization levels with caloxin 1b1 (gray bars in I/I0 units with von Kossa staining. The mineralization levels with caloxin 1b1 (gray bars in (D)) (D)) were 1.18 ± 0.04 I/I0 units with Alizarin red staining and 1.07 ± 0.01 I/I0 with von Kossa staining. were 1.18 ± 0.04 I/I0 units with Alizarin red staining and 1.07 ± 0.01 I/I0 with von Kossa staining. Statistically significant significant differences differences (in ((inB,D ())B, betweenD)) between columns columns (shown (shown by solid by lines) solid are lines) indicated are indicated by asterisks.asterisks. * *P P< < 0.05. 0.05.

3.4. Effects of PMCA Inhibitor on Detected mRNA Levels of Non-Collagenous Extracellular Matrix Proteins as Odontoblast Markers We analyzed the changes in the mRNA levels of non-collagenous extracellular ma- trix proteins, such as odontoblast markers, dentin matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP), following 3 days of HOB cell culture under physiological or high-pH conditions as well as isotonic or hypotonic conditions with or without 10 µM CE. High-pH stimulation significantly increased the mRNA levels of DMP-1 (Figure7A) and DSPP (Figure7B) compared to stimulation at pH 7.4. We did not observe any significant differences in the mRNA levels between hypotonic and isotonic stimulation; however, hypotonic stimulation tended to increase the mRNA levels of DMP-1 and DSPP compared to those under isotonic conditions. The PMCA inhibitor, CE, did not have any significant effects on their mRNA levels in the normal and high-pH as well as isotonic and hypotonic extracellular conditions in odontoblasts. Biomolecules 2021, 11, x 12 of 16

3.4. Effects of PMCA Inhibitor on Detected mRNA Levels of Non-Collagenous Extracellular Matrix Proteins as Odontoblast Markers We analyzed the changes in the mRNA levels of non-collagenous extracellular matrix proteins, such as odontoblast markers, dentin matrix protein-1 (DMP-1), and dentin sial- ophosphoprotein (DSPP), following 3 days of HOB cell culture under physiological or high-pH conditions as well as isotonic or hypotonic conditions with or without 10 µM CE. High-pH stimulation significantly increased the mRNA levels of DMP-1 (Figure 7A) and DSPP (Figure 7B) compared to stimulation at pH 7.4. We did not observe any significant differences in the mRNA levels between hypotonic and isotonic stimulation; however, hypotonic stimulation tended to increase the mRNA levels of DMP-1 and DSPP compared to those under isotonic conditions. The PMCA inhibitor, CE, did not have any significant Biomolecules 2021, 11, 1010 effects on their mRNA levels in the normal and high-pH as well as isotonic13 of 17 and hypotonic extracellular conditions in odontoblasts.

Figure 7. 7.The The changes changes in mRNA in mRNA level of level dentin of matrixdentin protein-1 matrix (DMP-1) protein-1 (A) (DMP-1) and dentin ( sialophos-A) and dentin sialophos- phoprotein (DSPP) (DSPP) (B) as(B markers) as markers specific specific for odontoblasts for odontoblasts in response toin high-pHresponse or hypotonicto high-pH or hypotonic stimulation with with or without or without CE in HOBCE in cells. HOB Real-time cells. RT-PCRReal-time was usedRT-PCR to quantify was used mRNA to levels quantify by mRNA levels measuringby measuring the increase the increase in fluorescence in fluorescence elicited by the elicit bindinged of by SYBR the green binding dye to of double-stranded SYBR green dye to double- DNA. Data were analyzed by the 2−[∆∆Ct] method, with β-actin as an internal control (which was stranded DNA. Data were analyzed by the 2-[ΔΔCt] method, with β-actin as an internal control (which positive in all samples). Each bar denotes the mean ± SE of 5 experiments under physiological or was positive in all samples). Each bar denotes the mean ± SE of 5 experiments under physiological high-pH condition and 3 experiments for isotonic or hypotonic conditions. The levels of mRNA were normalizedor high-pH to conditionβ-actin mRNA and levels. 3 experi Statisticallyments significant for isotonic differences or hypotonic between columnsconditions. (Shown The by levels of mRNA solidwere lines) normalized are marked to with β-actin asterisks. mRNA * P <0.05. levels. Statistically significant differences between columns (Shown by solid lines) are marked with asterisks. * P < 0.05. 3.5. Immunofluoresence Analysis of PMCA1 in HOB Cells We further observed the presence of the ubiquitously expressed PMCA1 [11,14] protein in HOB cells by immunofluorescence staining. Distinct PMCA1 immunoreactivity on HOB cells was observed throughout the cell membrane (Figure8). Biomolecules 2021, 11, x 13 of 16

3.5. Immunofluoresence Analysis of PMCA1 in HOB Cells We further observed the presence of the ubiquitously expressed PMCA1 [11,14] pro- tein in HOB cells by immunofluorescence staining. Distinct PMCA1 immunoreactivity on Biomolecules 2021, 11, 1010 HOB cells was observed throughout the cell membrane (Figure14 of 17 8).

Figure 8. Immunofluorescence analysis of PMCA1 protein in HOB cells. HOB cells were positive for FigurePMCA1 immunoreactivity8. Immunofluorescence (red). Nuclei are shown analysis in blue. of Scale PMCA1 bar: 100 µ proteinm. No fluorescence in HOB was cells. HOB cells were positive detected in the negative control (not shown). for PMCA1 immunoreactivity (red). Nuclei are shown in blue. Scale bar: 100 µm. No fluorescence 4. Discussion was detected in the negative control (not shown). We have demonstrated the pharmacological properties of PMCAs and their roles in the cellular functions of odontoblasts. We have also shown the detection of mRNAs en- 4.coding Discussion PMCAs and PMCA1 immunoreactivity in human odontoblasts. In mammals, four PMCAs are numbered (1–4) [14]. PMCA1 and PMCA4 are expressed ubiquitously, whereas PMCA2We and have PMCA3 demonstrated have tissue-specific distributionsthe pharmacological [11,14]. It has been reportedproperties that the of PMCAs and their roles in stereocilia of hair cells express PMCA2 at high levels, and PMCA2 defects are linked to theprofound cellular hearing functions impairment/loss of inodontoblasts. humans [12]. The ablation We have of the PMCA4 also geneshown causes the detection of mRNAs en- codingmale infertility PMCAs [12]. Although and PMCA1 the ablation immunoreactivity of the PMCA1 gene results in in earlyhuman embryonic odontoblasts. In mammals, four lethality (highlighting the essential role of PMCA1 in development, organogenesis, and PMCAshousekeeping are function numbered [12]), the deletion(1–4) of[14].PMCA1 PMCA1in the intestine and is associatedPMCA4 with are expressed ubiquitously, whereasreduced bone PMCA2 mineralization and [34 PMCA3]. In addition, have osteoblasts tissue-specifi and ameloblasts,c distributions which form [11,14]. It has been re- hard tissues and play roles in bone mineralization and amelogenesis, respectively, also portedexpress PMCA that [the16,17 stereocilia,35,36]. In ameloblasts, of hair PMCA cells activity express increases PMCA2 gradually at during high levels, and PMCA2 defects aredevelopmental linked processes,to profound suggesting hearing that PMCA impairment/l in ameloblasts is associatedoss in withhumans amel- [12]. The ablation of the ogenesis and the maturation of ameloblasts [16,36]. During odontoblast differentiation, PMCA4immunohistochemical gene causes analysis male using infertility monoclonal antibodies [12]. Although against erythrocyte the ablation PMCA of the PMCA1 gene results revealed a lack of immunoreactivity in the pre-odontoblasts located at the surface of the indental early papilla, embryonic while gradient lethality increases in (highlighting the immunoreaction the intensity essential were observed role of PMCA1 in development, organogenesis,in the period when the and odontoblast housekeeping became fully differentiatedfunction [12]), and in parallel the deletion to the pro- of PMCA1 in the intestine is gression of mineralization of dentin [17]. These previous results [16,17] are in line with associatedour results showing with the reduced functional expression bone mineralization of PMCA in odontoblasts. [34]. In addition,In addition, we osteoblasts and amelo- blasts,detected which mRNAs encodingform hard PMCA1, tissues PMCA3, and PMCA4, play whileroles the in level bone of detectable mineralization and amelogenesis, PMCA2 mRNA was significantly lower than those of its paralogs. Notably, it is well docu- respectively,mented that mRNA also levels express of ion transporters PMCA and channels[16,17,35,36]. do not perfectly In ameloblasts, correlate with PMCA activity increases graduallytheir protein levelsduring for reasons developmental including microRNA processes, regulation suggesting and post-transcriptional that PMCA in ameloblasts is asso- degradation. Although we have observed PMCA1 immunoreactivity in human odonto- ciatedblasts, further with studies amelogenesis to reveal detailed and protein the levelsmaturation and cellular of localization ameloblasts patterns [16,36]. During odontoblast differentiation,for PMCA subtypes inimmunohistoc odontoblasts, and to revealhemical changes analysis in levels/localization using duringmonoclonal antibodies against odontoblast differentiation, odontogenesis, and dentinogenesis, are of immediate interest. erythrocyteIn the present PMCA study, we revealed also found thata lack intracellular of immunoreactivity Ca2+ increased by hypotonic in the or pre-odontoblasts located at high-pH stimulation was extruded into the extracellular space by PMCA activity. PMCA the surface of the dental papill2+ a, while gradient increases2+ in the immunoreaction intensity generally has a high affinity for Ca (Kd ~0.2 µM) but a lower capacity for Ca trans- 2+ wereport than observed NCX [10,13 in,37 ].the This period allows for when the maintenance the odonto of lowblast [Ca ] ibecamein the resting fully differentiated and in par- 2+ allelstate [to13, 15the,37]. progression The steady-state baselineof mineralization [Ca ]i has been shown of dentin to be between [17]. pCaThese 6.4 previous results [16,17] are and 6.6 in rat incisor odontoblasts [38,39]. Thus, consistent with previous reports, our data 2+ insuggest line thatwith the roleour of PMCAresults may showing be to maintain the [Ca function]i below 100al nM expression in odontoblasts of PMCA in odontoblasts. In addition,under resting we conditions. detected mRNAs encoding PMCA1, PMCA3, and PMCA4, while the level of detectable PMCA2 mRNA was significantly lower than those of its paralogs. Notably, it is well documented that mRNA levels of ion transporters and channels do not perfectly correlate with their protein levels for reasons including microRNA regulation and post- transcriptional degradation. Although we have observed PMCA1 immunoreactivity in human odontoblasts, further studies to reveal detailed protein levels and cellular locali- zation patterns for PMCA subtypes in odontoblasts, and to reveal changes in levels/local- ization during odontoblast differentiation, odontogenesis, and dentinogenesis, are of im- mediate interest. In the present study, we also found that intracellular Ca2+ increased by hypotonic or high-pH stimulation was extruded into the extracellular space by PMCA activity. PMCA generally has a high affinity for Ca2+ (Kd ~0.2 µM) but a lower capacity for Ca2+ transport than NCX [10,13,37]. This allows for the maintenance of low [Ca2+]i in the resting state Biomolecules 2021, 11, 1010 15 of 17

The application of the non-selective PMCA inhibitors CE or caloxin 1b1 attenuated the 2+ 2+ decay of [Ca ]i after hypotonic or high-pH stimulation-induced [Ca ]i increases in both rat and human odontoblasts, showing that PMCA extrudes intracellular Ca2+ increased by membrane stretch or high-pH stimuli. Thus, PMCA participates in the maintenance 2+ of low [Ca ]i not only during rest but also after intracellular signaling events by stimuli to the dentin surface. Hypotonic and direct mechanical stimuli (which mimic membrane stretch and cell deformation of odontoblasts via dentinal fluid movement in response to multiple external stimuli to the dentin surface) activate Ca2+ influx via mechanosensitive ion channels, including TRP vanilloid (TRPV), TRP ankylin 1 (TRPA1), and Piezo1 channels, 2+ increasing [Ca ]i in rat and mouse odontoblasts [3,5,29]. The application of high-pH dental materials such as calcium hydroxide and mineral trioxide aggregates on the dentin surface increases the pH of the extracellular environment [40,41], which is detected by odontoblasts in rats and humans [6]. The high-pH environment activates TRPA1 channels and alkali- sensitive metabotropic receptors and stimulates Ca2+ release-activated Ca2+ channels that induce store-operated Ca2+ entry in odontoblasts [6,7]. We previously showed that the functional coupling between Ca2+ influx via high-pH- and mechanosensitive-TRP channels 2+ and NCX plays an important role in regulating [Ca ]i homeostasis and driving cellular functions in rat, mouse, and human odontoblasts, including the induction of reactionary dentin formation [1–3,6,9]. Thus, both NCX and PMCA likely play important roles in reactionary dentin formation by exporting Ca2+ to the mineralizing front after the external stimulation of the dentin surface or during the application of high-pH dental materials. In the mineralization assay with Alizarin red and von Kossa staining, the PMCA inhibitors CE and caloxin 1b1 significantly decreased the mineralization levels. Mineral- ization in odontoblasts cultured in mineralization medium without PMCA inhibitor (as a control experiment) mimicked the physiological or developmental conditions of dentino- genesis. Thus, these results also demonstrate that Ca2+ extrusion via PMCA is essential for dentin mineralization under physiological conditions. High pH, but not hypotonicity, significantly augmented DMP-1 and DSPP mRNA levels. Together with our previous results showing that high-pH stimulation increased mineralization levels via TRPA1 ac- tivation in HOB cells [6], it is concluded that extracellular alkaline conditions effectively promote dentinogenesis by increasing not only Ca deposition through the Ca2+ extrusion pathway, but also the secretion of non-collagenous extracellular matrix proteins to the mineralizing front. During hypotonic stimulation, Ca2+ extrusion following an increase 2+ in [Ca ]i was driven by PMCA activity; however, we did not observe any significant differences in levels of DMP-1 and DSPP mRNA under hypotonic conditions as compared to under isotonic conditions. Interestingly, PMCA inhibitors did not affect their mRNA levels in the normal-/high-pH and isotonic/hypotonic extracellular conditions, whereas PMCA activity mediated mineralization under physiological conditions by odontoblasts. Although further studies are needed, these results indicate that Ca2+ extrusion processes via PMCA and the secretion of extracellular matrix proteins are regulated by independent signaling pathways. In conclusion, this study demonstrates that PMCA contributes to the maintenance of 2+ [Ca ]i homeostasis in odontoblasts. PMCA was found to play critical roles not only in physiological dentin formation but also in the pathological tertiary (reactionary) dentin formation induced by multiple external stimuli applied to the dentin surface, as well as by the application of high-pH dental materials on the dentin surface. It has been reported that the activation of neural activity in the brain causes a rapid rise in extracellular pH by removing H+ from the external space. This has been proposed to arise from neuronal PMCA activity, which exchanges internal Ca2+ for external H+ [42]. Therefore, there is considerable interest in understanding whether PMCA activation leads to increased extracellular alkalinity in odontoblasts. Although further studies are needed to clarify the Ca2+-H+ exchanging ability of PMCA in odontoblasts, compounds that alter PMCA activity may be good candidates for inclusion in dental materials to promote dentinogenesis. Biomolecules 2021, 11, 1010 16 of 17

Author Contributions: Conceptualization, M.K., H.M., H.K., and Y.S.; Methodology, M.K., H.M., H.K., and Y.S.; Software, Y.S.; Validation, M.K., H.M., R.S., M.I., E.K., K.K., S.N., T.S., H.K., and Y.S.; Formal analysis, M.K., H.M., R.S., M.I., E.K., K.K., S.N., T.S., H.K., and Y.S.; Investigation, M.K., H.M., R.S., M.I., E.K., K.K., S.N., T.S., and H.K.; Resources, M.K. and H.M.; Data curation, M.K., H.M., R.S., M.I., E.K. and Y.S.; Writing—original draft preparation, M.K., H.M., and H.K.; Writing— review and editing, Y.S.; Visualization, M.K. and Y.S.; Supervision, Y.S.; Project administration, Y.S.; Funding acquisition, M.K. and Y.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by Grant-in-Aid 19K10117/19H038336 for Scientific Research from MEXT of Japan and a Private University Research Branding Project from MEXT of Japan (Multidisciplinary Research Center for Jaw Disease (MRCJD); Achieving Longevity and Sustainability by Comprehensive Reconstruction of Oral and Maxillofacial Functions). Institutional Review Board Statement: The study was approved by the Ethics Committee of Tokyo Dental College (300301, 190301, and 200301, approved on 01 April 2018, 2019, and 2020). Informed Consent Statement: Not applicable. Data Availability Statement: All data is contained within the article. Conflicts of Interest: The authors declare no conflict of interest.

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