Bull Group Int Rech Sci Stomatol Odontol. 53(1): 16-27 (2016) ORIGINAL RESEARCH ARTICLES EXPRESSION OF PHOSPHATE TRANSPORTERS IN OPTIMIZED CELL CULTURE MODELS FOR DENTAL CELLS BIOMINERALIZATION Laure Merametdjian1,2,3, Amandine David1,2, Nina Bon1,2, Greig Couasnay1,2, Jérôme Guicheux1,2,3,Céline Gaucher4,5, Sarah Beck-Cormier1,2 and Laurent Beck1,2* 1 INSERM, U791, LIOAD, Nantes, F-44042, France. 2 Université de Nantes, UFR Odontologie, UMR_S 791, Nantes, F-44042, France. 3 CHU Nantes, PHU 4 OTONN, Nantes, F-44042, France. 4 Dental School University Paris Descartes PRES Sorbonne Paris Cité, EA 2496, Montrouge, F-92120, France 5 AP-HP, Odontology Department, Hôpital Albert Chennevier, GHHM, AP-HP, Créteil, F-94010, France * Corresponding author: Laurent Beck, Ph.D. INSERM U791-LIOAD, Faculté de Chirurgie Dentaire 1, place Alexis Ricordeau, 44042 Nantes cedex 1, France, email: [email protected] Abstract / Résumé Keywords Phosphate is a key component of dental mi- phosphate, mineralization, ALC, M2H4 neral composition. The physiological role of membrane proteins of dental cells is suspec- Introduction ted to be crucial for mineralization mecha- The tooth is the most mineralized organ of nisms. Contrary to published data related the body and is composed of both calcified to calcium, data on regulation of phosphate tissues like dentin and cementum, and mine- flux through membrane of mineralizing cells ral acellular structures like enamel. For each are scarce. To address this lack of data, we dental structure, the mineralization process studied the expression of six membranous has specific features. Whereas enamel mine- phosphate transporters in two dental cell lines: ralization requires specific proteins such as a rat odontoblastic cell line (M2H4) and a mou- amelogenins or enamelins, dentin minerali- se ameloblastic cell line (ALC) for which we zation mechanisms are closer to those taking optimized the mineralizing culture conditions. place in bone by involving collagenous and La place essentielle du phosphate dans la non-collagenous matrix proteins. In addition, composante minérale de la dent laisse sup- dentin mineralization shows significant diffe- poser un rôle physiologique déterminant pour rences, between the outer mantle and the cir- les protéines membranaires permettant son cumpulpal dentin in term of mineral density, entrée dans les cellules dentaires. Contraire- and also between the intertubular and peritu- ment à celles disponibles pour le calcium, les bular areas in term of protein’s matrix com- données sur les molécules permettant la pro- position (Goldberg et al., 2011). Cementum duction et la régulation du flux de phosphate also displays its own specificities with some aux sites de formation du minéral par les ce- distinguable cellular/non cellular and fibrillar/ llules minéralisantes de la dent sont peu con- non fibrillar areas along the roots (Goncalves nues. Nous avons analysé dans cette étude et al., 2005). six transporteurs de phosphate membranai- Two main mineral deposition mechanisms res dans deux lignées cellulaires dentaires: are reported in the literature, namely, matrix une lignée d’odontoblastes de rat (M2H4) et vesicles budding from mineralizing cells, or une lignée améloblastique murine, les ALC, secretion of a non-mineralized extracellular pour laquelle nous avons optimisé les condi- matrix that will eventually become a mine- tions de culture minéralisantes. ralized structure in supersaturated calcium e16 Bull Group Int Rech Sci Stomatol Odontol. 53(1): 16-27 (2016) (Ca)-phosphate (Pi) environment in which mi- its deletion did not aggravated the phenoty- neral will nucleate (Veis et al., 2013). These pe of the Npt2a knockout mice (Miyamoto et mechanisms have been mainly described in al., 2011). In both models, although the dental bone, and the mineralization processes in- phenotype has not been explored in details, volved in dentin and enamel are much less the animals showed no major eruption failu- known. Nonetheless, the mineral part of any re or dental mineralization defects (L. Beck, mineralized tissue consists of Pi and Ca ions unpublished data). Invalidation of the Npt2b that will accumulate, be combined together gene in mice is embryonic lethal at E10.5 and stabilized in the form of hydroxyapatite (Shibasaki Y., BBRC 2009). A tissue-specific (Ca10(PO4)6(OH)2), eventually combined with invalidation in the intestine was performed, minor ions giving rise to biological apatites. revealing its essential role in the intestinal ab- The essential requirement of Ca and Pi for sorption of Pi (Sabbagh et al., 2009). When tooth mineralization raises the question of the mutated in humans, loss of Npt2b results in mechanisms underlying the production and lung and testis calcifications (Corut et al., regulation of ions fluxes at the mineral forma- 2006). Its role in tooth mineralization in vivo tion sites of the tooth. Whereas Ca transport has not been formally demonstrated. Howe- and regulation in the dental enamel have been ver, some studies have shown that Npt2b partially described (Hubbard M.J., 2000), Pi was expressed in odontoblasts and that its transport on the dental mineralization sites is expression correlated with that of the Phex still a large question mark. gene (Onishi et al., 2007). Interestingly, muta- In mammals, there are six identified Na-Pi co- tions of Phex in humans cause osteomalacia, transporters located at the plasma membra- impaired renal reabsorption of Pi, together ne and responsible for Pi entry into the cell. with an abnormal mineralization of cementum These transporters have been historically and dentin (Gaucher et al., 2009). Similarly to separated into three families, based on their SLC17 and SLC34 transporters, the involve- sequence similarities and tissue expression. ment of PiT1 and PiT2 in dental mineralization The first family, NPT1/SLC17A1, is expres- is poorly described. The invalidation of the sed mostly in kidney and liver, but also in the PiT1 gene in mice leads to a lethal phenotype brain. Although NPT1 displays a Pi trans- at mid gestation, and does not allow to study port activity in cultured cells in vitro, its phy- its physiological role in the tooth (Beck et al., siological function is now known as being a 2010). Although, the transgeni cover expres- cloride-dependent urate exporter (Iharada et sion of PiT1 in rats has suggested the involve- al., 2010). The second SLC34 family com- ment of PiT1 in enamel mineralization, the un- prises three members, among which Npt2a/ derlying mechanisms are unknown (Yoshioka SLC34A1 and Npt2c/SLC34A3 are primarily et al., 2011). Finally, a comprehensive study expressed in the proximal tubule of the kid- reported a strong expression of PiT2 in pulp ney, whereasNtp2b/SLC34A2 has a wider ex- cells, a fainter expression in ameloblasts and pression (mainly lung and intestine) (Wagner an absence of expression in odontoblasts at et al., 2014). The third family is represented all stages of development (Zhao et al., 2006). by PiT1/SLC20A1 and PiT2/SLC20A2, which In summary, despite the considerable need of are expressed in a large number of tissues Pi for tooth mineralization, knowledge on the (Forster et al., 2013). mechanisms and molecules involved in the In recent years, considerable progress has production and regulation of the Pi flux to the been made in determining the physiological sites of mineral formation by the mineralizing function of these transporters. By generating cells are very scarce. the first knockout mouse model for a Pi trans- As a first step to determine the functional in- porter, we have shown that Npt2a is the main volvement of Pi transporters in tooth mine- carrier responsible for the reabsorption of Pi ralization, the objective of this study was to by the kidney (Beck et al., 1998), at least in characterize the expression of the six known rodents. More recent studies have confirmed Pi transporters in ameloblast–ALC (Nakata these data and in addition, showed that the et al., 2003) and odontoblast-M2H4 (Ritchie function of Npt2c carrier, expressed in the et al., 2002) cell lines. To this aim, we used same segments of the nephron than Npt2a, the ALC mouse ameloblast-lineage (Nakata was to increase the reabsorption capacity of et al., 2003) and in the M2H4 rat odontoblast Pi during growth. However, the physiological (Ritchie et al., 2002) cell lines after optimiza- role of Npt2c in rodents remains minor, since tion of the culture conditions necessary to ob- e17 Bull Group Int Rech Sci Stomatol Odontol. 53(1): 16-27 (2016) tain a robust mineralization. I -coated (Rat Tail collagen I solution, BD Bioscience USA) culture plates, as described Material and Methods previously (Nakata et al., 2003; Takahashi et Cell culture and conditions al., 2007). The growth medium was changed The odontoblastic rat M2H4 cells, cloned from every 2 days. In order to induce mineraliza- the rat dental pulp cell line RPC-C2A, were tion, other media were evaluated including cultured as previously described (Magne et betaglycerophosphate (BGP, Sigma Aldrich al., 2004), in a maintenance medium con- G6251), ascorbic acid (Sigma Aldrich A4034), sisting of MEM (Minimal Essential Medium, ITS (insulin transferrin selenite, Sigma Al- Invitrogen 21090-022) containing 10% fetal drich) and dexamethasone (Sigma Aldrich). bovine serum (FBS, Pan Biotech GmbH), 1% RNA isolation and real time qPCR penicillin/streptomycin (Invitrogen) and 1% Total RNA was extracted from M2H4 and ALC L-glutamine (Invitrogen). Cells were subcul- cells using a Nucleospin® RNA II kit (Mache- tured once a week using trypsin/EDTA, and rey-Nagel, Germany) in accordance with the maintained at 37 °C in a humidified atmos- manufacturer’s instructions. After DNaseI phere of 5% CO2 in air. To induce odontoblast treatment, the RNA was quantified using a UV differentiation, MEM was switched to α-MEM spectrophotometer (Nanodrop ND-1000, Lab- (Invitrogen 22571-020) containing ascorbic tech, Palaiseau, France). acid. To induce extracellular matrix minerali- Real-time PCR was performed on a Bio-Rad zation, 3 mM inorganic phosphate (Pi) were CFX96 using SYBR®Select Master Mix (Life added to the culture medium on day 2.