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Expression and Localization of TREK-1 K+ Channels in Human Odontoblasts
H. Magloire1,*,
F. Lesage2,
M.L. Couble1,
M. Lazdunski2 and
F. Bleicher1
1 Laboratoire du Développement des Tissus Dentaires, EA 1892, IFR 62, Faculté d’Odontologie, Rue G. Paradin, 69372, Lyon cedex 08, France; and
2 Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UPR 441, Sophia Antipolis, 06560 Valbonne, France;
Correspondence: *corresponding author, magloire{at}laennec.univ-lyon1.fr
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ABSTRACT
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During tooth development, odontoblasts are the cells that form dentin and possibly mediate early stages of sensory processing in teeth. It is suggested that ion channels assist in these events. Indeed, mechanosensitive potassium currents, transducing mechanical stimuli into electrical cell signals, have been previously recorded in the human odontoblast cell membrane. Here, we show by RT-PCR that the mechanosensitive potassium channel TREK-1 (a member of the two-pore-domain potassium channel family) is overexpressed in these cultured cells compared with pulp cells in vitro. In situ hybridization showed that transcripts are detected in the odontoblast layer in vivo. The use of antibodies shows that TREK-1 is strongly expressed in the membrane of coronal odontoblasts and absent in the root. This distribution is related to the spatial distribution of nerve endings identified by labeling of the low-affinity nerve growth factor (NGF) receptor (p75NTR). These results demonstrate the expression of TREK-1 in human odontoblasts in vitro and in vivo.
Key Words: K+ channels • odontoblast • teeth
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INTRODUCTION
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Odontoblasts constitute a well-defined layer of tall, columnar, and strongly polarized cells along the interface between the dental pulp and the mineralized dentin tubules, where the proximal ends of the cells are running in a liquid phase, the so-called dentinal fluid. They play a central role during the formation of dentin in that they synthesize the organic matrix macromolecules and actively participate in the transportation and accumulation of calcium at the mineralization front (Linde and Lundgren, 1995). In addition to their critical role in dentinogenesis, odontoblasts can possibly mediate the cellular mechanisms underlying sensory processing in teeth, and previous studies (Davidson, 1993, 1994; Lundgren and Linde, 1997; Guo and Davidson, 1998) have suggested that ion channels could play a functional role in these events. Besides plasma membrane voltage-gated Ca2+channels (Seux et al., 1994; Lundgren and Linde, 1997; Davidson and Guo, 2000), Cl- and K+ channels have been found in isolated odontoblasts (Guo and Davidson, 1998). More recently, we have identified high-conductance Ca2+-activated potassium channels (KCa) displaying mechanosensitivity in the odontoblast cell membrane (Allard et al., 2000), suggesting that the fluid displacement within dentinal tubules following tooth stimulation could be transduced into electrical cell signals.
Molecular cloning has recently identified a structural class of mechano-gated K+ channels belonging to the family of potassium subunits with two-pore domains, named K2p channels (Lesage and Lazdunski, 2000). In mammals, the first K2p channel identified was TWIK-1 (Tandem of P domains in Weak Inward rectifier K+ channels; Lesage et al., 1996), and 13 related K2p channels have been cloned. The TWIK-Related K+ channel 1 (TREK-1) (Fink et al., 1996.) is a mammalian mechanosensitive K+ channel which shares most of the properties of the Aplysia neuronal S channel (Patel et al., 1998), a pre-synaptic background K+ channel. Human TREK-1 is found mainly in the brain, ovary, and small intestine. Given its polymodal activation by stretch, lipids, temperature, pH, or anesthetics (Patel et al., 2001), this channel could be involved in odontoblast response to similar stimuli. In this study, we reported the mRNA expression and localization of TREK-1 ion channels in human odontoblasts differentiated in vitro as well as their immunolocalization on cell membranes in vivo.
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MATERIALS & METHODS
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Odontoblast Cell Culture
We recently developed a unique cell culture system allowing for the differentiation of human dental pulp cells into odontoblasts at both the morphological and functional levels (Couble et al., 2000). Briefly, pulp cells obtained from sound human third molar germs (extracted for orthodontic reasons with the informed consent of the patients, in accordance with French legal requirements; article 672-1, Public Health Code). Specimens were grown in Eagle’s basal medium supplemented with fetal calf serum, ascorbic acid, antibiotics with and without 10 mM sodium β-glycerophosphate (βGP) for 2-3 wks. Addition of βGP to the culture medium induced odontoblast (Od) features in the cultured pulp cells (Pc). Cells (Od and Pc) were either used for immunolocalization or harvested for isolation of total RNA.
Total RNA Extraction and RT-PCR Analysis
Total RNA was extracted from the cultured cells (Od and Pc) by means of the RNeasy kit and protocol (Qiagen, Valencia, CA, USA). Purified RNA (200 ng) was reverse-transcribed and amplified in a Titan One Tube RT-PCR system (Roche, Mannheim, Germany). This system allowed for reverse transcription and amplification of TREK-1 and GAPDH, a housekeeping gene. The gene-specific primers for TREK-1 were forward 5'-ATTTGGAAACATCTCACCACGCACA-3' and reverse 5'-GATCCACCTGCAACGTAGTC-3' corresponding to bp positions 430-456 and 766-785. Primers for GAPDH were forward 5'-ACCACAGTCCATGCCATCAC-3' and reverse 5'-TCCACCACCCTGTTGCTGTA-3'. RT-PCR conditions were 35 cycles with an annealing temperature of 55°C. The PCR products (expected fragment sizes: TREK-1, 356 bp; GAPDH, 450 bp) were analyzed on a 2% agarose gel by electrophoresis.
In situ Hybridization
The material consisted of sound non-erupted human third molars or incisors extracted in the same conditions as described above. Immediately after extraction, the pulp tissue was carefully removed from the dentin walls and embedded in Tissue Tek OTC compound (EMS, Washington, PA, USA). The specimens were then immersed in liquid-nitrogen-cooled isopentane and stored frozen at -70°C. Cryostat sections (10 µm) were collected on 3-aminopropyltriethoxysilane-coated slides, air-dried, and kept frozen (-70°C) until treatment. For detection of the TREK-1 transcripts, in situ hybridization was performed by means of a single-stranded DNA probe (Bleicher et al., 1999) with a specific activity of about 2.8 x 106 cpm/pmol. The images were processed in Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA, USA).
Immunohistochemistry
Immediately after extraction, the pulp tissue was kept frozen, cryostat-sectioned, and incubated (dilution 1:500 in PBS-0.2% bovine serum albumin) for 45 min in the  -TREK-1 antibodies prepared and characterized as published previously (Bearzatto et al., 2000; Maingret et al., 2000a). After being washed, the sections were reacted with Cy3 goat anti-rabbit IgG (Interchim, Paris, France), washed again, mounted in glycerol, and observed under the fluorescence microscope. In control procedures, anti-TREK-1 antibodies were either omitted or pre-absorbed with the immunizing fusion protein, the other steps remaining unchanged. Other pulp specimens were rapidly fixed in 4% paraformaldehyde before being removed from the dentin, frozen, and routinely prepared for p75NTR identification (mouse anti-human p75NTR antibodies at dilution 1/40; Roche, Mannheim, Germany). Cultured odontoblasts were rapidly fixed with 4% formaldehyde, 0.05% Triton X-100 in PBS, and treated as above.
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RESULTS
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Analysis of mRNA expression by RT-PCR revealed that TREK-1 was clearly over expressed in cultured odontoblasts in contrast to pulp cells in vitro. The housekeeping gene GAPDH was amplified to control the equal amount of template RNA. The products ran at the expected size of 356 bp for TREK-1 and 450 bp for GAPDH (Fig. 1 ).
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Figure 1. Analysis of the RT-PCR product of TREK-1 channel mRNAs from cultured odontoblasts (O) and pulp cells (Pc) in vitro. Lane 1 contains the molecular-weight markers (standard VIII, Roche Molecular Biochemicals). The TREK-1 and GAPDH products migrate to a position in good agreement with their respectively predicted sizes of 356 bp and 450 bp.
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An TREK-1 immunoreactivity was strongly observed in the cultured odontoblasts (Fig. 2), with a specific staining underlining the plasma membrane, particularly between adjacent cell bodies. Immunoreactivity was not observed in the cell processes. On human dental pulp tissue in vivo (Fig. 3), the highest density of TREK-1 transcripts was detected in coronal odontoblasts and vascular smooth-muscle cells compared with pulp cells or root region, despite a moderate level of tissue labeling. In cryostat sections of extracted pulps (Fig. 4), TREK-1 protein clearly showed a different distribution, as a function of the position of the odontoblasts in the tooth. Thus, numerous positive dots underlined the surface membranes of the odontoblasts localized at the tip of the crown (Fig. 4a). In mid-crown regions, fewer positive immuno-signals were present, especially at the apical pole of the odontoblasts (Fig. 4b). In the root regions, no staining could be identified (Fig. 4c). Some blood vessel walls were also found to be positive (Fig. 4e).
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Figure 3. In situ hybridization (a,b,c) of a dental pulp section with TREK-1 probe. (a) Note the relative overexpression of transcripts in the coronal odontoblast layer (od) in contrast to pulp cells. (b) In the root region, no significant signal could be identified. (c) TREK-1 mRNA is also detected in the arterioles (ar).
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The use of p75NTR as a marker of nerve fibers (Hildebrand et al., 1995) revealed a distinct distribution of the nerve endings (Fig. 4d ) between crown and root, with a dense positive nerve marginal plexus beneath the odontoblast layer of the coronal pulp region and a marked decrease of nerves down to the root area.
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DISCUSSION
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In this study, our PCR experiments demonstrated the overexpression of TREK-1 mRNA in odontoblasts in cultures originating from human dental pulp compared with pulp cells in vitro. Additionally, the presence of the channel protein was confirmed with antibodies whose specificity has been established previously. TREK-1 expression was restricted to the plasma membranes of the cultured cells, similar to that in brain projection or inter-neurones of adult rats (Hervieu et al., 2001). More importantly, using in situ hybridization experiments, we showed that the TREK-1 gene was also overexpressed by cervical odontoblasts in vivo (the relatively weak signal is probably related to low turnover of the protein) compared with underlying pulp cells. Transcripts and protein were also detected in vascular smooth-muscle cells. This latter distribution is not surprising, because TREK-1 channels have been recently shown to be involved in the regulation of smooth-muscle relaxation (Koh et al., 2001). With the use of immunohistochemistry, TREK-1 was expressed by odontoblasts with a strong decreasing gradient from the cusp to the root region. This particular pattern could be related to the nerve fiber distribution, showing that the pulpal horn is more heavily innervated than cervical or root dentin (Byers, 1984). A similar pattern is also described for pulpal vasculature. In this respect, a close correlation could exist between pulpal nerve sprouting and the up-regulation of TREK-1 expression. The physiological role of this channel in coronal odontoblasts remains unknown, but we speculate that it could be stretch-activated when fluid moves in the dentinal tubules following tooth stimulation, thus generating a signal to afferent nerve terminals closely coiled around odontoblasts. Additionally, when expressed in Xenopus oocytes (Maingret et al., 2000b), this channel was shown to be opened reversibly by heat and other potent stimuli eliciting tooth pain and could be assumed to be a temperature sensor. The absence of TREK-1 transcripts and protein expression in the root regions could be related to the age of the tooth specimens not mature enough to have developed sensory axons. Nevertheless, this physiological feature highlights the morphological difference between crown and root odontoblasts previously described. On the other hand, the presence of TREK-1 in odontoblast cell membranes in vitro suggests a crown phenotype of these cultured cells and might be related to physiological roles characterizing the background K+ channels, such as the control of cellular volume and shape (Hamill and Martinac, 2001). This crucial physiological regulation should not be excluded in vivo, insofar as odontoblasts, partly included in the mineralized dentin tissue they elaborate throughout the life of the teeth, are continuously moving to the pulp core. The only use of functional specific channel blockers or openers (not yet identified) in conjunction with patch-clamp techniques will permit the clarification of their roles in wild cell types and particularly in odontoblasts. Indeed, it should be pointed out that TREK-1 electrophysiological properties have been successfully recorded when the gene is overexpressed in COS cells or Xenopus oocytes.
Finally, TREK-1 channels might also be involved in the K+ homeostasis of odontoblasts following dentin injury. This process generates modifications in the dentinal fluid flow, changes in local microcirculation of the pulp tissue, an increase in the pulp pressure, and, consequently, tissue ischemia. TREK-1 channel activation, such as in the brain (Lauritzen et al., 2000; Maingret et al., 2000a), could have an odontoblast-protective effect during this process.
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ACKNOWLEDGMENTS
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We thank Dr. Rochigneux, the Staff of the "Service de Stomatologie de l’hopital St Joseph" Lyon, and Dr. Exbrayat for collecting tooth samples. We are grateful to Lee Pape for grammatical review of the manuscript. This work was supported by the "Ministère de l’Education Nationale" (EA 1892-IFR 62). A preliminary report was presented at the VIIth International Conference on Tooth Morphogenesis and Differentiation (La Londe les Maures, June 16-21, 2001).
Received for publication September 3, 2002.
Revision received March 12, 2003.
Accepted for publication April 8, 2003.
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Journal of Dental Research, Vol. 82, No. 7,
542-545 (2003)
DOI: 10.1177/154405910308200711
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ABSTRACT
INTRODUCTION
