A Micro-evolutionary Change in Target Binding Sites as a Key Determinant of Ultrabithorax Function in Drosophila.

Hox genes encode Homeodomain-containing transcription factors, which specify segmental identities along the anterior-posterior axis. Functional changes in Hox genes have been directly implicated in the evolution of body plans across the metazoan lineage. The Hox protein Ultrabithorax (Ubx) is expressed and required in developing third thoracic (T3) segments in holometabolous insects studied so far, particularly, of the order Coleoptera, Lepidoptera and Diptera. Ubx function is key to specify differential development of the second (T2) and T3 thoracic segments in these insects. While Ubx is expressed in the third thoracic segment in developing larvae of Hymenopteran Apis mellifera, the morphological differences between T2 and T3 are subtle. To identify evolutionary changes that are behind the differential function of Ubx in Drosophila and Apis, which are diverged for more than 350 million years, we performed comparative analyses of genome wide Ubx-binding sites between these two insects. Our studies reveal that a motif with a TAAAT core is a preferred binding site for Ubx in Drosophila, but not in Apis. Biochemical and transgenic assays suggest that in Drosophila, the TAAAT core sequence in the Ubx binding sites is required for Ubx-mediated regulation of two of its target genes studied here; CG13222, a gene that is normally upregulated by Ubx and vestigial (vg), whose expression is repressed by Ubx in T3. Interestingly, changing the TAAT site to a TAAAT site was sufficient to bring an otherwise unresponsive enhancer of the vg gene from Apis under the control of Ubx in a Drosophila transgenic assay. Taken together, our results suggest an evolutionary mechanism by which critical wing patterning genes might have come under the regulation of Ubx in the Dipteran lineage.

A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes.

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein-protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context.

Bimolecular Fluorescence Complementation (BiFC) and Multiplexed Imaging of Protein-Protein Interactions in Human Living Cells.

Deciphering protein-protein interactions (PPIs) in vivo is crucial to understand protein function. Bimolecular fluorescence complementation (BiFC) makes applicable the analysis of PPIs in many different native contexts, including human live cells. It relies on the property of monomeric fluorescent proteins to be reconstituted from two separate subfragments upon spatial proximity. Candidate partners fused to such complementary subfragments can form a fluorescent protein complex upon interaction, allowing visualization of weak and transient PPIs. It can also be applied for investigation of distinct PPIs at the same time using a multicolor setup. In this chapter, we provide a detailed protocol for analyzing PPIs by doing BiFC in cultured cells. Proof-of-principle experiments rely on the complementation property between the N-terminal fragment of mVenus (designated VN173) and the C-terminal fragment of mCerulean (designated CC155) and the partnership between HOXA7 and PBX1 proteins. This protocol is compatible with any other fluorescent complementation pair fragments and any type of candidate interacting proteins.

LACC1 deficiency links juvenile arthritis with autophagy and metabolism in macrophages.

Juvenile idiopathic arthritis is the most common chronic rheumatic disease in children, and its etiology remains poorly understood. Here, we explored four families with early-onset arthritis carrying homozygous loss-of-expression mutations in LACC1. To understand the link between LACC1 and inflammation, we performed a functional study of LACC1 in human immune cells. We showed that LACC1 was primarily expressed in macrophages upon mTOR signaling. We found that LACC1 deficiency had no obvious impact on inflammasome activation, type I interferon response, or NF-κB regulation. Using bimolecular fluorescence complementation and biochemical assays, we showed that autophagy-inducing proteins, RACK1 and AMPK, interacted with LACC1. Autophagy blockade in macrophages was associated with LACC1 cleavage and degradation. Moreover, LACC1 deficiency reduced autophagy flux in primary macrophages. This was associated with a defect in the accumulation of lipid droplets and mitochondrial respiration, suggesting that LACC1-dependent autophagy fuels macrophage bioenergetics metabolism. Altogether, LACC1 deficiency defines a novel form of genetically inherited juvenile arthritis associated with impaired autophagy in macrophages.

The human HOXA9 protein uses paralog-specific residues of the homeodomain to interact with TALE-class cofactors.

HOX proteins interact with PBX and MEIS cofactors, which belong to the TALE-class of homeodomain (HD)-containing transcription factors. Although the formation of HOX-PBX complexes depends on a unique conserved HOX motif called hexapeptide (HX), the additional presence of MEIS induces a remodeling of the interaction, leading to a global dispensability of the HX motif for trimeric complex formation in the large majority of HOX proteins. In addition, it was shown that the anterior HOXB3 and central HOXA7 and HOXC8 proteins could use different alternative TALE interaction motifs, with or without the HX motif, depending on the DNA-binding site and cell context. Here we dissected the molecular interaction properties of the human posterior HOXA9 protein with its TALE cofactors, PBX1 and MEIS1. Analysis was performed on different DNA-binding sites in vitro and by doing Bimolecular Fluorescence Complementation (BiFC) in different cell lines. Notably, we observed that the HOXA9-TALE interaction relies consistently on the redundant activity of the HX motif and two paralog-specific residues of the HOXA9 HD. Together with previous work, our results show that HOX proteins interact with their generic TALE cofactors through various modalities, ranging from unique and context-independent to versatile and context-dependent TALE binding interfaces.

Human HOX Proteins Use Diverse and Context-Dependent Motifs to Interact with TALE Class Cofactors.

HOX proteins achieve numerous functions by interacting with the TALE class PBX and MEIS cofactors. In contrast to this established partnership in development and disease, how HOX proteins could interact with PBX and MEIS remains unclear. Here, we present a systematic analysis of HOX/PBX/MEIS interaction properties, scanning all paralog groups with human and mouse HOX proteins in vitro and in live cells. We demonstrate that a previously characterized HOX protein motif known to be critical for HOX-PBX interactions becomes dispensable in the presence of MEIS in all except the two most anterior paralog groups. We further identify paralog-specific TALE-binding sites that are used in a highly context-dependent manner. One of these binding sites is involved in the proliferative activity of HOXA7 in breast cancer cells. Together these findings reveal an extraordinary level of interaction flexibility between HOX proteins and their major class of developmental cofactors.

A systematic survey of HOX and TALE expression profiling in human cancers.

HOX and TALE genes encode homeodomain (HD)-containing transcription factors that act in concert in different tissues to coordinate cell fates and morphogenesis throughout embryonic development. These two evolutionary conserved families contain several members that form different types of protein complexes on DNA. Mutations affecting the expression of HOX or TALE genes have been reported in a number of cancers, but whether and how the two gene families could be perturbed together has never been explored systematically. As a consequence, the putative collaborative role between HOX and TALE members for promoting or inhibiting oncogenesis remains to be established in most cancer contexts. Here, we address this issue by considering HOX and TALE expression profiling in normal and cancer adult tissues, using normalized RNA-sequencing expression data deriving from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) research projects. Information was extracted from 28 cancer types originating from 21 different tissues, constituting a unique comparative analysis of HOX and TALE expression profiles between normal and cancer contexts in human. We present the general and specific rules that could be deduced from this large-scale comparative analysis. Overall this work provides a precious annotated support to better understand the role of specific HOX/TALE combinatorial codes in human cancers.

Exostosin 1 is expressed in human odontoblasts.

OBJECTIVE: Dental pulp is soft connective tissue maintaining the vitality of the tooth, while odontoblasts form the dentin. Our earlier DNA microarray analysis revealed expression of putative tumour suppressor exostosin 1 (EXT-1) in odontoblasts. EXT-1 is essential for heparan sulphate synthesis, which may play a role in the dentin mineralization. Since the absence of the functional EXT-1 causes bone tumours, expression in odontoblasts is interesting. Our aim was to analyse further the EXT-1 expression in human tooth. DESIGNS: DNA microarray and PCR techniques were used to study the EXT-1 expression in mature native human odontoblasts and pulp tissue as well as in newly-differentiated cultured odontoblast-like cells. Immunohistochemistry was performed to study EXT-1 protein in mature human teeth, teeth with incomplete root and developing teeth. RESULTS: Markedly higher EXT-1 was observed in mature odontoblasts than in pulp at mRNA level with DNA microarray and PCR techniques. Immunohistochemistry of mature tooth revealed EXT-1 both in odontoblasts and the predentin but not in the dentin. EXT-1 was also observed in the odontoblasts of incomplete root, but the localization of the staining was different. In developing foetal tooth, staining was detected in ameloblasts and the basal lamina. CONCLUSIONS: The detection of EXT-1 in both mature and newly-differentiated cells indicates a role in the odontoblast function, and EXT-1 staining in the predentin indicates a function in the dentin formation. Detection of EXT-1 in developing teeth indicates a role in tooth development.

Human odontoblast-like cells produce nitric oxide with antibacterial activity upon TLR2 activation.

The penetration of cariogenic oral bacteria into enamel and dentin during the caries process triggers an immune/inflammatory response in the underlying pulp tissue, the reduction of which is considered a prerequisite to dentinogenesis-based pulp regeneration. If the role of odontoblasts in dentin formation is well known, their involvement in the antibacterial response of the dental pulp to cariogenic microorganisms has yet to be elucidated. Our aim here was to determine if odontoblasts produce nitric oxide (NO) with antibacterial activity upon activation of Toll-like receptor-2 (TLR2), a cell membrane receptor involved in the recognition of cariogenic Gram-positive bacteria. Human odontoblast-like cells differentiated from dental pulp explants were stimulated with the TLR2 synthetic agonist Pam2CSK4. We found that NOS1, NOS2, and NOS3 gene expression was increased in Pam2CSK4-stimulated odontoblast-like cells compared to unstimulated ones. NOS2 was the most up-regulated gene. NOS1 and NOS3 proteins were not detected in Pam2CSK4-stimulated or control cultures. NOS2 protein synthesis, NOS activity and NO extracellular release were all augmented in stimulated samples. Pam2CSK4-stimulated cell supernatants reduced Streptococcus mutans growth, an effect counteracted by the NOS inhibitor L-NAME. In vivo, the NOS2 gene was up-regulated in the inflamed pulp of carious teeth compared with healthy ones. NOS2 protein was immunolocalized in odontoblasts situated beneath the caries lesion but not in pulp cells from healthy teeth. These results suggest that odontoblasts may participate to the antimicrobial pulp response to dentin-invading Gram-positive bacteria through NOS2-mediated NO production. They might in this manner pave the way for accurate dental pulp healing and regeneration.

Odontoblast physiology.

Odontoblasts are post-mitotic cells organized as a layer of palisade cells along the interface between the dental pulp and dentin. They are responsible for the formation of the physiological primary and secondary dentins. They synthesize the organic matrix of type I collagen and actively participate to its mineralization by secreting proteoglycans and non-collagenous proteins that are implicated in the nucleation and the control of the growth of the mineral phase. They also participate to the maintenance of this hard tissue throughout the life of the tooth by synthesizing reactionary dentin in response to pathological conditions (caries, attrition, erosion…). Besides these fundamental dentinogenic activities, odontoblasts were recently suspected to play a role as sensor cells. They are able to sense the bacteria invasion during caries and then to initiate the pulp immune and inflammatory response. They are also well equipped in ion channels implicated in mechanotransduction or nociception which make odontoblasts suitable candidates to sense external stimuli and to mediate tooth pain sensation.

Lipopolysaccharide-binding protein inhibits toll-like receptor 2 activation by lipoteichoic acid in human odontoblast-like cells.

INTRODUCTION: Previous studies have suggested that odontoblasts sense gram-positive bacteria components through Toll-like receptor 2 (TLR2) and trigger dental pulp immunity by producing proinflammatory cytokines. Currently, the factors that modulate odontoblast TLR2 activation are unknown. Our aim was to investigate lipopolysaccharide-binding protein (LBP) effects on the TLR2-mediated odontoblast response. METHODS: Human odontoblast-like cells were stimulated with lipoteichoic acid (LTA) (a TLR2 ligand), LBP, CD14 (a TLR2 cofactor), or various combinations of LTA/LBP, LTA/CD14, or LTA/CD14/LBP. CXCL8, IL6, and TLR2 gene expression was assessed by real-time polymerase chain reaction. CXCL8 and interleukin (IL)-6 production was determined by enzyme-linked immunosorbent assay in culture supernatants of cells stimulated with LTA, LTA/CD14, or LTA/CD14/LBP. LBP effects on nuclear factor kappa B (NF-κB), p38, JNK, ERK, STAT3, and p70S6 signaling pathways were determined in LTA-stimulated odontoblast-like cells with a multiplex biometric immunoassay. LBP effects were compared with specific inhibitors of these signaling pathways. LBP transcript and protein were investigated in vivo in healthy and inflamed dental pulps by real-time polymerase chain reaction and immunohistochemistry. RESULTS: Activation of CXCL8, IL6, and TLR2 gene expression and CXCL8 and IL-6 secretion in LTA- and LTA/CD14-stimulated odontoblast-like cells was significantly decreased by LBP. LBP inhibited NF-κB and p38 signaling pathways in LTA-stimulated cells in a similar way to NF-κB and p38 inhibitors. LBP transcript and protein were detected in vivo in inflamed dental pulps but not in healthy ones. CONCLUSIONS: These results demonstrate that LBP reduces TLR2-dependent production of inflammatory cytokines by odontoblast-like cells. We suggest that in this way it could modulate host defense in human dental pulp.

The involvement of SMILE/TMTC3 in endoplasmic reticulum stress response.

BACKGROUND: The state of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: We first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER). In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes. CONCLUSION/SIGNIFICANCE: In this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance.

Expression of NOD2 is increased in inflamed human dental pulps and lipoteichoic acid-stimulated odontoblast-like cells.

Human odontoblasts trigger immune response s to oral bacteria that invade dental tissues during the caries process. To date, their ability to regulate the expression of the nucleotide-binding domain leucine-rich repeat containing receptor NOD2 when challenged by Gram-positive bacteria is unknown. In this study, we investigated NOD2 expression in healthy and inflamed human dental pulps challenged by bacteria, and in cultured odontoblast-like cells stimulated with lipoteichoic acid (LTA), a Toll-like receptor (TLR) 2 agonist which is specific for Gram-positive bacteria. We found that NOD2 gene expression was significantly up-regulated in pulps with acute inflammation compared to healthy ones. In vitro, LTA augmented NOD2 gene expression and protein level in odontoblast-like cells. The increase was more pronounced in odontoblast-like cells compared to dental pulp fibroblasts. Blocking experiments in odontoblast-like cells with anti-TLR2 antibody strongly reduced the NOD2 gene expression increase, whereas stimulation with the synthetic TLR2 ligand Pam(2)CSK(4) confirmed NOD2 gene up-regulation following TLR2 engagement. These data suggest that NOD2 up-regulation is part of the odontoblast immune response to Gram-positive bacteria and might be important in protecting human dental pulp from the deleterious effects of cariogenic pathogens.

Cytokine production by human odontoblast-like cells upon Toll-like receptor-2 engagement.

Recent studies have suggested that odontoblasts are involved in the dental pulp immune response to oral pathogens that invade human dentin during the caries process. How odontoblasts regulate the early inflammatory and immune pulp response to Gram-positive bacteria, which predominate in shallow and moderate dentin caries, is still poorly understood. In this study, we investigated the production of pro- and anti-inflammatory cytokines by odontoblast-like cells upon engagement of Toll-like receptor (TLR) 2, a pattern recognition molecule activated by Gram-positive bacteria components. We used a highly sensitive Milliplex(®) kit for detecting cytokines released by cells stimulated with lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, or with the potent TLR2 synthetic agonist Pam2CSK4. We found that odontoblasts produce the pro-inflammatory cytokines interleukin (IL)-6 and CXCL8, as well as the immunosuppressive cytokine IL-10 in response to TLR2 agonists. GM-CSF, IFNγ, IL-1ß, IL-2, IL-4, IL-5, IL-7, IL-12(p70), IL-13 and TNF-α were not detected. These data indicate that TLR2 activation in human odontoblasts selectively induces production of mediators known to influence positively or negatively inflammatory and immune responses in pathogen-challenged tissues. We suggest that these molecules might be important in regulating the fine tuning of the pulp response to Gram-positive bacteria which enter dentin during the caries process.

Topical review. Dental pain and odontoblasts: facts and hypotheses.

Dental pain arises from exposed dentin following bacterial, chemical, or mechanical erosion of enamel and/or recession of gingiva. Thus, dentin tissue and more specifically patent dentinal tubules represent the first structure involved in dentin sensitivity. Interestingly, the architecture of dentin could allow for the transfer of information to the underlying dental pulp via odontoblasts (dentin-forming cells), via their apical extension bathed in the dentinal fluid running in the tubules, or via a dense network of trigeminal sensory axons intimately related to odontoblasts. Therefore, external stimuli causing dentinal fluid movements and odontoblasts and/or nerve complex responses may represent a unique mechanosensory system bringing a new role for odontoblasts as sensor cells. How cells sense signals and how the latter are transmitted to axons represent the main questions to be resolved. However, several lines of evidence have demonstrated that odontoblasts express mechano- and/or thermosensitive transient receptor potential ion channels (TRPV1, TRPV2, TRPV3, TRPV4, TRPM3, KCa, TREK-1) that are likely to sense heat and/or cold or movements of dentinal fluid within tubules. Added to this, voltage-gated sodium channels confer excitable properties of odontoblasts in vitro in response to injection of depolarizing currents. In vivo, sodium channels co-localize with nerve terminals at the apical pole of odontoblasts and correlate with the spatial distribution of stretch-activated KCa channels. This highlights the terminal web as the pivotal zone of the pulp/dentin complex for sensing external stimuli. Crosstalk between odontoblasts and axons may take place by the release of mediators in the gap space between odontoblasts and axons in view of evidence for nociception-transducing receptors on trigeminal afferent fibers and expression of putative effectors by odontoblasts. Finally, how axons are guided to the target cells and which kind of signaling molecules are involved is extensively discussed in this review.

Toll-like receptor 2 activation by lipoteichoic acid induces differential production of pro-inflammatory cytokines in human odontoblasts, dental pulp fibroblasts and immature dendritic cells.

Odontoblasts, dental pulp fibroblasts and immature dendritic cells (DCs) have been involved in the human dental pulp immune response to oral pathogens that invade dentine during the caries process. How they regulate the inflammatory response to Gram-positive bacteria remains nevertheless largely unknown. In this study we investigated the production of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (CXCL8) in these three cell types upon stimulation with lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria that activates the pattern recognition molecule Toll-like receptor 2 (TLR2). We observed that TNF-alpha gene expression was up-regulated in all LTA-stimulated cell types. IL-1beta gene expression was not or barely detectable in odontoblast-like cells and pulp fibroblasts when stimulated or not, but was expressed in immature DCs and increased upon stimulation. TNF-alpha and IL-1beta proteins were detected in DC culture supernatants but not in odontoblast-like cell and pulp fibroblast ones. CXCL8 gene and protein were clearly expressed and increased in the three cell types upon LTA stimulation. These data indicate that LTA-dependent TLR2 activation in odontoblasts and pulp fibroblasts, in contrast to immature DCs, does not lead to significant TNF-alpha and IL-1beta production, but that all three cell types influence the pulp inflammatory/immune response through CXCL8 synthesis and secretion.

Microtubule-associated protein 1b, a neuronal marker involved in odontoblast differentiation.

INTRODUCTION: Map-1B belongs to the family of proteins that govern the dynamic state and organization of microtubules within cells. MAP-1B is a microtubule-associated protein highly expressed during the development of the nervous system. Its expression, regulated by the fragile X mental retardation protein (FMRP), is essential to stabilize microtubules during the elongation of dendrites and neurites. Other microtubules-associated molecules such as tau or MAP2 seem to act similarly. The aim of this work was to identify the MAP-1B expression in in vitro and in vivo human odontoblasts during development and carious processes. The expression of MAP2 and tau was also studied. MATERIALS AND METHODS: In cultured cells, MAP-1B expression was analyzed by real-time polymerase chain reaction, flow cytometry, and Western blot. Its distribution was visualized by in situ hybridization and immunochemistry both in vitro and in vivo. The expression of FMRP, MAP2, and tau was identified by real-time polymerase chain reaction and immunochemistry. RESULTS: MAP-1B is specifically expressed in odontoblasts from adult third molars as well as incisor germs from human embryos. In adult carious teeth, it is also expressed in newly differentiated dentin-forming cells. In vitro, MAP-1B expression is related to the differentiation state of odontoblasts. MAP-1B clearly underlines the cellular architecture of cell bodies and processes of differentiated cells. FMRP, MAP2, and tau are also detected in vivo. CONCLUSION: On the basis of these data, MAP-1B could be considered as a new protein involved in the terminal differentiation of odontoblasts.

Spatial and temporal expression of KLF4 and KLF5 during murine tooth development.

OBJECTIVE: KLF4 and KLF5, members of the Krüppel-like factor (KLF) family, play key roles in proliferation, differentiation and apoptosis during development. In order to determine if these transcription factors are associated with tooth development, we examined the expression pattern of KLF4 and KLF5 during murine tooth development. DESIGN: In situ hybridization and immunohistochemistry were performed to detect the expression pattern of KLF4 and KLF5 from E12.5 to PN3 during murine tooth development. RESULTS: In situ hybridization analysis revealed that Klf4 was specifically expressed in polarizing odontoblasts from E16.5 (incisor) or E18.5 (first molar) to PN3. Immunohistochemistry staining showed that KLF4 was specifically expressed in both polarizing odontoblasts and ameloblasts at the same stages. KLF5 was mainly expressed from E18.5 to PN3 in secretory ameloblasts when enamel mineralization occurs and in secretory odontoblasts. However, an expression of KLF5 was also observed at earlier stages (E14.5 and E16.5) mainly in proliferating epithelial cells. CONCLUSIONS: These results suggest that the expression of KLF4 is closely correlated to the growth-arrest and the first step of odontoblast and ameloblast differentiation. Furthermore, KLF5 maybe involved in proliferation at the early stages of tooth development and related to mineralization of both enamel and dentin matrices at later stages.

Odontoblast: a mechano-sensory cell.

Odontoblasts are organized as a single layer of specialized cells responsible for dentine formation and presumably for playing a role in tooth pain transmission. Each cell has an extension running into a dentinal tubule and bathing in the dentinal fluid. A dense network of sensory unmyelinated nerve fibers surrounds the cell bodies and processes. Thus, dentinal tubules subjected to external stimuli causing dentinal fluid movements and odontoblasts/nerve complex response may represent a unique mechano-sensory system giving to dentine-forming cells a pivotal role in signal transduction. Mediators of mechano-transduction identified in odontoblast include mechano-sensitive ion channels (high conductance calcium-activated potassium channel--K(Ca)--and a 2P domain potassium channel--TREK-1) and primary cilium. In many tissues, the latter is essential for microenvironment sensing but its role in the control of odontoblast behavior remains to be elucidated. Recent evidence for excitable properties and the concentration of key channels to the terminal web suggest that odontoblasts may operate as sensor cells.