Activation of the Notch signaling pathway in response to pulp capping of rat molars H. Løvschall 1 , M. Tummers 2 , I. Thesleff 2 , E. -M. Füchtbauer 3 , K. Poulsen 4 1 Department of Dental Pathology, Operative Dentistry and Endodontics, Royal Dental College, Faculty of Health Sciences, University of Aarhus, Denmark, 2 Institute of Biotechnology, University of Helsinki, Helsinki, Finland, 3 Department of Molecular Biology, Faculty of Sciences, University of Aarhus, Aarhus, Denmark, 4 Department of Medical Microbiology and Immunology, Faculty of Health Sciences, University of Aarhus, Aarhus, Denmark The development of dental hard tissues is characterized by interactions between the dental epithelium and the mesenchyme. Numerous signal molecules are implicated in these interactions (1). Notch signaling operates through local cell–cell interactions. It is involved in a variety of developmental processes including odonto- genesis (2–5), angiogenesis (6), hematopoiesis (7), neuro- genesis (8), formation of skin appendages (9), anterior and posterior identification (10), and somite formation (11). Recent evidence showed reactivation of Notch signaling during the repair of tissue injury (9, 12–14). Cells communicate through extracellular Notch receptors and contacts to direct the differentiation of stem cell progenitors according to the states of their neighbors (15). The Notch-mediated cell communica- tion depends on the differential expression of ligands and Notch receptors on opposing cells. As develop- ment proceeds, the feedback regulation of local Notch signaling controls the differences between adjacent cells (11), as e.g. in Drosophila, where Notch-mediated lat- eral specification is responsible for the definition of borders between fields of cells (16). In mammals four extracellular receptors in the Notch family (Notch 1–4) have been described. They interact with membrane-bound ligands that are enco- ded by the Delta (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2) gene families. The signal induced by the binding of ligands, Delta or Jagged, initiates a cleavage resulting in the release of an intracellular Notch protein fragment (17). The transcriptional repressor, C-promotor binding factor 1 (CBF1), binds the Notch fragment. The resultant active Notch–CBF1 complex translocates to the nucleus and is expected to transactivate the transcription of target genes, inclu- ding the Hairy and Enhancer of Split (HES) loci, to yield a number of basic helix-loop-helix (bHLH) pro- teins (7). Hes1 is expected to direct tissue morpho- genesis by maintaining cells in an undifferentiated state. Taken together, the results indicate that the Notch-Hes pathway may play an essential role in the differentiation of several cell types (18). Tooth development involves an interaction between the oral epithelium and neural crest-derived cells. Some dental papilla cells commit themselves to the odontoblast lineage (19). Cells aligning along the basement mem- brane differentiate into odontoblasts. This is regulated by both cell–extracellular matrix interactions and soluble signals, including bone morphogenetic proteins (BMPs) and transforming growth factor-b (TGF-b). Similarities exist between the development and repair of dentin in mature teeth; however, the oral epithelial appendage is absent in the pulp. Studies on Notch signaling during the tissue regener- ation of injuries have been performed in different organs, including human skin (9) and rat carotid arteries (12). The Notch pathway is also involved in multiple aspects Løvschall H, Tummers M, Thesleff I, Fu ¨chtbauer E-M, Poulsen K. Activation of the Notch signaling pathway in response to pulp capping of rat molars. Eur J Oral Sci 2005; 113: 312–317. Ó Eur J Oral Sci, 2005 Notch signaling is an evolutionarily conserved pathway that controls the develop- mental choices made by individual cells. Cells communicate via Notch receptors and their ligands, which direct decisions on the fate of stem cells according to the states of their neighbors. In this study we explored Notch signaling after the pulp capping of adult first upper rat molars. The wound was capped with calcium hydroxide. In situ hybridization revealed an increased expression of Notch signaling genes on day 1, which showed a tendency to decrease on day 3. Notch1 increased in the subodonto- blast zone and close to the lesion limited to a few cells. Notch2 increased in pulp stroma surrounded by coronal odontoblasts. Notch1 and, especially, Notch3 expres- sion increased, corresponding to perivascular cell groups. A low increase of ligand expression was observed near the injury with Delta1 expression along the dentin wall and Jagged1 in the stroma. Expression of the downstream target, Hes1, was observed along the lesion and adjacent dentin walls. Hes5 expression was not observed. The results indicate that Notch signaling is activated in response to injury and associated with the differentiation of pulp cells into perivascular cells and odontoblasts. The findings are consistent with the concept that the Notch pathway controls stem cell fate during pulp regeneration. Henrik Løvschall, Department of Dental Pathology, Operative Dentistry and Endodontics, Royal Dental College, University of Aarhus, Vennelyst Boulevard 9, DK-8000 Aarhus C, Denmark Telefax: +45–86202202 E-mail: loev@odont.au.dk Key words: in situ hybridization; odontoblasts; pulp injury; stem cells; wound healing Accepted for publication April 2005 Eur J Oral Sci 2005; 113: 312–317 Printed in Singapore. All rights reserved Copyright Ó Eur J Oral Sci 2005 European Journal of Oral Sciences