The MAP Kinase Pathway Is Involved in Odontoblast Stimulation via p38 Phosphorylation Stephane Simon, DDS, MPhil,* †‡§jj Anthony J. Smith, jj Ariane Berdal, PhD,* †‡§ Philip J. Lumley, PhD, jj and Paul R. Cooper, PhD jj Abstract Introduction: We have previously shown that the p38 gene is highly expressed in odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein acti- vated by phosphorylation, when odontoblasts are stimu- lated such as during tertiary reactionary dentinogenesis. Methods: We stimulated immortalized, odontoblast- like MDPC-23 cells, alone or in combination, with heat- inactivated Streptococcus mutans, EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-b1, tumor necrosis factor-a (TNF-a), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear trans- location. Results: Our results suggest that the p38-MAP- Kinase pathway is activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phos- phorylation and enhanced nuclear translocation. Conclu- sions: Data indicate that odontoblast behaviour therefore potentially recapitulates that during active primary denti- nogenesis. (J Endod 2010;36:256–259) Key Words Dental pulp, dentinogenesis, MAP kinase, odontoblast D entinogenesis is the process of dentine secretion that continues throughout the life of a tooth. Primary and secondary dentinogenesis, occurring before and after eruption, respectively, are physiologic processes, whereas tertiary dentinogenesis, which can be either reactionary or reparative, occurs in response to injury (1). Primary and secondary dentins are histologically similar and are deposited at 4 and 0.4 mm/d, respectively. Reactionary dentinogenesis is the secretion of a tertiary dentine matrix by surviving odontoblasts in response to an appropriate stimulus (2). The dentin matrix is perme- able by virtue of its tubular structure, and, therefore, after injury to the tooth and/or subsequent restorative procedures, this may allow molecules to diffuse and contact the pulp. Such substances may include bacteria, toxins, and/or dentin matrix proteins (DMPs). Because the pulp is enclosed by a rigid, mineralized tissue shell, dentin matrix degradation by acid bacterial products begins before the disease process reaches the pulp. Notably, growth factors derived from the dentin have been shown to reach and stimulate the odontoblast layer, inducing new dentine secretion in those areas of the dentin-pulp complex that are in direct tubular connection with the traumatic agent (3). It has been suggested that, during tooth repair and tertiary reactionary dentinogen- esis, the healing process recapitulates developmental events (4). Transforming growth factor-b1 (TGF-b1) has been shown to play an important role in tooth development (5, 6), particularly in odontoblast differentiation (7, 8). Transforming Growth Factor b1 (TGF-b1) TGF-b1 is also sequestrated within the dentin matrix (9) and may be released during carious disease or by restorative agents commonly used in dentistry that dissolve mineralized components, such as EDTA, calcium hydroxide (10), or mineral trioxide aggregate (11). Notably, TGF-b1 has also been shown in vivo and in vitro to stimulate odontoblast behavior (12–14). It would be clinically valuable to be able to activate quiescent odontoblasts for dentine repair, and, thus, it is important to understand the regulatory control of cellular and molecular signaling in these cells. However, only limited information currently exists on these molecular events that occur during reactionary dentinogenesis. In a previous study, we provided evidence that changes in the secretory activity of odon- toblasts reflect differential transcriptional control and that, therefore, the transcriptome of the odontoblast evolves as the cell matures (15, 16). Among the dataset of genes whose expression changed during odontoblast maturation, we identified the p38 tran- script to be abundantly expressed in odontoblasts during primary dentinogenesis while being significantly down-regulated in secondary dentinogenesis. Based on these obser- vations, we hypothesized that p38 signaling might be involved in regulating odontoblast activity, and the present study aimed to determine whether the p38 protein is up-regu- lated and phosphorylated upon odontoblast stimulation. Materials and Methods Isolation of Dentin Matrix Proteins Dentin matrix proteins were isolated as previously described (10). Briefly, dentin matrix components were extracted from powdered dentin using 10% EDTA (pH = 7.2) supplemented with the protease inhibitors n-ethylmaleimide (10 mmol/L) (Sigma, Dorset, UK) and phenyl-methyl-sulphonyl fluoride (5 mmol/L) (Sigma). Extractions were performed with agitation over 14 days at 4  C and with the extraction solution changed daily. Extracts were transferred to dialysis tubing (Scientific Laboratory Supplies, Nottingham, UK) and dialyzed exhaustively for 10 days against repeated From the *INSERM, Centre de Recherche des Cordeliers, Paris, France; † Universite ´ Pierre et Marie Curie-Paris 6, Paris, France; ‡ Universite ´ Paris Descartes, Paris, France; § Team 5-Molecular Oral Physiopathology, Universite ´ Paris Diderot, Paris, France; and jj Oral Biology, School of Dentistry, University of Birmingham, Birmingham, United Kingdom. Address requests for reprints to Dr Ste ´ phane Simon, Labo- ratoire de Physiopathologie Orale Mole ´ culaire, INSERM, UMR S 872, Escalier B, 15-21 rue de l’Ecole de Me ´ decine, 75006 Paris, France. E-mail address: srs635@bham.ac.uk. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2009.09.019 Basic Research—Biology 256 Simon et al. JOE — Volume 36, Number 2, February 2010