Function of MAPK and downstream transcription factors in monomer-induced apoptosis Stephanie Krifka a , Karl-Anton Hiller a , Carola Bolay a , Christine Petzel a , Gianrico Spagnuolo b , Franz-Xaver Reichl c , Gottfried Schmalz a , Helmut Schweikl a, * a Department of Operative Dentistry and Periodontology, University Hospital Regensburg, University of Regensburg, D-93042 Regensburg, Germany b Department of Oral and Maxillofacial Sciences, University of Naples “Federico II”, Via S. Pansini 5, Napoli, Italy c Walther-Straub-Institute of Pharmacology and Toxicology and Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig-Maximilians-University of Munich, 80336 Munich, Germany article info Article history: Received 27 September 2011 Accepted 10 October 2011 Available online 26 October 2011 Keywords: TEGDMA Apoptosis MAPK inhibitors Transcription factor abstract The resin monomer triethylene glycol dimethacrylate (TEGDMA) disrupts vital cell functions, and the production of oxidative stress is considered a common underlying mechanism. The precise signaling pathways, however, that initiate monomer-induced effects, which disturb responses of the innate immune system, inhibit dentin mineralization processes, or induce apoptosis in target cells in vitro are still unknown. The present study provides insight into the causal relationship between TEGDMA-induced apoptosis and the activation of MAPK and transcription factors downstream using pharmacological inhibitors of the ERK1/2, p38 and JNK pathways. The endotoxin lipopolysaccharide (LPS; 0.1 mg/ml) was included as an inducer of MAPK activity in RAW264.7 mouse macrophages. Cell viability was decreased from 95% in untreated cultures to about 43% after a 24 h exposure to 3 mM TEGDMA. Inhibition of the ERK1/2 pathway by the MEK1/2 inhibitor PD98059 reduced cell viability to 84%. While apoptosis induced by TEGDMA remained unchanged, Western blot analyses revealed that the activation of ERK1/2 in the presence of TEGDMA was inhibited by PD98059. LPS-induced expression of activated transcription factors c-Jun, ATF-2, ATF-3 and phospho-Elk1 was decreased in cells co-treated with TEGDMA. This inhibition was more intense in the presence of PD98059, indicating that the MEK/ERK pathway is involved in the inhibition of the LPS-induced activation of transcription factors by TEGDMA. No clear effects of the p38 inhibitor SB203580 and the JNK inhibitor SP600125 on TEGDMA-induced apoptosis were detected. The antioxidant N-acetylcysteine (NAC) protected cells from TEGDMA-induced cell death, and inhibited the activation of ERK1/2, p38 and JNK by TEGDMA. Moreover, the TEGDMA-induced downregulation of the expression of the transcription factors c-Jun and ATF-2 was prevented as well. In conclusion, physiologically relevant concentrations of inhibitors differentially modified the expression of MAPK and transcription factors in cell cultures exposed to LPS and the monomer TEGDMA. The absence of a drastic effect of the MAPK pathway inhibitors on TEGDMA-induced apoptosis on the one hand, and the protective effect of NAC and PD98059 in particular on TEGDMA-induced MAPK activation and apoptosis on the other hand, leads to a new model for the role of MAPK in the regulation of cell homeostasis in monomer-exposed cells and tissues. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Dental restorative materials are a lasting source of bioactive compounds in various tissues of the oral cavity in short and long- term scenarios. Unreacted resin monomers are environmental stressors released from the organic matrix of dental composites after incomplete polymerization. Uncured hydrophilic monomers like 2-hydroxyethyl methacrylate (HEMA) or triethylene glycol dimethacrylate (TEGDMA) were identified along with a wide variety of monomers and additives in an aqueous environment [1,2]. Moreover, clinically polymerized composites are predisposed to biodegradation through enzyme activities in saliva [3]. The monomers released the most, HEMA followed by TEGDMA, cause specific stress responses in various eukaryotic cells in vitro [4]. It appears that the production of reactive oxygen species (ROS) is a first major response of cells to exposure to resin monomers. Most * Corresponding author. Fax: þ49 941 944 6025. E-mail address: helmut.schweikl@klinik.uni-regensburg.de (H. Schweikl). Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.10.026 Biomaterials 33 (2012) 740e750