From the Cover: Activation of NF-κB-Autophagy Axis by 2-Hydroxyethyl Methacrylate Commits Dental Mesenchymal Cells to Apoptosis

TOXICOLOGICAL SCIENCES, 157(1), 2017, 100–111 doi: 10.1093/toxsci/kfx023 Advance Access Publication Date: February 10, 2017 Research article Activation of NF-jB-Autophagy Axis by 2-Hydroxyethyl Methacrylate Commits Dental Mesenchymal Cells to Apoptosis Jing-jing Yu,1 Ling-xin Zhu,1 Jie Zhang, Shan Liu, Feng-yuan Lv, Xue Cheng, Guo-jing Liu, and Bin Peng2 State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, Wuhan University, Wuhan, China 1 These authors contributed equally to this study. 2 To whom correspondence should be addressed. Fax: (86) 27-87873260. E-mail: . ABSTRACT 2-hydroxyethyl methacrylate (HEMA) is the major resin monomer that is released from incomplete polymerized dental restorative and adhesive biomaterials during dental therapy. Autophagy and apoptosis are biologically connected and the relationship between autophagy and apoptosis is complex under various circumstances. This study aimed to determine whether autophagy is activated by HEMA and further explore the function of autophagy during the HEMA-induced apoptosis of dental mesenchymal cells (DMCs). We exposed DMCs to different concentrations of HEMA. Cell viability showed a time- and concentration-dependent decrease when exposed to HEMA. We showed that HEMA exposure increased autophagic vacuoles and the expression of autophagic biomarkers (Beclin1, Atg5 and LC3). Pre-incubated with autophagy inhibitors (3-methyladenine and chloroquine) significantly prevented HEMA-induced apoptosis. Interestingly, HEMA initiated nuclear factor-jB (NF-jB) expression and nuclear translocation, whereas the NF-jB inhibitor (Bay 11-7082) markedly suppressed HEMA-induced autophagic activation and apoptosis. As is consistent with the in vitro results, HEMA treatment resulted in dental pulp tissue toxicity and activation of typical autophagic vacuoles in the tooth slice organ culture model ex vivo. In summary, we demonstrated that NF-jB signaling functioned upstream of HEMA-inducecd autophagy in DMCs and that the activation of NF-jB-autophagy axis was responsible for HEMA-induced apoptosis. Our findings provide novel insights into the mechanisms of resin monomer-mediated dental pulp damage during dental treatment, highlighting the activation of NF-jB-autophagy axis as an important mechanism of HEMA-mediated apoptosis. Key words: 2-Hydroxyethyl methacrylate; autophagy; NF-jB; apoptosis; dental mesenchymal cells; tooth slice organ. Human dental pulp is a highly vascular tissue that is enriched with natural stem/progenitor cells (human dental mesenchymal cells, DMCs), which directly contribute to innate healing. DMCs are essential in tissue regeneration and in cell differentiation; thus, any clinical procedure that can disturb DMCs activity may cause severe alteration to pulp tissue homeostasis. Resin materials are widely used in dental restorative and adhesive biomaterials. However, along the resin biomaterial–DMC interface, monomers from incomplete polymerized resin materials can be released to the oral environment and dental pulp cavity and further disturb cell viability and differentiation (Chang et al., 2012, 2014; Lanza et al., 2009). 2-hydroxyethyl methacrylate (HEMA) is the major resin monomer that is released from modern dental composites in an aqueous environment (Van Landuyt et al., 2007). Characterized by relative hydrophilicity and low molecular weight, HEMA competes with water for penetration and infiltration into dentin, and copolymerizes with other monomers of resin composites. HEMA can easily flow into the network of the dentin organic matrix and diffuse through residual dentine, thus inducing various stress responses, such C The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. V All rights reserved. For Permissions, please e-mail: 100 YU ET AL. as cytotoxicity (Schweikl et al., 2006), apoptosis (Paranjpe et al., 2005), genotoxicity (Pawlowska et al., 2010), inhibition of mineralization and disturbance of innate immune functions, in eukaryotic cells (Andersson and Dahlgren, 2011; Bakopoulou et al., 2011). The mechanisms of toxicity by resin monomer HEMA on DMCs should be delineated, and these mechanisms could probably provide helpful insights in improving the biocompatibility of resin materials for dental mesenchymal tissues. Autophagy is an evolutionarily conserved and highly regulated ubiquitous cellular process that degrades intracellular components in response to stressful conditions. In autophagy, the characteristics of double-membrane or multi-membrane autophagosomes initially form by surrounding the parts of the cytoplasm and intracellular organelles. The outer membrane of autophagosomes subsequently fuses with lysosome, forming autophagolysosomes. Finally, the contents of the autophagolysosomes are subsequently digested by the lysosome enzyme (Levine and Kroemer, 2007; Mizushima et al., 2008). Among the identified autophagy-related genes, Beclin1, Atg5, Atg12, and microtubuleassociated protein 1 light chain 3 (LC3, a mammalian homolog of yeast Atg8) are essential for autophagy induction. Recent studies have found that autophagy and apoptosis are biologically connected (Yuan et al., 2014) and that the relationship between autophagy and apoptosis is complex under various circumstances. Several studies demonstrated that autophagy is an important process against apoptosis, whereas autophagy can promote apoptotic cell death in certain settings (Lum et al., 2005; Yang and Klionsky, 2010). However, no studies have reported on the autophagy induction capabilities of HEMA in dental pulp. In addition, whether the pro-apoptotic effects exerted by HEMA are associated with autophagy remains unknown. Previous reports stated that the NF-jB signaling pathway performs an essential function during resin monomer-mediated cell apoptosis (Samuelsen et al., 2007; Spagnuolo et al., 2004). The NFjB transcription factor is a member of the Rel/NF-jB family of transcription factors, consisting of heterodimers of various subunits, including p65/RelA, p50, cRel, p52 and RelB. The heterodimer p65–p50 is the most frequently observed association in numerous cell types. The NF-jB pathway participates in numerous essential biological processes, including cell survival, apoptosis, differentiation, and inflammation (Xiao, 2007). Recently, several studies have shown a direct association between the NFjB pathway and autophag, either promoting or inhibiting the process depending on the conditions (Fabre et al., 2007). In this study, we aimed to determine the autophagy induction potential by HEMA, as well as the possible interplay between autophagy and apoptosis in DMCs. The possible involvement of NF-jB signaling in HEMA-mediated autophagy and apoptosis was also investigated. Furthermore, to best mimick the effects of HEMA on dental pulp tissues, we first utilized an ex vivo tooth slice organ model (...truncated)