J Cancer 2019; 10(27):6915-6924. doi:10.7150/jca.31319

Research Paper

E-cigarette aerosols induce unfolded protein response in normal human oral keratinocytes

Eoon Hye Ji1*, Naseim Elzakra1*, Wei Chen1, Li Cui1, Eon S Lee2, Bingbing Sun3,4, Diana Messadi1, Tian Xia3, Yifang Zhu2, Shen Hu1✉

1. School of Dentistry, University of California, Los Angeles, CA 90095, USA
2. Fielding School of Public Health, University of California, Los Angeles, CA 90095, USA
3. Division of Nanomedicine, School of Medicine, University of California, Los Angeles, CA 90095, USA
4. State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Dalian University of Technology, 2 Linggong Road, 116024, Dalian, China
* Equal contribution

This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Ji EH, Elzakra N, Chen W, Cui L, Lee ES, Sun B, Messadi D, Xia T, Zhu Y, Hu S. E-cigarette aerosols induce unfolded protein response in normal human oral keratinocytes. J Cancer 2019; 10(27):6915-6924. doi:10.7150/jca.31319. Available from http://www.jcancer.org/v10p6915.htm

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Objective: Since the introduction in 2004, global usage of e-cigarettes (ECs) has risen exponentially. However, the risks of ECs on oral health are uncertain. The purpose of this study is to understand if EC aerosol exposure impacts the gene pathways of normal human oral keratinocytes (NHOKs), particularly the unfolded protein response (UPR) pathway.

Materials and methods: EC aerosols were generated reproducibly with a home-made puffing device and impinged into the culture medium for NHOKs. DNA microarrays were used to profile the gene expression changes in NHOKs treated with EC aerosols, and the Ingenuity Pathway Analysis (IPA) was used to reveal signaling pathways altered by the EC aerosols. Quantitative PCR was used to validate the expression changes of significantly altered genes.

Results: DNA microarray profiling followed by IPA revealed a number of signaling pathways, such as UPR, cell cycle regulation, TGF-β signaling, NRF2-mediated oxidative stress response, PI3K/AKT signaling, NF-κB signaling, and HGF signaling, activated by EC aerosols in NHOKs. The UPR pathway genes, C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4), X box binding protein 1 (XBP1), and inositol-requiring enzyme 1 alpha (IRE1α) were all significantly up-regulated in EC aerosol-treated NHOKs whereas immunoglobulin heavy-chain binding protein (BIP) and PRKR-like ER kinase (PERK) were slightly up-regulated. qPCR analysis results were found to be well correlated with those from the DNA microarray analysis. The most significantly changed genes in EC aerosol-treated NHOKs versus untreated NHOKs were CHOP, ATF4, XBP1, IRE1α and BIP. Meanwhile, Western blot analysis confirmed that CHOP, GRP78 (BIP), ATF4, IRE1α and XBP1s (spliced XBP1) were significantly up-regulated in NHOKs treated with EC aerosols.

Conclusion: Our results indicate that EC aerosols up-regulate the UPR pathway genes in NHOKs, and the induction of UPR response is mediated by the PERK - EIF2α - ATF4 and IRE1α - XBP1 pathways.

Keywords: E-cigarette aerosols, protein response, human oral keratinocytes