C/EBP-b Regulates Endoplasmic Reticulum Stress–Triggered Cell Death in Mouse and Human Models Ofir Meir, Efrat Dvash, Ariel Werman, Menachem Rubinstein* Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Abstract Endoplasmic reticulum (ER) stress elicits the unfolded protein response (UPR), initially aimed at coping with the stress, but triggering cell death upon further stress. ER stress induces the C/EBP-H variant Liver-enriched Activating Protein (LAP), followed by the dominant-negative variant, Liver Inhibitory Protein (LIP). However, the distinct role of LAP and LIP in ER stress is unknown. We found that the kinetics of the ER stress-induced expression of LIP overlapped with that of the cell death in mouse B16 melanoma cells. Furthermore, inducible over-expression of LIP augmented ER stress-triggered cell death whereas over-expression of LAP attenuated cell death. Similar results were obtained in human 293T cells. Limited vasculature in tumors triggers hypoxia, nutrient shortage and accumulation of toxic metabolites, all of which eliciting continuous ER stress. We found that LAP promoted and LIP inhibited B16 melanoma tumor progression without affecting angiogenesis or accelerating the cell cycle. Rather, LAP attenuated, whereas LIP augmented tumor ER stress. We therefore suggest that C/EBP-H regulates the transition from the protective to the death–promoting phase of the UPR. We further suggest that the over-expression of LAP observed in many solid tumors promotes tumor progression by attenuating ER stress–triggered tumor cell death. Citation: Meir O, Dvash E, Werman A, Rubinstein M (2010) C/EBP-b Regulates Endoplasmic Reticulum Stress–Triggered Cell Death in Mouse and Human Models. PLoS ONE 5(3): e9516. doi:10.1371/journal.pone.0009516 Editor: Gian Maria Fimia, INMI, Italy Received October 17, 2009; Accepted February 11, 2010; Published March 3, 2010 Copyright: 2010 Meir et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported in part by Merck-Serono International SA and by the M.D. Moross Institute for Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: A research Grant from Merck-Serono. A patent application assigned to Yeda Research & Development Co. Dr. Rubinstein confirms that this will not alter his adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors http://www.plosone.org/ static/policies.action#sharing. * E-mail: Menachem.rubinstein@weizmann.ac.il Introduction The endoplasmic reticulum is the site of post-translational processing (refolding, glycosylation and oxidative disulfide bond formation) of secreted and cytoplasmic membrane proteins. The ER is also the site of membrane biogenesis and helps maintain intracellular calcium ion and lipid homeostasis. Accumulation of client proteins or misfolded proteins, overload of free cholesterol, perturbations in calcium ion homeostasis, oxidative stress and xenobiotic toxins rapidly induce ER stress, which triggers an evolutionarily conserved cellular response, termed the Unfolded Protein Response (UPR) [1]. Initial UPR is aimed at coping with the stress by inducing ER chaperones and attenuating general protein translation. Persistent ER stress induces the expression of the C/EBP homologous protein (CHOP), which initiates the cell death machinery [2]. The mechanisms that regulate the transition from the protective phase to the death–promoting phase of the UPR are not known. ER stress induces the expression of the transcription factor C/ EBP-b [3,4] but there are contradictory reports on the role of C/ EBP-b in ER stress. Embryonic fibroblasts from C/EBP-b-deficient mice are less sensitive to ER stress–mediated cell death [5,6]. In contrast, other cell types from these mice exhibit enhanced sensitivity to cell death triggered by growth factor deficiency, exposure to toxins or carcinogens and other stresses [7–11]. Other studies found that C/EBP-b is pro-apoptotic [5,12–16]. Because the common C/EBP-b mRNA encodes both transcriptional activators (LAP* and LAP) and a dominant-negative, truncated form (LIP) [17], these opposing observations may be related to the LIP:LAP ratio. Furthermore, the LIP:LAP ratio is not constant, increasing in time, as the ER stress continues [18]. Therefore, further studies are required to determine the specific role of LAP and LIP in the cellular response to ER stress. ER stress has been associated with many diseases, where it leads to cell death [1,19]. Because neovascularization lags behind tumor cell proliferation, solid tumors are under continuous ER stress due to nutrient shortage, accumulation of cytotoxic metabolites and hypoxia. However, the cells adapt to this stress through the constitutive activation of the protective phase of the UPR (reviewed in: [2,20–22]). So far, the mechanisms that mediate this adaptation are not fully characterized. The various C/EBP-b isoforms are over-expressed in many types of tumors [8,23–32], but the specific role of LAP and LIP in tumor progression has not been studied. Recently, C/EBP-b was shown to support the survival of neurons under hypoxic conditions, which also trigger ER stress [33]; however, in these studies, both LAP and LIP were either over-expressed or were deleted, and therefore their individual function is not known. To study the role of C/EBP-b LAP and LIP in ER stress and in tumor progression, we utilized B16 melanoma clones that PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9516