The new murine hepatic 3A cell line responds to stress stimuli by activating an efficient Unfolded Protein Response (UPR) Barbara Guantario a , Alice Conigliaro b , Laura Amicone b , Yula Sambuy a , Diana Bellovino a,⇑ a National Research Institute on Food and Nutrition (INRAN), Rome, Italy b Department of Cellular Biotechnologies and Haematology, University ‘‘La Sapienza’’, Rome, Italy article info Article history: Received 10 June 2011 Accepted 26 September 2011 Available online 4 October 2011 Keywords: Cellular model Hepatocytes ER stress RBP4 abstract In the present study we have investigated the properties of a novel cell line (3A cells) obtained from the liver of 14.5 days post coitum (dpc) wild-type mouse embryo. 3A cells morphology was characterized by fluorescent localization of F-actin and b-catenin. The expression of specific genes and proteins essential to liver function in these cells was comparable or even more efficient then in the differentiated hepatocytic cell line MMH-D6. 3A cells also showed the capability to excrete molecules in extracellular spaces resem- bling functional bile canaliculi, glycogen storage activity and the ability to control retinol-binding protein 4 secretion in response to retinol deprivation. Their response to the exogenous stress stimulus induced by tunicamycin was analysed by PCR Pathway Array containing 84 genes involved in the Unfolded Protein Response (UPR). 3A cells were shown to activate the UPR following a typical stressful event, indicating that this cellular model could be further exploited to investigate hepatic proteins secretion and specific reaction to different injuries. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction The effort to establish a differentiated hepatic cell line able to express in vitro the most important liver functions (such as the syn- thesis of specific serum proteins, the metabolism of carbohydrates and lipids, and the modification and excretion of endogenous and exogenous molecules) has long been pursued in several laborato- ries and different model systems have been described to date (Fer- rini et al., 1997; Gebhardt et al., 2003; Hewitt et al., 2007). Such in vitro systems are essential to test a wide panel of molecules, both of natural or synthetic origin, with beneficial (i.e. nutrients, bioac- tive molecules, drugs) or detrimental (i.e. environmental pollu- tants, metals, toxins, etc.) effects on the organism, and to study the molecular mechanisms that govern their absorption, metabo- lism, secretion and catabolism in the liver. Primary hepatocytes are still the closest in vitro model for the liver. However, they have scarce and often unpredictable availability, limited growth activity and lifespan, and undergo early phenotypic alterations (Guguen- Guillouzo and Guillouzo, 2010). Conversely, hepatoma cell lines such as HepG2, despite representing a widely used model charac- terized by indefinite proliferative capacity, lack several important regulatory mechanisms and crucial liver functions such as the cytochrome P450 activities (Guguen-Guillouzo and Guillouzo, 2010) or micronutrients transport (Bellovino et al., 1999; Pisu et al., 2005). In this study we have characterized the 3A hepatic cell line, iso- lated from 14.5 dpc embryo of a wild type mouse strain that under- went spontaneous immortalization. 3A cells were tested for the maintenance of several liver key features essential for a functional hepatocyte model, including the regulation of retinol binding protein 4 (RBP4) secretion in re- sponse to vitamin A deficiency. In addition to structural and func- tional characteristics, we have tested their capability to overcome stressful conditions by activating the Unfolded Protein Response (UPR). The synthesis and secretion of large amounts of proteins such as hormones, antibodies or growth factors as well as various stresses, such as hypoxia, starvation, heat and drug treatment can interfere with proper protein folding in the endoplasmic reticulum (ER) (Kaufman et al., 2002) and consequently with protein secre- tion. These events lead to ER stress and accumulation of misfolded proteins that, in turn, can result in cell failure and death by apop- tosis. Cells are able to activate specific pathways in order to pre- vent and relieve ER stress, in particular the UPR pathway, through which protein synthesis is inhibited and misfolded pro- teins are (i) detected, (ii) sequestered and/or re-folded, (iii) eventu- ally degraded (Schroder and Kaufman, 2005; Ron and Walter, 2007). The correct functioning of this pathway ensures a proper response to exogenous stress, and is therefore essential for cells to exert efficiently their functions. A new, readily available cell model, easy to obtain and to maintain in culture, would therefore 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.09.020 ⇑ Corresponding author. Address: INRAN, Via Ardeatina 546, 00178 Rome, Italy. Tel.: +39 06 51494457; fax: +39 06 51494550. E-mail address: bellovino@inran.it (D. Bellovino). Toxicology in Vitro 26 (2012) 7–15 Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit