Activation of transcription factor AP-1 in response to thermal injury in rat small intestine and IEC-6 cells

Zhang et al. BMC Gastroenterology (2015) 15:83 DOI 10.1186/s12876-015-0309-z RESEARCH ARTICLE Open Access Activation of transcription factor AP-1 in response to thermal injury in rat small intestine and IEC-6 cells Yonghong Zhang1, Hong Zhao1, Tao Liu1, Changrong Wan2, Xiaoxi Liu1,2, Zhimin Gao1, Xiaolin Hou1, Linshu Jiang1*† and Fenghua Liu1*† Abstract Background: Our previous studies indicated that heat stress can cause significant damage to the intestinal epithelium and induce differential expression of many genes in rat small intestine. The transcription factors AP-1 and NF-κB, which act as important mediators by binding to specific DNA sequences within gene promoters, regulate the transcription of genes associated with immune regulation, stress response and cell fate. Methods: To determine whether AP-1 and NF-κB are involved in hyperthermia-induced injury in rat small intestine and IEC-6 cells, we investigated their activity, and the expression of related proteins, by electrophoretic mobility shift assays and western blotting, respectively. Results: Heat stress resulted in severe damage to the epithelium of the small intestine. The cell morphology and viability were obviously altered when IEC-6 cell was exposed to hyperthermia. AP-1 was activated in the small intestine of heat-stressed rats, as was phosphorylation of the JNK signaling pathway. In IEC-6 cell line, AP-1 activation in groups exposed to 42 °C for 1 h, 2 h and 4 h was significantly increased. In contrast, NF-κB was not activated in both in vivo and in vitro models. Conclusion: These results reveal that AP-1 is likely to play an important role in regulating gene transcription in rat small intestine and IEC-6 cells during exposure to heat stress. Keywords: AP-1, NF-κB, Heat stress, Rat small intestine, IEC-6 Background As one important physical stimulus, ambient temperature can evoke a series of drastic changes in biological function [1] including gastrointestinal injury and dysfunction [2]. Our previous studies have shown that heat stress can induce damage in the rat small intestine, along with differential expression of many genes associated with immune regulation and metabolism, and those encoding regulatory peptides [3]. A number of growth-related molecules (such as Gdf15, Gdf9, Ctgf, and Egfr) which are critical for cellular survival, proliferation and migration, have also been shown to be differentially expressed in response to hyperthermia-induced damage [4]. * Correspondence: ; † Equal contributors 1 Beijing Key Laboratory for Dairy Cow Nutrition, Beijing University of Agriculture, No. 7, Beinong Road, Changping District, Beijing 102206, P. R. China Full list of author information is available at the end of the article Transcription factors, important mediators involved in signal transduction, bind to specific DNA sequences within gene promoters, and thus regulate transcriptional activity. Both NF-κB and AP-1 are well known pleiotropic transcription factors that independently and/or complementarily regulate a large number of genes related to a wide range of functions, including immune regulation, proliferation, differentiation, and apoptosis [5, 6]. NF-κB is a ubiquitous transcription factor and a member of a family of proteins that are important regulators of a variety of responses. NF-κB exists as a dimer predominantly composed of p50 and p65 subunits, although it also contains other family members, such as RelB, cRel, v-Rel and p52 [7]. The activity of NF-κB is regulated by a family of IκB inhibitor proteins [8], which sequester NF-κB in the cytoplasm. In response to various external pathogenic stimuli, IκB is phosphorylated, ubiquitinated, © 2015 Zhang et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. BMC Gastroenterology (2015) 15:83 and subsequently degraded by a proteosome-dependent pathway. Degradation of IκB allows NF-κB to translocate into the nucleus, where it binds to specific promoter elements and induces gene transcription. AP-1 is a central switch to convert extracellular signals into genetic responses and to determine cell proliferation, differentiation, and apoptosis. AP-1 complex consists of homodimers and heterodimers formed by a group of transcription factors, including members of the Jun, Fos, and ATF families [9]. Previous studies indicate that the c-Jun/ATF-2 heterodimer is one of the main components of expression pathways associated with oncogenesis [9] and the extreme cellular stress of ischemia and reperfusion [10]. JNK is one member of the mitogen-activated protein kinase (MAPK) family, which play crucial roles in many responses [11]. JNK was initially described as a stress-induced protein kinase acting to phosphorylate the NH2-terminus of the transcription factor c-Jun; hence, this pathway is often referred to as the stress-activated protein kinase (SAPK) pathway [12]. Multiple stresses increase JNK activity including UV, rirradiation, cytotoxic drugs, ischemia and reactive oxygen species. JNK phosphorylates several transcription factors including c-Jun, ATF-2, and p53 [13], which in turn regulate the expression of genes mediating cell proliferation, differentiation or apoptosis. Many studies have shown that there is crosstalk between JNK1 and NF-κB [14]. To further explore the mechanism of gene expression involved in hyperthermia-induced damage and repair in the rat small intestine, we investigated the activity of transcription factors AP-1and NF-κB and determined the expression of proteins acting upstream in their respective pathways, using both in vivo and in vitro models. Methods Animals and treatments All protocols and procedures involving animals were approved by the Beijing University of Agriculture Institutional Animal Care and Use Committee, and conducted in accordance with the committee’s guidelines. 12 male Sprague–Dawley (SD) rats weighing 200 ± 20 g (obtained from Beijing Vital River Laboratory, Animal Technology Co., Beijing, China) were caged at 25 °C, with a 12 h light:dark cycle and free access to food and water for 7 days. Rats were then randomly divided into control or heat-stress groups (6 rats per group) and housed in an artificial climate chamber (HPG-400BX, Harbin Donglian Electronic Technology, Heilongjiang, China) under normal conditions (25 °C, 60 % relative humidity). Rats in the heat treatment group were exposed to 40 °C and 60 % RH from 11:00 to 13:00 for 3 consecutive days. The detail of heat-stress procedure was previously described by Yu et al. [4]. Rat rectal temperature was recorded daily before and a (...truncated)