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Role of BI-1 (TEGT)-Mediated ERK1/2 Activation in Mitochondria-Mediated Apoptosis and Splenomegaly in BI-1 Transgenic Mice

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Role of BI-1 (TEGT)-Mediated ERK1/2 Activation in Mitochondria-Mediated Apoptosis and Splenomegaly in BI-1 Transgenic Mice Biochimica et Biophysica Acta 1823 (2012) 876–888 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr Role of BI-1 (TEGT)-mediated ERK1/2 activation in mitochondria-mediated apoptosis and splenomegaly in BI-1 transgenic mice Jung-Hyun Kim a, Eung-Ryoung Lee a, Kilsoo Jeon a, Hye Yeon Choi a, Hyejin Lim a, Su-Jeong Kim a, Han-Jung Chae b, Seung Hwa Park c, SangUk Kim d, Young Rok Seo e, Jin-Hoi Kim a, Ssang-Goo Cho a,⁎ a Department of Animal Biotechnology, Animal Resources Research Center, and SMART-IABS, Konkuk University, Seoul, South Korea b Department of Pharmacology and Research, Chonbuk National University, Jeonju, South Korea c Department of Anatomy, College of medicine, Konkuk University, Seoul, South Korea d School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, South Korea e Department of Life Science and Institute of Environmental Medicine, Dongguk University, Seoul, South Korea article info abstract Article history: Bax Inhibitor-1 (BI-1) is an evolutionally conserved apoptotic suppressor and belongs to the BI-1 family Received 16 September 2011 of proteins, which contain BI-1-like transmembrane domains. As their cellular functions and regulatory Received in revised form 22 January 2012 mechanisms remain incompletely understood, we compared their anti-apoptotic properties. Forced expression Accepted 23 January 2012 of BI-1 resulted in the most effective suppression of stress-induced apoptosis, compared with other family Available online 28 January 2012 members, together with significant extracellular signal-regulated kinase (ERK)1/2 activation. BI-1-mediated ERK1/2 activation led to the suppression of mitochondria-mediated reactive oxygen species (ROS) production. Keywords: fi Bax inhibitor-1 Involvement of the ERK signaling pathway in BI-1-induced anti-apoptotic effects was con rmed by knockdown BI-1 family protein studies with ERK- or BI-1-specific siRNA. Moreover, we produced transgenic (TG) mice overexpressing BI-1, and ERK1/2 the relationship between ERK1/2 activation and the suppression of ROS production or apoptosis was confirmed Splenomegaly in mouse embryonic fibroblast (MEF) cells derived from these mice. Interestingly, we found that BI-1 TG mice Autoimmune response showed splenomegaly and abnormal megakaryopoiesis. Taken together, our results suggest that BI-1-induced ERK1/2 activation plays an important role in the modulation of intracellular ROS generation and apoptotic cell death and may also affect autoimmune response. © 2012 Elsevier B.V. All rights reserved. 1. Introduction superfamily member 6 (Fas) ligation [4].MAPKscontainp42/p44 extracellular signal-related kinase (ERK), c-Jun N-terminal protein Apoptosis is a necessary process for the control of cell growth kinase (JNK)/stress-activated protein kinase (SAPK), and p38 MAPK. and elimination of damaged or unwanted cells, and its dysregulation Several isoforms of MAPK have been characterized in mammals: has been implicated in many diseases, including neurodegenerative ERK1/2, 3/4, 5, and 7/8; JNK1, 2, and 3; and p38-α,-β,-γ,and-δ [5,6]. diseases and several types of cancer [1,2]. The fundamental process Each MAPK has been identified for its action in independent signaling of apoptosis is conserved in different species, including yeasts, worms, pathways leading to pleiotropic functions [7].Theseproteinsare mice, and humans [3]. Many studies have shown that all of these involved in the regulation of various biological processes, including organisms share similar apoptosis-regulating proteins and that the mitosis, proliferation, differentiation, and apoptosis [8,9].Although apoptotic process proceeds through 2 main pathways, the cytoplasmic the mechanism of MAPK regulation of apoptosis varies with cell and and mitochondrial pathways. Both of these pathways are regulated by tissue type, generally, activation of JNK/SAPK and p38 MAPKs leads several signaling proteins, including the B-cell lymphoma 2 (Bcl-2) to induction of apoptosis, whereas p42/44 ERK activity is associated family proteins, kinases, and caspases [1]. with suppression of apoptosis [7]. The anti-apoptotic effect of ERK1/2 The mitogen-activated protein kinase (MAPK) pathway, an important can be regulated by a cascade comprised of the Ras/Raf/MEK (MAPK/ apoptosis-regulating pathway, has been shown to regulate apoptosis ERK kinase) 1/2/ERK1/2 pathways [8,9]. induced by UV irradiation, tumor necrosis factor (TNF), or TNF receptor Bax inhibitor-1 (BI-1), also known as TEGT (testis enhanced gene transcript), is an anti-apoptotic protein, which has been reported to suppress cell death induced by Bax expression or other diverse stress ⁎ Corresponding author at: Department of Animal Biotechnolgy, Konkuk University, Seoul 143-701, South Korea. Tel.: +82 2 450 4207; fax: +82 2 455 1044. stimuli [10,11]. Its anti-apoptotic function is widely conserved in most E-mail address: [email protected] (S.-G. Cho). eukaryotes, including yeasts and plants, and even in prokaryotes 0167-4889/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2012.01.016 J.-H. Kim et al. / Biochimica et Biophysica Acta 1823 (2012) 876–888 877 [12–18], and recent studies have shown that BI-1 protects cells from Primers for C-terminal truncation of BI-1 included 5′-GGGAAGAATT- the reduction of calcium content in the endoplasmic reticulum (ER), CATGAACATATTTGATCGA-3′ and 5′-GGGAACTCGAGTCACTAGGACCGG ischemia–reperfusion injury, and ROS accumulation following ER TACT TA-3′. DNA fragments containing BI-1 or BI-1 ΔCweresubcloned stress [19–21]. Analysis of the amino acid sequence of BI-1 has led into a pEF mammalian expression vector containing an oligonucleotide to the suggestion that BI-1 is an intracellular membrane protein con- encoding an HA tag upstream of BI-1 and BI-1 ΔC. For RNA interference taining 6 or 7 transmembrane domains, and that these types of in- of BI-1 expression, a double-stranded oligonucleotide was designed tracellular membrane protein may have very important signaling and according to a common BI-1 sequence (Target sequence: 5′-CCCCGU- transport functions [22,23]. As a large number of disease-related muta- CAACGCAGCAGCA-3′ as previously described [26]) to allow formation tions have been found in intracellular membrane proteins and are of the hairpin structure in the expressed oligo-RNA. This was then reported to affect their stability, folding, and recognition in the mem- cloned into the pSilencer vector from Ambion (Austin, TX). ERK-1 and brane bilayer, intracellular membrane proteins, including BI-1, may be 2specific siRNAs (Cat. No. sc-29308) and control siRNA purchased important targets for therapeutic applications [24,25]. from Santa Cruz were used for RNA interference of ERK expression, Here, we studied the anti-apoptotic effects of several BI-1 family according to the manufacturer's protocol. proteins containing BI-1-like transmembrane domains and attempted to reveal the molecular mechanism of BI-1-mediated anti-apoptosis using cells and transgenic (TG) mice overexpressing BI-1. 2.4. Reverse transcriptase PCR (RT-PCR) 2. Materials and methods Total RNA was isolated from different tissues using TRIzol from Invitrogen (Carlsbad, CA) according to the manufacturer's instructions. 2.1. Antibodies and materials The first-strand cDNA was prepared from 2ug of total RNA isolated using reverse transcriptase with oligo-dT primer. cDNA was subjected Chemicals including etoposide, 2′,7′-dichlorodihydrofluorescein to PCR amplification with DNA primers selective for genes. PCR diacetate (H2-DCFDA), and N-acetylcysteine were purchased from was carried out using Hot-start Taq polymerase (Finnzymes, Espo, BioMOL (Plymouth Meeting, PA) and electrophoresis reagents Finland), as follows; for an initial denaturation step at 95 °C for and protein assay kits were from Bio-Rad (Hercules, CA). The primary 15 min, followed by 35 amplification cycles at 95 °C for 30 s, 51–59 °C antibodies used included those against hemagglutinin (HA, Cat. for 30 s and 72 °C for 30 s, and a final step at 72 °C for 5 min. All samples No. SC-805), green fluorescence protein (GFP, Cat. No. SC-8334), were run in triplicate. Primer sequences were as follows: TMBIM4 [F]: actin (Cat. No. SC-8432), cytochrome C (Cat. No. SC-13156), 5′-CATCCGAATGGCCTTTCTG-3′,TMBIM4[R]:5′-GTAGGTACAGGTT- voltage-dependent anion channel (VDAC, Cat. No. SC-8828), ERK AAGGGG-3′; FAIM2 [F]: 5′-GTACTGGGCATCCTATGCT-3′, FAIM2 [R]: (Cat. No. SC-153), JNK (Cat No. SC-7345), p38 (Cat. No. SC-7149), 5′-GTACTGGGCATCCTATGCT-3′; GHITM [F]: 5′-CAAGAATTGGGATCC- phospho-JNK (Cat. No. SC-6254), and poly (ADP-ribose) polymerase GGCG-3′, GHITM [R]: 5′-CCTTGACATACTGAGGCC-3′;BI-1[F]:5′- (PARP, Cat. No. SC-7148) (Santa Cruz Biotechnology, Santa Cruz, CA), GCAGGGGCCTATGTCCAT-3′,BI-1[R]:5′-AGGATGCTGGGGTTGACAGC- and phospho-p38 (Cat. No. 9211S), and phospho-ERK (Cat. No. 9106S) 3′; GFP-exo [F]: 5′-CTGAGCAAAGACCCCAACG-3′. The integrity of each (Cell Signaling, Danvers, MA). RNA sample was checked by RT-PCR with mouse glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) primers (GAPDH [F]: 5′-AGAA- 2.2. Analysis of the amino acid sequences of BI-1 family proteins CATCATCCCTGCATCC-3′, GAPDH [R]: 5′-CACCACCTTCTTGATGTC-3′)as an internal standard. Amino acid sequences of BI-1 family proteins were retrieved from GenBank (NCBI). Accession numbers of the protein sequences are as follows: TMBIM4 (transmembrane Bax inhibitor motif containing 4: 2.5. Cell culture and DNA transfection NP_057140.1), FAIM2 (Fas apoptotic inhibitory molecule 2: NP_036438.2), GHITM (growth hormone-inducible transmembrane Human embryonic kidney (HEK) 293 cells were cultured at 37 °C in protein: NP_055209.2), and BI-1 (Bax inhibitor-1: NP_003208.1). Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% The degree of sequence identity was determined using expected fetal bovine serum (FBS; Hyclone, Logan, UT) and 100 U/ml penicillin/ value calculation based on the GenBank division.
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