Differential Control of the CCAAT/Enhancer-binding Protein β (C/EBPβ) Products Liver-enriched Transcriptional Activating Protein (LAP) and Liver-enriched Transcriptional Inhibitory Protein (LIP) and the Regulation of Gene Expression during the Response to Endoplasmic Reticulum Stress

Elena Bevilacqua,Peter F. Johnson,Mithu Majumder,Chuanping Wang,Martin D. Snider,Calin-Bogdan Chiribau,Maria Hatzoglou,Colleen M. Croniger,Yi Li

doi:10.1074/jbc.m801046200

Abstract

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a stress response program that protects cells early in the response and can lead to apoptosis during prolonged stress. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta), is one of the genes with increased expression during ER stress. Translation of the C/EBPbeta mRNA from different initiation codons leads to the synthesis of two transcriptional activators (LAP-1 and -2) and a transcriptional repressor (LIP). The LIP/LAP ratio is a critical factor in C/EBPbeta-mediated gene transcription. It is shown here that the LIP/LAP ratio decreased by 5-fold during the early phase of ER stress and increased by 20-fold during the late phase, mostly because of changes in LIP levels. The early decrease in LIP required degradation via the proteasome pathway and phosphorylation of the translation initiation factor, eIF2alpha. The increased LIP levels during the late phase were due to increased synthesis and increased stability of the protein. It is proposed that regulation of synthesis and degradation rates during ER stress controls the LIP/LAP ratio. The importance of C/EBPbeta in the ER-stress response program was demonstrated using C/EBPbeta-deficient mouse embryonic fibroblasts. It is shown that C/EBPbeta attenuates expression of pro-survival ATF4 target genes in late ER stress and enhances expression of cell death-associated genes downstream of CHOP. The inhibitory effect of LIP on ATF4-induced transcription was demonstrated for the cat-1 amino acid transporter gene. We conclude that regulation of LIP/LAP ratios during ER stress is a novel mechanism for modulating the cellular stress response.

Highlights

The accumulation of unfolded proteins in the endoplasmic reticulum triggers a complex regulatory program that involves regulation of both transcription and translation [1]
Because translation of the C/EBP␤ mRNA produces two transcriptional activators, LAP1 and -2, and a transcriptional repressor, LIP (Fig. 1E), we first determined the levels of LAP and LIP in C6 cells treated with Tg for 0 –24 h, which acts by depleting endoplasmic reticulum (ER) Ca2ϩ stores [6]
The lack of induction is consistent with the regulation of scriptional activators LAP-1 and LAP-2 and the transcriptional these genes by ATF6 and XBP1, rather than ATF4

Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructs—pGL3-3xUPRE and pGL3-2xERSE luciferase reporter vectors were gifts from Dr R. Amino acid starvation was induced by incubation of cells in Met-, Cys-free high glucose DMEM supplemented with 10% dialyzed FBS as described previously [26]. DNA Affinity Pulldown—A biotinylated double-stranded DNA oligonucleotide containing two copies of the cat-1 AARE (5Ј-CGCGCGGCTGATGAAACCGGCTCGCGCGCGGCTGATGAAACCGGCTCG-3Ј) was used to isolate proteins from C6 cell nuclear extracts as described previously [32] with minor modifications. Extracts (500 ␮g of protein) were incubated streptavidin magnetic particles (Roche Applied Science) for 30 min at 4 °C; the beads were removed, and 100 ␮g/ml poly(dI-dC) was added to the supernatants for 30 min, followed by 100 pmol of the biotinylated oligonucleotide in binding buffer (12 mM HEPES-NaOH, pH 7.9, 4 mM Tris-HCl, pH 7.9, 12% glycerol, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol) for 1 h at 4 °C. The PCR primers used are listed in supplemental Table 1

RESULTS

LAP in nuclear extracts decreased

DISCUSSION

Differential Regulation of LAP and LIP Levels during ER