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Originally published In Press as doi:10.1074/jbc.M312170200 on February 9, 2004 Originally published In Press as doi:10.1074/jbc.M312170200 on January 12, 2004

J. Biol. Chem., Vol. 279, Issue 14, 13953-13961, April 2, 2004
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Gene Expression Changes Associated with the Endoplasmic Reticulum Stress Response Induced by Microsomal Cytochrome P450 Overproduction*

Elzbieta Szczesna-Skorupa{ddagger}§, Ci-Di Chen§, Hong Liu||, and Byron Kemper{ddagger}**

From the {ddagger}Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, the Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota 55455, and ||Aventis Pharmaceuticals Inc., Bridgewater, New Jersey 08807

Received for publication, November 6, 2003 , and in revised form, January 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of drug-metabolizing microsomal cytochromes P450 (P450s) results in a striking proliferation of the smooth endoplasmic reticulum (ER). Overexpression of P450s in yeast and cultured cells produces a similar response. The signals mediating this process are not known but probably involve signal transduction pathways involved in the unfolded protein response (UPR) or the ER overload response (EOR). We have examined the temporal response of specific genes in these pathways and genes globally to overexpression of P450 in cultured cells. Activity of NF{kappa}B, an EOR component, was substantially increased by overexpression of full-length P450 2C2 or a chimera with the 28-amino acid signal anchor sequence of P450 2C2 in HepG2 cells, and the activation correlated temporally with the accumulation of P450 in the cells. In the UPR pathway, activation of the transcription factor XBP1 by IRE1 also correlated with the accumulation of P450 in the cells, and in contrast, maximum activation of the BiP/grp78 promoter preceded the accumulation. Differential effects of expression of P450 on apoptosis were observed in nonhepatic COS1 and hepatic HepG2 cells. In COS1 cells, apoptosis was induced, and this correlated with sustained activation of the pro-apoptotic JNK pathway, induction of CHOP, and an absence of the increased NF{kappa}B activity. In HepG2 cells, JNK was only transiently activated, and CHOP expression was not induced. As assessed by DNA microarray analysis, up-regulation of signaling genes was predominant including those involved in anti-apoptosis and ER stress. These results suggest that both the EOR and UPR pathways are involved in the cellular response to induction of P450 expression and that in hepatic cells genes are also induced to block apoptosis, which may be a physiologically relevant response to prevent cell death during xenobiotic induced expression of P450 in the liver.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The endoplasmic reticulum (ER)1 represents the central organelle of the cell in which the crucial steps of folding and modification of proteins and selection for transport to other compartments take place. Conditions that affect ER homeostasis, or ER stress, may have dire consequences for the survival of cells and may induce one of several signal transduction pathways. Accumulation of misfolded proteins, increased secretory load, glucose deprivation, or perturbations of calcium homeostasis activates an intracellular signaling pathway referred to as the unfolded protein response (UPR) (1–3). Although different stress inducers use diverse pathways to activate selected target genes, there is considerable cross-talk between the pathways. Common targets include molecular chaperones, folding enzymes, and genes with promoters that contain a cis-acting element known as the ER stress-response element (ERSE) (1, 2).

In mammalian cells, ER stress-activated signal transduction is mediated by three ER transmembrane proteins as follows: 1) transcriptional activator ATF6, which upon ER stress is proteolytically released from the membrane and up-regulates ERSE-containing promoters; 2) translation initiation factor 2 kinase, PERK, which mainly attenuates translation, slowing down the accumulation of ER proteins and allowing the ER to accommodate its cargo load; and 3) the transmembrane kinase/endoribonuclease IRE1 (4–8). The luminal domain of IRE1 is the proximal sensor of misfolded protein accumulation, which induces activation of IRE1 via oligomerization and autophosphorylation (2). Activation of IRE1 up-regulates molecular chaperones, but under severe stress it also activates the c-JUN amino-terminal kinase (JNK), part of the apoptotic pathway (9). Similarly to its yeast analog, the cytosolic domain of activated IRE1 acts also as an endonuclease/ligase, splicing the mRNA of the transcriptionally inactive protein XBP1 (x box-binding protein), leading to the formation of a potent bZIP transcriptional activator (7, 8, 10). Although in yeast, IRE1 and its spliced product HAC play a central role in UPR, in mammalian cells its role in ER stress response is less clear, because in IRE1 null cells unimpaired UPR and up-regulation of chaperones occur (8, 9, 11). Both ire1 and xbp1 null mice show embryonic lethal phenotypes, implying physiological functions other than the ER stress response function for these genes (8–13).

ER expansion induced by the increased accumulation of properly folded ER resident proteins represents a unique case of the ER stress response and is usually referred to as an endoplasmic reticulum overload response (EOR) (3, 14). Overexpression of certain membrane proteins, like hydroxymethylglutaryl-CoA reductase, induces smooth ER proliferation without UPR activation (15). On the other hand, overexpression of cytochrome P450 in yeast does induce UPR and activation of chaperones (16–18). EOR has been shown to activate the transcription factor NF{kappa}B (19–21); however, the mechanism connecting the ER overload detection to the transcriptional activation in the nucleus is not understood, and both reactive oxygen species and ER-released calcium have been suggested as mediators connecting ER stress with the NF{kappa}B activation (1, 19, 20).

Because NF{kappa}B plays a major role in inflammatory and immune responses, EOR caused by accumulation of viral proteins may represent an antiviral immune response (19). Pathological ER accumulation of non-viral proteins, which occurs in diseases such as cystic fibrosis, Alzheimer's, or hereditary emphysema, is often associated with inflammation (21). The inflammation may be related to activation of NF{kappa}B and its role in inflammatory responses. However, it is not known whether NF{kappa}B activation plays a role in ER-nucleus signal transduction leading to an expansion of the ER in response to physiological stimuli, such as increased secretory activity or induction of microsomal enzymes.

Microsomal cytochromes P450 (P450) are highly inducible enzymes, whose ER accumulation results in extensive proliferation of smooth ER membranes (16–18, 22, 23), but the mechanism and signals involved in this process are not known. It is conceivable that ER accumulation of P450s activates not only EOR, but also a general ER stress response, like the UPR. If so, then changes might be expected in expression of genes enhancing ER folding capacity or in ER-nucleus signal transduction pathway genes that are responsible for membrane biosynthesis, cell survival, and adaptation to the changing size and content of the ER.

In the current study we have undertaken several approaches to analyze the cellular response, and the signals involved, to the accumulation of P450 2C2 in transiently transfected cells. Based on our biochemical studies and DNA microarray analysis, we conclude that there is a time-dependent switch from the early activation of ER stress-inducible chaperones and the JNK pathway followed by a delayed activation of NF{kappa}B in HepG2 cells. NF{kappa}B may have a role in regulating apoptosis-related genes to allow cell survival. The kinetics of splicing and up-regulation of transcription factor XBP1 parallels that of the NF{kappa}B activation and is correlated with the kinetics of P450 accumulation, consistent with its possible involvement in sustained ER expansion. Furthermore, by using DNA microarray analysis, we show that a significant number of the P450 up-regulated genes contain in their promoters potential binding sites for NF{kappa}B and XBP1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Tissue culture materials were purchased from Invitrogen. The enhanced GFP vector was from Clontech. The pNF{kappa}B-luciferase reporter plasmid, which contains five copies of the NF{kappa}B-binding site, was from Stratagene. The pBiP-luciferase reporter plasmid (6, 24) contains the rat grp78 (BiP) promoter region -457 to -39. Construction of all the P450 chimeras was described previously (25–27). The phRK-TK reporter vector and dual luciferase reporter activity assay kit were from Promega. PJNK antibody was from Santa Cruz Biotechnology.

RT-PCR—RNA was prepared from transfected cells cultured on 6-well plates with the Qiagen RNeasy kit and used in RT-PCR performed with the Omniscript RT kit (Qiagen). The following sets of primers were used for the PCR amplifications: for BiP, 5'-ATCACGCCGTCCTATGTCGC-3' and 5'-TCTCCCCCTCCCTCTTATCC-3'; for actin, 5'-TCCTCACCCTGAAGTACCCC-3' and 5'-CTCTTGCTCGAAGTCCAGGG-3'; for CHOP, 5'-AGTCATTGCCTTTCTCTTCG-3' and 5'-GGTGCAGATTCACCATTCGG-3'; and for xbp1, 5'-CCTTGTAGTTGAGAACCAGG-3' and 5'-GGGGCTTGGTATATATGTGG-3'.

Cell Culture, Transfection, and Luciferase Assay—COS1 and HepG2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and transfected with Lipofectamine, as described previously (26, 27). For luciferase reporter assays, cells were transfected on 24-well plates with the amounts of DNA indicated in the figures legends. For mock transfections and to compensate for the differences in the total DNA used, pCMV5 vector DNA was added. The assays were performed using the dual-luciferase reporter 1000 assay system according to the manufacturer's protocol (Promega). Relative luciferase activity was defined as the ratio of firefly luciferase activity to Renilla luciferase activity.

JNK Activation Assay—Cells transfected on 6-well plates were washed with PBS and lysed in RIPA buffer, and the lysates were clarified by 10 min of centrifugation. The protein concentration was measured with Micro-BCA reagents (Pierce). Lysates were analyzed by SDS-PAGE followed by Western blots probed with anti-phosphorylated JNK antibody and the horseradish peroxidase SuperSignal chemiluminescent detection system (Pierce).

Fluorescent Protein Concentration Assay—To measure the concentration of C2/GFP chimera in transfected cells, cells cultured in 6-well plates were washed with PBS, scraped, and collected by brief centrifugation. After additional washing with PBS, cells were resuspended in 1 ml of PBS and used for spectrofluorimetry, as described (28).

Analysis of Cell Viability—Apoptosis in transfected cells was analyzed by assaying plasma membrane permeability to Hoechst 33342 dye and flow cytometry (29). Transfected cells were resuspended in 1 ml of PBS and, after adding Hoechst 33342 solution to a final concentration of 0.9 µg/ml, were incubated for 3 min at 37 °C, after which propidium iodide was added to a final concentration of 0.6 µg/ml, and the incubation was continued for 5 min on ice. Cells were analyzed by flow cytometry with UV excitation at 351 nm, emission band pass filter 450/65, for Hoechst 33342 detection and 488 nm, 630/30 filter, for propidium iodide detection.

cDNA Microarray Analysis—Total RNA from transfected HepG2 cells was isolated using the Qiagen RNeasy kit. Double-stranded cDNA was synthesized with 5–8 µg of total RNA using the T7-oligo(dT) primer (Genset Oligos, Boulder, CO) and the SuperScript double-stranded cDNA synthesis kit (Invitrogen) following the manufacturers' protocols. Precipitated double-stranded cDNA was used as a template for cRNA synthesis with the BioArray RNA transcript labeling kit with Bio-11-CTP and Bio-16-UTP (Enzo, Farmingdale, NY). Biotin-labeled cRNA was then purified (RNeasy Mini Kit, Qiagen, Hilden, Germany), fragmented, and hybridized to the HumU133A oligonucleotide microarrays (Affymetrix, Santa Clara, CA). The arrays were washed and stained with streptavidin-phycoerythrin. Fluorescence intensities were measured with the GeneArray scanner. Affymetrix chip data (HG-U133A) were generated from two mock- and two P450-transfected samples from two different transfections. Gene expression ratios of P450- versus mock-transfected samples for each set were measured by the PFOLD algorithm (30) that is derived from a Bayesian estimation scheme for estimating the fold change of gene expression.

Searching for the Transcription Factors-binding Sites in the Promoter Sequences—Putative promoters of human transcripts were retrieved from Ensembl (www.ensembl.org) based on their transcript and protein annotation. A putative promoter sequence is defined as a 1.5-kb upstream sequence including start codon ATG when the ATG is within the first exon. A total of 22,867 putative promoter sequences were searched for XBP1 and NF{kappa}B-binding sites using a pattern matching program written in Perl.2


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of NF{kappa}B in HepG2 Cells—P450 2C2 is inserted into the ER membranes via its amino-terminal signal-anchor sequence, which also mediates its direct ER retention, leaving the major catalytic domain of the protein on the cytosolic side of the membrane (31). By using transiently transfected cells and immunomicroscopy, we have observed that increasing levels of this protein in the ER membranes induce significant expansion of this compartment (not shown). Because this process most likely induces EOR, we first wanted to establish whether in analogy with previously studied ER retained proteins (19–21), P450 accumulation also activates transcription factor NF{kappa}B.

To analyze activation of NF{kappa}B in cells expressing P450 2C2, we co-transfected HepG2 cells with plasmid encoding a firefly luciferase reporter gene fused to five NF{kappa}B recognition sites in the promoter region. We have shown before, using chimeras of different P450 domains and GFP, that although full-length C2/GFP and C1–28/GFP (containing the amino-terminal 28- amino acid signal-anchor transmembrane region) are directly retained in the ER (excluded from the transport vesicles), C1–21/GFP, with the short juxtamembrane linker region deleted, recycles through the retrieval pathway (26). We thus also tested whether these P450-GFP chimeras induce NF{kappa}B to the same level.

As Fig. 1 shows, 24 h after transfection, transcriptional activity of NF{kappa}B in transfected HepG2 cells was strongly enhanced in a DNA concentration-dependent manner. The level of the activation in cells expressing full-length C2/GFP was the same as in the cells expressing C2 (amino acids 1–490) without the GFP tag, encoded by plasmid C2/CMV5 (31), so that GFP was not responsible for NF{kappa}B activation. Although all three of the chimeras activated NF{kappa}B, activation was lowest in cells expressing C1–21/GFP. This suggests that either the juxtamembrane linker (amino acids 22–28) contributes to the response or that EOR is weaker when proteins accumulating in the ER undergo transport to the intermediate compartment. Consistent with the second possibility, we have also observed that activation of NF{kappa}B in cells expressing P450 2E1, which unlike P450 2C2 can be packaged into the transport vesicles and enters the retrieval pathway (32), is about half of that observed with 2C2 (not shown).



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  		FIG. 1.
Transcriptional activation of the NF{kappa}B promoter by P450 2C2 chimeras. HepG2 cells were co-transfected with the reporter plasmid NF{kappa}B/luciferase (25 ng), phRK-Tk (0.1 ng), and the indicated amounts of P450 expression vector DNA. The relative luciferase activity (expressed as a ratio of firefly luciferase activity to Renilla luciferase activity) was measured after 24 h of transfection. All values represent the averages of three independent experiments (done in triplicates) with standard deviations shown (error bars).

 
The importance of ER retention for NF{kappa}B activation is demonstrated by only marginal up-regulation of NF{kappa}B in cells expressing the chimera EGC (Fig. 1). This chimera consists of the ER-targeting secretory signal sequence of epidermal growth factor receptor (EGFR), followed by GFP, and has the 2C2 TM-(1–28) at its carboxyl terminus (25). We have shown previously that this protein is not retained in the ER but is transported to the cell surface, because the ER retention signal of the C2 TM-(1–28) is not functional in the carboxyl-terminal location (25, 27, 31). These results establish that whereas ER retention is required for the activation of NF{kappa}B, activation is not dependent on the expression of the full-length enzymatically active P450, as similar up-regulation is observed with chimera containing only C2 TM-(1–28), nor on the presence of the TM-(1–28), which is present in EGC. Furthermore, the lack of NF{kappa}B activation by the EGC protein, which is transported to the cell surface, is consistent with the up-regulation of NF{kappa}Bby the accumulation of an integral ER membrane protein, rather than by an increase in the load of the secretory pathway.

Time Course of NF{kappa}B and BiP Activation in Transfected HepG2 Cells—To establish if activation of NF{kappa}B represents an early ER stress response similar to UPR-activated factors, we analyzed the time course of NF{kappa}B activation in HepG2 cells transfected with the full-length P450 2C2. Activation of NF{kappa}B was minimal at the early time points (7 h after transfection) but continuously increased after that, reaching a maximum between 24 and 48 h after transfection (Fig. 2). In contrast, activation of the ERSE-containing BiP/grp78 promoter (promoter of the rat grp78 gene attached to the luciferase reporter) was already 20% of its maximum at 7 h after transfection, reached a peak by 16 h, and subsequently slightly decreased to about 50% of its peak by 48 h (Fig. 2). Induction of the BiP/grp78 promoter is a general response to ER stress, part of the UPR pathway, and is mostly mediated by the transcription factor ATF6 (5). ATF6 is activated via proteolysis, which permits a rapid response to ER stress. This suggests that unlike the rapid induction of UPR, activation of NF{kappa}B is delayed and more closely parallels the increase of P450 levels (Fig. 2), so that it is probably triggered by a pronounced accumulation of P450 in the ER, as might be expected for an EOR.



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  		FIG. 2.
Time course of the transcriptional activation of BiP/grp78 and NF{kappa}B promoters and of C2/GFP accumulation in transfected HepG2 cells. HepG2 cells were co-transfected with phRK-Tk (0.1 ng), 25 ng of either NF{kappa}B/luciferase vector DNA or BiP/luciferase vector DNA, and 100 ng of C2/GFP vector DNA for luciferase activity assays, or with 100 ng of C2/GFP vector DNA only for fluorescent protein quantification (bottom panel). At the indicated times after transfection, luciferase activity was measured and presented as in Fig. 1, and the fluorescence of C2/GFP in transfected cells was measured as described under "Experimental Procedures." Black and gray bars indicate P450- or mock-transfected cells, respectively.

 
Activation of CHOP and JNK Pathway in HepG2 and COS1 Cells—Continuous stress to cells can trigger apoptosis presumably to protect the organism from the deleterious effects of damaged cells. Apoptosis in response to the expansion of ER membrane as a result of accumulation of pathological proteins, for example, could be beneficial. In the case of P450 induction, however, a normal protein is accumulated in the ER that is part of a cellular defense against toxic chemicals in the body. In this case, apoptosis would be counterproductive. NF{kappa}B has been shown to play a role in the regulation of the programmed cell death pathway (apoptosis) (33), so it seemed possible that its activation in cells overexpressing P450 contributes to the survival of these cells. Preliminary studies using immunoelectron microscopy showed that although the expression of P450 in both HepG2 and COS1 cells induces strong ER proliferation, COS1 cells, but not HepG2 cells, even within 18 h after transfection, showed morphological changes characteristic of apoptosis (data not shown). Further analysis by flow cytometry of transfected COS1 cells in which nuclei were stained with Hoechst 33342 dye and propidium iodide demonstrated apoptosis in about 30–35% of P450-expressing COS1 cells 24 h after transfection. We thus considered the possibility that this effect may be related to a potential difference in NF{kappa}B activation in the two cell lines, if a function of NF{kappa}B in the EOR is antiapoptotic (33). Indeed, there was no detectable activation of the NF{kappa}B promoter in COS1 cells transfected with P450 (Fig. 3A). In contrast, a dose-dependent activation of the BiP promoter in transfected COS1 cells was similar to that in HepG2 cells (Fig. 3B). Thus, transfected COS1 cells mount a UPR but are unable to activate NF{kappa}B, which is correlated with increased apoptosis.



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  		FIG. 3.
The effect of P450 2C2 overexpression in COS1 cells on the transactivation of NF{kappa}B (A) and BiP/grp78 (B). COS1 cells were transfected with the indicated amounts of C2/GFP vector DNA and the appropriate reporter vector DNA, as in Fig. 1. 24 h after transfection luciferase activity assays were performed and are presented as described in the legend to Fig. 1.

 
During the ER stress response, cells can activate death pathways by activating an ER-localized caspase 12, by activating the JNK signaling pathway, or by inducing the gene for the transcription factor CHOP (also known as GADD153), which is mediated by ATF6 and PERK (1, 3, 14, 34). To test the apoptotic response after P450 accumulation in the ER, we analyzed two of these pathways in COS1 and HepG2 cells. By using RT-PCR, we compared the level of CHOP mRNA in transfected COS1 and HepG2 cells expressing P450. There was no induction of CHOP in HepG2 cells analyzed at either 24 or 48 h after transfection, whereas in COS1 cells the level of this factor was significantly elevated at both time points (Fig. 4). These results suggest that in COS1 cells the induction of CHOP may initiate their apoptotic response.



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  		FIG. 4.
CHOP induction in HepG2 and COS1 cells overexpressing P450 2C2. RNA was prepared from HepG2 and COS1 cells transfected with C2/CMV5 DNA for either 24 or 48 h and used for RT-PCR with CHOP, BiP, and actin-specific primers as described under "Experimental Procedures." PCR products were analyzed on 2% agarose gels and detected by ethidium bromide staining.

 
IRE1 activated by ER stress may also induce apoptosis by activation of the JNK pathway (9). ER stress-induced oligomerization of IRE1 leads to the clustering and binding of the cytosolic adaptor protein TRAF2 to the IRE1 carboxyl terminus, which recruits and activates both JNK and NF{kappa}B (9, 35). We assayed JNK activation by Western analysis of lysates from transfected HepG2 and COS1 cells using an anti-PJNK antibody which specifically recognizes phosphorylated JNK, an active form of JNK during ER stress. In HepG2 cells expressing P450, PJNK was transiently increased 24 h after transfection and was only slightly increased after 48 h (Fig. 5). In contrast, a sustained increase in PJNK at 24 and 48 h was observed in COS1 cells (Fig. 5). NF{kappa}B activation has been proposed to be involved in down-regulating the proapoptotic JNK pathway in other systems (3, 14, 21, 34). It is possible then that the persistent elevation of NF{kappa}B in HepG2 cells results in suppression of JNK by 48 h, whereas in COS1 cells, in which NF{kappa}B is not activated, JNK activation persists and apoptosis is favored.



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  		FIG. 5.
JNK activation in P450-expressing cells. HepG2 and COS1 cells were transfected with 100 ng of C2/CMV5 DNA for either 24 or 48 h. Cellular lysates (40 µg of total protein) were analyzed by Western blotting, using antibody specific for phosphorylated JNK.

 
XBP1 Splicing in Response to P450 Expression—Induction of the BiP/grp78 promoter and transient activation of the JNK pathway suggests that P450 overexpression activates both ATF6 and IRE1 pathways. The critical step of the IRE1 pathway induced by ER stress is activation of the transcription factor XBP1. Activated IRE1 mediates frame-switching splicing of the XBP1, which results in formation of a potent transactivator that up-regulates its own expression and that of molecular chaperones (7, 8, 10). To analyze whether P450 accumulation in the ER activates IRE1-mediated XBP1 splicing, we performed RT-PCR on RNAs isolated from transfected cells using primers that permit the detection of both spliced and unspliced (intron-containing) mRNAs (7). As a positive control, we treated cells with the calcium-ATPase inhibitor thapsigargin, which is known to rapidly induce splicing and up-regulation of XBP1 (3). In both COS1 and HepG2 cells, the spliced variant was predominant after thapsigargin treatment (Fig. 6). In mock-transfected HepG2 cells, only a trace amount of the smaller, i.e. spliced form of XBP1 was detected, and splicing was substantially increased in P450-transfected cells at 48 h, with a modest effect at 24 h (Fig. 6). In COS1 cells, the basal level of XBP1 was lower than in HepG2 cells, and although expression of P450 slightly induced XBP1 splicing 24 h after transfection, the spliced form was even lower than in mock-transfected cells at the 48-h time point (Fig. 6). A more detailed time course of the increased XBP1 splicing in HepG2 cells showed (Fig. 7) that there was a low level of splicing at 7 h after the transfection, followed by a continuous increase to a maximum at 48 h. The total amount of the XBP1 mRNA was increased about 2–2.5-fold in P450-expressing cells at 48 h.



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  		FIG. 6.
XBP1 splicing in P450-expressing cells. HepG2 and COS1 cells were transfected with C2/CMV5 DNA for either 24 or 48 h. Untransfected cells were treated with 0.3 µM thapsigargin for 4 h. Total RNA was prepared from the cells and used for RT-PCR analysis using XBP1 and actin primers. PCR products were separated on 4% polyacrylamide gels which were stained with ethidium bromide. The positions of the unspliced (442 bp) and spliced (416 bp) products are shown as U and S, respectively.

 



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  		FIG. 7.
Time course of XBP1 splicing in HepG2 cells expressing P450 2C2. HepG2 cells were transfected with C2/CMV5 DNA for the indicated time, after which total RNA was prepared and used for RT-PCR, as described in Fig. 6. Quantification of the spliced and unspliced forms of XBP1, separated on 4% acrylamide gels, was done using NIH Image software version 1.23. Background values were subtracted from all calculations. Average values of three independent experiments are shown. Black and gray bars indicate P450- or mock-transfected cells, respectively.

 
Thus, in HepG2 cells the kinetics of XBP1 activation, similar to that of NF{kappa}B, correlated with accumulation of the P450 in the ER, in contrast to BiP induction, which is a marker of the early stress response. This is more consistent with XBP1 playing a role in ER membrane expansion, rather than a stress response, as has been suggested (10, 12, 36).

Microarray Analysis of the P450 Expression Response—To better understand genome-wide expression changes associated with the overexpression of P450, we have utilized DNA microarray analysis. Total RNA from control and P450-expressing HepG2 cells was prepared 48 h after transfection and subjected to microarray hybridization with the HumU133A oligonucleotide microarrays, which contain 22,283 probe sets (qualifiers) and represent more than 18,000 transcripts derived from human genes. We chose to focus our studies at the later time point, 48 h after transfection, because this is the time of maximal NF{kappa}B activation, and the changes in gene expression would more likely be directly associated with strong P450 accumulation. Affymetrix chip data (HG-U133A) were generated from two independent experiments (two transfections). As determined by hierarchical clustering (Fig. 8), global changes in the expression levels detected between control and P450-expressing cells in two independent transfections were very similar, indicating good reproducibility of the two data sets.



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  		FIG. 8.
Two-way hierarchical clustering analysis of gene expression in mock-transfected HepG2 cells or cells transfected with P450 expression vectors. To estimate expression levels of qualifiers, biotinylated cRNA probes were hybridized to the HumU133A microarrays; the microarrays were stained with streptavidin-phycoerythrin, and fluorescence intensity for each qualifier was measured as described under "Experimental Procedures." The range of fluorescent intensities is represented by colors as shown in the color scale for raw fluorescent intensities of qualifiers at the bottom of the figure. A total of 22,283 qualifiers on the U133A chip were clustered. On the y axis, genes were clustered based on the correlation of raw fluorescent intensities for qualifiers among the four treatment groups. On the x axis, the four samples were clustered based on a comparison of the overall pattern of gene expression in each sample, and the total length of the vertical lines connecting the different samples is proportional to the differences in expression patterns. The mock samples are more similar to each other than to either of the two induced samples, and the reverse is also true, which demonstrates the reproducibility of the data between the independent DNA array analyses.

 
Before the pairwise comparison of the P450 samples versus the control samples, data were filtered for those genes that showed either an "absent" call on the chip (which suggest no expression) or a low intensity value (<100) across all tested samples. Among 22,283 qualifiers on the U133A chip, there were 9,851 qualifiers that were eliminated based on these criteria. A gene was selected as responding to P450-induced ER overload, if its expression showed more than a 2-fold change when compared with both control samples. Based on those criteria, we selected 129 genes whose expression was up-regulated, whereas 72 genes were down-regulated by P450 overexpression (Tables III and IV of the Supplemental Material). Functional classification of the genes was performed using the Gene Ontology data base and literature searches. Table III (Supplemental Material) shows to which cellular pathway or function these up-regulated genes belong.

It appears that P450 overexpression up-regulates a large number of cellular pathways, and a detailed understanding of the affected genes will require future analysis. In the present paper we focus on connections between the different pathways implicated in the UPR or EOR by our biochemical studies. The major groups of genes implicated in these responses with elevated expression represent the following: chaperones and stress response genes, kinases/phosphatases, and other signaling molecules; proteins involved in trafficking and protein degradation; apoptosis and regulation of cell-cycle; transcription factors, and fatty acid and amino acid metabolism (Table III in Supplemental Material). Up-regulation of genes involved in amino acid and lipid metabolism would be consistent with the increased membrane biosynthesis induced by P450 accumulation.

Among the transcription factors, we observed strong upregulation of the GADD45b gene (growth arrest and DNA damage-inducible gene) and the transcription factor gene ATF3. GADD45b is known to be induced by NF{kappa}B (its promoter has three NF{kappa}B-binding sites), and it inhibits the JNK pathway in the anti-apoptotic cascade (37). ATF3 is a stress-induced factor, activated via the IRE1 pathway, and is also known as the CHOP repressor, so that its anti-apoptotic action involves down-regulation of the CHOP gene (38–40). Thus, up-regulation of these genes is consistent with our biochemical data, which showed both strong activation of NF{kappa}B, lack of CHOP induction in transfected HepG2 cells, and only transient activation of the JNK pathway during P450 overexpression. It would thus appear that at least one of the functions of NF{kappa}B activation during P450-induced ER expansion involves inhibition of apoptosis, although other functions regulating adaptation of cells to the ER expansion are also possible.

Motif Search—Recently it has been established that the sequence previously known as an ATF6-binding motif, TGACGT(G/C)(A/G), is in fact recognized by XBP1 and has been renamed the UPRE motif (41). Because we have observed significant activation of XBP1 after P450 overexpression, we searched for the binding motif of this factor in the promoters (defined as 1.5 kb of sequence upstream of the first ATG) of the up-regulated genes. From 129 up-regulated genes, putative promoter sequences are available for 89 genes, and of those, 14 genes (16%) contain the XBP1-binding motif (Table I), whereas a genome-wide search for the same motif showed that in 22,867 putative promoter sequences, only 6.4% (1,473) of the genes contained that motif. We have also searched promoters of the down-regulated genes, and of the 66 available promoters 5 (7.6%) contained a potential XBP1-binding motif, which is close to 6.4% found in whole genome, further supporting the significance of the overrepresentation of this motif in the promoters of the up-regulated genes. As Table I shows, one of those genes containing a potential XBP1-binding motif is ATF3, shown previously to be activated via the IRE1 pathway (40). ATF3 can be induced by many cellular signals, and although it has been shown to be important for liver regeneration (12, 42), its specific role in the stress response is not understood. The XBP1-binding motif is also present in the promoter of LDLR, a gene involved in lipid metabolism, a process that most likely undergoes changes during the expansion of the ER membranes. The activity of LDLR is regulated by sterol levels, so its up-regulation during increased membrane biosynthesis is not surprising. However, functional studies will be needed to establish whether XBP1 in fact regulates the expression of the ATF3 or LDLR genes.


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  		TABLE I
Up-regulated genes with an XBP1-binding site in the promoter Strand, "sense," same as the genomic DNA; "antisense," reverse strand of genomic DNA. Position, the distance of the first nucleotide of motif to "A" of ATG.

 
The results of a similar search for the NF{kappa}B-binding site, GGGRNNYYCC, in the promoters of the up-regulated genes are shown in Table II. We have found that 30 up-regulated genes contain potential NF{kappa}B-binding sites, which represents 33.7% of the tested genes. However, this is only a slight overrepresentation when compared with 31% of such genes found in the genome-wide population. Interestingly, we have found six potential NF{kappa}B-binding sites in the promoter of the XBP1 gene, which raises an intriguing possibility that XBP1 gene may be a downstream target of NF{kappa}B.


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  		TABLE II
Up-regulated genes with NF{kappa}B-binding site(s) in the promoter region Strand, sense, same as the genomic DNA; antisense, reverse strand of genomic DNA. Position, the distance of the first nucleotide of motif to the A of ATG.

 
Although XBP1 and NF{kappa}B recognition sites are present in the up-regulated genes, it is possible that some of these sites are not functional. Thus, only future functional studies will unequivocally establish the role of these transcription factors in the up-regulated genes expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our studies show that the cellular response of HepG2 cells to overexpression of P450 involves signaling pathways in both the UPR and EOR. In the UPR response, one set of activated genes is designed to correct the ER congestion and the presence of abnormal proteins to permit survival of the cell, while at the same time other genes are activated which leads to cell death. Whether apoptosis or survival is the ultimate outcome presumably depends on the severity of the insult and whether it can be corrected before an irreversible commitment to cell death. In the case of overexpression of P450 in the liver in response to a xenobiotic stimulus, survival of the hepatocytes is critical to its physiological detoxification function. The activation of NF{kappa}Bin the EOR response may be a key response for cell survival in this situation. Based on the induction of BiP, the HepG2 cell initially mounts a transient UPR response in the first few hours of the increased expression and later mounts a sustained EOR. NF{kappa}B is generally considered to be a cell survival factor that activates anti-apoptotic genes and suppresses pro-apoptotic ones (14). In the HepG2 cells this is expressed as a down-regulation of the apoptotic JNK pathway and repressed expression of the apoptotic gene CHOP. This idea is strongly supported by the observation that in the non-hepatic COS1 cell line, the early UPR occurs, but activation of NF{kappa}B is absent. The activated JNK pathway is sustained; CHOP expression is induced, and the cells are directed toward apoptosis. In HepG2 cells, the UPR activates pathways that can lead to either cell survival or cell death. The further activation of the EOR pathway may have the important effect of tilting the bias toward cell survival.

The activation of NF{kappa}B by overexpression of P450 in HepG2 cells correlates with the accumulation of P450 in the cells. Furthermore, this activation was strictly dependent on retention and accumulation of the protein in the ER. Similar NF{kappa}B induction was observed in cells expressing either a full-length P450 protein or the chimera containing only the amino-terminal signal-anchor TM sequence. This is consistent with earlier observations showing that for several ER-retained proteins the signal for both NF{kappa}B activation and ER proliferation is contained in the membrane-inserting peptide (3, 21, 43). Studies in yeast also concluded that the amino-terminal transmembrane signal-anchor of P450 was sufficient to induce ER membrane proliferation (16).

The dominant role of the ER retention function of the TM, rather than a specific signal sequence within the TM for ER membrane proliferation, is evident for the chimeric proteins C1–28/GFP and EGC. Both of the proteins contain the same 28-amino acid membrane-spanning sequence, yet only C1–28/GFP, which is directly retained in the ER, strongly activates NF{kappa}B, whereas EGC, which is transported to the plasma membrane, shows only minor activation. This observation is also consistent with NF{kappa}B activation in response to the accumulation of the ER-retained membrane protein rather than the increased volume of the ER-Golgi traffic. Interestingly, we observed that the chimeric protein C1–21/GFP, with the juxtamembrane linker region deleted, was a much weaker activator of the NF{kappa}B than C1–28/GFP although both proteins accumulate to a very similar level (C1–28/GFP is directly retained in the ER, whereas C1–21/GFP undergoes recycling between the ER and pre-Golgi (26)). This raises the possibility that the linker region may be involved in EOR signal transduction, which would be reminiscent of the report that the juxtamembrane region of aldehyde dehydrogenase is important for the ER membrane proliferation (44). Alternatively, the role of the linker may be to segregate P450 to a different subdomain of the ER which prevents recycling and results in a stronger stimulus for the EOR.

Studies in yeast have led to conflicting conclusions concerning the role of the IRE1 pathway in P450-induced ER proliferation. Takewaka et al. (18) showed that IRE1 was necessary for ER proliferation and P450 accumulation in yeast expressing P450 ALK1 (CYP 52A3). However, according to other studies, overexpression of P450 52A3 in yeast induced both the KAR2 gene and membrane proliferation, but membrane proliferation did not depend on the IRE1 gene. The authors concluded that induction of KAR2 in yeast is secondary to the ER membrane proliferation and maintains the same chaperone concentration in the increased volume of the ER (17).

Our results, showing that overexpression of P450 2C2 in HepG2 cells leads to the induction of the BiP/grp78 promoter and splicing of XBP1, are consistent with the activation of both ATF6 and the IRE1 pathways. According to the most recent models activation of the BiP and XBP1 promoters is mediated by ATF6, whereas the splicing of XBP1 mRNA is the exclusive function of IRE1 (7). In contrast to the yeast data, we have observed that the activation of the UPR chaperone BiP/grp78 promoter occurred as an early stress response, preceding maximal accumulation of the P450 proteins in HepG2 cells, whereas IRE1-mediated activation of the XBP1 gene paralleled the increase in P450 accumulation. Thus, the kinetics of the activation of these two UPR mediators is different. This result is consistent with other studies that indicate that IRE1 activation is not required for the UPR response and may be a delayed response in the UPR which induces ER-associated degradation of proteins (41). Furthermore, XBP1, which is activated by IRE1-mediated splicing of its mRNA, is known to play a role in cellular adjustments requiring substantial ER expansion, such as hepatocyte development and B-cell differentiation (12, 13). The IRE1 pathway thus is a prime candidate for inducing the proliferation of the ER.

The analysis of global gene changes in response to overexpression of P450 in HepG2 cells by DNA microarray analysis was generally consistent with the activation of both the UPR and EOR pathways. The most prominent functional group of the up-regulated genes is represented by signaling molecules, suggesting that multiple pathways are involved in adaptation of cells to the P450-induced EOR. We detected strong up-regulation of the anti-apoptotic GADD45b gene, which is known to be induced by NF{kappa}B and whose anti-apoptotic effect may depend on suppressing the JNK pathway (45). Moreover, one of the strongest activated transcription factors was stress-inducible factor ATF3, which is a negative regulator of CHOP and p53 and protects cells from apoptosis (39). Its significant up-regulation in the HepG2 cells overexpressing P450 may be involved in the suppression of CHOP activation and preventing apoptosis.

Up-regulation of genes for mostly cytosolic chaperones (HSP40, HSP70B, and DNAJ40), some of which have potential NF{kappa}B-binding sites in their promoters, may be related to their role in enhancing the folding capacity of the ER proteins exposed mostly on the cytosolic side of the membrane, like microsomal P450s, which essentially lack any luminal domain.

Up-regulation of the genes involved in lipid and amino acid metabolism (the latter ones most likely resulting from the activation of the PERK pathway) is consistent with the expected increase in membrane synthesis in response to the P450-induced EOR. Significant up-regulation of the LDLR, whose transcription is regulated by the level of sterols, may be related to the increased demand for cholesterol resulting from the proliferation of the smooth ER membranes. As in DNA microarray analysis of the UPR in HeLa cells (46), up-regulation of most genes involved in ER protein translocation and transport was not observed in HepG2 cells. This may be related to the fact that P450 expression results mainly in expansion of only smooth ER. Most interesting, in the class of genes involved in the intracellular transport, only SEC24B, related to the SEC24 gene family, was induced. Up-regulation of SEC24 may be of significance in view of the recent data suggesting that COPII components may be involved in the ER stress response and/or in the segregation of retained proteins to ER subdomains (47, 48).

We have not detected with microarray analysis up-regulation of the XBP1 gene, although our RT-PCR data showed a 2–2.5-fold increase in the total mRNA level 48 h after transfection. Most likely, this is due to the lower sensitivity of the microarray hybridization procedure and our stringent selection conditions, because XBP1 induction is only border-like for the required minimal 2-fold increase on microarray.

To examine whether the activation of NF{kappa}B and IRE1-mediated activation of XBP1 by P450 expression could be detected by analyzing the global expression of target genes for these transcription factors, we analyzed the promoters of the up-regulated genes. Although genes with XBP1-binding motifs were overrepresented in induced genes, those with NF{kappa}B-binding sites were not. Most interesting, multiple NF{kappa}B sites were present in the XBP1 promoter suggesting a cross-regulation between the EOR and UPR pathways. Several of the genes with XBP1-binding motifs were candidates for genes involved in ER stress response or proliferation such as LDLR, ATF3, MCL1, DUSP1, MAPK, and RAI3. Likewise, induced genes containing NF{kappa}B-binding motifs also include genes involved in apoptosis, lipid metabolism, and cytosolic chaperones. Whether any of these genes is in fact transcriptionally regulated by XBP1 and/or NF{kappa}B will require additional studies.

These data are consistent with the following model of the cellular response to the increasing P450 accumulation in the ER. The early response to the P450 synthesis involves induction of the UPR and general ER stress-response chaperone BiP (ATF6 pathway) and the IRE1 pathway, as indicated by a slight increase in the XBP1 splicing. Continuous P450 accumulation and increasing ER overload result in sustained activation of IRE1 and activation of NF{kappa}B. IRE1 may have a dual role of inducing ER proliferation and activation of the apoptotic JNK pathway by recruiting TRAF2. NF{kappa}B down-regulates the JNK pathway and also up-regulates some anti-apoptotic genes, ensuring survival of the hepatic cells with an expanding ER compartment which is the desired physiological outcome.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM35897, the Minnesota Lions Macular Degeration Center, and the Minnesota Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Tables III and IV. Back

§ Both authors contributed equally to this work. Back

** To whom correspondence should be addressed: University of Illinois at Urbana-Champaign, 407 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-1146; E-mail: byronkem{at}life.uiuc.edu.

1 The abbreviations used are: ER, endoplasmic reticulum; P450, cytochrome P450; UPR, unfolded protein response; EOR, endoplasmic reticulum overload response; ERSE, endoplasmic reticulum stress response element; GFP, green fluorescent protein; TM, transmembrane domain; LDLR, low density lipoprotein receptor; JNK, c-Jun NH2-terminal kinase; PBS, phosphate-buffered saline; RT, reverse transcriptase; PJNK, phosphorylated JNK. Back

2 H. Liu, unpublished data. Back



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