Regulation of BiP gene expression by cyclopentenone prostaglandins through unfolded protein response element.

Δ12-Prostaglandin (PG) J2, a cyclopentenone prostaglandin, plays a role in various stress responses. BiP, a stress-inducible chaperone protein, is implicated in protein folding and translocation in endoplasmic reticulum and induced in the condition of accumulation of unfolded proteins. Here, we examined the effect of Δ12-PGJ2 on the expression of the BiP gene. Δ12-PGJ2 markedly stimulated the expression of the BiP gene in a time- and concentration-dependent manner in HeLa cells. This stimulation was specific for cyclopentenone PGs among various PGs. Cycloheximide pretreatment completely inhibited the Δ12-PGJ2-induced expression of the BiP gene, suggesting that the effects on nascent protein synthesis are involved in the signaling mechanism. Δ12-PGJ2 markedly stimulated the promoter activity of the 5′-flanking region of the BiP gene through the unfolded protein response element. Furthermore, Δ12-PGJ2 stimulated the enhancer activity of the 3′-half of the unfolded protein response element, and this stimulation required three nucleotides within this region. Gel mobility shift assay demonstrated that this region was occupied with two specific nuclear protein factors with different mobilities in the control cells, and Δ12-PGJ2 induced the dissociation of the protein-DNA complex with lower mobility. These findings indicate that Δ12-PGJ2 stimulates the expression of BiP gene through the 3′-half of the unfolded protein response element.

⌬ 12 -Prostaglandin (PG) J 2 , a cyclopentenone prostaglandin, plays a role in various stress responses. BiP, a stress-inducible chaperone protein, is implicated in protein folding and translocation in endoplasmic reticulum and induced in the condition of accumulation of unfolded proteins. Here, we examined the effect of ⌬ 12 -PGJ 2 on the expression of the BiP gene. ⌬ 12 -PGJ 2 markedly stimulated the expression of the BiP gene in a time-and concentration-dependent manner in HeLa cells. This stimulation was specific for cyclopentenone PGs among various PGs. Cycloheximide pretreatment completely inhibited the ⌬ 12 -PGJ 2 -induced expression of the BiP gene, suggesting that the effects on nascent protein synthesis are involved in the signaling mechanism. ⌬ 12 -PGJ 2 markedly stimulated the promoter activity of the 5-flanking region of the BiP gene through the unfolded protein response element. Furthermore, ⌬ 12 -PGJ 2 stimulated the enhancer activity of the 3-half of the unfolded protein response element, and this stimulation required three nucleotides within this region. Gel mobility shift assay demonstrated that this region was occupied with two specific nuclear protein factors with different mobilities in the control cells, and ⌬ 12 -PGJ 2 induced the dissociation of the protein-DNA complex with lower mobility. These findings indicate that ⌬ 12 -PGJ 2 stimulates the expression of BiP gene through the 3-half of the unfolded protein response element.
Eicosanoids are oxygenated metabolites of arachidonic acid and are regarded as modulators of cellular functions in various physiological and pathological processes (1). Eicosanoids are divided into two groups, conventional eicosanoids and cyclopentenone-type prostaglandins (PGs) 1 , according to their mechanisms of action. Cyclopentenone PGs, such as ⌬ 12 -PGJ 2 and PGA 1 , exert a variety of biological actions, including cessation of cell growth, cell differentiation, and development (2,3). Among them, the prominent action of cyclopentenone PGs is a stress response. Cyclopentenone PGs are produced in response to various stress stimuli and then are actively transported into cells and induce the expression of various stress-related protein genes, including those of a family of cytosolic heat shock proteins (HSPs) (4,5), ribosome-inactivating protein (6), and heme oxygenase (7). The PG-induced stress protein gene expression has been shown to require de novo protein synthesis (5,8). The requirement of de novo protein synthesis for the cyclopentenone PG-induced gene expression suggests that the site of stress actions of the PGs may be on the regulation of nascent protein processing. However, the molecular characterization of stress actions of cyclopentenone PGs has been hardly carried out.
BiP is a member of the HSP family of molecular chaperones and plays an important role in the translocation of nascent proteins across the endoplasmic reticulum (ER) membrane and in their subsequent folding and assembly (9). Although BiP mRNA is synthesized constitutively, transcription of the BiP gene is induced by the accumulation of unfolded proteins in the ER, which is experimentally induced by various stress treatments, including depletion of ER Ca 2ϩ stores, reducing environment, and block of protein glycosylation (10). The promoter region of the yeast BiP gene has been identified to contain a regulatory domain, unfolded protein response element (UPR) that responds to the accumulation of unfolded proteins in the ER (11), which is highly conserved among various species (12). Thus, BiP is a key regulator for newly synthesized protein homeostasis, and the gene expression is critically controlled by an unfolded protein response pathway. From these findings, we postulated that the stress actions of cyclopentenone PGs may be correlated with an unfolded protein response linked to BiP gene expression. Therefore, we examined the effects of the PGs on the BiP gene expression. We report here that cyclopentenone PGs induce the expression of the BiP gene through UPR.
Northern Blots-Total RNA from HeLa cells was isolated using an Isogen RNA isolation kit (Nippon-gene, Tokyo), and 5 g of each RNA was separated by electrophoresis on a 1.5% agarose gel, transferred to a nylon membrane (Hybond-N, Amersham Corp.), and hybridized with a 32 P-labeled BamHI/SalI fragment of pSV-BiP (13). The same filter was rehybridized with 32 P-labeled glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA probe (Clontech). Hybridization was carried out at 65°C in 6 ϫ SSC, and the filter was washed at 65°C in 2 ϫ SSC. The filter was autoradiographed with x-ray film (Fuji RX). The radio-activity was determined with a Fuji BAS 2000 imaging analyzer (Fuji, Tokyo).
Construction of Plasmid DNA-Two different sizes of the 5Ј-flanking region of the human BiP gene (nucleotide residues Ϫ126 to ϩ90 and Ϫ92 to ϩ90, relative to the transcription start site) were obtained by means of the polymerase chain reaction from genomic DNA prepared from HeLa cells, as described previously (14). The polymerase chain reaction product was first constructed into pCR TM -II vector (Invitrogen), and then the HindIII/XbaI insert was constructed into pCAT-Basic Vector (Promega) to generate plasmid Ϫ126CAT and Ϫ92CAT, containing each 5Ј-flanking region of the BiP gene, upstream of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. The sequence of the cloned 5Ј-flanking region was confirmed by sequence analysis by the dideoxynucleotide chain-termination method. For the functional analysis of enhancer activity of UPR using a heterologous promoter, synthesized DNA fragments, encoding various mutated UPR, were inserted into tk-CAT vector, which carries the thymidine kinase (tk) promoter upstream from the CAT gene (15), and the DNA fragments were located upstream from the tk promoter in the forward and reverse directions.
Transfection and CAT Assay-HeLa cells were transiently transfected with the plasmid DNA constructs by the DEAE-dextran method (16). After the cells (3 ϫ 10 6 cells/assay) had been incubated for 30 min at 37°C in 0.5 ml of serum-free high glucose Eagle's minimal essential medium containing 5 g of plasmid DNA and 10 g of DEAE-dextran (Pharmacia Biotech Inc.), they were incubated for 10 min at 37°C with a dimethyl sulfoxide-hypertonic solution (28 mM Tris-HCl, pH 7.5, containing 0.4 M sucrose, 8% polyethylene glycol 4000, 84 mM NaCl, and 10% dimethyl sulfoxide). They were then cultured in high glucose Eagle's minimal essential medium containing 10% calf serum. Reactions were started by the addition of the test agents. After incubation for the indicated times, cellular extracts were prepared by four cycles of freezing and thawing. The CAT assay was carried out as described previously (16). Cellular extracts containing equal amounts of protein were treated at 65°C for 10 min to inactivate deacetylase and then incubated for 4 h at 37°C with 1-[ 14 C]deoxychloramphenicol (50 nCi) and 0.1 mg/ml acetyl CoA. The reaction mixtures were extracted with ethyl acetate and then separated on a silica TLC plate (F-1500, Schleicher & Schuell). After development of the TLC plate, the radioactivity was quantitated with a Fuji BAS 2000 imaging analyzer (Fuji, Tokyo). CAT activity was normalized as to ␤-galactosidase activity due to cotransfected Rous sarcoma virus-␤-galactosidase, which was assayed as described previously (16).
Gel Mobility Shift Assay-A complementary pair of DNA fragments encoding the 3Ј-half region of UPR (Ϫ109 to Ϫ99) was synthesized and then annealed as a probe. The fragment was radiolabeled at the 5Ј-ends with [␣-32 P]dCTP using Klenow fragment. The probe was purified from free nucleotides by Probe Quant G-50 micro column (Pharmacia). Nuclear extracts were prepared by the detergent lysis method of Brock et al. (17). The nuclear extracts (10 g) were dialyzed against a binding buffer (25 mM Hepes-NaOH (pH 7.9), containing 0.5 mM EDTA, 50 mM KCl, 10% glycerol, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) for 3 h at 4°C and incubated with 2 g of poly(dI-dC) and 1 ng of a 32 P-labeled probe (10,000 cpm) for 30 min at 30°C in the binding buffer. The reaction mixtures were electrophoresed on native 4% polyacrylamide gels at 4°C at 150 V for 2 h in 50 mM Tris-HCl (pH 8.5), containing 380 mM glycine and 2 mM EDTA. The gels were dried on Whatman 3MM paper and then autoradiographed with x-ray film (Fuji RX).

RESULTS
Induction of BiP mRNA by ⌬ 12 -PGJ 2 -We examined the effect of ⌬ 12 -PGJ 2 on the expression of the BiP gene in HeLa cells by Northern blot analysis. As shown in Fig. 1A, ⌬ 12 -PGJ 2 progressively increased the mRNA level of BiP, the level reaching 6-fold of the basal level by 24 h. ⌬ 12 -PGJ 2 caused marked accumulation of the mRNA in a concentration-dependent manner, the maximum being reached at 10 M (Fig. 1B). On the other hand, the mRNA level of G3PDH did not change.
We next examined the specificity of stimulation of the BiP gene expression for various PGs. As shown in Fig. 2A, ⌬ 12 -PGJ 2 and ⌬ 7 -PGA 1 markedly increased the mRNA level of BiP. PGA 1 also increased it, but the level was very low. However, the other PGs had no ability to accumulate the mRNA. We further examined the effects of various stress inducers on the expression of the BiP gene. As shown in Fig. 2B, tunicamycin and A23187 strongly increased the mRNA level of BiP. Arsenite also elevated the mRNA level, but diethylmaleate showed very low activity. ⌬ 12 -PGJ 2 was the most potent inducer of the BiP mRNA when it was compared with these inducers.
The BiP gene has been shown to be induced by the accumulation of improperly folded proteins newly synthesized in the ER (10). Then, we examined whether the ⌬ 12 -PGJ 2 -induced expression was dependent on de novo protein synthesis. As shown in Fig. 3, cycloheximide pretreatment strongly abolished the ⌬ 12 -PGJ 2 -and tunicamycin-induced accumulation of BiP mRNA, suggesting that de novo protein synthesis is necessary for ⌬ 12 -PGJ 2 -induced expression of the BiP gene.
Stimulation of Promoter Activity of the 5Ј-Flanking Region of the BiP Gene by ⌬ 12 -PGJ 2 -To determine whether UPR is the cis-regulatory element for ⌬ 12 -PGJ 2 -induced expression of the BiP gene, we examined the effect of ⌬ 12 -PGJ 2 on the transient  Fig. 4, ⌬ 12 -PGJ 2 markedly stimulated the promoter activity of Ϫ126CAT, containing UPR, by about 10-fold, but Ϫ92CAT without UPR had lost the ⌬ 12 -PGJ 2 responsiveness, indicating that the stimulation of promoter activity by ⌬ 12 -PGJ 2 is mediated by UPR. We further examined the effects of several inducers of BiP on the promoter activities of two plasmids. As shown in Fig. 5, ⌬ 12 -PGJ 2 , arsenite, and tunicamycin selectively stimulated the promoter activity of Ϫ126CAT, while A23187 slightly stimulated the activities of both plasmids. Thus, UPR is involved in the induction of BiP gene expression by ⌬ 12 -PGJ 2 , arsenite, and tunicamycin but not by A23187. Among these inducers, ⌬ 12 -PGJ 2 caused the most potent stimulation of the promoter activity.
To dissect the ⌬ 12 -PGJ 2 responsive domain in the UPR sequence, we constructed the UPR sequence mutated at either the 5Ј-or the 3Ј-half fused to another CAT gene with a heterologous promoter, tk promoter, and then examined the effects of ⌬ 12 -PGJ 2 on the enhancer activities of the mutant genes. As shown in Fig. 6, ⌬ 12 -PGJ 2 stimulated the enhancer activity of the 5Ј-half-mutated UPR in the forward or reverse orientation but not of the 3Ј-half-mutated UPR, indicating that the ⌬ 12 -PGJ 2 responsive element is located in the 3Ј-half of UPR. To identify more precise nucleotides responsible for the ⌬ 12 -PGJ 2 response within the 3Ј-half UPR, we mutated three nucleotides in the 3Ј-half UPR, which are highly conserved among various UPRs (11). This point mutation completely abolished the ⌬ 12 -PGJ 2 -induced stimulation of the enhancer activity of the 3Јhalf of UPR. These results indicate that these three nucleotides in the 3Ј-half of UPR are essential for the stimulation of the enhancer activity.
Identification of Associated Transcription Factor on UPR-To identify a nuclear protein that specifically recognizes this region of UPR, we carried out a gel mobility shift assay using the DNA fragment of the 3Ј-half UPR sequence. As shown in Fig. 7, three retarded bands were detected, and the formation of two protein-DNA complexes with the intermediate and low mobilities were competed for by an excess of unlabeled 3Ј-half of UPR fragment but not by that of the three nucleotidemutated 3Ј-half of UPR fragment, indicating that these two nuclear protein factors specifically recognize the three nucleo-tides of the sequence of 3Ј-half of UPR. Two specific protein-DNA complexes were observed in the control cells, but ⌬ 12 -PGJ 2 induced dissociation of the protein-DNA complex with the lower mobility but not the major complex with the intermediate mobility.
DISCUSSION ⌬ 12 -PGJ 2 exerts stress responses through the synthesis of a variety of stress proteins (2,3). Among the ⌬ 12 -PGJ 2 -induced protein syntheses, the mechanism for the synthesis of cytosolic HSPs has been well characterized (4,5). ⌬ 12 -PGJ 2 induces the marked expression of the HSP genes through activation of heat shock factors, which bind to the heat shock element located in the 5Ј-flanking region of the HSP gene (8). On the other hand, the 5Ј-flanking region of mammalian BiP gene does not contain a heat shock element, and thus the heat shock factor is not a regulatory transcription factor for expression of the BiP gene (14). Here, we demonstrated that ⌬ 12 -PGJ 2 stimulated the promoter activity of the 5Ј-flanking region of the BiP gene and then induced the BiP mRNA in HeLa cells, which was sensitive to cycloheximide (Figs. 1, 3, and 4). This action was mediated by UPR, which is a cis-acting element for accumulation of unfolded proteins. This is a new example of cyclopentenone PG-induced gene expression through a cis-regulatory element other than a heat shock element.
Among various PGs, the induction of BiP mRNA is specific for cyclopentenone PGs (Fig. 2). Furthermore, we showed that ⌬ 12 -PGJ 2 and ⌬ 7 -PGA 1 induced it much more potently than PGA 1 among the cyclopentenone PGs. Generally, the biological actions of PGA 1 and PGA 2 are much weaker than those of ⌬ 12 -PGJ 2 and ⌬ 7 -PGA 1 (2,3). Cyclopentenone PGs are thiolbinding molecules. We recently reported that the binding of ⌬ 12 -PGJ 2 to the intracellular particulate fractions is N-ethylmaleimide sensitive, indicating that the binding sites of ⌬ 12 -PGJ 2 are thiol groups (18). PGA 1 or PGA 2 forms a monoconjugate with a thiol (19,20), but ⌬ 12 -PGJ 2 or ⌬ 7 -PGA 1 can form a bis-conjugate with two thiols (21,22). Furthermore, the binding of PGA 1 to synthetic polymer-supported thiols as the model of thiol-containing proteins is reversible, but that of ⌬ 12 -PGJ 2 or ⌬ 7 -PGA 1 is irreversible (23). The stronger induction of the BiP mRNA by ⌬ 12 -PGJ 2 and ⌬ 7 -PGA 1 may be ascribed to their ability to form stable bis-conjugates with thiol groups. Arsenite is also a thiol-binding molecule and forms a metal-protein complex. Arsenite markedly stimulated promoter activity of the 5Ј-flanking region of the BiP gene through UPR and then induced the BiP mRNA (Figs. 2 and 5), suggesting that its binding to the thiol groups of proteins is involved in the activation of promoter activity through UPR. Thiol reactive agents, including cyclopentenone PGs and arsenite, may induce the BiP gene expression by interaction with the thiol groups of proteins, and this interaction may induce unfolded protein response.
We identified three nucleotides within the 3Ј-half of the UPR sequence that are essential to the ⌬ 12 -PGJ 2 -stimulated enhancer activity (Fig. 6). These nucleotides are highly conserved in UPR sequences of BiP genes of various species from yeast to human and in UPR sequences of GRP94, another ER-resident chaperone (11). Thus, the 3Ј-half of the UPR sequence, including these three nucleotides, is a crucial element for enhancer activity of UPR. We further identified two specific nuclear proteins, which bind to the 3Ј-half of UPR, and these putative transcription factors specifically recognized the three nucleotides in the 3Ј-half of UPR sequence, which are essential for the ⌬ 12 -PGJ 2 -induced stimulation of enhancer activity of UPR, indicating that these factors regulate the ⌬ 12 -PGJ 2 -induced BiP gene expression (Fig. 7). The homologous UPR binding activity was also reported to be constitutive, and no differences between control and stressed cells were found in both yeast and mammalian cells (24,25). On the other hand, we revealed here that the two nuclear factors stably bound to the 3Ј-half of UPR in the basal condition and that ⌬ 12 -PGJ 2 induced dissociation of the factor with the lower mobility from the DNA (Fig. 7). Thus, there are two types of nuclear factors for UPR: one nuclear factor constitutively binds to UPR, and the binding of the other factor is regulated by ⌬ 12 -PGJ 2 , indicating that the two factors are functionally different. The ⌬ 12 -PGJ 2 -induced dissociation of the factor may lead to the ⌬ 12 -PGJ 2 -induced stimulation of enhancer activity of UPR. The dissociated factor may play an important role in suppression of the enhancer activity of UPR.
We demonstrated here that ⌬ 12 -PGJ 2 induced expression of the BiP gene through UPR. This suggests that the stress actions of cyclopentenone PGs are mediated by unfolded protein response. Expression of the BiP gene is induced following exposure of eukaryotic cells to diverse conditions, which are related to pathophysiological stress (10). This response has been suggested to be important for protein homeostasis, from the folding and assembly of newly synthesized proteins to their repair or degradation, during the pathogenesis of disease states, including tissue injury and inflammation (26). Cyclopentenone PGs are final products of the arachidonate cascade, and the cascade serves endogenous stress molecules, cyclopentenone PGs, in such stress conditions. The induction of BiP by the PGs through UPR may play an important role in cytoprotective regulation of protein folding in the stress conditions. This study will contribute not only to our understanding of the BiP gene expression mechanism but will also facilitate elucidation of the molecular mechanisms of cyclopentenone PG actions.
FIG. 6. Enhancer activity of UPR using tk promoter. A, sequences of the mutant DNA fragments used for enhancer activity are shown. Wild-type sequence of the UPR is shown in capital letters. Bold letters represent the mutated sequences of the 3Ј-or 5Ј-half of the UPR region (3ЈmCAT and 5ЈmCAT), and underlined bold letters indicate further point-mutated nucleotides of 5Ј-half-mutated UPR (pm5ЈmCAT). B, indicated DNA fragments were inserted into tk-CAT vector in the forward (F) or reverse (R) direction. After cells (3 ϫ 10 6 cells) had been transiently transfected with the constructed vectors, they were treated for 12 h with (f) or without (Ⅺ) 10 M ⌬ 12 -PGJ 2 . The CAT assay was performed for cellular extracts, as described under "Experimental Procedures." The values are expressed as -fold of the untreated cell value of tk-CAT and are the means for three independent experiments, which varied by less than 10%. The value for the acetylated chloramphenicol of the untreated cells transfected with tk-CAT was 0.279 Ϯ 0.04%.  N, lanes 1-3) 10 M ⌬ 12 -PGJ 2 for 12 h, nuclear extracts were prepared. The gel mobility shift assay was performed using the 32 P-labeled DNA fragment in the absence (lanes 1 and 4) or presence of a 50-fold excess of the unlabeled 3Ј-half of UPR (lanes 2 and 5) or the three nucleotide-mutated 3Ј-half of UPR (lanes 3 and 6), as described under "Experimental Procedures." I and II represent specific nuclear protein-DNA complexes, and NS shows nonspecific binding. The results are representative of three independent experiments that yielded similar results.