©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Requirement of Tyrosine- and Serine/Threonine Kinases in the Transcriptional Activation of the Mammalian grp78/BiP Promoter by Thapsigargin (*)

(Received for publication, August 8, 1994; and in revised form, October 21, 1994)

Xianjun Cao Yanhong Zhou Amy S. Lee (§)

From the Department of Biochemistry and Molecular Biology and the Norris Cancer Center, University of Southern California School of Medicine, Los Angeles, California 90033-0800

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Depletion of endoplasmic reticulum (ER) Ca store by thapsigargin (Tg) in mammalian cells induces a set of ER protein genes known as the glucose-regulated proteins. Recently, IRE1p, a transmembrane protein postulated to have a serine/threonine kinase activity, has been identified as required for the induction of ER resident proteins genes in Saccharomyces cerevisiae. To investigate whether IRE1p can stimulate mammalian grp transcription, a stable Chinese hamster ovary cell line containing amplified copies of IRE1p has been created. The IRE1p expressing transfectants exhibited a modest (2-fold) enhancement of both the basal and Tg induced level of grp78 and grp94, two coordinately regulated grp genes. Using okadaic acid as a specific inhibitor for the endogenous serine/threonine protein phosphatase activities, a mild (2-fold) stimulative effect was observed for Tg induction of grp78 transcription. The okadaic acid potentiating effect requires a 50-base pair region in the vicinity of the grp78 TATA element. In contrast, the transcriptional activation of grp78 by Tg is almost totally eliminated by genistein, a tyrosine kinase inhibitor. The grp core, the C3 and C1 elements which are major Tg response elements of the rat grp78 promoter, are also major targets of the inhibitive effects of genistein.


INTRODUCTION

Thapsigargin (Tg) (^1)is a tumor promoter whose intracellular target has been identified as the endoplasmic reticulum (ER) Ca-ATPase (Jackson et al., 1988; Thastrup et al., 1990). Through its inhibition of the enzyme, Tg specifically discharges the ER calcium store. Within the ER resides a set of proteins known as the glucose-regulated proteins (GRPs) (Lee, 1992). GRP78, a 78-kDa protein also referred to as the immunoglobulin heavy chain protein (BiP), is one of the best characterized GRPs with protein chaperoning and calcium binding properties (Pelham, 1986; Little et al., 1994). Although GRP78 is constitutively expressed at a low basal level in many different tissues, its transcription is highly inducible by a variety of stress conditions, particularly those which perturb ER function (Lee, 1987; Watowich et al., 1988; Kozutsumi et al., 1988; Lee, 1992; Liu et al., 1992; Price et al., 1992). In mammalian cells, one of the most potent inducers for grp78 is chronic depletion of ER calcium stores by Tg or the calcium ionophore A23187 (Resendez et al., 1986; Drummond et al., 1987; Li et al., 1993). The induction process is independent of a rise in cytoplasmic Ca; furthermore, it requires a prolonged period of (2 h) of treatment and is sensitive to cycloheximide. Thus, the signaling mechanism for the activation of grp78 transcription involves more components than merely the efflux of Ca from the ER.

In examining the regulatory elements of the rat grp78 promoter which mediate the Tg and A23187 induction response, two functional domains have been identified (Wooden et al., 1991; Li et al., 1993). One region spans the promoter sequence from -159 to -110 and contains the grp core element which is conserved from yeast to human (Resendez et al., 1988) and a C3 element (Li et al., 1993). The other region spans -109 to -74 and contains a CCAAT motif (C1) most proximal to the TATA element (Wooden et al., 1991). Both regions are able to confer Tg and A23187 inducibility to a heterologous promoter upon transient transfection into mammalian cells (Li et al., 1993). Using ligation-mediated PCR, we found that specific changes in factor occupancy occurred after stress induction and that the major changes occur within the grp core element (Li et al., 1994). The factor which exhibits specific binding to this region appears to be constitutive, suggesting that during stress, the factor either undergoes a conformational change or has dissociated from inhibiting elements, resulting in the changes in the in vivo footprinting pattern.

The discovery that chronic depletion of Ca from the ER store could lead to grp78 transactivation correlated with factor interaction changes at the grp78 promoter, raises the important question of intracellular compartment signaling. What are the mediators of this response? The transcription of grp78 in fibroblast cells is partially inhibited by W-7 which inhibits Ca/calmodulin-regulated enzyme activities (Resendez et al., 1986). Other studies suggest an involvement of tyrosine and serine/threonine kinases and phosphatases. In the mammalian system, pretreatment of NIH3T3 cells with genistein, an isoflavone which specifically inhibits a number of tyrosine kinases, reduces Tg induction of grp78 mRNA levels, whereas okadaic acid (OA), a specific inhibitor of serine/threonine protein phosphatases type 1 (PP-1) and type 2A (PP-2A) (Schönthal, 1992), enhances Tg induction of grp78 (Price et al., 1992). In the yeast system, a IRE1/Ern1 gene encoding for a transmembrane serine/threonine kinase has recently been identified as required for the transcriptional induction of KAR2, the yeast homologue of grp78, in response to the accumulation of malfolded protein in the ER (Cox et al., 1993; Mori et al., 1993). It has been postulated that IRE1p is the proximal sensor of events in the ER and that binding of ligands causes transduction of information across the ER membrane, leading to activation of a specific set of transcription factors. IREp1 is of very low abundance in yeast cells. Its ligand and the involvement of IRE1p/Ern1p in Ca signaling of grp78 have not yet been determined.

Since IRE1p is the first and the only serine/threonine kinase identified so far which can affect KAR2/grp78 transcription, we created a mammalian expression vector for the yeast IRE1p and selected for stable transfectants containing amplified copies of the yeast gene. We report here that expression of IRE1p in hamster ovary cells is correlated with a 2-fold increase in the basal and Tg-induced levels of the endogenous grp78 mRNA. In mammalian cells, grp78 is coordinately regulated at the transcriptional level with grp94, which encodes a 94-kDa GRP, through common cis-regulatory elements and trans-acting factors (Chang et al., 1987; Liu and Lee, 1991). As in the yeast system where IRE1 also induces other ER resident protein genes (Cox et al., 1993), the mammalian IRE1p transfectants also exhibit 2-fold increase in the basal and Tg-induced levels of grp94 mRNA.

Because of the modest effect observed with overexpression of the yeast IRE1p protein in mammalian cells and the inherent difficulties associated with expression of a heterologous protein, we investigated the respective contributions of serine/threonine protein phosphatases and tyrosine kinases toward the Tg induction of grp78 in two different mammalian cell lines. Our results indicate that genistein eliminates the response almost completely. We further demonstrate that the inhibitive effect of genistein is at the level of transcription. We found that the grp core, the C3 and C1 elements which are the major Tg response elements of the rat grp78 promoter (Li et al., 1993), are also major targets of the inhibitive effects of genistein. The tyrosine kinase activity essential for the Tg induction of grp78 is likely to be distinct from EGFR, since lavendustin-A (L-A), a potent inhibitor for EGF-associated tyrosine kinase activity (Onoda et al., 1989), is without effect.

Using OA as a specific inhibitor for serine/threonine protein phosphatase activities, a mild (about 2-fold) stimulative effect was observed for Tg induction of grp78 mRNA levels. Nonetheless, the OA stimulative effect is not mediated through the Tg response elements; rather, it requires sequences in the vicinity of the grp78 TATA element unrelated to the known OA response elements. Thus, in the mammalian system, although a serine/threonine kinase activity may potentiate slightly the grp78 Tg response, a genistein-sensitive activity acting through the Tg response elements is the critical component of the signal transduction system that leads to transcription activation of grp78.


MATERIALS AND METHODS

Cell Cultures and Inhibitor Treatments

K12 cells, which are hamster fibroblast temperature-sensitive mutants derived from Wg1A as described previously (Lee, 1981), were maintained in Dulbecco's modified Eagle's medium containing 10% of cadet calf serum. NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium containing 10% of fetal calf serum. For the inhibitor studies, cells were grown to 90% of confluence in the above media containing 1% antibiotics (50 µg/ml penicillin, 50 µg/ml streptomycin, and 100 µg/ml of neomycin) and then preincubated with the inhibitors (genistein, lavendustin-A, and okadaic acid) (Life Technologies, Inc.) for 15 min in the fresh medium. Afterwards, the thapsigargin (Sigma) was added to incubate for another 7 or 16 h. DG44 cells, a double mutant of Chinese hamster ovary cell lines, lack the hamster dihydrofolate reductase (DHFR) gene (gift of Dr. Lawrence Chasin, Columbia University). These cells were maintained in the minimal essential alpha medium containing L-glutamine, ribonucleotides, and deoxyribonucleosides (Life Technologies, Inc.) supplemented with 10% of fetal calf serum (Urlaub et al., 1986). The DHFR transformants can be selected in the same medium without the nucleosides (alpha-minus).

Northern Blotting

After treatment with protein kinase inhibitors and thapsigargin for 7 h, the cells were washed twice in phosphate-buffer saline. Total cellular RNA was extracted following isohigh method as described previously (Zhong et al., 1994). 10 µg of RNA was loaded to 1% agarose, 2.2 M formaldehyde gels. The probe, a grp78 cDNA fragment (1.5 kb) from p3C5 digested with EcoRI and PstI, was labeled with the hexamer method to specific activities of 2 times 10^8 cpm/µg. p3A10, the entire plasmid, was labeled with the same method and used to assess the amount of loaded RNA (Lin and Lee, 1984). Conditions of the RNA blot and hybridization were described previously (Lee et al., 1983). Autoradiograms were quantitated by the densitometry to obtain relative levels of the grp78 and grp94 mRNA.

Plasmids

Plasmids(-456)CAT and(-355)CAT, previously referred to as pE43 and p3K, respectively (Chang et al., 1987), contain the rat grp78 promoter at deletion end points of -456 and -355. The construction of the plasmids (-375/-88)MCAT, (-159/-MCAT, (-109/-74)MCAT has been described (Li et al., 1993). Plasmid MCAT contains the minimal MMTV promoter fused to the chloramphenicol acetyltransferase (CAT) gene (Kim and Lee, 1989). pRSV-IRE was constructed by inserting the full-length of IRE1 gene (3.3 kb) into SalI and BamHI sites of pRSV-1 (gift of Dr. C. Gorman) (Li et al., 1992). The full-length of IRE1 gene was generated by the PCR using the plasmid pCS110 (Cox et al., 1993; gift of Dr. P. Walter, University of California, San Francisco) as template. pBluescripts KS (pBS) was purchased from the Stratagene. pBS-IRE contains the IRE1 gene derived from pRSV-IRE digested with SalI and BamHI.

In Vitro Transcription and Translation

The transcription of IRE1 gene from pBS-IRE was carried out in 20 µl of reaction mixture following the T3 transcription kit protocol (Amersham Corp.). The RNA was precipitated, and further translated in the 25 µl of reaction mixture to synthesize [S]methionine-labeled IRE1 protein (IRE1p) using a rabbit reticulocyte translation system (Promega). 5 µl of translation reaction mixture was separated on 8% SDS-PAGE.

Transient Transfection and CAT Assay

A total of 2 times 10^6 NIH3T3 cells/100-mm dish was plated 20 h before transfection. 10 µg of test plasmid was co-transfected with 5 µg of PCH110, an expression vector for beta-galactosidase (Hall et al., 1983), with the calcium phosphate precipitation method in the presence of 3 µg of carrier HeLa genomic DNA. After 24 h the cells were treated with either thapsigargin (300 nM), genistein (140 µM), okadaic acid (100 nM), or lavendustin-A (50 nM or 1 µM) alone or in combination of two reagents for 16 h. For the cells that were treated with two reagents, okadaic acid, genistein, or lavendustin-A was added 15 min prior to the addition of thapsigargin. The cells were further incubated in the presence of both reagents for 16 h before protein extraction. The protein concentration of the cell extract was determined by Bio-Rad protein assay. The CAT activities were measured at the linear range after normalization to the beta-galactosidase activities. The developed TLC plates were quantified on an Ambis Radioanalytic Imaging System (Ambis System, San Diego, CA) and exposed to films for autoradiography. Most plasmids were transfected two to five times independently.

Generation of Stable Transfectants

10 µg of pRSV-1 or pRSV-IRE was transfected into DG44 cells by using the calcium phosphate precipitation method. Twenty-four h after transfection, the cells were changed into the alpha-minus medium and grown for 14 days. The surviving colonies were exposed to 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 µM MTX for the further selection as described previously (Li and Lee, 1991). The resistant colonies were pooled together and maintained in the alpha-minus medium with the different concentrations of MTX.

Genomic DNA Hybridization

The stable transformants were grown to 90% confluence and washed twice in phosphate-buffer saline. The genomic DNA was extracted by using the previously described method (Li et al., 1989). 5 µg of each genomic DNA and 0.1 µg of pRSV-IRE were digested with EcoRI. The probe, consisting of the full-length IRE1 gene from SalI and BamHI double digestion of pRSV-IRE, was labeled by the hexamer method with specific activities of 2 times 10^8 cpm/µg. Conditions of the DNA blot and hybridization were described previously (Li et al., 1989).

Western Blot

The stable transformants in 100-mm dishes were grown to 90% confluence in the presence of 5.0 µM MTX and washed twice in phosphate-buffer saline. Cells were resuspended in 100 µl of lysis buffer (8 M urea, 10% Nonidet P-40 and 5% beta-mercaptoethanol). The supernatants were separated on 8% SDS-PAGE. The IREp was detected by using a rabbit polyclonal antisera (gift of C. Shamu and P. Walter, University of California, San Francisco) directed against the N terminus of IRE1p (1:2000 dilution) following Immun-Life Chemiluminescent Assay Kits protocol (Bio-Rad).


RESULTS

Expression of the Yeast IREp in Mammalian Cells

Recently, a 1115-amino acid transmembrane protein encoded by IRE1/ERN1 in Saccharomyces cerevisiae has been isolated from genetic screens based on its ability to activate KAR2/grp78 transcription (Cox et al., 1993; Mori et al., 1993). The cytoplasmic C terminus of this protein carries an essential kinase activity related to a cdc2/CDC28 kinase family and is postulated to be a serine/threonine kinase (Mori et al., 1993). Although it is not known whether a homologue of IRE1 exists in mammalian cells, with its discovery in the yeast system, it is possible to test whether IRE1p is functional in mammalian cells in activating grp78 transcription. Our approach is to obtain stable mammalian cell lines where the yeast IRE1 gene is amplified to produce high levels of IRE1p and to test for its ability to enhance grp78 expression over the endogenous level.

For this purpose, the yeast IRE1p coding sequence was obtained by engineering PCR primers directly flanking its ATG translation initiation codon and the termination codon (Fig. 1). After PCR amplification using the yeast genomic clone pCS110 as a template, the 3.3-kb fragment containing the IRE1p coding sequence was subcloned into the multiple cloning site located between the Rous sarcoma virus (RSV) long terminal repeat and an SV40 poly(A) site of the DHFR expression vector pRSV-1 (Li et al., 1992). This mammalian expression vector for IRE1p is referred to as pRSV-IRE. For purpose of in vitro transcription, the same IRE1p coding sequence was subcloned into a T3 vector and is referred to as pBS-IRE.


Figure 1: Construction of the expression plasmids. Plasmid pCS110 containing the yeast IRE1 gene was used to PCR-amplify a 3.3-kb full-length of IRE1 gene with SalI and BamHI sites at two ends. The IRE1 gene was then subcloned into pRSV-1 immediately after the LTR promoter to generate the pRSV-IRE plasmid, in which there is a selection marker, DHFR gene under the control of SV40 promoter. This plasmid was used to generate the stable cell line for the expression of IRE1 gene. The IRE1 gene derived from pRSV-IRE was further subcloned into pBS to generate the pBS-IRE plasmid. The pBS-IRE was used as a template in in vitro transcription under the direction of a T3 promoter.



Prior to transfection into mammalian cells, we established that the IRE1p coding sequence obtained from PCR was capable of translating into a protein close to the 127-kDa size predicted for IRE1p. In vitro transcribed IRE1p RNA was translated in a rabbit reticulocyte system in the presence of [S]methionine. As shown in Fig. 2A, only the sample containing the IRE1p mRNA was capable of synthesizing a protein band of about 130 kDa. Thus, the PCR DNA sequence is devoid of cryptic termination codons. Next, pRSV-IRE and as a control, the vector pRSV-1, were transfected into DG44 cells, which is a CHO cell line devoid of DHFR activity. Stable transfectants were selected, and the transfected plasmids were amplified by subjecting the transfected cells to increasing concentrations of MTX. Southern blot analysis of genomic DNA prepared from these cells revealed that at MTX concentrations of 0.2 µM and higher, there was notable amplification of IRE1p coding sequence in the pRSV-IRE transfectants (Fig. 2B). By comparing the band intensities with the control plasmid ran in parallel in the same DNA blot, we estimated the copy number of the IRE1p coding sequence in the stable transfectants was about 50. To confirm that pRSV-IRE stable transfectants were expressing IRE1p, total protein extract was prepared from stable transfectants selected by 5 µM MTX, as well as from pRSV-1 transfectants subjected to the same MTX selection. To detect IRE1p, a Western blot was performed using antisera raised against the N terminus of IRE1p. In the protein sample from the pRSV-IRE transfectants, an immunoreactive band of 130 kDa was detected. This band was not observed in the control sample from pRSV-1 transfectants (Fig. 2C). These results establish that in the pRSV-IRE stable cell line where the IRE1p coding sequence was highly amplified, sufficient amounts of IRE1p was produced to allow detection by immunoreactivity with antibody specific for IREp.


Figure 2: IRE1p expression in vitro and in vivo. A, [S]methionine-labeled luciferase (lane 1) and IRE1p (lane 2) were synthesized in the in vitro rabbit reticulocyte system and analyzed by 8% SDS-PAGE and autoradiography. As a negative control (lane 3), a translation reaction mixture was performed without any mRNA. The arrow indicates the location of [S]methionine-labeled IRE1p. B, pRSV-1 and pRSV-IRE were stably transfected into DG44 cell lines. Genomic DNA were isolated from DG44 (lane 1), pRSV-1 stable transfectants selected by 5 µM MTX (lane 2) and pRSV-IRE transfectants selected by increasing concentrations (0.005-5.0 µM) of MTX (lanes 3-6). The DNA was digested with EcoRI and separated on the agarose gel, blotted onto the filter, and hybridized with the IRE1 gene. In lane 7, the pRSV-IRE plasmid was digested with EcoRI. C, the IRE1p expression in pRSV-1 (lane 1) and pRSV-IRE (lane 2) stably transfected cells selected by 5 µM MTX was analyzed by Western blot using antisera against yeast IRE1p. The arrow indicates the location of IREp.



Endogenous grp78 and grp94 mRNA Levels in IRE1p-expressing Transfectants

To test whether expression of IRE1p has any effect on grp expression, total cytoplasmic RNA was isolated from both the pRSV-IRE and pRSV-1 transfectants under nontreated and Tg-treated conditions. The level of grp78 mRNA was measured by Northern blot analysis (Fig. 3A), and the fold induction was determined after normalization with a nonvariant control transcript 3A10 (Lin and Lee, 1984; Li et al., 1993) (Fig. 3B). We observed that in the IRE1p-expressing cells, the basal and Tg-induced levels of grp78 mRNA were approximately 2-fold higher than the control cells. An interesting characteristic of ire1 negative mutants in yeast is that not only is KAR2/grp78 transcription downregulated, transcription activation of other ER resident protein genes such as protein disulfide isomerase is also negatively affected (Cox et al., 1993). In mammalian cells, there is tight coordinate regulation of the grp78 gene with the grp94 gene (Lee, 1987). To test whether expression of IRE1p in mammalian cells would also affect grp94 expression, the level of grp94 mRNA in the pRSV-IRE and pRSV-1 stable transfectants was also examined. As in the case of grp78 mRNA, the level of grp94 mRNA was increased by about 2-fold in nonstressed and Tg-stressed cells (Fig. 3, A and B). Overall, our results indicate that amplification of the yeast IRE1p gene in mammalian cells and overexpression of its protein only showed a modest stimulative effect on the grp78 and grp94 mRNA levels.


Figure 3: Relative mRNA levels of grp78 and grp94 in the stably transfected cells after thapsigargin treatment. A, total RNA were isolated from pRSV-1 and pRSV-IRE stably transfected cells selected by 5 µM MTX. At 16 h prior to RNA extraction, cells were untreated(-) or treated (+) with 300 nM of thapsigargin. The RNA blots were hybridized with grp78, grp94, and 3A10 cDNA probes. The autoradiogram is shown. B, levels of grp78 and grp94 mRNA from stably transfected cells were quantitated by densitometry and normalized against the invariant 3A10 mRNA level. The mRNA levels for grp78 and grp94 from pRSV-1 stably transfected cells without treatment of thapsigargin were set as 1. The fold induction by Tg is indicated. RNA levels from cells treated with (+) or without(-) thapsigargin are plotted.



Sensitivity of Tg Induction of grp78 to Genistein and Okadaic Acid

We next investigated the respective contributions of endogenous serine/threonine protein phosphatases and tyrosine kinases toward the Tg induction of grp78 through the use of their specific inhibitors. Two mammalian cell lines (NIH3T3 and K12 hamster fibroblast cells) were tested for the effect of genistein and okadaic acid on the induction of grp78 by Tg. In the inhibitor studies, the cells were pretreated with the inhibitors, and subsequently Tg was added for 7 h, since the maximum level of grp78 mRNA accumulation occurred after 6-h of addition of 300 nM of Tg for K12 cells (Li et al., 1993). RNA blot analysis, as shown in Fig. 4A, was performed to quantitate the grp78 mRNA level which was normalized against 3A10. The relative RNA levels for both cell lines are summarized in Fig. 4B. Consistent with the previous study (Li et al., 1993), there was a 12-15-fold increase in grp78 mRNA cells after Tg treatment. OA or genistein alone only showed minor effect on grp78 mRNA basal level expression. The most striking result is that when genistein, an inhibitor for tyrosine kinase activity, was added in combination with Tg, there was a drastic reduction in the grp78 mRNA induction level. In contrast, OA, a serine/threonine protein phosphatase inhibitor, in combination with Tg only showed a slight potentiating effect in K12 cells, whereas in NIH3T3 cells, a 2-fold increase was detected. Based on these observations, we conclude that in mammalian cells, an endogenous tyrosine kinase activity sensitive to genistein is a critical component of the transactivation mechanism for the Tg induction of grp78. Serine/threonine protein phosphorylation sensitive to OA may also contribute partially to the overall grp78 induction level in NIH3T3 cells.


Figure 4: Effect of genistein and okadaic acid on the thapsigargin induced accumulation of grp78 mRNA. A, total cytoplasmic RNA was isolated from untreated K12 cells (C); cells treated with 300 nM thapsigargin (Tg) and cells pretreated with genistein then Tg added (G + Tg), with 100 nM of okadaic acid (OA), or pretreated with OA then Tg added (OA + Tg). RNA blot analysis for grp78 and 3A10 mRNA levels were performed. The autoradiograms are shown. B, the levels of grp78 mRNA from K12 and NIH3T3 cells after treatment with okadaic acid (OA), genistein (G), or thapsigargin (Tg) or in combinations (OA +Tg or G + Tg) were determined by densitometry and normalized against mRNA levels of 3A10. The levels of grp78 mRNA in untreated NIH3T3 or K12 cells (C) were set as 1. The fold induction for each of the treatment conditions is shown.



The Genistein and OA Effect Are Mediated through the grp78 Promoter

The change in grp78 mRNA levels when cells were pretreated with genistein or OA prior to addition of Tg could be due to changes in the transcriptional rate of the grp78 gene or due to changes in the stability of the grp78 mRNA. To address this issue, we examined whether genistein and OA could exert the same effects on the expression of a marker gene linked to a grp78 promoter. Previously, we have shown(-456)CAT, which contains a 418-bp fragment spanning -456 to -38 of the rat grp78 promoter linked to a CAT gene, is able to confer a 5-6-fold induction of CAT activity following Tg induction of K12 transfectants harboring this plasmid (Li et al., 1993). In this study, using NIH3T3 cells as recipient cells in transient transfection assays, a similar result was observed such that the CAT activity was enhanced about 6-fold when the cells were induced by Tg (Fig. 5, A and B). Compared with the nontreated cells with basal level set as 1, OA and genistein treatment alone resulted in 1.4- and 0.8-fold, respectively. When OA-treated cells were induced by Tg, the induction was increased to 12-fold. In contrast, genistein treatment eliminated almost completely the Tg response. Since the CAT activities are comparable with the endogenous grp78 mRNA level upon addition of OA or G in the Tg induction (compare Fig. 4B and 5B), we conclude that the major effect of genistein and OA on grp78 Tg induction is at the transcriptional level and is mediated through the grp78 promoter elements.


Figure 5: Effect of genistein and okadaic acid on the Tg induction of grp78 promoter activity. A, NIH3T3 cells were transiently transfected with(-456)CAT and subjected to treatments with OA, genistein, and Tg alone or in combinations (OA + Tg or G + Tg). The autoradiogram of the CAT assays is shown. The positions of the chloramphenicol (CM) and its acetylated forms (3Ac and 1Ac) are indicated. B, the CAT conversion in each sample was quantitated by an Ambis Radioanalytic Imaging System. The level of the promoter activity in untreated cells (C) was set as 1. The fold induction for the various treatments are shown.



The Inhibitive Effect of Genistein Acts through the grp78 Tg Response Elements

Since genistein treatment eliminated almost completely the Tg response, we tested whether the grp78 Tg response elements are targets for the genistein mediated inhibitive effect. A panel of grp78 promoter fragment/CAT fusion genes previously demonstrated to be able to respond to or confer Ca depletion stress was utilized (Kim and Lee, 1989; Li et al., 1993). As shown in Fig. 6,(-456)CAT contains multiple arrays of regulatory elements upstream of the TATA element located at -55. The important elements for Tg induction previously identified by 5` deletion, linker scanning, and internal deletion mutants include the core at -150, the C3 element at -125, and the C1 element at -95 (Wooden et al., 1991; Li et al., 1993). There are additional upstream enhancer elements such as 12-O-tetradecanoylphorbol-13-acetate response element, cAMP response element, and additional CCAAT elements; however, they do not contribute to grp78 stress inducibility (Alexandre et al., 1991). The results of all transient transfections are summarized in Table 1and some representative CAT assays are shown in Fig. 7.


Figure 6: Schematic drawings of the grp78/CAT and grp78/MCAT fusion genes used in the transient transfection assays. The 5` and 3` end points of the grp78 promoter fragments used are indicated. The positions of the core, C3, C1, and TATA elements are indicated. The small triangles indicate upstream enhancer elements. The hatched lines in the MCAT constructs represent the promoter sequence of the mouse mammary tumor virus (MMTV). The MCAT plasmid contains the promoter fragment (-105 to +104) of the MMTV fused to the CAT reporter gene. The target regions for the inhibitive effect(-) of genistein (G) and okadaic acid (OA) and stimulative effect (+) of OA during Tg induction are bracketed.






Figure 7: Differential effect of genistein and okadaic acid on the Tg induction of grp78/CAT and grp78/MCAT fusion genes. Left panels, comparison between(-355)CAT and (-375/-88)MCAT. Right panel, comparison between (-109/-MCAT and the minimal MMTV promoter driven CAT (MCAT). NIH3T3 cells were transiently transfected with these plasmids and subjected to the various treatment conditions (C, Tg, OA, G, OA + Tg, G + Tg). The autoradiograms of the CAT assays are shown. The position of chloramphenicol (CM) and its acetylated forms (3Ac and 1Ac) are shown.



We next tested (-375/-88)MCAT, which contains 287 bp of the grp78 promoter fused to a minimal MMTV promoter. The results showed that the 4-fold induction of CAT activity by Tg was completely eliminated when the cells were treated with genistein in combination with Tg, whereas genistein alone showed no effect (Table 1). Thus, genistein response elements are located within this 287-bp region. One of the target domains spans -159 to -110, since duplicate copies of this subfragment when linked to the minimal MMTV promoter (-159/-110)CAT was able to confer a 8-fold induction by Tg. However, this induction was reduced to 1.6-fold when genistein was added (Table 1). This region contains the 3`-half of core and C3 element. Another target region spans -109 to -74 and it contains the C1 element. Duplicate copies of this subfragment linked to the minimal MMTV promoter (-109/-74)CAT were able to confer a 13-fold induction by Tg. Addition of genistein and Tg completely eliminated the induction ( Fig. 7and Table 1).

To demonstrate that the inhibitive effect of genistein on grp78 transcription is not due to a general toxic effect of tyrosine kinase inhibitors and to delimit the tyrosine kinase activity targeted by genistein, we tested the effect of L-A, a potent inhibitor for EGF receptor-associated tyrosine kinase activities (Onoda et al., 1989) on(-456)CAT and(-355)CAT. Two concentrations (50 nM and 1 µM) of L-A were used. The results, as shown in Table 1, demonstrated that L-A, either added alone or in combination with Tg, did not have any effect.

The Potentiating Effect of Okadaic Acid Requires a 50-bp Region in the Vicinity of the grp78 TATA Sequence

In examining the response of (-375/-88)MCAT, (-159/-110)MCAT and (-109/-74)MCAT to OA treatment in combination with Tg, we discovered that instead of a stimulative effect as observed for(-456)CAT, OA actually reduced slightly the Tg induction. Thus, the Tg induction for the three plasmids listed decreased from 4-, 8- and 13-fold to about 2-, 6-, and 8-fold, respectively ( Fig. 7and Table 1). These results indicate that in NIH3T3 cells, the enhancing effect of OA on grp78 transcription is not mediated by the known Tg response elements of the grp78 promoter.

Since OA exerts a potentiating effect on(-456)CAT but not on (-375/-88)MCAT (Fig. 7), the target for the stimulation either lies in the upstream region spanning -456 and -375 or in the downstream region spanning -88 to -38 (Fig. 3). The upstream region contains basal enhancer elements and the downstream region contains the sequence surrounding the grp78 TATA element at -55 but excludes transcription initiation located at (+1) (Li et al., 1993). To differentiate between these two possibilities, we first compared the response between(-457)CAT and(-355)CAT. Our results show that these two plasmids responded nearly identically to the treatment of Tg and OA (Table 1). Thus, the upstream sequence between -456 and -355 is not needed for the potentiating effect as both plasmids showed that OA enhanced the Tg induction from about 6-12-fold. Next the response between(-355)CAT and (-375/-88)MCAT was compared. While both plasmids showed a 4-5-fold induction by Tg, OA in combination with Tg stimulated the(-355)CAT activity to 11-fold and reduced (-375/-88)MCAT activity to 2-fold ( Fig. 7and Table 1). The minimal MMTV promoter (MCAT) was relatively unaffected by Tg or the various inhibitors, either added alone or in combinations with Tg ( Fig. 7and Table 1). Our results show that the OA stimulative effect of grp78 Tg induction requires a 50-bp downstream region surrounding the grp78 TATA sequence. Previously, we demonstrated that this same region, when fused to a CAT reporter gene, is almost devoid of any basal promoter activity and is not inducible by Ca depletion stress (Resendez et al., 1988).


DISCUSSION

With the recent discovery that in the S. cerevisiae a serine/threonine kinase (IRE1p) plays a critical role in activating the ER protein gene transcription in response to malfolded protein accumulation stress, the question arises as to whether protein phosphorylation is also utilized as a mechanism for the induction of the mammalian ER protein genes. In this report we focused on grp78 gene expression which is induced to high levels by thapsigargin following chronic depletion of the ER Ca store and provide several lines of evidence that protein phosphorylation and dephosphorylation are involved in grp transactivation.

Our first approach is to engineer the coding sequence of IRE1p into a mammalian expression vector and create a stable CHO cell line with amplified copies. This was achieved, and a cell line harboring about 50 copies of the yeast gene was established. In these cells, sufficient amounts of IRE1p were expressed to allow identification with specific antibody against the protein. Nonetheless, under these favorable conditions, the enhancement for grp78 mRNA level in stressed and nonstressed cells was about 2-fold. Could this slight increase in grp78 mRNA levels be due to the serine/threonine kinase activity of the transfected gene? The fact that the 2-fold induction we observed is similar to that reported for S. cerevisiae when the same protein is overproduced over the endogenous level (Mori et al., 1993) suggests that this might be the physiological limit for this protein in a normal cell background. Furthermore, the co-induction of other ER protein genes such as grp94 in the IRE1p expressing cells is consistent with the action of IRE1p in yeast cells. On the other hand, expression of a heterologous protein has its inherent problems which are difficult to resolve in a mammalian system. For example, the modest enhancement of the grp mRNA levels could have been caused by improper insertion of IRE1p into the designated cellular compartment, thus diminishing the capacity of IRE1p to fully induce the grps. Another complication is that the portion of IRE1p located in the endoplasmic reticulum might not fold properly. This effect could have elicited a mild grp stress response. Although amino acids essential for the kinase activity have been identified within IRE1p (Mori et al., 1993), to create an equivalent CHO cell line with the amplified mutant genes at the same level of expression as the wild type gene to differentiate between a 2-fold effect is technically difficult, if not impossible. The functional significance of IRE1p in animal cells is best evaluated when the fold induction of the transfected wild-type protein can be manipulated so that it is sufficiently high to allow mutational analysis. This may be possible using recipient cells with the homologous mammalian gene deleted. However, this requires the confirmation that such a gene exists in mammalian cells and its subsequent isolation. Based on the current limitations, we focused our effort into examining the contribution of endogenous protein kinases and phosphatases in the Tg induction of grp78.

Potent protein kinase inhibitors have been used widely as biochemical tools to study the function of kinase and phosphatase activities in regulating gene expression in mammalian cells. In this study we use a panel of specific inhibitors to determine their effect on grp78 induction by Tg. The majority of intracellular phosphatase activity has been attributed to PP-1 and PP-2A, and OA has the ability to inhibit both enzyme activities. Generally, PP-2A is inhibited more efficiently than PP-1 (Cohn, 1989). In our experiments, we discovered that OA only exhibits a mild potentiating effect on grp78 transcription. Since the effect is more pronounced at 100 nM of OA than 50 nM(^2), PP-1, rather than PP-2A, may be the affected phosphatase. OA has been reported to stimulate a large number of gene systems, including that of the heat shock gene (Schönthal et al., 1991; Guy et al., 1992; Chang et al., 1993). Since OA strongly increases the heat shock hsp70 promoter activity, it was proposed that the heat-induced transcriptional activation of the heat shock gene is associated with the phosphorylation of component(s) of the transcriptional complex and that OA enhances this phosphorylation (Chang et al., 1993). Based on the optimal concentrations of OA, PP-2A, as opposed to PP-1, was implicated in this induction process. Thus, although grp and hsp genes are both stress-inducible genes and share some common induction conditions (Lee, 1987; Watowich and Morimoto, 1988), their response to OA during stress is not identical.

In examining the grp78 promoter sequence which is required for the OA stimulative effect on Tg induction in NIH3T3 cells, we discovered that it does not act through the Tg response elements. Rather, it exerts a mild inhibitive effect on those elements. A 50-bp region in the vicinity of the grp78 TATA sequence is required for the OA stimulative effect of Tg induction. Interestingly, no sequence resemblance was found with known target sites of OA action (Wera et al., 1993; Kharbanda et al., 1993; Chauhan et al., 1994; Hyun et al., 1994), and the majority of the previously described OA response elements are upstream regulatory sequences. Thus, the grp78 target may represent a novel site. However, OA does not have to act directly on the transcription factors which bind to the 50 bp region. For example, it could inhibit a serine/threonine protein phosphatase activity which modifies the activity of any intermediatory regulatory components. Nonetheless, our finding that OA only exerts a modest effect on the Tg induction of grp78 and that the response does not involve the Tg response elements prompted us to investigate whether kinases other than that of the serine/threonine type are more critical elements for the Tg induction process in mammalian cells.

Our experiments indicate that genistein, a tyrosine kinase inhibitor, almost totally eliminated the Tg induction response. Furthermore, we have found that the grp core, the C3 and C1, which are major targets for Tg induction, are also major targets of the inhibitive effect exerted by genistein. Thus, genistein acts either directly on the transcription factors which interact with these promoter regulatory elements, or it modifies other targets in the intermediary pathways leading to the activation of the nuclear transcription factors. Recent examples suggest that genistein can exert its effect both ways. In the T cell antigen receptor signaling system, genistein was used to identify a tyrosine kinase activity which down-regulates specifically calcium-mediated signaling at a point downstream of the tyrosine kinase p56 but upstream of calcium mobilization (Baldari and Telford, 1994). Genistein, on the other hand, blocks the transcriptional stimulation of interferon-induced genes by inhibiting tyrosine phosphorylation of the transcription factor ISGF3 which binds directly to the interferon-stimulated response element found in the promoter of inducible genes (Gutch et al., 1992). In the grp78 promoter, the core, C3, and C1 act in concert to enhance grp expression (Wooden et al., 1991). Thus, it is possible that upon stress induction, as in the case of ER Ca depletion, a common tyrosine kinase activity is activated which leads to the modification of the binding factors or their co-activators, resulting in grp78 induction.

Among kinase inhibitors, genistein is a specific inhibitor for tyrosine kinases but not serine and threonine kinases and other ATP analogue-related enzymes (Akiyama et al., 1987). Thus, it has minimal effect on the enzyme activities of cAMP-dependent protein kinases, protein kinase C, phosphorylase kinase, 5`-nucleotidase and phosphodiesterase. While it is highly potent against tyrosine kinases such as the EGF receptor, pp60 and pp110, it is also capable of inhibiting tyrosine kinases other than these three well characterized ones. Since the targets for genistein can be broad, we initiated experiments to determine which type of tyrosine kinase activity is involved in the Tg induction of grp78. We found that treatment of the cells with lavendustin-A, a specific inhibitor for EGF receptor-associated tyrosine kinase, neither stimulates nor inhibits the Tg response, suggesting that the tyrosine kinase activity mediating the grp response to Tg is likely to be distinct from that of the EGF receptor type. Based on our findings, we hypothesize that a tyrosine kinase activity is essential for grp induction by Tg. It is also possible that genistein inhibits grp induction through a yet unknown mechanism. The identification of the genistein sensitive components, their subcellular localizations and regulatory mechanism will contribute significantly to the understanding of how ER protein genes respond to Ca depletion stress in mammalian cells.


FOOTNOTES

*
The work was supported by United States Public Health Service Grant R37 CA27607 from the National Cancer Institute (to A. S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 213-764-0507; Fax: 213-764-0094.

(^1)
The abbreviations used are: Tg, thapsigargin; ER, endoplasmic reticulum; GRP, glucose-regulated proteins; PCR, polymerase chain reaction; OA, okadaic acid; PP-1 and -2, protein phosphatases type 1 and type 2; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor: L-A, lavendustin-A; DHFR, dihydrofolate reductase; kb, kilobase pair(s); CAT, chloramphenicol acetyltransferase; bp, base pair(s); MMTV, murine mammary tumor virus; PAGE, polyacrylamide gel electrophoresis; MTX, methotrexate; RSV, Rous sarcoma virus.

(^2)
X. Cao, Y. Zhou, and A. S. Lee, unpublished results.


ACKNOWLEDGEMENTS

We acknowledge Cory Gorman for the gift of the pRSV-1 plasmid, Larry Chasin for the DG44 cells, Caroline Shamu and Peter Walter for the pCS110 plasmid and the IRE1p antisera. We thank Axel Schönthal, Wilfred Li, and Meera Ramakrishnan for critical review of the manuscript.


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