©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Generation of a Mammalian Cell Line Deficient in Glucose-regulated Protein Stress Induction through Targeted Ribozyme Driven by a Stress-inducible Promoter (*)

Edward Little , Amy S. Lee (§)

From the (1) 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

GRP94 is an endoplasmic reticulum (ER) localized glycoprotein with Cabinding and protein chaperoning properties. Using a ribozyme driven by a stress-inducible promoter and targeted against grp94 mRNA, we have generated a cell line deficient in its ability to induce GRP94. The effect of the ribozyme is mediated by the cleavage of the grp94 message just downstream of the initiation codon, and not by an antisense effect, as determined by the level of intact grp94 mRNA. Unexpectedly, this cell line's ability to induce GRP78 is also impaired. Transient overexpression of recombinant human lysosomal hydrolase - L-iduronidase in the ribozyme expressing cells indicates that the secretion ratio of this enzyme is reduced by about 6-fold. Additionally, the ribozyme expressing cells showed increased sensitivity to Cadepletion from ER caused by either A23187 or thapsigargin, an ER-Ca-ATPase inhibitor, but not to tunicamycin. These combined results show that the induction of GRP94 may play important roles in ER to nuclear signaling, protein sorting and secretion, and specific protection against Cadepletion stress.


INTRODUCTION

The endoplasmic reticulum (ER)() is the cellular organelle where proteins, lipids, and complex carbohydrates that are destined for transport to the Golgi apparatus, the plasma membrane, lysosomes, or the cell exterior are synthesized and processed. The ER is also an important store for intracellular Ca. Residing within the ER is a class of proteins known as the glucose-regulated proteins (GRPs) (Lee, 1987; Hightower, 1991; Little et al., 1994). The GRPs are ubiquitously expressed stress-inducible proteins that are related to the heat shock proteins (HSPs) (Watowich and Morimoto, 1988; Wooden and Lee, 1992). GRP94, a 94-kDa protein also referred to as ERp99 (Mazzarella and Green, 1987) or endoplasmin (Koch et al., 1986), is an abundant ER glycoprotein. It shares 50% amino acid identity to HSP90, the 90-kDa member of the heat shock protein family (Mazzarella and Green, 1987). The function of HSP90 as a molecular chaperone is exemplified by its ability to bind to a wide range of cellular proteins, including that of the glucocorticoid receptor and pp60src (Hutchison et al., 1993).

Recent evidence suggests that GRP94 may also possess protein chaperoning activity since its synthesis is induced by the accumulation of malfolded proteins in the ER (Kim et al., 1987; Kozutsumi et al., 1988). GRP94, together with other ER molecular chaperones, has been found to form stable complexes with viral and cellular proteins in the ER. For example, a mutant form of the herpes simplex virus 1 glycoprotein B with altered conformation accumulates in the ER. GRP94 and GRP78, a 78-kDa ER lumen protein, stably complexed with the mutant gB but not with the fully processed viral protein (Navarro et al., 1992; Ramakrishnan et al., 1995). Similarly, major histocompatibility complex class II molecules expressed in the absence of the invariant chain remained in an immature form in the ER and are associated with GRP94 and ERp72, another member of the GRP family (Schaiff et al., 1992). Recently, GRP94 has also been shown to participate in normal protein folding and assembly. In the ER, GRP78 and GRP94 act in tandem on folding intermediates of the newly synthesized immunoglobulin chains, with GRP94 assisting at a more advanced step in their processing (Melnick et al., 1992, 1994). In another study, GRP94 has been demonstrated to be associated with an ATPase activity and contain tightly bound peptides (Li and Srivastava, 1993).

Other lines of evidence strongly suggest that GRP94 and other ER lumen proteins are major Ca-binding proteins. While the binding affinity for Cais low, GRP94 and GRP78 can bind superstoichiometric amounts of Ca(Koch et al., 1986), of which the ER is a major store. The GRP94 and GRP78 are also present in the sarcoplasmic reticulum vesicles of cardiac and skeletal muscles, another organelle known to contain high levels of Ca(Milner et al., 1992; Volpe et al., 1992; Cala and Jones, 1994). Thus, as low affinity but high capacity Ca-binding proteins, GRPs may play significant roles in protein trafficking (Gething and Sambrook, 1992) and intracellular Cahomeostasis (Brostrom et al., 1990).

GRP94, while expressed constitutively in most cell types under normal growth conditions, is highly induced in stressed cells. The most potent inducing reagents are those that disrupt the ER function (Lee, 1987, 1992; Little et al., 1994). This includes depletion of intracellular Caby the calcium ionophore A23187 (Resendez et al., 1986; Drummond et al., 1987) and thapsigargin, which specifically inhibits the ER Ca-ATPase (Li et al., 1993). Another powerful inducer of GRP94 is the blockage of N-linked protein glycosylation through treatment of cells with tunicamycin or temperature-sensitive mutation of the oligosaccharide transferase (Lee et al., 1986; Kim et al., 1987). With these and other stress treatments, there is a 10- and 25-fold increase in the transcriptional rates of grp94 and grp78, respectively. Furthermore, the two genes appear to be coordinately regulated at the transcriptional level through common trans-acting transcription factors recognizing common promoter elements (Lee, 1987; Chang et al., 1989). For instance, amplification of a transfected plasmid containing the common promoter regulatory elements led to the titration of the trans-acting factors from the endogenous genes. This resulted in a concomitant reduction of GRP94, GRP78, and ERp72, slower growth rates, and increased sensitivity of cells to the calcium ionophore A23187 (Li and Lee, 1991).

To dissect the functional contribution of the individual GRPs and to understand their coregulation mechanism, it is necessary to inhibit the expression of each of the GRPs and study the resulting phenotypes. Here we report the successful use of a targeted ribozyme driven by a stress-inducible grp promoter to modulate the level of grp94 mRNA in stressed and non-stressed mammalian cells. Ribozymes are small RNAs that can catalytically cleave a phosphodiester bond in the target RNA (Forster and Symons, 1987; Koizumi et al., 1988). While engineered ribozymes have been used successfully to reduce gene expression in vivo, in many of these studies it is not clear that gene expression has been reduced by the cleaving activity of the ribozyme or by its inherent antisense activity (Castanotto et al., 1992). Further, the use of tissue-specific or inducible promoters to modulate ribozyme expression in inhibiting cellular gene expression has only been explored recently (Zhao and Pick, 1993; Efrat et al., 1994). We show here that a ``hammerhead'' ribozyme targeted against the grp94 transcript is able to cleave the grp94 RNA both in vitro and in vivo. Upon stress treatment, induced higher level of the ribozyme is able to significantly reduce the level of intact grp94 mRNA, leading to minimal stress induction of the GRP94 protein. To our surprise, in the transfected cells which exhibit impaired GRP94 induction, the level of stress-inducible grp78 mRNA and protein is reduced by half.

With the establishment of these stable cell lines, we examined the role of GRP94 as a molecular chaperone in protein trafficking. The highly glycosylated enzyme - L-iduronidase (EC 3.2.1.76) is translated into the ER and later processed in the Golgi (Rome et al., 1978). The majority of this protein is normally targeted to the lysosome by means of a mannose 6-phosphate marker, but a small fraction is secreted. Overproduction of this protein in mammalian cells causes it to be selectively secreted at a higher rate (Kakkis et al., 1994). In overexpressing this human recombinant protein in the ribozyme expressing cells, we observed that the secretion ratio of this enzyme is much impaired. Additionally, we compared the protective roles of GRPs in calcium homeostasis and glycosylation block. The increased sensitivity to A23187 and thapsigargin but not tunicamycin in ribozyme expressing cells shows that GRP94 plays an important protective role in cells under Castress conditions, but may not be sufficient to confer protection against glycosylation block stress. The possible role of GRP94 as a mediator in the grp stress response is also discussed.


MATERIALS AND METHODS

Cloning of the Full-length Rat grp94 cDNA

The gt10 library used to isolate the full-length rat grp94 cDNA clone was generously provided by Dr. Jonathon Howard (Cambridge Research Station). About 10bacteriophage plaques were transferred to nitrocellulose membranes (Scheider and Schuell) in duplicate. Prehybridization, hybridization, and washing conditions were as described previously (Ting and Lee, 1988), except the probe used was the 1-kb BamHI- EcoRI fragment of the hamster grp94 cDNA from the plasmid p4A3 (Lee et al., 1983). This fragment was hexamer-labeled (Feinberg and Vogelstein, 1983) to a specific activity of approximately 5 10cpm/µg. Nineteen plaques were detected and purified with two more rounds of plaque hybridization. The largest insert was identified by EcoRI digestion of the positive clones and subcloned into the EcoRI site of pBluescript II KS(Stratagene). The 5` and 3` ends of the cDNA were sequenced using the dideoxy chain termination method (Sanger et al., 1977) to verify the initiation codon and poly(A) addition signal. This clone was designated pBS94.

Plasmid Constructions

The plasmid pRc/RSV was purchased from Invitrogen, pUC8 from New England Biolabs, and pSP65 from Promega.

The pRc/GRP plasmid was derived from the pRc/RSV plasmid. It was constructed by removing the RSV LTR promoter from the pRc/RSV plasmid by digestion with NruI and HindIII and purifying the 4.7-kb vector fragment from low melting point agarose. The proximal 1.3 kb of the rat grp78 promoter was isolated from the plasmid p2I (Resendez et al., 1985) by digestion with PstI and BssHII and followed by purification from low melting point agarose. The two DNA fragments were blunt end-ligated. Correct insertion of the new promoter was verified by restriction digests.

The plasmid pUC-ribo1 was constructed as follows: 1 nmol each of 2 oligo-nucleotides, 5`-TAATACGACTCACTATAGG-3`, and 5`-ATGAGGGTTTCGTCCTCACGGACTCATCAGCTGTGGGTCCCTATAGTGAGTCGTATTA-3`, were used. The underlined region indicates the T7 polymerase site. These oligomers were annealed by heating to 85 °C for 3 min, then cooling to room temperature. The resulting hemi-duplex was filled in by Klenow DNA polymerase and kinased by T4 polynucleotide kinase. The double-stranded DNA was eluted by crush-soak method from an ethidium bromide-stained 6% polyacrylamide gel and ethanol-precipitated. This fragment was cloned into the SmaI site of pUC8.

pSP65-ribo1 was constructed by cloning the 93-bp HindIII- EcoRI fragment from pUC-ribo1 into the HindIII- EcoRI-digested pSP65.

pRc/RSV-ribo1 was constructed by cloning the 72-bp BamHI- EcoRI fragment from pUC-ribo1 into the NotI-digested pRc/RSV.

pRc/GRP-ribo1 was constructed by cloning the 72-bp BamHI- EcoRI fragment from pUC-ribo1 into the NotI-digested pRc/GRP. The correct orientation of the ribozyme in each of the expression plasmids was confirmed by DNA sequencing.

In Vitro Ribozyme Assay-Two µg of EcoRI linearized pUC-ribo1 was transcribed with T7 DNA polymerase in a buffer containing 40 m M Tris HCl, pH 8.0, 50 m M NaCl, 8 m M MgCl, 2 m M Spermidine, 200 n M ATP, CTP, and GTP, 20 n M UTP, 20 µCi of [P]UTP (DuPont NEN), 150 n M dithiothreitol, 40 units of RNasin. The target of the ribozyme catalyzed cleavage was transcribed from the FokI-digested pBS94 essentially as above, but with 40 units of T3 RNA polymerase, 80 n M UTP, and 30 µCi of [P]UTP. The target RNA was purified by PAGE and excising the 260 nt band (detected by exposing the wet gel to Kodak X-OMAT film) and eluted by crush-soak method.

The in vitro cleavage reaction generally contained approximately 0.1 pmol OF target RNA and 10-fold excess of ribozyme, incubated in 10 µl of 50 m M Tris-HCl, pH 7.5, 20 m M MgCl, 1 m M EDTA at 50 °C for 1, 2, and 4 h. One-fifth volume of 0.5 M EDTA was added as the reactions were completed to stop the reactions. The reaction mixes were kept at -80 °C until all reactions were complete. The products were separated on a 6% PAGE, which was then dried and exposed to film at -80 °C for 16 h.

Cell Culture

The NRK cell lines, their culture conditions, and DNA transfection protocols have been previously described (Resendez et al., 1985). 400 µg/ml G418 was added 2 days after transfection. Survivors were pooled after 3 weeks and maintained under the same selection conditions.

Cell survival studies were performed by seeding 2,000 cells/10-cm dish in duplicate for each of the concentration of drugs used as described previously (Li and Lee, 1991). Growth rates of each construct were measured as described previously (Li and Lee, 1991), except that 50,000 cells were seeded per 10-cm dish. Each experiment was repeated twice.

DNA Isolation and Southern Blot Analysis

Cells were seeded in 15-cm dishes and harvested after reaching 100% confluence. DNA was extracted as described previously (Li and Lee, 1991). Ten µg of the respective DNA was digested with PvuII or EcoRI at 10 units/µg DNA at 37 °C for 16 h. These digests were run on a 1.2% agarose gel and transferred to a nitrocellulose membrane. The DNA was cross-linked to the membrane by UV irradiation (Stratagene UV Stratalinker 2400). Prehybridization, hybridization, and washing conditions were as described previously (Li and Lee, 1991).

The probe made to detect the constructs was generated by digesting the pRc/GRP-ribo1 plasmid with EcoRI and isolated from a 1% low melting agarose gel. The 1 kb band was then excised and labeled directly via hexamer labeling to an approximate activity of 5 10cpm/µg.

RNA Isolation and Northern Blot Analysis

Cells were seeded in duplicate in 15-cm dishes. Once cell density reached approximately 70-80% confluence, the media was replaced with fresh media. To one dish of each set was added the appropriate GRP inducing agent (7 µ M A23187 or 1.5 µg/ml tunicamycin). After 16 h, the media was removed, and the cells washed three times with phosphate-buffered saline. Conditions for RNA isolation, gel electrophoresis, and blot hybridization have been described (Lee et al., 1983). The first probe was generated by digesting the pBS94 clone with NlaIII and isolating the 1 kb band from a low melting point agarose gel. This probe contained approximately 900 bp of plasmid sequence and the first 104 bp of the rat grp94 cDNA. Grp78 cDNA (Lee et al., 1983) and glyceraldehyde-3-phosphate dehydrogenase cDNA (Tso et al., 1985) probes were also used. Probes were hexamer labeled to an approximate specific activity of 5 10cpm/µg.

RNase Protection Analysis

For detection of ribozyme transcript, an SP6 antisense probe was synthesized from HindIII-linearized pSP65-ribo1 and hybridized to total RNA samples, and analyzed essentially as described (Naeve et al., 1992), with the exception that the hybridization temperature was 42 °C. Protected fragments were separated on 6% sequencing gels, which were then dried and exposed to film at -80 °C with an intensifying screen. The transcribed probe was 105 nt, and a protected 72-nt fragment was expected from the in vivo transcribed ribozyme. As a control, the ribozyme was also transcribed in vitro with T7 polymerase (40 m M Tris-HCl, pH 8.0, 50 m M NaCl, 8 m M MgCl, 2 m M Spermidine, 200 n M NTP, 150 n M dithiothreitol, 40 units of RNasin, 40 units of T7 RNA polymerase) and hybridized with the probe. The expected protected size for the in vitro transcribed ribozyme was a 48-nt fragment.

Protein Isolation and SDS-PAGE

Seventy % confluent cells were labeled with 100 µCi of [S]methionine (DuPont NEN) in duplicate in 10-cm dishes in 5 ml of methionine-free media containing 10% dialyzed fetal calf serum. One dish from each set was treated with the appropriate GRP inducing agent (7 µ M A23187 or 1.5 µg/ml tunicamycin). Proteins were isolated as described previously (Li et al., 1992) and separated on an 8% SDS-PAGE as described by Laemmli (1970) using 10cpm from each sample.

Western Analysis

Proteins from non-labeled control and A23187-induced cells from the pRc/GRP and pRc/GRP-ribo1 cell lines were isolated and resuspended in RIPA buffer (10 m M Tris-HCl, pH 7.5, 150 m M NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 m M phenylmethylsulfonyl fluoride, and 1 µg/ml pepstatin). Quantitation of the protein was determined by the Bio-Rad Protein assay. For GRP94, 40 µg from each cell line were separated on denaturing SDS-PAGE and transferred electrophoretically to Immun-Lite Blotting Membrane (Bio-Rad). For GRP78, 120 µg of total protein was required. Equal loading of the protein samples was confirmed by Coomassie Blue staining. The protocol described in the Immun-Lite Assay kit (Bio-Rad) was used for protein detection with the exception that blocking was performed in 2.5% non-fat dry milk in 1 TBS (20 m M Tris-HCl, pH 7.5, 500 m M NaCl) for GRP78. For GRP94, 1% non-fat dry milk and 0.5 TBS were used. The primary antibodies were rat monoclonal anti-GRP94 and mouse monoclonal anti-GRP78 antibodies (Stressgen) at dilutions of 1:1,000 and 1:500, respectively. Alkaline phosphatase-conjugated goat anti-rat antibody (Oncogene Science) and goat anti-mouse (Bio-Rad) at dilutions of 1:3,000 were used as the secondary antibodies.

Enzyme Assays

7.5 10cells from each of the cell lines were seeded in 10-cm dishes in triplicate. Two days later, the cells were transiently cotransfected with 10 µg each of the human - L-iduronidase expression plasmid pRc/CMV-huId (Kakkis et al., 1994) and the -galactosidase expression plasmid pRc/CMV--gal (both are a gift of Dr. Emil Kakkis, UCLA Harbor) by the calcium phosphate method as described previously (Wooden et al., 1991). Two days after transfection aliquots of the media were removed from each dish and assayed for secreted - L-iduronidase activity (Kakkis et al., 1994). The cell pellets were split to assay for intracellular - L-iduronidase and -galactosidase activities (Wooden et al., 1991) as described. The latter was used to standardize the transfection efficiencies of each dish of cells.


RESULTS

Ribozyme Targeted against grp94

A full-length rat grp94 cDNA was isolated from a gt10 cDNA library and sequenced. Based on its 5` terminus sequence, a hammerhead type ribozyme (Sarver et al., 1990) was designed such that cleavage of the grp94 transcript by the ribozyme would occur close to the AUG initiation codon (Fig. 1). Sequencing of the grp94 cDNA further downstream revealed no in frame AUG codons for over 300 bases (data not shown). Thus, the truncated transcript, even if it remains stable, will not be able to be translated into GRP94. Additionally, if the ribozyme were to act through antisense base pairing with the transcript, it is likely to block translation initiation because of steric hindrance by the ribozyme at the region surrounding the AUG codon. This ribozyme, designated ribo1, contains 11 conserved nucleotides that make up three of the four conserved regions of the catalytic core. The remaining part of the catalytic center is derived from the grp94 mRNA at the GUC nucleotides starting at position 106. Cleavage is expected to occur 3` to the cytosine residue, six nucleotides downstream from the initiation codon.

First, to test whether ribo1 could act as predicted in vitro, a ribozyme was generated by in vitro transcription. This was done using both a hemiduplex DNA oligomer containing a T7 polymerase site, and the EcoRI-linearized pUC-ribo1 plasmid, into which the ribozyme oligomer had been cloned. Yield from the pUC-ribo1 plasmid was much greater than from the hemiduplex; consequently all experiments used the pUC-ribo1 plasmid to generate ribozyme transcripts. The target RNA, a 260-nt transcript encoding the 5` terminus of the grp94 transcript, was also generated by in vitro transcription. This transcript should be cleaved into two fragments of 176 and 84 nt by the ribo1 (Fig. 2 A). The integrity of the ribozyme and the target RNA after mock reactions is shown in Fig. 2 B, lanes 2 and 3, and the results of the cleavage reaction after 1, 2, and 4 h are shown in lanes 4-6. After 1 h of incubation with the ribozyme, the 260-nt target RNA was being cleaved to generate the expected products. The reaction was essentially complete after 4 h. Based on these positive results, ribo1 was engineered into expression vectors to test its ability to inhibit grp94 expression in mammalian cells.


Figure 2: Ribozyme catalyzed RNA cleavage in vitro. A, schematic drawing of target RNA for in vitro ribozyme cleavage reaction. Target RNA includes 68 nt of pBS KS II+ polylinker sequence and the 5` 192 nt of grp94 mRNA. The initiation codon and cleavage site are indicated. B, target and ribozyme RNA were transcribed in vitro using [P]UTP. Lane 1, x-174- HinfI marker; lane 2, ribozyme; lane 3, target RNA; lanes 4-6, ribozyme catalyzed cleavage reaction after 1, 2, and 4 h, respectively. After 1 h, the expected fragments of 176 and 84 nt are observed with a corresponding decrease of target signal intensity. By 4 h, the reaction is complete.



Stress Inducibility of Ribo1 in Mammalian Cells

The grp94 gene is constitutively expressed at low basal levels. However, upon treatment of cells with the calcium ionophore A23187 or tunicamycin, there is about a 10-fold increase in its transcript levels. To overcome this, we inserted ribo1 into two mammalian expression vectors (Fig. 3). One vector is the commercially available plasmid pRc/RSV which uses the RSV LTR as the promoter to drive the transcription of ribo1. Since in many mammalian cell types the RSV LTR has proven to be a strong viral promoter, the high level of ribo1 produced constitutively might be sufficient to counteract the induced level of grp94 mRNA. In the other approach, we created a new expression vector by replacing the constitutive RSV LTR promoter with the proximal 1.3 kb of the rat grp78 promoter. This grp promoter fragment contains a large number of basal enhancing elements and confers full stress inducibility to chimeric gene products (Resendez et al., 1985; Lin et al., 1986). This expression vector, designated pRc/GRP-ribo1, is identical to pRc/RSV-ribo1 with the exception that pRc/GRP-ribo1 should direct high basal level as well as stress-induced high level expression of ribo1. Both vectors contain a neomycin resistance gene driven by the SV40 early promoter that allows selection of stable transfectants.

Transfection of these constructs into NRK cells followed by selection with G418 resulted in stable integration of the plasmids, as demonstrated by genomic blot analysis (Fig. 4). Two convenient restriction sites, EcoRI and PvuII, were utilized to analyze the integration profile with the 1-kb EcoRI fragment from pRc/GRP-ribo1 (Fig. 4 A) as a probe. Comparison of the genomic blots with EcoRI-digested pRc/GRP-ribo1 plasmid shows the copy number of the integrated plasmids to be approximately 2 in all three cell lines (Fig. 4 B and data not shown). Southern blot analysis of these cells also benefitted from the presence of a PvuII site within the ribo1 oligomer. Because of this new site, genomic DNA from pRc/GRP-ribo1 and pRc/GRP transfectants digested with this enzyme yielded different patterns. As expected, pRc/GRP and pRc/GRP-ribo1 shared a common 1.1-kb fragment; however, the 0.5-kb fragment from pRc/GRP was cleaved to a 0.3-kb fragment in pRc/GRP-ribo1 due to the internal PvuII site from ribo1 (Fig. 4 C). Similar results were obtained for the pRc/RSV-ribo1 transfectants (data not shown). Thus, the integration of the appropriate plasmids in the respective cell lines was confirmed.


Figure 4: Southern blot analysis of NRK cell lines. A, schematic drawing of restriction sites of the plasmids and the region hybridizing to the probe. The probe used is a 1-kb EcoRI fragment prepared from pRc/GRP-ribo1. B, EcoRI-digested DNA from NRK, pRc/GRP, and pRc/GRP-ribo1 cells, respectively. Lanes 4 and 5 show EcoRI-digested pRc/GRP-ribo1 plasmid equivalent to 3 and 1 copy, respectively. C, PvuII-digested DNA from NRK, pRc/GRP, and pRc/GRP-ribo1 cells, respectively.



Ribozyme expression in the transfectants was detected by RNase protection assays, and the expected sizes of the protected regions hybridizing to the in vivo and in vitro synthesized ribozyme are shown in Fig. 5 A. Total cytoplasmic RNA was prepared from pRc/GRP, pRc/GRP-ribo1, and pRc/RSV-ribo1 transfectants. For the first two transfectants, RNA was also prepared from the same cells treated with A23187 for 16 h prior to the extraction of RNA. As expected, the in vitro ribozyme protected a band of 48 nt (Fig. 5 B, lane 2). For the in vivo samples, the expected protected band is a 72-nt fragment which was observed most prominently from the pRc/GRP-ribo1 samples ( lanes 5 and 6), and also at a low level from that of pRc/RSV-ribo1. As expected, this band was absent from samples prepared from the vector pRc/GRP transfectants ( lanes 3 and 4) and from tRNA ( lane 8). These results demonstrate that: ( a) the ribozyme is expressed only in pRc/GRP-ribo1 and pRc/RSV-ribo1 cells; ( b) the rat grp78 promoter confers an 8-fold induction in ribozyme levels in response to 7 µ M A23187; and ( c) even in non-induced conditions, the rat grp78 promoter is about 2-fold stronger than the RSV LTR promoter in these pooled transfectants with similar integrated copies of the two plasmids.


Figure 5: RNase protection assay to detect ribo1. A, schematic drawing of the ribozymes and the probe used for the RNase protection assay. The probe was transcribed from HindIII-linearized pSP65-ribo1 using [P]UTP, generating a 105-nt transcript. This probe protects a 48-nt fragment from the in vitro ribozyme and a 72-nt fragment from the in vivo ribozyme. B, 30 µg of total RNA from each of the transfectants were hybridized with labeled probe and digested with RNase T1. Lane 1, x-174- HinfI marker; lane 2, In vitro transcribed ribozyme; lanes 3 and 4, RNA from control and A23187-treated pRc/GRP cells; lanes 5 and 6, RNA from control and A23187-treated pRc/GRP-ribo1 cells; lane 7, RNA from untreated pRc/RSV-ribo1 cells; and lane 8, tRNA. The untreated cells are denoted with (-) and the treated samples (+).



Inhibition of GRP Stress Induction in Ribozyme Expressing Cells

To test whether the expression of ribozyme results in inhibition of GRP protein synthesis, NRK and the pRc/GRP and pRc/GRP-ribo1 cells were grown under stress and non-stress conditions. The proteins were isolated from [S]methionine-labeled cells and analyzed by SDS-PAGE (Fig. 6, A and B). The GRP94 and GRP78 band intensities were quantitated and normalized against that of actin in the same sample. Our results indicate that in non-stressed cells, the basal level of the GRPs was similar in the three cell lines tested. In tunicamycin-treated cells where N-linked protein glycosylation was blocked, the response of NRK and pRc/GRP cell lines was nearly identical, showing a 5-6-fold induction in GRP94 and GRP78 synthesis. In contrast, the pRc/GRP-ribo1 cell line displayed only a 1.5-fold induction (Fig. 6 A). To our surprise, we observed that the level of GRP78 induction was also reduced from 6- to 7-fold to approximately 4-fold in the GRP-ribo1 cells. Similarly reductions in GRP94 and GRP78 synthesis were observed for A23187-treated pRc/GRP-ribo1 cells (Fig. 6 B). In the pRc/GRP cells transfected with vector alone, GRP94 and GRP78 induction was 5.5- and 7.5-fold, respectively. After A23187 treatment, their induction levels were 1.5- and 3-fold, respectively, in the pRc/GRP-ribo1 cells. While the near complete inhibition of GRP94 induction was expected in the pRc/GRP-ribo1 cells, the partial down-regulation of the non-targeted GRP78 protein is intriguing since there is no sequence identity between the 5` terminus of the grp94 and grp78 transcript (this study and Chang et al., 1987). Therefore, it is unlikely that ribo1 could base pair with the grp78 transcript resulting in its cleavage or blockage of translation.

Coomassie Blue staining and Western blots were performed to determine the steady state levels of GRP94 and GRP78 in pRc/GRP-ribo1 and pRc/GRP cells. As shown in Fig. 6 C, the Coomassie Blue-stained bands corresponding to GRP94 and GRP78 were normally induced by A23187 treatment of the pRc/GRP cells. In contrast, the level of GRP94 and GRP78 induction was reduced in the A23187-treated pRc/GRP-ribo1 cells. Western blots were performed to determine more accurately the levels of GRP94 and GRP78. Using a monoclonal antibody against GRP94, we detected that the basal level of GRP94 in pRc/GRP-ribo1 was 25% less than the control pRc/GRP cells under non-stressed conditions (Fig. 6 D). After A23187 treatment, a 3.5-fold induction of GRP94 steady state level was observed in pRc/GRP cells. For the ribozyme expressing cells, the fold of induction was reduced to 1.5-fold. Using a monoclonal antibody against GRP78, a 20% reduction in basal and 50% reduction in A23187-induced levels of GRP78 were observed in the pRc/GRP-ribo1 cells (Fig. 6 D).


Figure 6: Measurement of GRP levels. A and B, analysis of in vivo labeled protein by SDS-PAGE. [S]Methionine-labeled protein extracts from NRK, pRc/GRP, and pRc/GRP-ribo1 cells were isolated and separated on 8% SDS-PAGE. A, lanes 1 and 2, protein from control and tunicamycin-treated NRK cells; lanes 3 and 4, protein from pRc/GRP cells; lanes 5 and 6, protein from pRc/GRP-ribo1 cells. B, lanes 1 and 2, protein from control and A23187-treated pRc/GRP cells; and lanes 3 and 4, protein from pRc/GRP-ribo1 cells. C, Coomassie Blue staining of protein samples. Equal amounts (40 µg) of each protein sample were separated on 8% SDS-PAGE and stained with Coomassie Blue. Lane 1, protein size marker ( M); lanes 2 and 3, protein from pRc/GRP cells; lanes 4 and 5, protein from pRc/GRP-ribo1 cells. D, Western blot analysis. Non-labeled protein extracts were separated on 8% SDS-PAGE. Monospecific antibodies against GRP94 and GRP78 were used as primary antibodies. Lanes 1 and 2, protein from pRc/GRP cells; and lanes 3 and 4, protein from pRc/GRP-ribo1 cells. The untreated samples are denoted with (-) and the A23187 treated samples (+). The autoradiographs are shown. The locations of GRP94 and GRP78 are indicated.



In Vivo Cleavage of the Target grp94 Transcript by the Ribozyme

To examine the mechanism whereby ribo1 reduces the expression of GRP94 and GRP78, total cytoplasmic RNA was prepared from pRc/GRP and pRc/GRP-ribo1 cell lines treated or non-treated with A23187. First, we examined the effect of ribo1 on grp94 mRNA levels by using a 5` terminus probe that hybridizes only to the non-cleaved, full-length grp94 transcript (Fig. 7 A). The level of the grp94 mRNA in each sample was quantitated by densitometry (Fig. 7 B). After normalization against the level of the non-A23187-inducible GAPDH transcript, the pRc/GRP cell line showed an 8-fold induction of intact grp94 mRNA. The basal level of the grp94 mRNA in the pRc/GRP-ribo1 cells is slightly reduced, and the overall induction level is about 3-fold in these cells. The amount of intact grp94 message in the A23187 treated pRc/GRP-ribo1 cell is reduced by 75% compared to control cells. Thus, the expression of ribo1 results in substantial loss of the full-length grp94 mRNA and is likely due to the targeted cleavage by the ribozyme.


Figure 7: RNA levels of the NRK transfectants. A, Northern analysis of pRc/GRP and pRc/GRP-ribo1 cell lines. 10 µg of total RNA from cells under control (-) and A23187-treated (+) conditions were hybridized with P-labeled probes. The grp94 probe is a fragment that contains only the 5` 104 bp of the grp94 cDNA. Thus, it can only detect intact, uncleaved grp94 mRNA. The other probe is the GAPDH cDNA. B, relative levels of uncleaved grp94 and grp78 mRNA from control (-) and A23187-treated (+) pRc/GRP and pRc/GRP-ribo1 cells normalized against the level of GAPDH transcripts. The graph represents results averaged from two to three independent RNA measurements.



In the case of grp78, the level of grp78 induction dropped from 15-fold in the pRc/GRP cells to about 8-fold in the pRc/GRP-ribo1 cells (Fig. 7 B). This 50% reduction in grp78 transcript level was consistently observed in independent RNA blot analyses and directly correlates with the 50% drop in the GRP78 protein level in the stress-treated cells. These results show that an inhibition of GRP94 synthesis in stressed cells results in reduced grp78 transcript level.

Suppression of GRP Levels Correlates with a Decrease in Secretion Ratio of Recombinant - L-Iduronidase

GRP94 and GRP78 have both been implicated as a molecular chaperones in the ER. The creation of pRc/GRP-ribo1 allowed us to determine whether suppression of GRP levels in these cells affected ER protein trafficking. In this study, we utilized recombinant human - L-iduronidase as a model. This lysosomal enzyme offers interesting properties since it undergoes modifications in both the ER and golgi. When overexpressed in mammalian cells, the enzyme is secreted into the media and the activity of the properly folded protein can be conveniently measured fluorometrically (Kakkis et al., 1994). The NRK, pRc/GRP, and pRc/GRP-ribo1 cells were transiently transfected with an expression plasmid of the human - L-iduronidase driven by the strong constitutive CMV promoter. A CMV-driven -galactosidase expression plasmid was cotransfected to normalize for transfection efficiencies. For each cell line, the - L-iduronidase activity which was secreted into the media and remained intracellularly were measured. The results from multiple transfection experiments were summarized in Table I. These experiments reveal that the parental cell line NRK and vector control cell line pRc/GRP produced similar units of - L-iduronidase enzyme activity and secreted similar amounts. However, in the pRc/GRP-ribo1 cell line, the total enzyme activity recovered was about half of the control cells. Strikingly, the ratio of the secreted versus intracellular activity in the pRc/GRP-ribo1 cells was 6-fold lower than pRc/GRP and 8-fold lower than NRK.

Deficiency in GRP Induction Correlates with Lower Viability after CaStress

Other phenotypes of the NRK, pRc/GRP, and pRc/GRP-ribo1 cells were examined. The growth rates of the three cell lines were compared under normal culture conditions (Fig. 8). There was little difference in the growth rates among these cells, with each line showing a doubling time of about 14 h.

To determine the effect of reduced induction of GRP94 and GRP78 toward resistance to calcium stress, the three cell lines were treated with increasing concentrations of A23187 (Fig. 9 A) or thapsigargin (Fig. 9 B) for 16 h. Subsequent cell viability was measured by the ability of the cells to form colonies 7-10 days after the stress treatment. The NRK and the pRc/GRP cells which showed normal induction of GRP94 and GRP78 tolerated the stress treatments relatively well, with about 70% survival up to the 20 µ M range for A23187 and 600 n M of thapsigargin. In contrast, the pRc/GRP-ribo1 cells with impaired GRP induction showed increased sensitivity to A23187 throughout the whole range being tested. At 20 µ M of A23187, a survival rate of 45% was observed (Fig. 9 A). In the case of thapsigargin treatment where the ER Castore was being depleted, a similar decrease in cell survival for the pRc/GRP-ribo1 cells was observed (Fig. 9 B). Thus, suppressed GRP induction results in lower viability after Castress.


Figure 9: Resistance of NRK transfectants to stress. A, colony survival analysis after treatment with A23187. The cells described above were treated with 0, 1, 2, 7, 10, and 20 µ M A23187 for 16 h. Survival was measured by the ability to form colonies after 7 days of growth in normal media. B, colony survival analysis after treatment with thapsigargin. As above, except that cells were treated with 0, 30, 100, 300, and 600 n M thapsigargin. C, ;Colony survival analysis after treatment with tunicamycin. As above, except that cells were treated with 0, 0.1, 0.5, 1.0, 1.5, and 3.0 µg/ml tunicamycin.



Since the GRPs have protein chaperoning activity which could account for their protective roles in cells, we next tested whether the induction of GRP94 and GRP78 is important for cell survival after treatment of the cells with increasing concentrations of tunicamycin. The blockage of protein glycosylation by tunicamycin is expected to result in the accumulation of underglycosylated proteins in the ER, thus creating a pool of malfolded or non-functional proteins which cannot exit to the Golgi and the cell surface. Surprisingly, despite the fact that GRP94 and GRP78 levels were suppressed in pRc/GRP-ribo1 cells as compared to the NRK and pRc/GRP transfectants (Fig. 6), there was no apparent increase in sensitivity of the ribozyme expressing cell line to this stress treatment throughout the whole range of tunicamycin tested (Fig. 9 C). Thus, in contrast to the Castress situation, the failure to fully induce GRP94 and GRP78 has no effect on cell survival when underglycosylated proteins are synthesized in the mammalian cells.


DISCUSSION

In mammalian cells, glucose starvation and depletion of calcium from the ER lead to the specific induction of the GRPs. Of special interest is GRP94 which is a highly abundant glycoprotein with sequence similarity to HSP90. GRP94 is encoded by a single copy gene in mammalian cells (Lee et al., 1983). Under a variety of stress conditions, it is coregulated with GRP78, also known as the immunoglobulin heavy chain binding protein, BiP (Pelham, 1986). So far the homologue of GRP94 has not been isolated from the lower eukaryotes.

In this study, we explore the use of the ribozyme technology to modulate GRP94 expression. As the function of GRP94 is not well defined in mammalian cells, the creation of a mammalian cell line deficient in GRP94 expression and/or induction will be a useful tool to dissect its role in cell growth and resistance to stress. Additionally, a recent study has linked specific enhanced expression of GRP94 to greater tumorigenicity (Menoret et al., 1994). In a rat colon carcinoma model, it appeared that tumorigenic clones show higher resistance to glucose starvation and tunicamycin treatment and were able to synthesize larger amounts of GRP94 than the non-tumorigenic clones. In contrast, there was no difference in the induced levels of GRP78 synthesis between tumorigenic and non-tumorigenic clones. Therefore, the development of molecular tools able to specifically modulate GRP94 expression will also have important implications in cancer gene therapy.

Because of the highly inducible nature of GRP94 by stress, we utilized a strong cellular promoter from the coregulated grp78 gene to drive the expression of the ribozyme. The inducible promoter driving ribozyme expression was chosen for the following reasons: (i) the endogenous grp78 promoter, being stronger than the grp94 promoter, should express ribozyme at a higher level and (ii) grp78 induction is usually higher than that of grp94. Therefore, under stress conditions, the amount of ribozyme being synthesized will potentially be comparable to or higher than the endogenous grp94 mRNA. Our in vivo expression studies indicate that the grp78 promoter is capable of conferring stress inducibility to the ribozyme. At the RNA level, the amount of grp94 mRNA was reduced by 75% in the stressed cells expressing the ribozyme. This reduction was due to cleavage at the 5` terminus, as predicted by the ribozyme design and our in vitro test results. While the cleaved 5` grp94 fragment is probably rapidly degraded, the remainder of the grp94 transcript can be detected in RNA blots.() The rapid degradation of the 5` fragment is likely due to its small size and the lack of the stabilizing signals such as the 3`-UTR and the poly(A) tail present in the longer transcript. In comparison to the commonly used viral RSV-LTR, the grp78 promoter is about 2-fold stronger in non-stressed cells. This estimation is based on similar copy number of the integrated plasmids and that the large number of pooled transfectants should neutralize the position effect. Thus, in designing ribozyme expression in mammalian cells, the cellular grp78 promoter offers an alternative to the widely used viral promoters.

In examining the effect of ribozyme expression on the growth rates of the transfected cells, we did not observe any difference with the non-transfected cells under normal culture conditions. We note that the basal level of grp94 mRNA is reduced by about 50% in the ribozyme expressing cell line, and, by criterion of Western blot with monospecific GRP94 antibody, the steady state level of GRP94 is reduced by about 25%. One explanation for a residual basal level of GRP94 is that it is required for cell viability under normal cell growth and that the stable transfectants which survive are able to express a low basal level of GRP94. This result predicts that a complete knockout of GRP94 function may result in cell lethality. In the case of GRP78 where a yeast homologue known as KAR2 has been isolated, mutation of this gene is lethal (Rose et al., 1989).

In stress-induced pRc/GRP-ribo1 cells, the ability to induce GRP94 is severely compromised. Interestingly, the level of the non-targeted GRP78 is also reduced. Although the level of reduction is less drastic than that of GRP94, a 50% decrease in GRP78 was consistently observed. This inhibitive effect on the grp78 mRNA and protein level cannot be due to transcriptional factor competition because the pRc/GRP cell line which contains the identical grp promoter vector as pRc/GRP-ribo1 did not show this effect. Thus, the inhibition is dependent on ribo1 expression and GRP94 reduction. This observation, although unusual, is consistent with previous studies demonstrating that there is coregulation between GRP78 and GRP94. Thus, GRP78 overexpressing cells treated with tunicamycin or A23187 exhibited a reduced induction of endogenous grp78 and grp94 mRNA as compared to wild-type Chinese hamster ovary cells (Dorner et al., 1992). In two separate studies, suppression of GRP78 expression by a grp78 antisense vector also resulted in reduced stress induction of GRP94 at the mRNA level (Li et al., 1992; Sugawara et al., 1993). Here we showed that reduction of GRP94 stress induction reciprocally down-regulates GRP78 expression. It is possible that GRP94 could act as one component of the signal transduction systems partially mediating grp78 transcription during stress. It has been reported that GRP94 undergoes conformational alteration in A23187-treated cells (Kang and Welch, 1991). In addition, GRP94 exhibits both lumenal and transmembrane protein properties (Kang and Welch, 1991). Thus, GRP94, by itself or in concert with other interacting molecules, such as transmembrane kinases or phosphatases, could transmit the signal from the ER to nucleus to regulate GRP transcription. One such transmembrane kinase, IRE1, has been identified in yeast to mediate the grp78 stress response (Cox et al., 1993; Mori et al., 1993). Recently, calreticulum, a ER calcium-binding protein which also possesses a nuclear translocation signal, has been shown to be able to modulate gene expression by its association with the glucocorticoid receptor (Burns et al., 1994).

In examining the phenotypes of the ribozyme expressing cell line, we report here a 6-fold decrease in the secretion ratio of the enzyme. Partial reduction in the secretion ratio of recombinant - L-iduronidase was also observed in a Chinese hamster ovary cell line with suppressed GRP78 and GRP94 induction by antisense (Li et al., 1992).() Since - L-iduronidase is a highly modified protein trafficking through the ER, Golgi, lysosome, and the secretion pathway, future studies in the ribozyme expressing cells can determine which step(s) of its processing/trafficking pathway is blocked due to the lower level of the GRPs. Previously, it was demonstrated that in mammalian cells a decrease in GRP78 expression resulted in increased secretion of recombinant proteins such as Factor VIII (Dorner et al., 1992). These combined results indicate that for some recombinant proteins, their secretion is impeded by high levels of GRPs. However, for other proteins, high levels of GRPs are required for efficient secretion.

In the ribozyme expressing cells, we observed a 50% reduction in the - L-iduronidase enzymatic activity. Attempts to measure directly the amount of the enzyme have met with technical difficulties, probably due to the low amount of the protein being synthesized in the transiently transfected cells. Nonetheless, since the level of transfected -galactosidase activity driven by the same CMV promoter was nearly equivalent in the ribozyme expressing and control cells, we speculate that translation of the transfected genes was not affected. Rather, suppression of the GRP levels may have affected folding/processing of the recombinant enzyme, resulting in lower activity.

In the cell survival studies, the NRK and the cell lines transfected with the vector alone showed a higher resistance to the calcium ionophore A23187 and the ER CaATPase inhibitor thapsigargin than the ribozyme expressing cells. The higher resistance correlates with the higher induced levels of GRP94 and GRP78. Since GRP94 is known to be major calcium-binding protein in the ER, its increased synthesis could protect the cells from by helping to maintain Cahomeostasis. Our studies would lend support to the hypothesis that GRP94, by itself or in conjunction with GRP78 or other molecules not identified in this study, plays a protective role in cells under calcium stress conditions. In contrast, the lack of induction of GRP94 and lower induction of GRP78 did not increase tunicamycin sensitivity over that of the control cells, implying that although the GRPs are induced by glycosylation-block stress, they either do not confer a protective role in this form of stress or that their induction alone is not sufficient to mitigate the lethality caused by this stress. Because of the multiple functions ascribed to the GRP94 in protein processing, antigen presentation, and tumor progression, the availability of a targeted ribozyme against grp94 and the establishment of a stable cell line deficient in GRP induction should facilitate further investigations into the physiology of GRPs.

  
Table: Recombinant human - L-iduronidase activities recovered from transiently transfected cells

The levels of - L-iduronidase activity were normalized with respect to transfection efficiency. The secretion ratio was determined by dividing the enzymatic activity secreted into the media by the intracellular activity retained in the cell pellet for each cell line. The secretion ratio for NRK was set as 1.0. The table summarized the results obtained from two independent transfection experiments each performed in triplicate. One unit corresponds to the activity catalyzing the hydrolysis of 1 nmol of substrate/h. The standard deviations of the calculated percentages and secretion ratio are shown.



FOOTNOTES

*
This research was supported in part 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.

The abbreviations used are: ER, endoplasmic reticulum; GRPs, glucose-regulated proteins; HSPs, heat shock proteins; kb kilobase(s); cpm, counts/min; RSV, Rous sarcoma virus; LTR, long terminal repeat; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; nt, nucleotide(s); CMV, cytomegalovirus.

E. Little and A. S. Lee, unpublished results.

E. D. Kakkis and A. S. Lee, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Jonathan Howard (Institute of Animal Physiology and Genetics Research, Cambridge Research Station, United Kingdom) for the rat gt10 cDNA library and Joseph Landolph (University of Southern California School of Medicine) for the cDNA plasmid for glyceraldehyde-3-phosphate dehydrogenase. We are indebted to Dr. Emil Kakkis (UCLA) for his generous supply of the plasmids pRc/CMV-HuId and pRc/CMV--gal and his expert advice on the recombinant - L-iduronidase system. We thank Colin Jamora for technical assistance with the immunoblots. We are grateful to Dr. John Rossi (City of Hope, CA) for consultations on ribozyme design and helpful discussions. We thank Dr. Axel Schönthal and Meera Ramakrishnan for critical review of the manuscript.


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