From the
GRP94 is an endoplasmic reticulum (ER) localized glycoprotein
with Ca
The endoplasmic reticulum (ER)
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
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 Ca
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
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
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
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.
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
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.
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.
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).
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 Ca
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.
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
In the ribozyme expressing cells, we observed a
50% reduction in the
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 Ca
The levels
of
We thank Drs. Jonathan Howard (Institute of Animal
Physiology and Genetics Research, Cambridge Research Station, United
Kingdom) for the rat
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
binding 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 Ca
depletion 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 Ca
depletion stress.
(
)
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).
-binding proteins. While the binding affinity for
Ca
is 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 Ca
homeostasis (Brostrom et
al., 1990).
by 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).
-
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 Ca
stress 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.
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 10
bacteriophage 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
10
cpm/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.
, 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.
, 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.
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).
10
cpm/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
10
cpm/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 10
cpm 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 10
cells
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.
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.
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.
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.
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
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
-
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 Ca
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.
Stress
store 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 Ca
stress.
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.
(
)
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.
-
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.
-
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.
ATPase
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 Ca
homeostasis. 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
-
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.