1 Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109; and 2 Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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Pancreatic
secretagogues enhance acinar protein synthesis at physiological
concentrations and inhibit protein synthesis at high concentrations. We
investigated the potential role in this process of the eukaryotic
translation initiation factor (eIF)2B. Cholecystokinin (CCK) at
10-100 pM did not significantly affect eIF2B activity, which
averaged 35.4 nmol guanosine 5'-diphosphate exchanged per minute per
milligram protein under control conditions; higher CCK concentrations
reduced eIF2B activity to 38.2% of control. Carbamylcholine chloride
(Carbachol, CCh), A-23187, and thapsigargin also inhibited eIF2B and
protein synthesis, whereas bombesin and the CCK analog JMV-180 were
without effect. Previous studies have shown that eIF2B can be
negatively regulated by glycogen synthase kinase-3 (GSK-3). However,
GSK-3 activity, as assessed by phosphorylation state, was inhibited at
high concentrations of CCK, an effect that should have stimulated,
rather than repressed, eIF2B activity. An alternative mechanism for
regulating eIF2B is through phosphorylation of the -subunit of eIF2,
which converts it into an inhibitor of eIF2B. CCK, CCh, A-23187, and
thapsigargin all enhanced eIF2
phosphorylation, suggesting that
eIF2B activity is regulated by eIF2
phosphorylation under these
conditions. Removal of Ca2+ from the medium enhanced the
inhibitory action of CCK on both protein synthesis and eIF2B activity
as well as further increasing eIF2
phosphorylation. Although it is
likely that other mechanisms account for the stimulation of acinar
protein synthesis, these results suggest that the inhibition of acinar
protein synthesis by CCK occurs as a result of depletion of
Ca2+ from the endoplasmic reticulum lumen leading to
phosphorylation of eIF2
and inhibition of eIF2B.
protein translation; cholecystokinin; glycogen synthase kinase-3; intracellular calcium
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INTRODUCTION |
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CCK is a major gastrointestinal hormone regulator of exocrine pancreatic function (45). In addition to its role of stimulating pancreatic exocrine secretion, it has been shown to regulate pancreatic protein synthesis, mitogenesis, and gene expression (44). In vivo, CCK exerts both short-term stimulatory effects on pancreatic protein synthesis and long-term trophic effects on pancreas size and digestive enzyme content (4, 6, 38). In vitro, in addition to its action to acutely stimulate pancreatic acinar cell secretion, CCK regulates the synthesis of pancreatic protein in a dose-dependent manner, with a characteristic biphasic response (2, 24, 25). These observations suggest that hormonal modulation of enzyme synthesis may be coordinated with the regulation of secretion. With short-term stimulation, there is no change in mRNA levels for digestive enzymes, and the major regulatory effect appears to be at the level of polysomes associated with the rough endoplasmic reticulum actively translating digestive enzyme mRNAs (27, 31, 32). Recent studies have begun to elucidate the signaling pathways regulating translational control in the pancreas. We have demonstrated that CCK activates the regulatory step in translation initiation that is critically dependent on the mRNA binding protein eIF4E that binds to the cap structure at the 5' end of the mRNA and mediates assembly of an initiation-factor complex termed eIF4F. Assembly of this complex can be regulated by eIF4E-binding proteins (4E-BPs), which inhibit eIF4F complex assembly. In normal rats in vivo, both exogenous and endogenous CCK enhance phosphorylation of eIF4E and, more importantly, the release of eIF4E from its binding protein PHAS-I (4E-BP1) (2, 3). CCK also activates the 70-kDa ribosomal protein S6 kinase (p70s6k) (1) as a result of the same signaling pathway leading to the phosphorylation of PHAS-I.
The other major regulatory step in the translation initiation pathway
is the binding of the initiator methionyl-tRNAi to the 40 S
ribosomal subunit regulated by the eukaryotic initiation factor-2
(eIF2) (13, 34). eIF-2 is a heterotrimer composed of ,
, and
subunits and forms a ternary complex with
initiator methionyl-tRNAi and GTP. This complex binds to
the 40 S ribosomal subunit forming the 43 S species. After the 60 S
ribosomal subunit joins the 43 S complex, the eIF2-bound GTP is
hydrolyzed to guanosine 5'-diphosphate (GDP) and the eIF2-GDP complex
is released from the ribosome (13). For eIF2 to promote
another round of initiation, GDP must be exchanged for GTP, a reaction
catalyzed by the guanine nucleotide exchange factor, eIF-2B
(13). In some cells, including skeletal muscle and Swiss
3T3 cells, the global stimulation of protein synthesis is mediated by
enhancing eIF2B activity. Insulin can regulate this activity through
activation of the phosphatidylinositide 3-kinase (PI3K) pathway that
leads to phosphorylation and inactivation of glycogen synthase kinase-3
(GSK-3) (21, 43) and finally to the dephosphorylation
(activation) of eIF2B
(10, 42). A second regulatory
pathway for eIF2B involves the phosphorylation of serine residue 51 in
eIF2
. Once this site has been phosphorylated, eIF2 has an increased
affinity for eIF2B, converting eIF2 from a substrate to a competitive
inhibitor of eIF2B (36, 39). Phosphorylation of eIF2
is
also a major mechanism whereby environmental stress (12,
36), nutrient deprivation (5, 13, 30, 41), or viral
infection (9) shuts off protein synthesis. Depletion of
calcium from the endoplasmic reticulum also correlates with inhibition
of protein synthesis and increased eIF2
phosphorylation (18,
39).
In the current study, we evaluated the activity of eIF2B in isolated
pancreatic acini in vitro in response to CCK and other acinar cell
secretagogues. Although no evidence was found for eIF2B stimulation
contributing to secretagogue stimulation of protein synthesis, enhanced
phosphorylation of eIF2 and inhibition of eIF2B were found to
correlate with inhibition of protein synthesis induced by high doses of
CCK and alterations in intracellular calcium.
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MATERIALS AND METHODS |
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Materials.
Sulfated CCK octapeptide was from Research Plus (Bayonne, NJ); bombesin
(BBS) was from Bachem (Torrance, CA); and carbamylcholine chloride
(Carbachol, CCh), soybean trypsin inhibitor (SBTI), wortmannin, GDP,
and dithiothreitol were obtained from Sigma (St. Louis, MO). Chromatographically purified collagenase was from Worthington Biochemicals (Freehold, NJ); goat anti-rabbit and anti-mouse IgG conjugated to horseradish peroxidase and enhanced chemiluminescence (ECL) reagent were from Amersham Pharmacia Biotech (Piscataway, NJ);
minimal essential amino acids were from GIBCO (Grand Island, NY); 10 and 12% Tris · HCl precast gels and low range prestained SDS-PAGE standard markers were from Bio-Rad (Hercules, CA);
nitrocellulose membranes were from Schleicher & Schuell (Keene, NH);
and A-23187, thapsigargin, and cycloheximide were from Calbiochem (La
Jolla, CA). L-[35S]methionine (1,175 Ci/mM) and [3H]GDP (11.3 Ci/mM) were from NEN Life
Science Products (Boston, MA); the scintillation liquids Bio-Safe II
and Filtron-X were from Research Products International (Mount
Prospect, IL) and National Diagnostics (Atlanta, GA), respectively; 25 mm nitrocellulose filter disks (HAWP) were from Millipore (Bedford,
MA); ampholytes "resolyte" 4-8 were from BDH Laboratory
Supplies (Poole, England) and urea (ultra pure) was from ICN
Biomedicals (Aurora, OH). GSK-3 monoclonal antibody was from
Upstate Biotechnology (Lake Placid, NY), phospho-GSK-3/
(Ser21/9) polyclonal antibody was from New England
Biolabs (Beverly, MA), and Phospho-eIF2
(Ser51)
polyclonal antibody was from Research Genetics (Huntsville, AL). An
eIF2
monoclonal antibody, originally developed by Dr. E. C. Henshaw, that recognizes both phosphorylated and unphosphorylated forms
of eIF2
was also used. Purified eIF2 and eIF2B were prepared from
rat liver as described (15, 19).
Preparation of pancreatic acini. Pancreatic acini were prepared by collagenase digestion (1) of pancreases of 125- to 150-g male Sprague-Dawley rats. Acini were suspended in incubation buffer, consisting of a HEPES-buffered Ringer solution supplemented with 11.1 mM glucose, Eagle's minimal essential amino acids, 0.1 mg/ml SBTI, and 10 mg/ml BSA, and equilibrated with 100% O2.
Methionine incorporation into pancreatic protein. To measure total net protein synthesis in acinar cells, L-[35S]methionine incorporation into protein was evaluated as described previously (2). After 1-h preincubation in HEPES-buffered Ringer, aliquots of isolated acini (1 ml) were incubated with agonists for 60 min at 37°C with gentle shaking. During the last 15 min of incubation, 2 µCi/ml of L-[35S]methionine were added to the incubation medium. Incubation was terminated by dilution with 2 ml of 154 mM/l NaCl at 4°C. After centrifugation at 300 g for 3 min, acinar pellets were resuspended in 0.5 ml water and sonicated. Samples were precipitated with 10% TCA at 4°C. The precipitates were washed twice with ice-cold 10% TCA and dissolved in 200 µl of 0.1 N NaOH, and radioactivity in the insoluble material was measured in Bio-Safe II scintillation medium. All samples contained an equal amount of water and NaOH to ensure equal quenching. Background samples contained NaOH to control for chemiluminiscence.
Measurement of eIF2B activity. Determination of eIF2B activity in pancreatic acini was performed as described by Kimball et al. (14), who measured the rate of exchange of [3H]GDP, present in an exogenous eIF2 · [3H]GDP complex for free, nonradiolabeled GDP. To prepare acinar samples after experimental treatment, acini were rinsed in ice-cold PBS, centrifuged, and resuspended in 150 µl of lysis buffer consisting of 45 mM HEPES, pH 7.4, 0.375 mM magnesium acetate, 95 mM potassium acetate, 0.075 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium vanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The samples were then sonicated for 10 s and centrifuged for 15 min at 12,000 rpm at 4°C in a microcentrifuge. Supernatants were assayed for guanine nucleotide exchange activity. Briefly, 35 µl of a prepared binary complex, which was assembled by incubation of purified eIF2 (14) with 1.3 µM [3H]GDP, was combined with a mixture consisting of 35 µl of acini "homogenate," 87.5 µl of water, and 140 µl of buffer A (62.5 mM MOPS, pH 7.4, 209 µM GDP, 2.5 mM magnesium acetate, 125 mM potassium chloride, 1.25 mM dithiothreitol, and 250 µg/ml bovine serum albumin). The reaction was initiated by combination of these reactants and transferred to a 30°C water bath. At four time points (0, 1, 2, and 3 min), a 60-µl aliquot was removed and placed into tubes containing 2.5 ml of ice-cold wash buffer (50 mM MOPS, pH 7.4, 2 mM magnesium acetate, 100 mM potassium chloride, 1 mM dithiothreitol). The contents were then mixed and immediately filtered under suction through a nitrocellulose filter disk and rinsed with 5 ml of ice-cold wash buffer. Filters were dissolved in 7 ml of Filtron-X. The guanine nucleotide exchange activity was measured as a decrease in eIF2 · [3H]GDP complex bound to the filters and expressed as nanomoles GDP exchanged per minute per milligram acinar protein or as a percentage of the control group.
Evaluation of the phosphorylation state of eIF2.
The phosphorylation state of eIF2
was determined by two different
methods. In the first method, the relative amount of eIF2
in the
phosphorylated form was quantitated by protein immunoblot analysis
using an affinity-purified antibody that specifically recognizes
eIF2
phosphorylated at Ser51. For this analysis,
aliquots of acinar lysates were heated for 5 min in a SDS sample buffer
at 100°C, and after cooling they were resolved in a 10% SDS-PAGE
gel, transferred to nitrocellulose followed by Western blot analysis
using the anti-phospho eIF2
polyclonal antibody (1:1,500), and
detected by ECL. Quantification was performed using Multi-Analyst
software (Bio-Rad). To ensure equal loading, the same membranes were
stripped and reprobed for total eIF2
using a monoclonal antibody to
eIF2
diluted 1:500, which was also detected by ECL.
Quantitation of eIF2 and eIF2B
.
The contents of eIF2 and eIF2B in pancreatic acinar cells were
determined by protein immunoblot analysis using as standards eIF2
and eIF2B
expressed in and purified from Sf21 cells (7, 16). The eIF2B
antibody was raised against eIF2B, which was purified from rat liver as described previously (19).
Evaluation of the phosphorylation state of GSK-3.
As an indicator of GSK-3 activity, acinar samples in SDS buffer were
resolved in a 10% SDS-PAGE gel followed by Western blot analysis using
an anti-phospho GSK-3/
(Ser21/9) polyclonal antibody
(1:1,000) and detected by ECL. Quantification was performed using
Multi-Analyst software. To ensure equal loading, the same membranes
were stripped and reprobed with a GSK-3
/
monoclonal antibody
(1:1,000), which was also detected by ECL. GSK-3
is the predominant
form in acinar cells and was the form whose phosphorylation was
quantitated. Similar qualitative changes were seen in GSK-3
. A
decrease in GSK-3 activity is indicated when phosphorylation is
increased with respect to basal samples.
Statistical analysis. Data are represented as means ± SE and were obtained from at least four separate experiments. Statistical analysis was carried out by Student's t-test, unless otherwise indicated, as calculated by the SigmaStat program. Differences with P < 0.05 were considered significant.
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RESULTS |
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CCK stimulation and inhibition of protein synthesis.
To study the effect of CCK on pancreatic protein synthesis, we first
evaluated the levels of L-[35S]methionine
incorporation into TCA-precipitable protein at different concentrations
of CCK. As in previous studies of acini prepared from diabetic rats
(2, 25), CCK stimulated protein synthesis of rat acini in
a dose-dependent manner (Fig. 1),
describing a biphasic curve similar to the one described for in vitro
secretory studies with acute preparations of pancreatic acini. Low
concentrations of CCK, from 3 pM and above, increased
L-[35S]methionine incorporation into
TCA-precipitable protein with a maximum effect at 100 pM, where
synthesis was 157.8 ± 12.8% of control. Higher concentrations of
CCK (1 and 10 nM) decreased the incorporation of radiolabeled amino
acid to 60.4 ± 9.4% of basal at 10 nM (Fig. 1).
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CCK effects on translation initiation factors eIF2B and eIF2.
Because modulation of eIF2B activity is known to be one of the most
important regulatory points in translation initiation (13), we studied the effect of different concentrations of
CCK on eIF2B activity. Extracts of control pancreatic acini exhibited an eIF2B activity of 35.4 ± 2.7 nmol · min
1 · mg
1 protein
(n = 10). A small, but not significant, increase in
eIF2B activity was seen at CCK concentrations that were stimulatory for
protein synthesis (10 and 100 pM) (Fig.
2A). This insignificant increase was also observed when data for 100 pM CCK were pooled from
all of the experiments carried out for this study (41.0 ± 2.4 nmol · min
1 · mg
1,
n = 18). By contrast, a clear and significant reduction
in eIF2B activity was seen at concentrations of CCK (from 1 to 100 nM) that inhibited protein synthesis, reaching a minimum of 14.4 ± 2 nmol · min
1 · mg
1 at 100 nM
CCK (Fig. 2A).
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CCK effects on GSK-3 phosphorylation.
Another mechanism by which eIF2B activity can be regulated is through
phosphorylation of eIF2B by GSK-3, which results in inhibition of
its guanine nucleotide exchange activity. In the present study, GSK-3
regulatory phosphorylation increased in response to 100 pM to 100 nM
CCK, reaching a maximum of 432 ± 51% (Fig. 3). This increased phosphorylation would
be expected to inhibit GSK-3 activity and might thereby increase eIF2B
activity. However, we observed either a nonsignificant increase or a
reduction in eIF2B activity at these concentrations of CCK (Fig. 3).
Therefore, GSK-3 is not likely to be involved in the inhibition of
eIF2B. Moreover, acini treated with wortmannin to stimulate GSK-3 also showed no change in eIF2B activity (100.0 ± 14.4% in basal,
91.7 ± 17.4% at 100 pM CCK, 58.3 ± 0.1% at 1 nM CCK, and
30.6 ± 5.6% at 10 nM CCK, which is comparable to samples
stimulated with CCK alone).
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Effects of high concentrations of various agonists on protein
synthesis, eIF2B activity, and eIF2 phosphorylation.
To establish whether there is a parallelism between secretagogue
agonists mediating inhibition of eIF2B activity and protein synthesis,
we studied the effect of high concentrations of other well-known
pancreatic secretagogues, including CCh, BBS, and the CCK analog
JMV-180 on protein synthesis, eIF2B activity, and eIF2
phosphorylation. As expected, and like in previous studies
(25), CCh at 1 mM had an inhibitory effect on protein
synthesis, inhibiting L-[35S]methionine
incorporation to pancreatic protein to 44.8 ± 7.0% (Fig.
4A). CCh at this concentration
also inhibited eIF2B activity to 50% of the basal, comparable to the
effect of CCK at 10 nM (Fig. 4B). CCh also increased both
eIF2
phosphorylation and the proportion of eIF2
in the
phosphorylated form, although these were lower than the effect of CCK
at 10 nM (Fig. 4C and Table 1). In agreement with previous
studies (28, 29), neither 1 µM of BBS nor 1 µM of
JMV-180 inhibited acinar protein synthesis (Fig. 4A). These
later agents also had no effect on either eIF2B activity (Fig.
4B) or eIF2
phosphorylation (Fig. 4C and Table 1). Together, these results suggest that the inhibition of protein synthesis caused by CCK and CCh likely occurs through a common pathway
that involves an increase on eIF2
phosphorylation and inhibition of
eIF2B activity.
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Calcium effects on protein synthesis, eIF2B activity, and eIF2
phosphorylation.
CCK and CCh have similar effects on protein synthesis, eIF2B activity,
and eIF2
phosphorylation at high concentrations. These secretagogues
share a common intracellular mechanism to increase free cytoplasmic
calcium by mobilizing calcium from intracellular stores and by
activating the influx of extracellular calcium. Because it is known
that depletion of calcium from intracellular stores increases eIF2
phosphorylation and inhibits protein synthesis in other cell types
(18, 33, 39), we first studied the effect of the ionophore
A-23187 and the inhibitor of the microsomal Ca2+-ATPase
thapsigargin on protein synthesis, eIF2B activity, and eIF2
phosphorylation. The results show that both agents strongly inhibited
protein synthesis to 4.9 ± 0.2% (thapsigargin) and to 9.4 ± 3.5% (A-23187) of the basal value (n = 3-5).
They also inhibited eIF2B activity to <30% (Fig.
5A) and increased severalfold
eIF2
phosphorylation (Fig. 5B) and the percentage of
phosphorylated eIF2
to ~50% (Table 1), compared with basal
values. Thus the inhibition of protein synthesis caused by releasing
calcium from intracellular stores could be due to the inhibition of
eIF2B activity and an increase on eIF2
phosphorylation.
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Relative amount of eIF2B and eIF2 in pancreatic acinar cells.
The mechanism through which eIF2
phosphorylation causes an
inhibition of eIF2B activity involves an increased affinity of eIF2B
for phosphorylated compared with unphosphorylated eIF2. The increased
affinity of eIF2B for phospho-eIF2
results in the effective
sequestration of eIF2B into an inactive complex, with phospho-eIF2
inhibiting eIF2B on an approximately equimolar basis. Thus to inhibit
eIF2B activity completely, an equimolar amount of phospho-eIF2
must
be present. To establish whether the changes in eIF2
phosphorylation
shown in Figs. 2B, 4C, 5B, and
6B could account for the observed changes in protein
synthesis and eIF2B activity, the molar concentrations of eIF2 and
eIF2B were determined. As shown in Fig.
7, the molar ratio of eIF2/eIF2B is 3:1
in acinar cells as determined by Western blot analysis of acinar
samples using purified eIF2
and eIF2B
as standards (Fig. 7,
A and B). The molecular mass of the proteins used
as standards is greater than the native proteins, because the eIF2
and eIF2B
were expressed in Sf21 cells with an 8-amino acid
extension at the NH2 terminus to aid in purification. None
of the agonists or treatments of the acini modified the quantities of
either eIF2
or eIF2B
(Fig. 7A) (data not shown for
BBS), thapsigargin, or A-23187 treatments. These results suggest that a
significant portion of the inhibitory effect on eIF2B activity could be
related to the phosphorylated eIF2
.
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DISCUSSION |
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The present study was designed to evaluate the regulation of the
guanine nucleotide exchange factor eIF2B and its competitive inhibitor,
phosphorylated eIF2, by CCK and other pancreatic secretagogues. Stimulation of acinar protein synthesis at low concentrations of CCK
(10-100 pM) was not correlated with stimulation of eIF2B activity.
CCK at high concentrations (1-100 nM) by contrast inhibited both
protein synthesis and eIF2B activity. CCh at high concentrations also
inhibited protein synthesis and eIF2B activity. Both CCK and CCh
increased eIF2
phosphorylation, whereas high concentrations of BBS
and JMV-180 had no significant effect on these parameters. Both
depletion of calcium from intracellular stores caused by incubating
acini in the presence of thapsigargin or the ionophore A-23187 and
removing calcium from the incubation medium by addition of EGTA
inhibited protein synthesis and eIF2B activity and increased eIF2
phosphorylation. These results suggest a common inhibitory pathway for
CCK and CCh that could be related to changes in intracellular calcium levels.
Results of the present study using acini from normal young rats showed a biphasic dose-response curve for the inhibition of protein synthesis by CCK, with a maximum stimulatory effect of ~150% of basal levels at 100 pM CCK. In previous studies with diabetic rats, the maximum stimulatory effect was found at the same concentration of CCK (100 pM), but it reached higher values, ~200-250% of basal (2, 24, 25). Because insulin also stimulates protein synthesis in pancreatic acini, the greater effect of CCK observed in acini from diabetic rats has been interpreted as due to the much lower levels of insulin in the incubation medium (2, 24, 25, 26, 31). Thus in control animals, acinar protein synthesis is already upregulated by insulin and the relative increase caused by CCK is therefore reduced. Using acini from diabetic rats therefore has the advantage of yielding a greater CCK stimulatory effect (25). However, it has the disadvantage of altering the animal's normal physiology and other protein synthesis-related mechanisms. Therefore, in the present study, we used normal rats to avoid the complications of experimental diabetes including hyperglycemia and hyperlipidemia. An example of an effect of diabetes could be the differences seen at supramaximal concentrations of CCK. In normal rats, the inhibitory phase of the CCK curve reaches lower levels of protein synthesis (50% below basal) than in diabetic animals (2, 24).
Previous studies have established that the acute regulation of protein
synthesis in pancreas and in pancreatic acinar cells occurs at the
translational level (2, 3, 31, 32). Activity of eIF2B can
be rate limiting for protein synthesis, and an increase/decrease in
eIF2B activity is usually correlated with an increase/decrease in the
rate of protein synthesis (22, 23, 43). In our study, however, concentrations of CCK shown to enhance protein synthesis did
not stimulate eIF2B activity significantly. This effect might be
explained by the fact that basal levels of eIF2B activity were already
high, suggesting that intracellular signaling pathways regulating eIF2B
activity are constitutively active in acini or that an increase in
eIF2B activity is masked by the inhibitory effect of phosphorylated
eIF2 at stimulatory doses of CCK. Alternatively, the increase seen
in total protein synthesis at 100 pM CCK could be related to the
stimulation of other regulatory steps in translation initiation, as
seen in our previous studies (2). From these studies, we
know that CCK at stimulatory doses increases the phosphorylation and
activation of the p70s6k, the phosphorylation of
PHAS-I, and the formation of the eIF4F initiation complex. These
mechanisms are known to be downstream of PI3K and mTOR. However, eIF2B
is not regulated through the PI3K/mTOR pathway in acinar cells, because
neither the PI3K inhibitor worthmannin nor the mTOR inhibitor rapamycin
(data not shown) had an effect on eIF2B activity, despite the fact that
they blocked protein synthesis (2). The stimulatory effect
of CCK in pancreatic acinar protein synthesis could thus be related to
a stimulation of the PI3K pathway, thereby activating
p70s6k and eIF4F assembly, without a significant
effect on eIF2B activity. In fact, GSK-3 (one of the known eIF2B
kinases) is phosphorylated and inactivated by CCK (Fig. 3) through the
PI3K-PKB pathway (data not shown). In our case, however, GSK-3
inhibition would not activate eIF2B at stimulatory doses of CCK,
because first, eIF2B was already activated in basal acini and second,
stimulatory doses of CCK didn't significantly increase eIF2B activity.
eIF2B could be constitutively activated by another kinase such as
casein kinase-I or -II (20, 23, 35) or through changes in
the redox state of pyridine dinucleotides, because reduced pyridine
dinucleotides upregulate eIF2B activity (11).
Inhibition of protein synthesis is characterized by disaggregation of
polysomes, with a concomitant increase in free ribosomal subunits and
monomers and a decrease in the rate of peptide-chain initiation
(18, 32). At the first regulatory step of translation, phosphorylation of eIF2 immediately reduces the level of functional eIF2 and limits initiation events on all cellular mRNAs within the
cell. Control through reversible phosphorylation of eIF2
provides
the cell with an efficient and rapid means to respond to a variety of
different stimuli (12). In this study on pancreatic acini
in vitro, we show how the eIF2-eIF2B complex is involved in the
inhibition of protein synthesis by high doses of CCK or CCh. The
inhibition seen in the protein synthesis experiments is correlated with
the inhibition of eIF2B activity and an increase in the phosphorylation
of eIF2
, transforming eIF2
into an inhibitor of eIF2B (20,
34). The molar ratio of eIF2 to eIF2B in pancreatic acini is 3:1
(Fig. 7), similar to most eukaryotic cells, where this ratio is
2-5:1 (17). Because eIF2
(P) inhibits eIF2B on an
approximately equimolar basis (17), and we found that
there are three molecules of eIF2
per one of eIF2B, we could suggest that phosphorylation of eIF2
accounts for most of the inhibitory effect seen in eIF2B activity. In fact, the proportion of eIF2
in
the phosphorylated state increases with increasing concentrations of
CCK until a value (at 10 nM CCK) almost three times the one in the
basal group (Fig. 2C). Overall, these results suggest that phosphorylation of eIF2
can account completely for the observed inhibition of eIF2B activity (Fig. 2B) at 10 nM CCK. As a
result, the inhibition of protein synthesis at 10 nM CCK to ~60% of
control could be directly related to the inhibition of eIF2B, although eIF2B activity was inhibited to a greater extent (40% of basal). That could indicate that another mechanism (i.e., through regulating the eIF4F complex formation) compensates some of the eIF2B inhibitory effect at this concentration of CCK. An apparent conundrum is that
eIF2
is also partially phosphorylated at 10 and 100 pM CCK (Fig.
2B), although no inhibition of eIF2B activity or protein synthesis is observed. Instead, eIF2B activity and protein synthesis started decreasing at 1 nM CCK, suggesting that at low concentrations of CCK, some other pathway(s) is activated that could be affecting eIF2B activity. One possible mechanism could involve changes in the
phosphorylation of the
-subunit of eIF2B, which might counteract the
inhibition caused by eIF2
phosphorylation (17). The
putative kinase activity involved is not known but is unlikely to be
GSK-3. If GSK-3 were the only regulatory mechanism for eIF2B activity, we would expect that the activation of GSK-3 occurring at high concentrations of CCK would inhibit, rather than stimulate, eIF2B activity.
Protein synthesis is inhibited in a variety of cell types as a result
of an increase in calcium release from ER or other intracellular stores
(12, 18, 33, 39). For some types of cells, the release of
calcium from the ER is sufficient to inhibit protein synthesis
(18). The triggering mechanism for the inhibition of
protein synthesis in acini could be the release of calcium from
intracellular stores induced by some agonists. In unstimulated pancreatic acinar cells, intracellular calcium concentration
([Ca2+]i) is maintained by a balanced array
of influx, efflux, and intracellular sequestration mechanism. High
concentrations of CCK rapidly release (within 1-2 s) intracellular
calcium and increase [Ca2+]i from 100 to
500-1,000 nM (45). In our study, we demonstrate that,
in addition to increasing [Ca2+]i, high
concentrations of CCK (1-100 nM) and CCh (1 mM) inhibited protein
synthesis and eIF2B activity and also increased eIF2 phosphorylation. These results give clear evidence for a
calcium-related regulation of protein synthesis. Results obtained with
high concentrations of BBS and the CCK analog JMV-180 confirm this
calcium-related theory, because BBS initiates similar intracellular
messengers, as does CCK, but is ~10-fold less potent than CCK and
does not induce as large a rise in intracellular calcium
(29). In the case of the CCK analog JMV-180, it's effect
on calcium release is also only 50-60% as efficacious as CCK and
may be induced through a different pathway (44).
Depletion of intracellular calcium inhibits protein synthesis in a
variety of cell types (18, 33, 39). We have shown that
depleting calcium from the ER or from other intracellular stores,
either by A-23187, thapsigargin, and calcium mobilizing agonists or
chelation of extracellular calcium inhibits protein synthesis and eIF2B
activity. The release of calcium induced by thapsigargin and A-23187
induced a severalfold increase in eIF2 phosphorylation and
phosphorylation of ~50% of total eIF2
. Since the ratio
eIF2
/eIF2B is 3:1, these results clearly show that a direct effect
of the phosphorylation of eIF2
can account for the inhibition of
eIF2B activity. Moreover, calcium-free media plus EGTA also inhibited
protein synthesis at all assayed CCK concentrations (from 10 pM to 10 nM), having a stronger inhibitory effect when combined with high
concentrations of CCK. Thus the trigger for the inhibition of protein
synthesis could be the depletion of the ER-sequestered calcium
(18). In general, alterations in
[Ca2+]i alter ER homeostasis and induce ER
stress. This phenomenon induces a short-term response at the
translational level and promotes a long-term adaptation to survival or
apoptotic cell death through changes in gene expression
(12). Our results suggest that pancreatic acinar cells
adapt to short-term stress induced by reduction in calcium stores by
inhibiting protein synthesis of pancreatic enzymes. The inhibition of
protein synthesis associated with high concentrations of CCK could be
an adaptive and protective mechanism. One of the most frequently used
mechanisms for translational control is the reversible phosphorylation
of eIF2, and the most likely kinase associated to phosphorylation of
the eIF2
in pancreas would be one of the ER stress-signaling kinase
such as pancreatic eIF-2
kinase, known alternatively as PEK or PERK
(8, 37). Although PEK/PERK could have a direct role in
pancreatic acinar cell ER stress, it is possible that multiple
eIF2
kinases, including PKR (double-stranded RNA-dependent protein
kinase) may signal in response to separate as well as overlapping ER
stresses (12, 40).
In summary, we conclude that CCK inhibition of protein synthesis in rat
pancreatic acinar cells is mediated through the inhibition of eIF2B
activity, and this effect is most likely related to depletion of
intracellular calcium and activation of an eIF2 kinase. This phenomenon could be related to pathophysiological effects in the case
of acute pancreatitis, where the inhibition of pancreatic digestive
enzyme synthesis could stop, to some extent, the proteolytic enzyme
autoactivation and autodigestion cascade.
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ACKNOWLEDGEMENTS |
---|
We thank Lynne Hugendubler for her assistance and instruction in the eIF2B assay.
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FOOTNOTES |
---|
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52860 (to J. A. Williams), DK-15658 (to S. R. Kimball), and DK-34933 (to the Michigan Gastrointestinal Peptide Center).
Address for reprint requests and other correspondence: M. D. Sans, 1301 E. Catherine St., 7737 Med Sci II, Ann Arbor, MI 48109-0622 (E-mail address: mdsansg{at}umich.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00274.2001
Received 20 June 2001; accepted in final form 2 November 2001.
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