(Received for publication, December 11, 1995; and in revised form, February 28, 1996)
From the
Glucocorticoids are known to promote the formation of zymogen
granules in acinar cells of the exocrine pancreas in vivo as
well as in vitro. To gain insight into the mechanism of this
regulation, we studied the effects of glucocorticoids on the synthesis
of two components of the secretory granule membrane, the glycoprotein 2
(GP-2) and the -glutamyl transpeptidase (GGT). It was demonstrated
that following adrenalectomy, degranulation of pancreatic acinar cells
is accompanied by a sharp decrease in GGT and GP-2 synthesis as
measured by mRNA and protein accumulation. The decline of GGT synthesis
was prevented by glucocorticoid replacement therapy, whereas GP-2
synthesis could be maintained with either glucocorticoid or estradiol
treatment. These in vivo observations were corroborated and
extended in an in vitro study using AR42J pancreatic cells.
With this cell line, it was demonstrated that dexamethasone induces the
formation of zymogen granules and the accumulation of a specific GGT
transcript (mRNA III) by decreasing its degradation rate. At the same
time, the GP-2 mRNA level was not modified by the hormonal treatment.
These data demonstrate that glucocorticoids exert a positive control on
the GGT expression in pancreatic cells at a post-transcriptional level.
GGT, an enzyme of the glutathione metabolism, could play a significant
role in protein packaging in secretory cells.
In eukaryotic cells, newly synthesized secretory proteins are sequestered in the rough endoplasmic reticulum and delivered to the Golgi apparatus. At the trans-side of the Golgi, most of the secretory proteins are packaged into secretory granules, a storage compartment. Upon stimulation, the granules move to the cell apex and release their content by exocytosis(1, 2) .
Despite a
considerable amount of progress in our understanding of the
intracellular trafficking of secretory proteins, the mechanisms by
which they are packaged and maintained in an osmotically inactive form
in the zymogen granule (ZG) ()is unknown. Possible clues for
the elucidation of these processes come from experiments which reported
that adrenalectomy of male rats caused a marked depletion of ZG in the
pancreas acinar cells, an effect which was reversed by treatment with
the synthetic glucocorticoid, triamcinolone(3, 4) . It
was also found that a dexamethasone treatment of the AR42J rat
pancreatic cells induces the appearance of ZG(5) . These
observations led us to the hypothesis that glucocorticoids were
directly involved in the packaging of secretory proteins and ZG
formation.
Glucocorticoids could influence the packaging of
secretory proteins and ZG formation through the ZG membrane protein
content. The latter membrane is a highly specialized membrane; it
contains, roughly, 10 major proteins and shows a rather limited
spectrum of polypeptides(6, 7) , with a significant
overlapping composition to membranes from other exocrine glands,
irrespective of the nature of stored proteins(8) . The
glycoprotein 2 (GP-2) is one of the major components of this membrane;
it is a glycosyl phosphatidylinositol-anchored protein (9, 10) that is released in the pancreatic ducts upon
cleavage with a phosphatidylinositol-phospholipase. The released GP-2
polymerizes and forms a fibrillar network(11, 12) .
The physiological role of GP-2 is not yet defined, but this fibrillar
network may form a barrier to colonization of the pancreatic duct by
intestinal bacteria. In addition, recent studies have shown that GP-2
shares some antigenic determinants and structural homology with a
phospholipase A(13) .
-Glutamyl transpeptidase
(GGT) is another glycoprotein component of the secretory granule
membranes, which has been used as a marker to follow their isolation by
subcellular fractionation(8) . In the exocrine pancreas, we
localized GGT, which is a type II membrane protein, in the ZG membrane,
the apical membrane and pancreatic juice (14) where it is
associated with ``pancreasomes'' (12, 15) .
GGT hydrolyzes glutathione, which may affect the disulfide bridges of
the proteins with high cysteine content, like GP2, along the secretory
pathway(16) . Biochemical and immunocytochemical observations
have indicated that both GP-2 and GGT are continuously released by the
pancreas acinar cell in the pancreatic juice, and, hence, a substantial
fraction of these proteins do not follow its host membrane that
recycles after exocytosis(11, 12, 15) . This
implies a de novo synthesis of these proteins and their
insertion into the recycled membrane as new granules form. If these
proteins are essential for protein packaging and ZG formation,
therefore, one could postulate an increase in their rate of synthesis
when ZG formation is induced by glucocorticoids(4) .
The study of the expression of these two very different membrane proteins is now greatly facilitated as a result of cloning and sequencing of their respective cDNAs(10, 17) . In the rat, GGT is encoded by a single copy gene (18) transcribed into several mRNA species (mRNAs I to V) according to the tissues(19, 20, 21, 22, 23) . The GP-2 protein is also encoded by a single copy gene, but only one species of mRNA is transcribed(10, 24) . In this work, we show that in acinar cells, glucocorticoid treatment induces in parallel ZG formation and GGT expression. The GGT regulation occurs at a post-transcriptional level since glucocorticoids induce the accumulation of the GGT mRNA III transcript by decreasing its degradation. We also provide evidence that ZG formation can be induced independently of GP-2 expression.
The labeled GGT complementary RNA
(cRNA) probes, which hybridize specifically to the GGT mRNA I
(cRNA-17), mRNA II (cRNA-12), mRNA III (cRNA-3), or to all the GGT mRNA
types (cRNA-139A), were synthesized by in vitro transcription
of the GGT plasmids pGEM-17, pGEM-12, FL2, or pGEM4-139 as
described elsewhere (19, 22) . These plasmids were
linearized by NsiI (pGEM-17), HincII (pGEM-12), or SacI (FL2 and pGEM4-139) prior to their transcription
from the T7 promoter in the presence of
[-
P]UTP, except for FL2 which was
transcribed from its SP6 promoter. The GP-2 cRNA probe was obtained
from a GP-2 recombinant plasmid (10) kindly provided by Dr. C.
Rindler. The coding sequence, from the nucleotide 174 to the nucleotide
1607, was subcloned into pGEM-4 (Promega, Madison) and digested by EcoRI and BamHI. The recombinant plasmid was then
linearized by EcoRI and transcribed from its T7 promoter. The
-actin probe was obtained by labeling the 800-bp HindIII-KpnI cDNA fragment(30) , using the
megaprime labeling kit (Amersham, France) in the presence of 50 µCi
of [
-
P]dATP.
Figure 1:
Effect of dexamethasone and estradiol
on granulation of rat pancreatic acinar cells (800
magnification). A, sham-operated rats. Zymogen granules are
numerous on the luminal side of the acini. B, adrenalectomized
rats. The number of granules decreases in each cell. C,
adrenalectomized rats treated by estradiol. The hormone does not modify
the degranulation induced by adrenalectomy. D,
adrenalectomized rats treated by glucocorticoids. Regranulation is
observed in all acini; the number of zymogen granules is higher than in
control cells from sham-operated rats.
Figure 2:
Effect of dexamethasone on granulation of
AR42J cells. A, control AR42J cells. Few ZG are found in these
cells where nucleus (NU) occupy a large volume ( 9900). B, AR42J cells treated with 10
M dexamethasone during 48 h. An increase in the number of ZG (arrowhead) and in the cytoplasmic volume can be observed
(
9900). RER, rough endoplasmic
reticulum).
Figure 3: Effect of dexamethasone on GGT activity in AR42J cells. Cells in exponential phase of growth were cultured with or without dexamethasone (100 nM) for different periods of time, up to 72 h. GGT activities were obtained from two independent experiments where the assays were carried out in duplicate. Open squares, control cells; solid circles, dexamethasone-treated cells.
GP-2 protein level was measured by the Western blot technique on protein extracts prepared from pancreas and AR42J cells. The general pattern of the proteins appears to be similar among the different samples (Fig. 4). Immunoblotting with GP-2 polyclonal antibodies reveals a single band in control pancreatic extracts. This band exhibits an apparent molecular weight of 78,000 that corresponds to the glycosylated GP2. This polypeptide cannot be detected in the pancreas of adrenalectomized animals, but it reappears following a glucocorticoid replacement therapy (Fig. 4). No GP-2 polypeptide can be detected in AR42J protein extracts even when granule formation has been stimulated by glucocorticoids (Fig. 4).
Figure 4:
Western blot analysis of GP-2 protein in
rat pancreas and AR42J cells. Left panel, Coomassie Blue
staining of protein extracts that are PANCREAS, CONTROL (sham-operated rats), ADX (adrenalectomized rats), ADX + DEX (adrenalectomized rats + dexamethasone),
and AR42J, DEX (10M),
DEX (10
M) (cells treated by 10
and 100 µM dexamethasone during 48 h), CONTROL (cells cultured in absence of dexamethasone). Right
panel, detection of GP-2 protein on the Western blot using rabbit
GP-2 antibodies. Molecular mass standards (Bio-Rad Labs, Mississauga,
Ontario, Canada) are 97.4, 66.4, 45.0, 31.0, 21.5, 14.4
kDa.
Figure 5:
Northern blot analysis of rat GGT mRNA in
rat pancreas and AR42J cells. Poly(A) RNA (5 µg)
were separated on an agarose denaturing gel and blotted onto a nylon
membrane. The blots were hybridized to the GGT cRNA-139A probe that
hybridizes to the coding region (A), to the actin cDNA probe (B), to the GGT cRNA-3, a specific probe for the GGT mRNA III (C), and to the GGT cRNA-12, a specific probe for the mRNA II (D). Autoradiography was performed with two intensifying
screens at -80 °C for three days. The samples were obtained
from fetal liver (FETAL LIVER), from AR42J cells cultured in
the absence (-DEX), or in the presence of 100 nM dexamethasone (+DEX) during 48 h and from pancreas
from rats with different glucocorticoid status. SHAM, sham-operated; ADX, adrenalectomized; ADX + DEX, adrenalectomized + dexamethasone; ADX + EST, adrenalectomized + estradiol.
To determine whether the GGT mRNA III accumulation in pancreatic cells was under a direct control by glucocorticoids, GGT mRNA accumulation was measured in the AR42J cells. When cultured in the absence of hormone, these cells express the 2.4-kb GGT mRNA at a low level, as revealed by the hybridization to the cRNA-139A probe (Fig. 5A). Addition of 100 nM dexamethasone to the culture medium for 2 days induces a strong increase in the expression of this mRNA species (Fig. 5A), identified as the GGT mRNA III species by hybridization to the cRNA-3 probe (Fig. 5C). Hybridization to the cRNA-12 probe does not reveal any clear signal in AR42J cells (Fig. 5D). Control hybridization to an actin probe shows that equal amounts of RNA were loaded on both lanes (Fig. 5B). Northern blot analysis clearly shows that the increase in the GGT enzyme activity induced by glucocorticoids in pancreatic cells results from the accumulation of the GGT mRNA III.
GP-2 mRNA expression was analyzed in AR42J cells, pancreas, and in other rat tissues. A strong signal corresponding to the 1.9-kb GP-2 mRNA (10) is obtained in the pancreas from sham-operated rats (Fig. 6A). This signal is markedly decreased in the adrenalectomized group, whereas treatment with dexamethasone increases the GP-2 mRNA to a level that is higher than in the control group. Replacement therapy with estradiol also prevents a decrease of GP-2 mRNA level in adrenalectomized rats. A control hybridization with the actin probe (Fig. 6B) does not reveal inequal mRNA loading. No GP-2 mRNA sequences are detected in samples from fetal liver (Fig. 6A) or in adult liver, lactating mammary gland, small intestine, and kidney (data not shown). In AR42J cells, the basal level of GP-2 mRNA is low and does not change significantly following a 48-h treatment by 100 nM dexamethasone (Fig. 6A). It can be noticed that in the dexamethasone-treated sample, the GP-2 mRNA migrates slightly faster than in the control sample, whereas actin mRNA migrate at the same rate in both samples (Fig. 5B). There is no clear explanation for these data; it can only be speculated that both GP-2 mRNA could derive from different transcripts, initiated at different sites on the GP-2 gene, or from the same primary transcript, by alternate splicing or use of different polyadenylation sites.
Figure 6:
Northern blot analysis of GP2 mRNA in rat
pancreas and AR42J cells. Poly(A) RNA (5 µg) were
separated on an agarose denaturing gel and blotted onto a nylon
membrane. The blots were hybridized to the GP-2 cRNA probe (A)
or to the actin cDNA probe (B). Autoradiography was performed
with two intensifying screens at -80 °C for 3 days except the
fetal liver and pancreas samples in A (5 h). The samples were
obtained from fetal liver (FETAL LIVER), from AR42J cells
cultured in the absence (-DEX), or in the presence
(+DEX) of 100 nM dexamethasone during 48 h and
from pancreas from rats with different glucocorticoid status. SHAM, sham-operated; ADX, adrenalectomized; ADX + DEX, adrenalectomized + dexamethasone; ADX + EST, adrenalectomized +
estradiol.
Figure 7:
Dexamethasone effects on GP-2 and GGT gene
transcription. AR42J cells were pretreated with 100 nM dexamethasone for 0, 12, or 48 h; nuclei were isolated and allowed
to continue transcription in the presence of
[-
P]UTP. The newly transcribed labeled RNAs
were hybridized to a nylon membrane where a GP-2-specific cDNA
sequence, pGEM (negative control), the 470-bp GGT genomic
sequence described under ``Experimental Procedures,'' and an
amylase-specific cDNA sequence have been immobilized. A,
autoradiograms were exposed with two intensifying screens at -80
°C for 10 h. B, densitometric analysis of the
autoradiogram.
Figure 8: Decay of GGT mRNA III accumulation in AR42J cells treated with actinomycin D. Cells were cultured for 48 h with or without 100 nM dexamethasone prior to the addition of actinomycin D (time 0) at the concentration of 1.25 µg/ml and maintained in culture for up to 8 h. The amount of GGT mRNA III was assessed by an RNase protection assay, using 10-30 µg of RNA extracted from dexamethasone-treated cells and 30-40 µg of RNA extracted from control cells, after different times of treatment with actinomycin D. A, autoradiograms of the GGT-protected sequences obtained from a typical experiment (30 µg RNA) and separated on an acrylamide gel. The undigested probe (362 nucleotides) was loaded on the gel along with the dexamethasone-treated samples. The length of the protected cRNA probe fragment was 294 bases. Autoradiograms were exposed with two intensifying screens, at -80 °C, for 4 days (samples from control cells) and without screen for 24 h (samples from dexamethasone-treated cells). B, GGT mRNA III decay curves. Curves were drawn from average values obtained from three to six independent experiments (± S.E.). For each condition (+ DEX or -DEX), the mRNA accumulation remaining after different times of exposure to actinomycin D is expressed as a percentage of the mRNA level before the addition of the drug (time 0: 100%) and plotted against time on a semi-logarithmic scale. Open triangles, dexamethasone-treated cells; solid circles, control cells.
To gain insight into the mechanisms of ZG formation in pancreatic acinar cells, we investigated the influence of glucocorticoids and estradiol on the expression of GGT and GP-2 proteins in the rat pancreas in vivo and in vitro, using the AR42J pancreatic carcinoma cell line. Adrenalectomy caused a marked depletion of zymogen granules that was prevented by glucocorticoid replacement therapy (Fig. 1) as described previously(4) . In adrenalectomized animals we observed a decrease in the GP-2 mRNA level that cannot be related to a specific effect of glucocorticoids since it can also be prevented by estradiol (Fig. 6). The amount of GP-2 protein in pancreas is correlated to the GP-2 mRNA level under different glucocorticoid status (Fig. 4). In AR42J cells, however, glucocorticoid treatment, which promotes extensive granulation of these cells (Fig. 2), induces neither the GP-2 mRNA nor the GP-2 protein level ( Fig. 4and Fig. 6). These data show that granule formation can occur independently of GP-2 synthesis. This fact is corroborated by the absence of GP-2 mRNA in the lactating mammary gland (data not shown), an organ very active in protein secretion. In a recent study, Dittié and Kern (34) reached similar conclusions based on the following observations. 1) GP-2 mRNA was undetectable in fetal pancreas during the differentiation period, including when ZG formation takes place. 2) GP-2 mRNA synthesis does not occur in parotid and adrenal glands. 3) Glucocorticoids are without effect on GP-2 mRNA accumulation in AR42J cells.
In contrast to GP-2, GGT expression is clearly under a direct positive control of glucocorticoids. This is supported by a decrease of both enzyme activity and mRNA level in adrenalectomized animals and by prevention of these effects with dexamethasone but not with an estradiol replacement therapy (Fig. 5). This direct effect of the hormone can be confirmed in vitro in AR42J cells that respond to dexamethasone by a marked increase in the GGT activity (Fig. 3). Northern blot analysis clearly showed that the increase in mRNA induced by glucocorticoids, observed both in vivo and in vitro, results from an accumulation of the GGT mRNA III (Fig. 5), transcribed from GGT promoter III on the GGT gene(22) . The induction of the GGT expression by glucocorticoids is not due to a direct effect of the hormone receptor complex onto the palindromic target sequence, previously identified on the GGT promoter III(22) , or to the participation of glucocorticoid-regulated factors that could act at the level of the GGT gene. In fact, no hormonal effect can be observed on the transcription rate of the GGT promoter III in AR42J cells after a 48-h glucocorticoid exposure (Fig. 7), conditions which induce markedly the GGT mRNA III accumulation. Under the same conditions, we observed an increase in the amylase gene transcription rate that is known to be mediated by a glucocorticoid-induced factor (35) . In contrast, our data show that the glucocorticoid treatment has a strong stabilizing effect on the GGT mRNA III by preventing the rapid decay observed in control cells (Fig. 8). The effect of glucocorticoids on GGT activity is prevented by the anti-glucocorticoid RU-38486. This indicates that the effect requires binding of the hormone to its receptor and subsequent interaction of this complex with a glucocorticoid-responsive gene encoding a protein that is able to modulate the GGT transcript degradation rate. This effect on mRNA stability, which would require RNA and protein synthesis, is most consistent with the long lag observed between addition of the hormone and induction of GGT activity. Such a post-transcriptional effect of the glucocorticoids on mRNA half-life has already been reported for insulin(36) , growth hormone, and its receptor(37) . The mechanisms of this regulation are not yet clearly known, but the presence in these mRNAs of unique sequences that could act as a target for glucocorticoids stabilizing or destabilizing factors has been proposed.
In contrast to GP-2, GGT synthesis parallels the zymogen granule content in pancreatic cells in vivo and in vitro in AR42J cells. During development, patterns of GGT and amylase expression are biphasic and comparable(38) . This developmental profile corresponds to the massive granule formation that occurs before birth and at the weaning period (39) and has been associated to the fluctuating levels of glucocorticoids(40) . All these data support the view that GGT protein expression is associated with zymogen granule formation. The quantitative differences observed between the extent of granulation and GGT activity in the pancreas from adrenalectomized rats subjected to glucocorticoid therapy indicate that a partial recovery of GGT activity might be sufficient to obtain optimal granulation. However, the precise roles for GGT and its substrate, glutathione (GSH), in the secretory process are still conjectural. Glutathione is the major redox buffer in the cell, and its role in protein secretion has been emphasized(41) . Moreover, it has been shown that the GHS/GSSG ratio, which exhibits a value greater than 30 in the cytoplasm, drops to nearly 5 in the secretory pathway(16) , a ratio suitable for an optimal oxidative protein folding catalyzed by a disulfide isomerase (42) . The mechanism maintaining this low GSH/GSSG ratio remains elusive. We speculate that GGT, by initiating GSH degradation into cysteinyl glycine in the granule, may contribute to keeping it in a more oxidized state, since cysteinyl glycine can easily undergo a rapid nonenzymatic oxidation(43, 44) .
In conclusion, this study shows that ZG formation induced by glucocorticoids is closely linked to GGT expression. The localization of this enzyme in the granules of the pancreas and other secretory glands(8) , as well as its induction in the lactating mammary gland (45) , suggests that glucocorticoids may play a significant role in the packaging process by regulating the expression of GGT in these glands. The glucocorticoids appear now to have a well integrated function interacting at different levels of the secretory process, synthesis of secretory proteins as reported for amylase (35) or casein(46) , increase in the number of cholecystokinin receptors(47) , and also synthesis of protein components of secretory vesicles, as demonstrated in this study.