(Received for publication, July 28, 1995)
From the
We recently reported that gastrin and glycine-extended
progastrin processing intermediates (G-Gly) exert growth-promoting
effects on AR4-2J cells (derived from rat pancreas) via
interaction with distinct receptors. In this study we sought to
investigate the mechanisms by which gastrin and G-Gly stimulate cell
proliferation. While gastrin increased
[Ca]
in AR4-2J
cells, G-Gly had no effect. Similarly, G-Gly had no effect either on
basal and 10
M vasoactive intestinal
polypeptide-stimulated cAMP generation, although gastrin is known to
inhibit cAMP generation. Gastrin dose dependently stimulated
AR4-2J cell mRNA content of both c-fos and
c-jun, two genes known to function in regulating cell
proliferation, but G-Gly had no effect. Gastrin also induced the
expression of luciferase in AR4-2J cells transfected with a
construct consisting of a luciferase reporter gene coupled to the serum
response element of the c-fos gene promoter. In similar
fashion, gastrin stimulated the activity of mitogen-activated protein
kinase, an enzyme known to mediate the induction of the c-fos serum response element in response to growth factor stimulation.
Although G-Gly had none of these effects of gastrin in AR4-2J
cells, it stimulated activity of c-Jun amino-terminal kinase, an enzyme
known to phosphorylate and transcriptionally activate c-Jun. These data
support the notion that gastrin stimulates cell proliferation by
inducing c-fos and c-jun gene expression, while G-Gly
acts by post-translationally regulating early gene transcriptional
activation. Our studies represent a novel model in which both the
precursor and the product of a key processing reaction, peptide
-amidation, act cooperatively to stimulate cell proliferation via
distinct receptors linked to different signal transduction pathways.
Although characterized as a stimulant of gastric acid secretion (1) , the peptide hormone gastrin also exerts growth-promoting
effects on normal and malignant gastrointestinal
tissues(2, 3, 4) . The structure of gastrin
is similar to numerous other polypeptides of the brain and gut in that
its carboxyl-terminal amino acid is amidated, and the amide moiety was
thought to be an absolute requirement for biological
activity(5, 6, 7, 8, 9) .
Recently, however, we have identified that glycine-extended
intermediates of progastrin post-translational processing (G-Gly), ()the substrate for formation of carboxyl-terminally
amidated gastrins, exert growth-promoting effects of their own via
interaction with distinct receptors(10) , introducing the novel
concept that glycine-extended intermediates of prohormone processing
reactions have independent and hitherto unrecognized important
biological functions. While some of the intracellular events that are
responsible for the growth-promoting effects of gastrin (11, 12) have been described, virtually nothing is
known about the signal transduction pathways that are activated by
G-Gly.
Growth factor stimulation of cell proliferation involves the
activation of numerous protein kinases, which, in turn, regulate the
expression and the transcriptional activation of the early response
genes c-fos and c-jun(13) . These genes take
part in different programs of cell activation, and they play an
important role in the propagation of mitogenic signals from the cell
surface to the
nucleus(13, 14, 15, 16) .
Accordingly, we examined whether gastrin and G-Gly regulate the
expression and the transcriptional activation of c-fos and
c-jun. Our studies demonstrate that while gastrin stimulates
c-fos and c-jun gene expression, G-Gly
post-translationally regulates early gene transcriptional activation.
These data support a novel model in which both the precursor and the
product of a key processing reaction, peptide -amidation, act in
concert to stimulate cell proliferation via distinct receptors linked
to different signal transduction pathways.
Since mobilization of intracellular Ca is
an essential signal transduction mechanism that mediates the action of
gastrin in AR4-2J cells(27) , we investigated the effects
of gastrin and G
-Gly on
[Ca
]
mobilization. As depicted
in Fig. 1A, in a fashion identical to our observations
in canine gastric parietal cells(17) , gastrin (10
M) increased
[Ca
]
, while
G
-Gly had no effect at any of the doses tested
(10
-10
M).
Similarly G
-Gly
(10
-10
M) had no
effect on either basal or 10
M vasoactive
intestinal polypeptide-stimulated cAMP generation, while, in contrast,
gastrin has been shown previously to inhibit cAMP generation in the
AR4-2J cells (18) (Fig. 1B). We then
examined whether the growth stimulatory effects of gastrin and
G
-Gly on AR4-2J cells are associated with
induction of the early response genes c-fos and
c-jun. As depicted in the Northern blot in Fig. 2,
gastrin (10
-10
M)
induced c-fos-specific mRNA in a dose-dependent fashion, with
a maximal effect detected at a dose of 10
M. In contrast, no induction was noted when the cells
were incubated with identical doses of G
-Gly.
Similar results were obtained when the RNA was hybridized with a
c-jun c-DNA probe (data not shown). Since growth
factor-induced protein kinases are known to target a specific DNA
regulatory element present in the promoter of the c-fos gene,
known as the serum response element (SRE)(13) , we investigated
the effects of gastrin and G
-Gly on c-fos gene transcription regulated by the SRE. For these experiments we
transfected the AR4-2J cells with luciferase reporter plasmids
containing the c-fos SRE upstream of the thymidine kinase (TK)
gene minimal promoter and the luciferase reporter gene. As depicted in Fig. 3, 10
M gastrin induced SRE
transcriptional activity 5-fold (5.0 ± 1.8-fold induction over
control, mean ± S.E., n = 4), but
G
-Gly did not, suggesting that only gastrin is
able to regulate early gene expression. Cells transfected with a
plasmid containing the TK gene minimal promoter but devoid of the SRE
exhibited a 1.4-fold induction in response to treatment with gastrin
(1.4 ± 0.1-fold induction over control, mean ± S.E., n = 11).
Figure 1:
Effect of
G-Gly on
[Ca
]
mobilization and
cAMP generation in AR4-2J cells.
[Ca
]
was measured in
Fura-2-loaded AR4-2J cells stimulated with increasing
concentrations of G
-Gly followed by a
10
M dose of G17-NH
(A). cAMP levels were measured in cells treated with
increasing concentrations of G
-Gly in the presence (hatched bars) or absence (solid bar) of
10
M of vasoactive intestinal polypeptide (B).
Figure 2:
Effect of G17-NH and
G
-Gly on c-fos gene expression in
AR4-2J cells. Aliquots of total RNA extracted following exposure
of the cells to G17-NH
or G
-Gly, were
examined by Northern blot analysis using a
P-labeled cDNA
probe for c-fos. The autoradiograms were controlled for RNA
quantity by hybridization of the RNA with a cDNA probe encoding the
ubiquitous enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Similar results were obtained when the RNA was
hybridized with a c-jun cDNA probe (data not shown). These
data were reproduced in two other separate
experiments.
Figure 3:
Effect of G17-NH and
G
-Gly on SRE transcriptional activity in
AR4-2J cells. Cells were transfected with the plasmids SRE-Luc (A) and TK-luc (B) and treated with 10
M G17-NH
and 10
M G
-Gly. Data are expressed as -fold induction
over control, mean ± S.E. *, p < 0.05. RLU,
relative light units.
Since MAPK activation is required for
induction of the SRE in response to growth factor
stimulation(28) , we tested the effect of
G-Gly on MAPK activity using in-gel kinase assays.
As shown in Fig. 4, while gastrin induced MAPK activity,
G
-Gly had no effect. JNK is known to phosphorylate
the amino terminus of c-Jun and to induce c-Jun transcriptional
activation in response to growth factor stimulation (29) .
Accordingly, we examined the effect of gastrin and
G
-Gly on AR4-2J cell JNK activity. For these
experiments we co-transfected the AR4-2-J cells with the
Gal4-cJun expression vector and the 5xGal luciferase reporter plasmid.
In this system, Gal4-cJun can transactivate and stimulate luciferase
activity only if the c-Jun amino terminus is phosphorylated by JNK.
G
-Gly dose dependently induced c-Jun
transcriptional activity with a maximal stimulatory effect achieved at
a dose of 10
M (4.4 ± 1.1-fold
induction over control, mean ± S.E., n = 4) (Fig. 5A). When we compared the effects of gastrin
(10
M) and G
-Gly
(10
M) using a larger number of
observations (n = 20), we noted that JNK activity was
also weakly stimulated by gastrin, although this effect was much less
potent than the response observed with G
-Gly (1.8
± 0.2 versus 3.2 ± 0.4-fold induction over
control, in the presence of gastrin and G
-Gly,
respectively) (Fig. 5B). The specificity of our
luciferase assay data was confirmed by solid state JNK assays, in which
we used AR4-2J cell extracts and GSH-agarose beads to which
GST-c-Jun was bound. In this system, 10
M
G
-Gly was able to stimulate JNK activity (Fig. 5C), confirming that G
-Gly
is responsible for c-Jun post-translational modification by
phosphorylation and activation.
Figure 4:
Effect of G17-NH and
G
-Gly on MAPK activity in AR4-2J cells. MAPK
activity was measured by in-gel assays in cells stimulated with
increasing concentrations of G
-Gly followed by a
10
M dose of G17-NH
. Identical
results were obtained in two other separate
experiments.
Figure 5:
Effect of G17-NH and
G
-Gly on JNK activity in AR4-2J cells. Cells
were transfected with the Gal4-cJun expression vector and the 5xGal
luciferase reporter plasmid and treated with G17-NH
and
G
-Gly (n = 4) (A). The
effects of G17-NH
(10
M) and
G
-Gly (10
M) were
compared using a larger number of observations as shown in B.
Co-transfection of the AR4-2J cells with the expression vector
Gal4-cJun(AA) (Ser
and Ser
mutated to
Ala
and Ala
) and the 5xGal luciferase
reporter plasmid gave only background luciferase activity (data not
shown). Data are expressed as -fold induction over control, mean
± S.E. *, p < 0.05. The specificity of the
luciferase assay data was confirmed by solid state JNK assays, in which
10
M G
-Gly stimulated
phosphorylation of GST-c-Jun(1-79) but not of GST-GST (C). These data were reproduced in three other separate
experiments. RLU, relative light
units.
Although characterized primarily as a stimulant of gastric
acid secretion(1) , gastrin is a potent growth factor for both
normal and malignant gastrointestinal
tissues(2, 3, 4) . Gastrin is initially
synthesized as a large precursor that is post-translationally processed
to form mature carboxyl-terminally amidated
gastrin(5, 6, 7, 8, 9) .
Despite the observation that G-Gly achieves plasma levels roughly
equivalent to those of gastrin(30) , its physiological
relevance, other than to serve as the immediate precursor for gastrin
synthesis, has remained obscure. Recently, however, we reported that
both G-Gly and gastrin acting on distinct receptors are equally potent
in stimulating the proliferation of the rat pancreatic carcinoma cell
line AR4-2J(10) . In addition, although G-Gly has
essentially no acute effect on gastric acid secretion, it appears to be
a potent inducer of the H,K
-ATPase
-subunit gene expression, indicating that it could function to
potentiate gastric acid secretagogue action by enhancing expression of
the gene responsible for H
generation(17) .
Thus, both the substrate and the product of the terminal progastrin
processing reaction appear to have important biological functions
through the activation of separate receptors. The elucidation of the
signal transduction pathways that are responsible for the numerous
physiological actions of gastrin has been the focus of intense
investigation. In the AR4-2J cells, in particular, signaling
through the gastrin/CCK
receptor is linked to the
simultaneous inhibition of cAMP generation and to the activation of
membrane phospholipid turnover(27) . In contrast, all that is
known about G-Gly signaling is that, in the gastric parietal cell, it
appears to activate cellular protein tyrosine kinases through a pathway
that is independent of [Ca
]
mobilization (17) . In this study, we have attempted to
define further the signaling pathways that are responsible for the
growth-promoting effect of G-Gly using AR4-2J cells as our model.
The process of cellular proliferation is under the control of a
complex cascade of phosphorylation reactions that is triggered by the
interaction of growth factors with their specific cellular receptors (13) . One of the best characterized pathways linked to the
control of cellular growth is known to involve the activation of the
serine-threonine protein kinase Raf through its interaction with the
small GTP-binding protein Ras(13) . Raf is responsible for the
phosphorylation and activation of a dual specificity protein kinase
(mitogen-activated protein/extracellular signal-related kinase kinase
(MEK)), which in turn phosphorylates both serine and tyrosine residues
in a family of serine-threonine protein kinases known as
MAPKs(13) . The MAPKs phosphorylate numerous cellular proteins,
including transcription factors such as p62 or Elk-1 that
play an important role in the transcriptional regulation of the
promoter of the early response gene c-fos through the SRE (13, 28) . These early response genes propagate
mitogenic signals from second messengers to the nucleus and activate or
repress the next set of genes in the biological programs initiated by
the extracellular signals(13, 16) . The activity of
the best characterized members of this family of genes, c-fos and c-jun, is extensively regulated by phosphorylation
catalyzed by specific kinases such as JNK, which appears to act in
concert with the MAPK pathway in the complex regulation of early gene
function(29, 31, 32, 33) .
In our
report we have demonstrated that gastrin can induce
[Ca]
mobilization, stimulate
c-fos and c-jun gene expression, and induce MAPK
activation, confirming previous reports suggesting that its
growth-promoting effects could be mediated at least in part by the
activation of these signaling pathways(11, 12) . In
contrast, G-Gly had no effect on cAMP generation,
[Ca
]
mobilization, early gene
expression, and MAPK activation, indicating that G-Gly acts on a
receptor distinct from the gastrin/CCK
receptor, which is
linked to different signaling systems. Thus, we tested the hypothesis
that the growth stimulatory effect of G-Gly could involve JNK induction
and subsequent early gene transcriptional activation by
phosphorylation. For these experiments we took advantage of chimeric
Gal4-cJun proteins that are capable of transactivating and stimulating
transcription only if the c-Jun amino terminus is phosphorylated by
JNK(19) . In this system, G-Gly clearly was active and potent
in stimulating c-Jun transcriptional activity. These findings support
the notion that while gastrin is responsible for the induction of early
gene expression, most likely through the activation of the MAPK
pathway, G-Gly post-translationally activates c-Jun by stimulation of
JNK. Further studies are required to establish the exact chain of
upstream protein kinases that are activated by the G-Gly receptor and
in the end are able to target JNK. Nevertheless, our studies present a
novel model in which both the precursor and the product of a key
processing reaction,
-amidation, act in concert to stimulate cell
proliferation via distinct receptors linked to different but
complementary signal transduction pathways.