(Received for publication, May 18, 1995; and in revised form, July 20, 1995)
From the
Expression of the pancreatitis-associated protein I (PAP I), an
exocrine pancreatic protein, increases rapidly and strongly in acinar
cells during the acute phase of pancreatitis. This is reminiscent of
the response to stress of acute phase proteins. We have previously
demonstrated that serum factors from rats with acute pancreatitis, but
not from healthy rats, could induce endogenous PAP I gene expression in
the acinar cell line AR-42J (Dusetti, N., Mallo, G., Dagorn, J.-C.,
Iovanna, J. L. (1994) Biochem. Biophys. Res. Commun. 204,
238-243). In the present work, we have evaluated the influence of
several mediators of inflammation on rat PAP I gene transcription in
these cells. Tumor necrosis factor induced an increase in PAP I
mRNA expression, and interferon
caused an even greater increase
in PAP I mRNA level. These stimulations were antagonized by
dexamethasone. Interleukin (IL)-1, IL-6, or dexamethasone alone were
ineffective. Combinations of IL-1 with IL-6 or dexamethasone were also
ineffective. IL-6 and dexamethasone together induced a marked
stimulation of PAP I gene transcription, and this effect was slightly
attenuated by IL-1. To analyze the cis-regulatory elements
responsible for the induction of transcription, we fused a 1.2-kilobase
segment of the rat PAP I promoter to the chloramphenicol
acetyltransferase (CAT) gene as reporter. The resultant chimeric DNA
was transfected into AR-42J cells. Addition of IL-6 or dexamethasone
was ineffective, whereas their mixture increased the CAT activity 12
times. Progressive deletions of the PAP I promoter were then fused to
the CAT gene, and the constructs were transfected to AR-42J cells. A
12-fold increase in CAT activity was seen upon IL-6/dexamethasone
treatment with constructs containing more than 274 base pairs upstream
from the cap site. In that region, two sequences are similar to the
canonical IL-6 response element. Site-directed mutagenesis of these
regions strongly decreased induction, showing that they were
functional. PAP I should therefore be classified among acute phase
proteins of class 2, whose expression is increased by IL-6 acting in
combination with glucocorticoids.
The acute phase of pancreatitis is characterized by a pattern of
changes in the expression of secretory proteins(1) . Whereas
expression of most pancreatic enzymes decreases, mRNA levels of the rat
pancreatitis-associated protein (PAP) ()increase
dramatically. Recently, we have described two other PAP-related mRNAs
and named the corresponding proteins PAP II and PAP
III(2, 3) . In consequence, the original PAP became
PAP I. Like PAP I, PAP II and III are induced in pancreas during the
acute phase of pancreatitis. The sequences of the genes encoding rat
PAP I, II, and III have been recently determined (2, 4, 5) . All three genes are organized in
six exons, and similarities observed in their coding sequences extend
to their 5`-flanking regions. In addition, the three genes have been
located to the same position on chromosome 4q33-34(6) ,
suggesting that they derived from the same ancestral gene by gene
duplication.
In fact PAP I was not detectable in the pancreas of
healthy animals. It could be evidenced in pancreatic juice 6 h after
induction of an experimental acute pancreatitis, reached a maximum
during the acute phase (12-48 h), and disappeared during
recovery(7) . The rapid and strong induction of the PAPs is
unique among secretory proteins and reminiscent of the response to
stress of acute phase proteins. Recently, we have demonstrated the
presence of factors in serum from rats with acute pancreatitis, but not
from healthy rats, capable of inducing PAP I gene expression in the
pancreatic acinar cell line AR-42J. In addition, the cis-acting element was localized within the 1.2 kilobases
upstream region of the transcription start site(8) . It has
long been known that the changes occurring in the liver and in other
organs during the acute phase response are coordinated by signals
generated at the site of injury, among which several cytokines have
been well characterized, including IL-1, IL-6, TNF, IFN
,
leukemia inhibitory factor, IL-11, and oncostatin M. These proteins are
locally produced by the tissue and by circulating mononuclear cells in
response to prototype inflammatory stimuli and can elicit the diverse
biological effects characteristic of the acute phase response.
Interestingly, during the acute phase of pancreatitis, levels of
cytokines are strongly increased in serum(9) . In the current
study, we have evaluated the respective contributions of several
cytokines and of dexamethasone to the transcriptional induction of the
rat PAP I gene in vitro, using a rat pancreatic acinar cell
line.
Figure 5: Identification of two functional IL-6REs within the PAP I promoter by site-directed mutagenesis. A, nucleotide substitutions in p-274/+10PAPI-CAT plasmid. B, AR-42J cells were transfected with 20 µg of plasmids p-274/+10PAPI-CAT, pmut1-274/+10PAPI-CAT, pmut2-274/+10PAPI-CAT, or pmut3-274/+10PAPI-CAT. Thirty-six hours after transfection of AR-42J cells, IL-6 (100 units/ml) and dexamethasone (100 nM) were added to the culture medium. Cells with no hormones added were taken as controls. Specific induction by IL-6/dexamethasone was calculated as the ratios of the values from induced and control cells. Values represent the means (± standard error) of six independent transfection experiments.
Figure 1:
Induction of PAP I
mRNA accumulation by cytokines and dexamethasone treatment. Forty-eight
hours after placing AR-42J cells in culture, cytokines and
dexamethasone, alone or in combination, were added to the culture
medium. After 24 h total RNA was isolated, submitted to electrophoresis
(15 µg/lane) through a formaldehyde-agarose gel, transferred to a
nylon membrane, and hybridized to P-labeled cDNAs specific
for the PAP I and
-actin mRNA. Results for IL-1, IL-6, and
dexamethasone are given in panelA. Results for
IFN
, TNF
, and dexamethasone are given in panelB; results with dexamethasone and the combination
IL-6/dexamethasone, from an experiment run in parallel, are given as
controls. Concentration of cytokines and dexamethasone are provided in
the corresponding tables. Lanes7 and 13 in panelA and lane9 in panelB refer to experiments where no cytokines or
dexamethasone were added.
Figure 2:
Induction of PAP I/CAT hybrid gene
expression by IL-6 and dexamethasone. AR-42J cells were transfected
with 20 µg of the p-1253/+10PAPI-CAT hybrid gene and
treated with IL-6 (100 units/ml) and dexamethasone (100 nM)
individually or in combination. CAT activities were quantitated using a
phase extraction procedure. CAT activity was normalized for
transfection efficiency, using the ratio of CAT activity to
-galactosidase activity. Values represent the means of six
independent transfection experiments (± standard error). In each
experiment, CAT activities were expressed relative to the level of CAT
activity in untreated control cells, which was assigned a value of
1.0.
Figure 3:
Deletion analysis of the rat PAP I
promoter. Numbers in plasmid names refer to the position of
first and last nucleotides of the PAP I gene. Relative CAT activity
(± standard error) in extracts from AR-42J cells transfected
with the corresponding plasmids was measured. CAT activity was
normalized for transfection efficiency, using the ratio of CAT activity
to -galactosidase activity. Values represent the means of six to
nine independent transfection experiments. Values were expressed as
percentage of the p-1253/+10PAPI-CAT
activity.
A 12-fold increase in CAT activity was seen upon IL-6/dexamethasone treatment of cells transfected with constructs containing more than 274 base pairs of 5`-flanking sequence (Fig. 4). Deletion to position -180 led to a 3-4-fold drop in induction. Finally, a 2-fold induction was observed when we transfected with p-118/+10PAPI-CAT and p-61/+10PAPI-CAT constructs but not with p+10/-1253PAPI-CAT.
Figure 4: Localization of IL-6/dexamethasone response regions in PAP I promoter. AR-42J cells were transfected with 20 µg of the plasmids described in Fig. 3. Thirty-six hours after transfection, AR-42J cells were incubated with medium alone (control) or with IL-6 (100 units/ml) in association with dexamethasone (100 nM). Specific induction by IL-6/dexamethasone was calculated as the ratio of the values from induced and control cells. Values represent the means (± standard error) of four to seven independent transfection experiments.
Induction of an experimental pancreatitis causes a more than
200-fold increase in PAP I mRNA expression during the acute phase of
pancreatitis(7) . PAP I mRNA accumulation reaches a maximum 6 h
after induction, with a kinetics probably controlled by the cascade of
events taking place during the acute phase. That cascade includes the
activation of monocytes and macrophages and the synthesis and secretion
of inflammatory mediators eventually transported to the target cells.
In support of that hypothesis, we have recently demonstrated the
presence of factors in serum from rats with pancreatitis, but not from
healthy rats, capable to induce PAP I gene expression(8) . The
present work was carried out primarily to localize the cis-regulatory elements in the PAP I gene and to characterize
their response to several acute phase mediators including IL-1, IL-6,
IFN, TNF
, and dexamethasone.
The cytokines tested in this
study had very different effects on PAP I gene expression in AR-42J
cells (Fig. 1). The most striking result was the strong
stimulation of the association IL-6/dexamethasone, and the limited
stimulation by IFN or TNF
, compared to the absence of effects
of IL-1 or IL-6. Another intriguing finding was the inhibition by IL-1
of IL-6/dexamethasone stimulation. However, a growing number of reports
show that expression of acute phase protein genes is not always
mediated by single cytokines but by combinations of several cytokines (15, 16, 17) or by cytokines in association
with cofactors such as glucocorticoids(16) . It was also shown
that one cytokine may modulate the effect of other
cytokines(17, 18) . These findings suggest that
specific responses of a cell to various inflammatory stimuli are
mediated by specific combinations of cytokines and/or glucocorticoids.
Although an important regulatory function during the acute phase
reaction has been attributed to glucocorticoids and IL-6(17) ,
at the utilized doses, dexamethasone and IL-6 alone were unable to
induce PAP I gene expression in AR-42J cells ( Fig. 1and Fig. 2). Then, two mechanisms may account for the synergy
between IL-6 and dexamethasone. First, glucocorticoid and IL-6 response
elements might be localized in close vicinity on the PAP I promoter. In
that instance, interaction of the nuclear factors binding the two
transcription activator elements might enhance individual responses
that would be otherwise too weak to be observed. Such a situation was
reported for the
-acid glycoprotein(19) .
Second, dexamethasone could stimulate IL-6 receptor synthesis in AR-42J
cells, resulting in an increased number of IL-6 receptors at the cell
surface. This was already observed in hepatic
cells(20, 21) . That mechanism might apply to the PAP
I gene since, in the hepatic carcinoma cell line HepG2 transfected with
p-1253/+10PAPI-CAT, CAT activity could be strongly induced
(25-fold) by IL-6 alone (data not shown). Hence, the IL-6 enhancer
element of the PAP I promoter does not require glucocorticoids to be
active. More studies are, however, necessary to understand the
synergistic effect of IL-6 and dexamethasone on the PAP I gene
induction in AR-42J cells.
The mechanism by which IL-1
down-regulates the stimulation by IL-6 associated with dexamethasone is
also unknown. IL-1 has already been shown to inhibit IL-6 induction of
the endogenous T kininogen in rat primary hepatocytes(22) , but
IL-1 and IL-6 can also act independently (additive effect) or
synergistically in the regulation of other acute phase genes such as
-acid glycoprotein, haptoglobin, hemopexin, complement
C
, and serum amyloid A(17) . Therefore, relative
positions of the different enhancer sequences are likely to influence
the effect of cytokines acting in combination. A mechanism involving
interaction of IL-1 with expression of the IL-6 receptor, as suggested
for dexamethasone, cannot be ruled out, although such regulation has
never been reported in other systems. However, inhibition by
interaction of IL-1 with the IL-6 receptor is unlikely because the two
cytokines have their own specific membrane receptors.
PAP I gene
expression was significantly induced by IFN or TNF
, although
100-fold less than with IL-6 and dexamethasone. Other genes induced by
TNF
are also induced by
IFN
(23, 24, 25) . This may be due to the
ability of TNF
and IFN
to activate the same transcription
factors, such as interferon regulatory factors 1 and
2(26, 27) . A similar PAP I mRNA induction was
obtained with 100, 500, or 1000 units/ml IFN
, but an inhibitory
effect was observed when we incubated the cells in presence of more
than 500 units/ml TNF
(Fig. 1), suggesting a toxic effect
of this cytokine. Addition of dexamethasone to these cytokines
inhibited induction, as already reported for other
genes(28, 29, 30, 31, 32, 33, 34) .
Again, the opposite effect of dexamethasone on IL-6 and TNF
or
IFN
underscores that understanding the mechanism of effector
action requires a detailed topological analysis of promoter sequences.
We have chosen to address in this study the molecular mechanism of IL-6 and dexamethasone stimulation of the PAP I promoter. Analysis of the promoter sequence revealed the presence in two positions of the potential IL-6 response element of type 2 (CTGGGA), previously identified in several acute-phase genes (17, 18, 35) and shown to be functional by mutation analysis(36) . Demonstration that the two IL-6REs identified in the PAP I promoter were indeed functional was obtained by mutation and transfection assays (Fig. 5). However, these are not the only cis-elements involved in the IL-6/dexamethasone response of PAP I. Another cis-cytokine response element, localized between -61 and +10, is responsible for a 2-fold induction. This has been shown previously in other IL-6-activated cellular genes(37) . For instance, Baumann et al.(38) have demonstrated for several acute phase proteins that IL-6 acts directly through an IL-6RE but also indirectly by increasing expression of C/EBPs, which in turn stimulates acute phase proteins gene expression.
Acute phase proteins have been divided
into two subclasses according to their pattern of regulation by
cytokines(17) . The synthesis of class 1 acute phase proteins (e.g. -acid glycoprotein, C-reactive protein,
haptoglobin, and serum amyloid A) is induced by IL-1 or combinations of
IL-1 and IL-6, whereas the genes for class 2 acute phase proteins (e.g.
-macroglobulin,
-antichymotrypsin, and fibrinogen) are mainly
regulated by IL-6 and glucocorticoids. PAP I is therefore an additional
member of the second group of acute phase proteins, with the original
feature of being a secretory protein.
Finally, it is interesting to note that whereas the PAP I gene is expressed as an acute phase protein in pancreas, it is constitutively expressed by the epithelial cells of the intestinal tract(39, 40) . The PAP I promoter is therefore complex. It confers to the gene the capacity of being regulated along several pathways, the switch between pathways being possibly under the control of tissue-specific elements.