Transcription of the Rat Serine Protease Inhibitor 2.1 Gene in Vivo: Correlation with GAGA Box Promoter Occupancy and Mechanism of Cytokine-Mediated Down-Regulation
Anne Emmanuelle Simar-Blanchet,
Catherine Legraverend,
Jean Paul Thissen1 and
Alphonse Le Cam
Laboratoire INSERM U376 Hôpital Arnaud de Villeneuve
34295 Montpellier cedex 05, France
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ABSTRACT
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Two GH-response elements (GHREs) and a single
glucocorticoid (GC)-response element were found to regulate activity of
the rat serine protease inhibitor 2.1 gene (spi 2.1)
promoter in vitro. To assess the physiological relevance of
these observations, we have investigated the relationship existing
between the level of spi 2.1 gene transcription, structural
modifications of the chromatin, and in vivo nuclear
protein-promoter interactions monitored by genomic footprinting, in
control, hypophysectomized, and inflamed rats. We also addressed the
mechanism of inflammation-mediated gene down-regulation. We found that
a high level of spi 2.1 gene transcription correlates
with hypersensitivity of the promoter to deoxyribonuclease I (DNase
I) and maximal occupancy of the GAGA box (GHRE-I). The failure of
GAGA-box binding proteins (GAGA-BPs) to interact with the GAGA box
appears to result from an impairment in GH action due to its absence
(i.e. hypophysectomized animals) or to the appearance of a
cytokine-mediated GH-resistant state (i.e. inflamed rats)
in liver. Unlike the GAGA box, signal transducer and activator of
transcription (STAT) factor-binding sites included in the GHRE-II were
never found to be protected against DNase I attack but displayed a
differential DNase I reactivity depending on the level of gene
transcription. Alterations in DNase I reactivity of the GC-response
element region suggest that GC receptor-GC complexes may associate, in
a transient manner, with the promoter in the actively transcribing
control state. Taken together, our studies suggest a mechanism of
spi 2.1 gene activation in vivo whereby the
GH-dependent chromatin remodeling caused by or concomitant to the
recruitment of GAGA-box binding proteins is the first compulsory and
presumably predominant step.
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INTRODUCTION
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Functional promoter analysis mainly relies on the introduction of
plasmid constructs bearing mutated or deleted versions of any given
promoter placed in front of a reporter gene, in cells supporting its
activity. Concomitantly, direct in vitro binding studies
[i.e. footprinting and electrophoretic mobility shift
assays (EMSA)] provide interesting information on nuclear proteins
that have the potential to interact with functionally relevant sites.
However, whether or not specific DNA-protein complexes that assemble in
cell-free systems can also be formed in vivo in the context
of intact chromatin remains a major question that must be answered
before ascribing any physiological significance to such macromolecular
complexes. A typical example of this problem is represented by
GH-mediated formation of signal transduction activator of transcription
(STAT)-DNA complexes that have been shown to play a major role in GH
action in vitro, in cell culture systems (1, 2, 3, 4, 5, 6). However,
their in vivo function remains still to be demonstrated
since direct evidence that GH also triggers their formation in living
animals have not yet, to our knowledge, been obtained. Resolving this
issue is not trivial and requires the use of powerful, high resolution
methods such as in vivo footprinting by ligation-mediated
PCR (LMPCR) (7).
During the last decade, we have extensively studied the rat serine
protease inhibitor (spi) gene system, which comprises three
members (8) that are differentially expressed, depending on the
physiological situation. Two of them, spi 2.1 and 2.2, are
maximally expressed in normal animals and are down-regulated during
acute inflammation (9). In contrast, the third one, spi 2.3,
is virtually silent in control animals and is transiently induced
during the acute-phase response (9). Regulation of expression of
the spi 2.3 gene is complex. It involves positive
promoter-regulatory elements including several interleukin-6-response
sites and a glucocorticoid (GC)-response element (GRE) (10), and a
negative regulatory element located in the 3'-untranslated region (11),
which most likely keeps the gene silent in normal rats. Two types of
effectors, GH and GC, appear to be responsible for the high level of
spi 2.1 (and 2.2) gene expression in normal animals (12). We
(4, 13) and others (5) have characterized a distal GH-response element
(GHRE-II) in the spi 2.1 promoter which, in cultured
hepatocytes, accounts for approximately 50% of the GH response (4).
This element contains two potential binding sites for STAT-5 (5), a
protein that belongs to a class of transcription factors activated upon
tyrosine phosphorylation by a GH-activated Janus kinase (Jak2) (for a
review, see Refs. 14 and 15). We have recently identified the second
spi 2.1 promoter GH-response element (GHRE-I). This element
also referred to as the GAGA box is structurally unrelated to
STAT-binding sites (16) and behaves, at least in cultured hepatocytes,
as a bifunctional enhancer controlling basal transcription and
responsible for part of the GH response (4, 16). In addition to these
GHREs, the spi 2.1 promoter also contains a GRE, which,
in vitro, binds specifically GR receptor-GC complexes
(GR-GC) and entirely mediates GC response, and several
CCAAT/enhancer-binding protein (C/EBP)-binding sites that contribute to
basal (i.e. hormone-independent) promoter activity (4, 13).
Specific binding of transcription factors (i.e. C/EBP,
STATs, GR-GC complexes and GAGA box-binding proteins, GAGA-BPs) to
their cognate sites in the spi 2. 1 promoter has been
demonstrated by EMSA and in vitro footprinting (4, 16, 17).
However, the physiological role of such factors remains questionable
since identical in vitro footprinting patterns were obtained
with liver nuclear extracts from control, hypophysectomized (hypox), or
inflamed rats (17), which correspond to high, negligible, and
intermediate levels of spi 2.1 gene expression, respectively
(13). To address this issue, we have used genomic footprinting to
examine changes in site occupancy occurring in vivo within
the proximal spi 2.1 promoter region as a function of the
level of gene transcription. Concomitantly, we have analyzed
deoxyribonuclease I (DNase I) sensitivity of the promoter region to
evaluate structural chromatin alterations that take place during gene
activation. The data suggest that transcription of the spi
2.1 gene in vivo strictly correlates with occupancy of the
GAGA box presumably triggered by or associated to a GH-dependent
chromatin remodeling event. In addition, our studies largely unravel
the mechanism of down-regulation of the spi 2.1 (and 2.2)
gene that takes place during acute inflammation.
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RESULTS
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Comparison of the Level of spi 2.1 Gene Transcription
in Control, Hypophysectomized, and Acutely Inflamed Rats
Chances of establishing a relationship between binding of
transcription factors to specific promoter sites and gene expression
in vivo are greatly enhanced by the possibility to analyze
conditions featuring significantly different levels of transcriptional
activity. Figure 1
shows that such a
requirement is met with the spi 2.1 gene. The high level of
transcription observed in control animals, in which it exceeds that of
the albumin gene, was decreased by more than 95% upon hypophysectomy,
which, in contrast, did not affect transcriptional activity of albumin
and ß-actin genes. In acutely inflamed rats, spi 2.1 gene
transcription was also severely depressed. The 5- to 7-fold decrease in
signal intensity observed in the run-on assay, in fact, underestimates
the inflammation-mediated decrease in spi 2.1 gene
transcription. Indeed, because of the extensive degree of sequence
identity (more than 95%) between the two cDNAs in their 5' and central
regions (9), the spi 2.1 probe used also recognized the
spi 2.3 transcript, which was essentially undetectable in
control animals but is induced during inflammation (Refs. 9 and 10; see
also Fig. 7
). As previously observed, acute inflammation negatively
affected albumin gene transcription but had no effect on the expression
of the ß-actin gene (10).

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Figure 1. Nuclear Run-On Assay
Nascent in vivo liver RNA transcripts contained in
nuclei from control (CONTROL), hypophysectomized (HYPOX), or 18-h
inflamed (INFLAMED) rats were elongated in vitro in the
presence of [ -32P]UTP, purified, and hybridized to
various plasmids bearing cDNA sequences from spi 2.1,
albumin, or ß-actin genes immobilized
on a nylon membrane. Empty vector (pUC 19) was used as a blank control.
After hybridization performed at 42 C for 48 h, the membrane was
treated with RNases A and T1, extensively washed, and autoradiographed
at -80 C with an intensifying screen for 3 days.
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Figure 7. Northern Blotting Analysis of spi 2
mRNAs in Cultured Hepatocytes: Effects of GH and Cytokines
Forty eight-hour-old cultured hepatocytes were incubated for 24 h
in the absence or presence of rGH (500 ng/ml), without or with 10 ng/ml
of various cytokines used alone or in combination. Total RNAs were then
extracted and analyzed as described in the legend to Fig. 7 .
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High Levels of spi 2.1 Gene Transcription Correlate
with the Appearance of DNase I Hypersensitivity within the Promoter
Region
Changes in level of transcription are generally associated
with alterations of the chromatin structure, more particularly in
regions thought to be important for transcription in vivo.
In eukaryotic genomes, chromatin remodeling has been often detected as
sites of DNase I hypersensitivity (for a review, see Ref.18). Figure 2
shows that digestion of genomic DNA
with BamHI generated a 2.6-kb fragment (panel A) spanning
the -862/+1725 gene region (panel B). DNase I treatment of liver
nuclei from normal rats led to the appearance of three minor fragments
of approximately 0.8, 0.75, and 0.67 kb that were not detected in DNA
isolated from hepatic nuclei of hypox animals. This revealed the
presence of three DNase I-hypersensitive sites (HSS I, II, and III)
that were seen only when the gene is efficiently transcribed and that
map in the proximal promoter region containing the two GHREs previously
identified by functional in vitro studies (4, 5, 13). This
is consistent with the observation made by Yoon et al. (19),
who first reported a GH-dependent induction of DNase I-hypersensitive
sites in the 5'-flanking spi 2.1 gene region. A similar
analysis has also been performed with liver nuclei from inflamed rats,
yielding results comparable to those obtained with nuclei from hypox
animals (data not shown).

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Figure 2. DNase I Sensitivity of the spi 2.1
Gene Promoter Region in Control and Hypox Rats
A, Appearance of DNase I-hypersensitive sites in the proximal
5'-flanking region of the spi 2.1 gene. Hepatic nuclei
prepared from control (lanes 17) and hypox (lanes 813) rats were
exposed to varying concentrations of DNase I as described in
Materials and Methods. Purified DNA was then digested
with BamHI and subjected to electrophoresis. On the
autoradiogram, arrows indicate the position of the
2.6-kb fragment covering the -862/+1725 gene region and that of the
three fragments of approximately 0.80, 0.75, and 0.67 kb, generated by
the action of 6 µg/ml DNase I on control nuclei but not on nuclei
from hypox animals. Size of fragments was determined using mol wt
markers run alongside. B, Diagram summarizing the organization of the
-862/+1725 BamHI spi 2.1 genomic
fragment, the location of the probe used for Southern analysis, and the
putative position of the DNase I HSS detected in control nuclei.
Open rectangles indicate the position of the two GHREs
(see Fig. 3 ) identified in the promoter.
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In Vivo DNase I Footprinting of the spi 2.1
Promoter
The results presented in Fig. 2
suggested that chromatin
remodeling occurred in the spi 2.1 promoter region, when
switching from a situation of very low level (i.e. hypox
rats) to a situation of very high level (i.e. control
animals) of gene transcription. However, they did not provide any
information on the relationship that may exist between the degree of
occupancy of putative functional sites within the promoter in
vivo and the level of gene transcription. Our previous in
vitro binding studies (i.e. EMSA and footprinting
experiments) have not allowed us to correlate changes in the level of
spi 2.1 gene transcription in vivo with changes
in DNA-protein interactions more particularly in the region of the GAGA
box (17) which proved to be the major spi 2.1 promoter
regulatory element in isolated hepatocytes (4, 16). We thus turned to
the genomic footprinting technique that allows a precise analysis of
nuclear protein-DNA interactions in vivo. Figure 3
displays the results of an analysis of
the lower strand of the spi 2.1 promoter region from
positions -160 to +10 relative to the cap site. This region
was previously found to contain the elements required for maximal basal
as well as hormone-stimulated promoter activity in cultured hepatocytes
(4, 13). A comparison of patterns of bands obtained with naked DNA
(in vitro) and with the three DNase I-treated nuclei-derived
templates (in vivo) revealed major differences throughout
the promoter. In control rats, a unique region encompassing the GAGA
box and presumably extending toward the 3'-region, as suggested by the
slight decrease in intensity of the -37/38 and -26/-27 bands, was
strongly protected against DNase I attack. In contrast, essentially no
protection of the GAGA element occurred in hypox or acutely inflamed
rats. It should also be noted that hypophysectomy and, to a lesser
extent, inflammation induced a strong DNase I-reactive site at
positions -37/-38 (presumably a doublet) and weaker sites at
positions -26/-27 and +10. Unlike what was observed with the GAGA
box, no DNase I reactivity of the adjacent C/EBP site spanning the -66
to -58 region was detected, regardless of the situation analyzed. This
obviously precludes drawing any conclusion on the in vivo
role of this site shown to be functional in vitro (4, 13).

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Figure 3. In Vivo DNase I Footprinting of the
spi 2.1 Gene Promoter
Lower strand analyses of LMPCR products revealed with the
32P-labeled oligonucleotide L3. The (G+A) lane corresponds
to naked DNA chemically cleaved at purine residues and the IN
VITRO lane corresponds to naked DNA treated with DNase I. The
three other lanes (IN VIVO) correspond to templates
obtained from DNAse I-treated liver nuclei from normal (CONT),
hypophysectomized (HYPOX), or LPS-treated (INFL) rats. The major
spi 2.1 promoter elements found to be functional in
cultured hepatocytes and shown on the diagram at the lefthand
side are: the TATA box (-32/-26), the proximal GHRE
(GHRE-I/GAGA box, -45/-57), the most active C/EBP site (-58/-66),
the GRE (-73/-88), and the distal GHRE (GHRE-II, -104/-149) that
contains the two putative STAT-5-binding sites (-124/-132 and
-139/-147). The DNase I-protected region (PR) observed only with the
in vivo control template and the major reproducible
changes detected between either IN VITRO and IN
VIVO templates, or between the three IN VIVO
templates are indicated by arrows at the righthand side
of the figure.
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In addition to the GAGA box, two other promoter regions appeared to be
differentially affected by hypophysectomy and acute inflammation. The
first one includes the GRE, which was previously defined as the
-88/-74 motif, on the basis of its functional activity (4) and its
structural similarity with the consensus GRE sequence (20). In
agreement with what has been observed for interactions of other genes
with GR-GC complexes (21, 22), no footprint (i.e.
protection) was observed in the spi 2.1 GRE region (Fig. 3
)
which, however, displayed marked differences in DNase I reactivity as a
function of the level of gene transcription. Thus, a strong reactive
site occurring at position -86 within the GRE undetected with a naked
template (i.e. in vitro) and a minor one flanking
this motif occurring at position -92 or -93 (or possibly both) also
seen in vitro became apparent in control animals but almost
totally disappeared upon hypophysectomy or inflammation. In contrast,
enhanced DNase I reactivity was detected at positions -90, -95, -96,
and -98 (i.e. 5' to the GRE) in nuclei from hypox and, to a
lesser extent, inflamed animals, compared with both in vivo
controls and naked DNA. Finally, the fairly strong nuclease reactive
site found at position -80 in DNase I-treated naked DNA and in DNA
isolated from control nuclei digested with the enzyme was significantly
attenuated in hypox and acutely inflamed rat nuclei.
The second region differentially affected in both
pathophysiological models relative to the control encompasses the
spi GHRE-II (4) and includes the potential STAT-5 binding
sites present at positions -124 to -132 and -141 to -147. In
addition to other minor differences that proved poorly reproducible,
two major observations were consistently made: 1) unlike what was
observed with the GAGA box, no clear protection of any of the
STAT-binding sites could be detected, whatever the state of the animal,
by comparing in vitro and in vivo footprint
patterns; 2) in the in vivo patterns, several DNase
I-hyperreactive sites (positions -126, -128, and -137) were visible
with hypox and, although much less pronounced (positions -126 and
-128), inflamed rat liver nuclei-derived templates but not with
controls. In addition, two minor nuclease-reactive sites (positions
-118 and -120) that were seen with both a naked template and DNA
prepared from DNase I-digested control nuclei became barely detectable
upon hypophysectomy or inflammation. Figure 4
shows a similar analysis performed with
the upper strand. The most striking observation made with naked DNA as
well as with templates derived from DNase-treated nuclei was the almost
complete lack of DNase I reactivity of the GAGA box region and, as
already noted with the lower strand, of its 5'-flanking C/EBP site. As
observed with the lower strand, no clear protection but rather
significant changes in DNase I reactivity of the promoter were detected
in the GRE region. Thus, a set of hyperreactive sites internal to the
GRE (positions -82 and -77 to -72) that were not observed with naked
DNA but could be seen with templates prepared from control nuclei, were
undetectable except for two of them (positions -74 and -73), in
templates prepared from nuclei of hypox or inflamed rats. In addition,
two other minor changes including the appearance of a weak reactive
site at position -84 and attenuation of stronger ones at positions
-92 and -91 were also detected in this region, with in
vivo hypox and inflamed rat-derived templates compared with
controls or in vitro DNase I-treated DNA.

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Figure 4. In Vivo DNase I Footprinting of the
spi 2.1 Gene Promoter
Upper strand analysis of LMPCR products revealed with the
32P-labeled oligonucleotide L*3. Legend is identical to
that in Fig. 3 .
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As observed with the lower strand, no significant in
vivo protection of the GHRE-II region could be observed with the
upper strand, regardless of the situation analyzed. However, the
enhanced DNase I reactivity of the most 3'-STAT-5-binding site region
(increased signal at positions -128 and -127) described above
with the lower strand (Fig. 3
) and specifically seen with hypox and
inflamed rat-derived templates was also observed. Several other
nuclease-reactive sites were induced in the two pathophysiological
animal models examined, both in this distal region of the
spi 2.1 promoter (positions -165, -106, and -104) and in
a region closer to the cap site (positions -34 to -32). It
is also remarkable that the cap site region (positions +1 to
-15) displayed enhanced DNase I reactivity in all in vivo
conditions analyzed, compared with naked DNA.
Analysis of GAGA-BPs and STAT-5 Contents of Liver Nuclei
by EMSA
The almost complete absence of detectable in
vivo interactions between GAGA-BPs and the GAGA box in hypox and
acutely inflamed animals revealed by genomic footprinting could be
merely due to a decrease in amounts of active GAGA-BPs in nuclei
preparations. To test this hypothesis, we made extracts from liver
nuclei prepared in the same way as those used for DNase I treatment and
analyzed by EMSA the capacity of nuclear proteins to bind to the GAGA
box. Figure 5A
shows that no significant
qualitative or quantitative difference could be detected between
control, hypox, and inflamed rat liver nuclear extract preparations. As
already observed with control extracts (16), formation of a major (a)
and a minor (b) complex, previously shown to represent specific binding
of nuclear factors to the spi GAGA box, occurred with all
three different sets of nuclear proteins.

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Figure 5. EMSA Analysis of GAGA-BP and STAT-5 Content of
Liver Nuclei
A, Ten micrograms of liver nuclear proteins prepared from normal
(CONTROL), hypophysectomized (HYPOX), or LPS-treated (INFLAMED) rats
were incubated with a 32P-labeled single-copy GAGA box
probe (GA-ext). Bound and free probes were separated by electrophoresis
on a 5% native polyacrylamide gel and detected by autoradiography. The
region of the gel containing the specific (a and b) and nonspecific (c)
complexes are shown. B, Ten micrograms of liver nuclear proteins
prepared from normal rats were preincubated at 4 C for 1 h with
preimmune serum (PI) or antibodies (1 µg purified Ig per assay)
directed against STAT-5 b ( -STAT-5 b). A labeled oligonucleotide
probe representing the STAT-5 ß-casein gene promoter-binding site was
then added, and incubation was allowed to proceed for 15 min at RT.
Bound and free probes were separated by electrophoresis on a 4% native
polyacrylamide gel and detected by autoradiography. Region of the gel
containing the specific DNA-protein complexes (thin
arrows) and immune complexes (IC, wide arrows)
are shown.
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An intriguing observation arising from genomic footprinting of
the spi 2.1 promoter was the absence of any protection of
the GHRE-II region against DNase I attack even when the gene is
maximally transcribed (i.e. in control animals). This was
indeed unexpected in the context of GH action since this region has
been shown to bind STAT-5 in vitro and activate GH-dependent
transcription ex vivo in transfected cells (5, 6). A
possible explanation to this apparent lack of stable STAT-5 in
vivo binding could be the absence of active forms of this
transcription factor that might have leaked out or be inactivated
(i.e. by dephosphorylation) during nuclei preparation. The
EMSA performed with liver nuclear extracts from control rats shown in
Fig. 5B
does not support this hypothesis. Indeed, significant amounts
of STAT-5 present in control nuclear extracts bind to a specific STAT-5
probe derived from the ß-casein gene promoter. In addition to STAT-5,
C/EBP factors massively represented in liver nuclei have also been
shown to bind the spi 2.1 GHRE-II in vitro (17).
However, based on our genomic footprinting data, they do not appear to
form detectable stable complexes with this promoter region in
vivo.
In Vivo Effect of Lipopolysaccharides (LPS)
and in Vitro Effect of Cytokines on GH-Dependent or
Independent spi 2 mRNA Levels
An important observation made in the genomic
footprinting experiments was that the lack of protection of the GAGA
box was associated with a very strong down-regulation of the
spi 2.1 gene in inflamed animals. Since GH appears to
strictly control spi 2.1 GAGA box occupancy in
vivo (see Discussion), a perturbation occurring in the
GH transduction pathway subsequent to the development of a GH-resistant
state could account for this observation. In keeping with this
possibility, some of us recently showed that treatment of primary
hepatocytes with interleukin (IL)-1ß or tumor necrosis factor-
(TNF
) (23), which are two major lymphokines implicated in the liver
acute-phase response (for a review, see Refs. 24 and 25), or injection
of inflammatory compounds (LPS) to living animals (26) strongly
inhibited GH-dependent insulin-like growth factor-I (IGF-I) message
synthesis. It thus became important to check whether this type of
effect could represent a more general phenomon and apply to the
spi 2 gene system. To this aim, we investigated the effect
of LPS (in vivo) or cytokines (in vitro) on GH
action on the level of spi 2 messages. As previously
observed (9), acute inflammation reduced by about 50% spi
2.1 and 2.2 mRNA liver steady-state levels and, inversely, induced the
spi 2.3 message (Fig. 6A
, compare lanes 13 to lanes 46; and Fig. 6B
). Injection of GH alone
slightly increased the level of spi 2.1 and 2.2 messages and
had no effect on that of spi 2.3 (Fig. 6
, A and B).
Interestingly, the inflammatory drug largely prevented GH to restore
normal spi 2.1 and 2.2 mRNA levels (Fig. 6A
, lanes 1012;
and Fig. 6B
).

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Figure 6. Northern Blotting Analysis of spi 2
mRNAs in Vivo: Effects of LPS and GH
Separate groups of six rats were injected with saline, LPS, or bGH or a
combination of both as described in Materials and
Methods. Total liver RNAs were then purified, resolved on a 1%
denaturing agarose gel, and transferred onto a nylon membrane. Blots
were hybridized with a spi 2.1 cDNA probe that revealed
the spi 2.3 (the 2.3-kb band) and spi 2.1
+ spi 2.2 mRNA species (the 1.8-kb band). Panel A,
Autoradiogram of a gel in which RNAs from three different animals per
group were analyzed. Panel B, The amount of spi mRNA was
quantitated by scanning the autoradiograms. Each bar
represents the mean +/- SEM (n = 6).
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Unlike what we previously observed with hepatocytes maintained in
primary culture on a plastic support (13), cells cultured on matrigel
in the absence of GH retained a fairly high level of expression of
spi 2.1 and 2.2 messages (Fig. 7
, lane 1). This level could be
increased, however, by GH by 2- to 3-fold (lane 2), an effect that was
almost completely obliterated by IL-1ß (lanes 3 and 4), TNF
(lanes
7 and 8), or a combination of both agents (lanes 11 and 12). In
contrast, IL-6 did not appear to signicantly influence GH action on
spi 2.1 and 2.2 messages but, as expected (10), strongly
induced the spi 2.3 mRNA (lanes 5 and 6). The latter, which
was already slightly expressed under unstimulated conditions, was also
increased by IL-1ß (compare lanes 1 and 3) but not TNF
(compare
lanes 1 and 7). This effect of IL-1ß, which was not previously
observed by using a different culture system [i.e. plastic
dishes and a serum-containing medium (10) instead of matrigel-coated
dishes and a serum-free medium, this study], can presumably be
accounted for by the presence of a nuclear factor
B (NF-
B) site
within the spi 2.3 promoter (8, 10). IL-1 has indeed been
shown to activate NF-
B (for a review, see Ref.27), which, in turn,
stimulates transcription of specific genes such as the nitric oxide
synthase gene in pancreatic ß-cells (28). To further characterize the
antagonistic action of IL-1ß on the GH effect, we performed
dose-dependent studies. Induction of spi 2.1 and 2.2 mRNA
species by GH in cultured hepatocytes was inhibited in a dose-dependent
manner by IL-1ß, with a maximal effect (nearly 100% inhibition)
occurring with 10 ng/ml (Fig. 8
, panel
A). A similar relationship was also observed for induction of the
spi 2.3 mRNA. Production of spi 2.1 and 2.2
messages proved highly sensitive to GH since a concentration of 100
ng/ml (
5 nM) was maximally efficient (Fig. 8
, panel B).
However, regardless of the GH concentration used, the effect was
totally inhibited by IL-1ß (Fig. 8
, panel B). Thus, data obtained by
analyzing IGF-I (23, 26) and spi 2 messages (present study)
strongly support the notion that acute inflammation, through the action
of IL-1ß (and possibly TNF
), causes the appearance of a strong
GH-resistant state in the hepatocyte.

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Figure 8. Dose-Dependent Effects of IL-1ß and GH on
spi 2 mRNAs in Cultured Hepatocytes Analyzed by Northern
Blotting
Panel A, Forty eight-hour-old cultured hepatocytes were incubated for
24 h in the absence or presence of rGH (500 ng/ml) with increasing
concentrations of IL-1ß. Panel B, Cells were incubated in the
presence of increasing concentrations of rGH, without or with IL-1ß.
Total RNAs were then extracted and analyzed as described in the legend
to Fig. 7 .
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DISCUSSION
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The rat spi 2.1 gene represents one of the very rare
examples of a GH-regulated gene for which the main features of in
vivo regulation can be largely reproduced in cultured hepatocytes
with promoter constructs (4, 13). However, regulatory mechanisms
inferred from in vitro studies do not account for all
in vivo observations. Thus, a significant level of basal
(i.e. GH- and GC-independent) promoter activity accounted
for by a role of C/EBP sites was detected in primary hepatocytes (4, 13) whereas in vivo, in hypox animals lacking GH and GC, the
gene was shown to be almost totally silent (12). Similarly,
dexamethasone proved to be as efficient as GH in activating the
spi 2.1 promoter in vitro (4, 13) but failed to
reinduce gene expression in hypox rats (12). These discrepancies raised
questions on the role of C/EBP sites and the GRE as regulatory elements
in living animals. Another major, as yet unresolved, question pertains
to the role played in vivo by the two spi 2.1
promoter GHREs characterized in vitro (4, 5, 13). The data
presented in this paper, which for the first time demonstrate a
correlation between in vivo promoter occupancy and the rate
of transcription of a GH-regulated gene, introduced major information
that largely reconciles in vitro and in vivo
observations and provides answers to some of the aforementioned
questions.
The most striking observation arising from genomic footprinting
analysis of the spi 2.1 promoter is the strict correlation
existing between the stable interaction of nuclear proteins with the
proximal GHRE (GHRE-I) recently identified as the GAGA box (16), and
the level of gene transcription in vivo. Such a correlation
fully agrees with the observation that, in cultured hepatocytes, a
mutation of the GAGA box that is lethal for both basal and hormone
(i.e. GH and GC)-dependent promoter activities (4) also
abolishes binding of as yet unidentified GAGA-BPs (16). This also
further supports the notion that the GAGA box, a purine-rich promoter
motif fully conserved in all three spi 2 genes (8), is a key
control element working both in vivo and in vitro
as an on/off transcriptional switch.
Clearly, transcriptional activation of the spi 2.1 gene
in vivo is strictly GH-dependent, since only GH can
reinduce gene expression in hypox rats (12). This strongly
suggests that proper assembly of GAGA-BPs-GAGA box complexes,
which is mandatory for spi 2.1 gene promoter activity
in vivo, requires that the signal initiated by GH binding to
its cell surface receptors is normally processed to the nuclei. Thus,
in any situation where a severe impairment in the GH-signaling pathway
occurs, formation of GAGA-BPs-GAGA box complexes should be prevented
and spi 2.1 gene transcription shut off. This is the case,
as expected, in hypox rats due to the absence of GH, but also in
acutely inflamed animals. Indeed, lipopolysaccharides (in
vivo) and inflammatory cytokines (IL-1ß and TNF
, ex
vivo) induce a strong GH-resistant state in hepatocytes,
presumably leading to a blockade of GAGA box-GAGA BPs complex formation
and subsequent inactivation of the spi 2.1 gene. Cytokines
have generally been found to directly regulate transcription of
acute-phase genes via their specific promoter response elements (24, 25). Our data reveal that cytokines can also regulate acute-phase gene
expression indirectly, by disturbing the functioning of a key promoter
element such as the spi GAGA box.
How GH contributes to bringing GAGA-BPs to the promoter, however,
remains an open question. Experiments performed with cell-free systems
strongly suggest that binding of GAGA-BPs (see Fig. 5A
) and subsequent
transcriptional activation (16) can occur independently of GH. This
seems to indicate that only the recruitment of GAGA-BPs, presumably
linked to the chromatin-remodeling event shown to take place during
spi 2.1 gene activation, requires GH in vivo. At
least two hypotheses can be put forward: 1) GH might first activate a
chromatin-remodeling system independent of GAGA-BPs that would locally
alter the nucleosomal structure and permit binding of those
transcription factors to the promoter; 2) GAGA-BPs themselves could
bear a chromatin-remodeling activity stimulated by GH (e.g.
through posttranslational modifications). In keeping with the latter
possibility, trans-displacement of histones has been
proposed as one mechanism for trancription factor-targeted generation
of a nucleosome-free region in chromatin (29). Assessing these
hypotheses must await the cloning and characterization of GAGA-BPs that
should allow setting up reconstitution experiments.
Unlike what was observed with the spi 2.1 GHRE-I
(i.e. the GAGA box), we never detected any protection of the
STAT-5-binding sites contained in the GHRE-II in vivo. This
was true in control animals, despite the fact that a significant level
of active nuclear STAT-5 could be demonstrated, and also in hypox rats
treated for 2 h with GH, a condition under which the nuclei
content of active STAT-5 exceeds that in controls (C. Legraverend,
unpublished results). This, however, does not eliminate the possibility
that STAT-5 can bind to the GHRE-II in living animals. It is indeed
conceivable that activation of STAT-5 by GH could lead to a transient
interaction with its cognate binding sites that would not be detected
by the genomic footprinting technique but that could subsequently
modify the local chromatin structure and contribute to transcriptional
activation. Such a structural modification appears to take place upon
gene activation, at least at the level of the more proximal
STAT-5-binding site, as evidenced by the decrease in local DNase I
reactivity detected with control templates. However, an alternative
explanation to such a change in DNase I reactivity that was also
observed in other parts of the promoter when switching from a
transcriptionally inactive (i.e. hypox or inflamed rats) to
a transcriptionally active (control animals) state could be the
displacement or the structural alteration of a nucleosome. Thus, our
genomic footprinting data do not provide a clear answer to the question
of whether STAT-5 can form a complex with the spi 2 GHRE-II
in vivo. Whatever the answer to this question might be,
however, it should be kept in mind that STAT-5 could contribute to
transcriptional activation of the spi 2.1 promoter without
directly contacting the DNA. This transcription factor has indeed been
shown to associate with GR receptors and potentiate their effect
in vitro (30).
The absence of detectable C/EBP footprints, whatever the in
vivo situation analyzed, appears at first to be totally
paradoxical, based on our previous in vitro footprinting
assays (4, 17) and heterologous transactivation experiments (11).
Unlike what is happening in vitro (see EMSA in Fig. 8
), the
results of genomic footprinting clearly indicate that there is no
stable interaction of C/EBP with the -130 to -122 site internal to
the GHRE-II, in vivo. As pointed out for STAT-5, however, we
cannot formally rule out that transient and/or very loose binding of
C/EBP might occur in the context of intact chromatin and confer some
functionality to this site in vivo. Due to the lack of DNase
I sensitivity of the other potentially important C/EBP-binding site
immediately flanking the GAGA box (-66/-58), it is not possible to
exclude this site as being functional in vivo.
Unlike what has been observed in vitro (4), no
protection of the spi 2.1 promoter GRE could be observed
in vivo by genomic footprinting, either with control rats
having normal circulating GC levels or with inflamed animals known to
overproduce GCs (24, 25). However, a differential alteration in DNase I
reactivity of the GRE region was observed as a function of the level of
gene transcription by the technique of in vivo footprinting.
The most striking change is represented by the presence of several
DNase I-hyperreactive sites mapping within the GRE or in its close
vicinity in chromatin from control liver nuclei and their absence in
naked DNA and in chromatin from nuclei of hypox or inflamed rats.
Similar changes in reactivity to DNase I occurring upon gene activation
mediated by GCs have been observed with the rat tyrosine
aminotransferase promoter (21) and the mouse mammary tumor virus long
terminal repeat (22). They have been interpreted as the signature of a
transient association of GR-GC complexes with GREs. Thus, the
possibility seems to exist that those hypersensitive sites specifically
detected in the GRE region in an actively transcribing state
(i.e. control animals) are, at least in part, the
consequence of an interaction with GR-GC complexes.
The genomic footprinting patterns of bands generated within the
GRE region by DNase I treatment of nuclei isolated from hypox and
inflamed rat liver clearly differed from those obtained with control
nuclei but, surprisingly, were qualitatively similar. This was rather
unexpected, considering that hypophysectomy and acute inflammation
represent two opposed situations with respect to endogenous levels of
circulating GCs (i.e. almost total absence of hormone in
hypox rats vs. enhanced levels in inflamed rats). This makes
it unlikely that GR-GCs complexes mediate changes occurring in DNase I
reactivity in the spi 2.1 gene GRE region in either of these
animal models compared with controls. Those alterations could more
likely be related to an overall modification of the nucleosomal
structure occurring in the promoter upon gene activation and revealed
by the appearance of DNase I-hypersensitive sites. Further studies
involving in vitro reconstitution of the nucleosomal
structure will be necessary, however, to assess this hypothesis.
In conclusion, we would like to propose a mechanism for
spi 2.1 gene activation in vivo that includes
first, a GH-dependent remodeling of the chromatin in the promoter
region caused by, or concomitant to, the recruitment of GAGA-BPs. This
would then allow association of GR-GC complexes with the GRE, which in
turn could boost GAGA-BP binding through a dynamic interplay, thus
accounting for the potentiating effect of GCs on GH action. At this
point, the contribution of STAT-5 to GH-mediated spi 2.1
gene activation in vivo remains to be clarified. It should
be pointed out, however, that unlike activation of STAT-5, which
appears to be sex-dependent (31), functioning of the GAGA box as well
as expression of spi 2.1 and 2.2 genes are essentially
identical in the male and the female (our unpublished observations).
This strongly argues for a predominant role of the GAGA box in
controlling GH-dependent regulation of spi 2 gene expression
in vivo. Finally, a very important outcome of these studies
is the elucidation of the indirect mechanism of cytokine-mediated
down-regulation that accounts for the transcriptional shut-off of
spi 2.1 and 2.2 gene expression induced by acute
inflammation.
 |
MATERIALS AND METHODS
|
---|
Materials
[
-32P]ATP (3000 Ci/mmol) and
[
-32P]UTP (800 Ci/mmol) were obtained from Du Pont NEN
(Paris, France). Vent Exo- and Amplitaq DNA polymerases
were purchased from New England Biolabs (Beverly, MA) and Perkin Elmer
(Paris, France), respectively, and DNase I (3000 U/mg) was from
Worthington (Freehold, NJ). Collagenase (0.65 U/mg) was from Boehringer
Mannheim (Mannheim, Germany) and bacterial (Escherichia
coli) LPS were obtained from Life Technology (Cergy-Pontoise,
France). Bovine GH (bGH) was a kind gift of Mosanto (St-Louis, MO). Rat
GH (rGH) (NIH-B-13, AFP-87401) was a gift from the National Institute
of Diabetes and Digestive and Kidney Diseases, and recombinant murine
IL-1ß, IL-6, and TNF
were purchased through R & D systems
(Abingdon, U.K.). Antibodies to STAT-5b were purified Ig preparations
obtained from Santa Cruz Biotechnology Co. (Santa Cruz, CA). Nylon
membrane (Hybond N+) was from Amersham Life Science
(Amersham, U.K.). Female Wistar rats were obtained from the Katholieke
Universiteit Leuven (Heverlee, Belgium) and male Wistar rats were
purchased from IFFA-CREDO (lArbresle, France).
Probes used for EMSA listed below (genomic sequences are in
uppercase letters) include: (i) an oligonucleotide (GA-ext)
(8) containing the spi GAGA box (underlined) and
bearing its natural 6 and 7 nt-long 5'- and 3'-flanking sequences,
respectively: 5'-tcgaTTCCTAAGAGGAGGGAGGAGCCTTTGGT; (ii) an
oligonucleotide comprising the high-affinity STAT-5-binding site
(underlined) present in the ß-casein gene promoter (32):
5'-gTCCCTTAATTCCAAGAAGTCC.
The following spi 2.1 promoter-derived oligonucleotides were
used in genomic footprinting to amplify: (i), the upper strand:
L1 (-238/-218), 5'-GTACACTCTACTTTTGCTTTG;
L2 (-211/-187): 5'-TCCCACTTTCCTCATTGACTTTGAC;
L3 (-201/-170): 5'-CTCATTGACTTTGACCACTCAATAAATAAAAGG;
and (ii), the lower strand:
L11 (+73/+56), 5'-TGATACCAGCCAGCTGCC;
L21 (+50/+28), 5'-CTGTGTTTGCTGACACCTGATGT;
L31 (+42/+18), 5'-GCTGACACCTGATGTTCAGGGTTGT.
It should be mentioned that, despite the extensive sequence
conservation of the three spi 2 gene family members, more
particularly in the promoter region (8), sets of oligonucleotides used
in our experiments were totally specific, since they allowed exclusive
amplification of the spi 2.1 sequence. The linker used was
the same as that described by Mueller and Wold (7).
Nuclear Run-On Assays
Nuclei isolated from the liver (33) of normal, hypox, or
LPS-treated (killed 18 h after a single ip injection of 750
µg/100 g body weight) adult male rats were used to evaluate the
in vivo rate of transcription initiation. The procedure for
run-on analysis, which includes incubation of the nuclei with
[
-32P]UTP, purification of total cellular RNA, and
hybridization to templates immobilized on a nylon membrane, has been
previously described (34). The following plasmids were used as
hybridization templates: pUC19 bearing 0.52 kb of the spi
2.1 cDNA 5'-region (9); pBR322 bearing 1.6 kb of the mouse ß-actin
cDNA (35); pBR322 containing 1.2 kb of the rat albumin cDNA (36). The
amount of labeled transcripts used for hybridization corresponded to
20 x 106 trichloroacetic acid-precipitable
counts.
EMSA
Liver nuclear extracts were prepared from control, LPS-treated
(18 h), or hypox rats, according to the method of Gorski et
al. (33) as modified by Sierra (37). The following minor changes
were introduced: 0.5 mM phenymethylsulfonylfluoride (PMSF),
14 µg/ml aprotinin, 1 mM Na3VO4,
and 10 mM NaF were added to the homogenizing buffer; 0.1
mM PMSF, 0.1 mM Na3VO4,
and 0.1 mM NaF were added to the nuclear lysis buffer; and
0.1 mM Na3VO4 and 0.1
mM NaF were added to the dialysis buffer. Double-stranded
oligonucleotides were end-labeled using T4 polynucleotide kinase and
[
-32P]ATP. Probes were purified on nondenaturing
polyacrylamide gels before use. The incubation mixture (20 µl)
contained 10 µl liver nuclear extracts (10 µg proteins diluted in
dialysis buffer), 1 µl (2 µg)
poly(deoxyinosinic-deoxycytidylic)acid, 1 µl (100 µg) BSA, 4 µl
of 5x buffer (25 mM MgCl2, 20 mM
spermidine, 4.4 mM dithiothreitol, 0.25 mM
EDTA, 50% glycerol, 0.25 mM
Na3VO4, 0.25 mM NaF), and 2 µl
water or antibodies. After a 15-min preincubation at room temperature
(RT), the labeled probe (
0.2 ng of oligonucleotide, 50100.000 cpm)
was then added in 2 µl, and the binding reaction was allowed to
proceed for 15 min at RT. For supershift experiments, nuclear proteins
were first incubated with preimmune or immune serum for 1 h at 4
C. Samples were electrophoresed for 3 h at RT on 5% nondenaturing
polyacrylamide gels in 0.5 x TBE (44.5 mM Tris base,
44.5 mM boric acid, 1 mM EDTA) at 130 V.
In Vivo Footprinting of the spi 2.1
Promoter
Preparation of DNase I-Treated Templates
Nuclei isolated from the liver of control, hypox, or inflamed
(i.e. LPS-treated for 18 h) rats were incubated for 10
min on ice in a mixture containing 10 mM Tris-HCl (pH 7.4),
15 mM NaCl, 60 mM KCl, 0.15 mM
spermine, 0.5 mM spermidine, 0.5 mM
CaCl2 and 1 mM MgCl2, in the
presence of DNase I (10 µg/ml). Reactions were stopped by adding 4
vol of a lysis solution containing 1 mg/ml proteinase K, 30
mM EDTA, and 2.5% SDS, and nuclear lysates were incubated
for 4 h at 45 C. Naked DNA was concomitantly treated with DNase I.
Genomic DNA was purified as described by Rigaud et al. (21)
by phenol extraction and was ethanol precipitated and dissolved in
water.
Genomic Sequencing with the LMPCR Procedure
This was performed using a modification of the method of Mueller and
Wold (7) described by Grange et al. (38), except for
ligation of the linker that was carried out for 1.5 h at RT, in
the presence of 15% polyethylene glycol (39). Extension of primers 1
(L1 or L11) was performed at 50 C, exponential amplification (30
cycles) with primers 2 (L2 or L21) was performed at 66 C, and extension
(9 cycles) of 32P-labeled primers 3 (L3 or L31) for
detection of LMPCR products was performed at 68 C. Chemical sequencing
reactions were performed by the Maxam and Gilbert procedure (40).
Labeled products were separated on 6% polyacrylamide-urea sequencing
gels that were dried and autoradiographed without an intensifying
screen for 1224 h at RT. Autoradiographs shown in Results
are raw data from a representative experiment. In in vivo
footprinting, only alterations in the pattern of bands that have been
reproduced with at least three independent template preparations have
been considered as real.
Analysis of DNase I-Hypersensitive Sites
Conditions used to incubate nuclei isolated from the liver of
control or hypox male rats with increasing concentrations of DNase I
(020 µg/ml) and the procedure to purify genomic DNA were as
described for the preparation of DNase I-treated templates for LMPCR.
DNA samples (25 µg) were then digested with BamHI, and
fragments were resolved on a 1% agarose gel and transferred onto a
nylon membrane. The membrane was hybridized to a uniformly
32P-labeled single-stranded RNA probe corresponding to the
BamHI-PstI fragment (positions -862 to -479) of
the spi 2.1 gene (8) and submitted to autoradiography at
-80 C for 48 h with an intensifying screen.
Animal Treatment, Hepatocyte Culture, and Northern Blotting
Analysis of spi 2 mRNAs
All animal studies were conducted in accord with the principles
and procedures outlined in "Guidelines for Care and Use of
Experimental Animals." Four groups (n = 6/group) of 4-week-old
female Wistar rats were used to analyze the effect of LPS and GH on
liver spi 2 mRNA levels in vivo. The first group
received one ip injection of LPS (750 µg/100 g body weight) and one
sc injection of saline; the second group received one sc injection of
bGH (200 µg/100 g body weight) and one ip injection of saline; the
third group was administered LPS and bGH; and the last group received
two injections of saline. All animals were killed 10 h after
injections.
Rat hepatocytes cultured on matrigel-coated plates for 48 h in
serum-free medium were exposed during 24 h to cytokines and rGH at
different doses. Procedures used to culture hepatocytes and extract
total RNAs from the liver of adult rats or from cultured cells were as
previously described (23). For Northern blotting analysis, 20-µg
samples of total RNA were resolved on 1% denaturing agarose gels and
transferred onto nylon membranes (Hybond) by vacuum blotting. Blots
were then hybridized under standard conditions with a random-primed
32P-labeled spi 2.1 cDNA probe (fragment
601800) that was previously shown to recognize all spi
messages (9). The 2.3-kb band corresponds to the spi 2.3
mRNA species, whereas the 1.8-kb band represents undistinguishable,
size-matched spi 2.1 and 2.2 messages which, on the basis of
the abundance of corresponding proteins, appeared to be equally
represented in the normal liver (12).
 |
ACKNOWLEDGMENTS
|
---|
The technical assistance of Josiane Verniers for hepatocyte
culture and of Dominique Defalque for in vivo experiments is
acknowledged. We are deeply indebted to Thierry Grange for his help in
setting up the genomic footprinting method, his advice throughout these
studies, and his critical reading of the manuscript. We thank Michel
Raymondjean and Claude Sardet for reading the manuscript and Laurent
Charvet for art work.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Alphonse Le Cam, INSERM U376, Hopital Arnaud de Villeneuve, 371 Rue du Doven Gaston Giraud, Montpellier, Cedex 05 France 34295.
1 Present address: Unité de Diabétologie et Nutrition,
School of Medicine, The University of Louvain, B-1200, Brussels,
Belgium. 
Received for publication September 10, 1997.
Revision received December 15, 1997.
Accepted for publication December 16, 1997.
 |
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