Departments of 1 Pediatrics and 3 Biochemistry and 2 Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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The rat serine
protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2)
and negative (Spi 2.1) acute phase reactants, facilitating modeling of
regulation of hepatic acute phase response (APR). To examine the role
of signal transducer and activation of transcription (STAT) proteins in
the divergent regulation of these model genes after induction of APR,
we evaluated the proximal promoters of the genes, focusing on STAT
binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can
bind to a -activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To
create an in vitro model of APR, primary hepatocytes were treated with
combinations of cytokines and hormones to mimic the hormonal milieu of
the whole animal after APR induction. Incubation of primary rat
hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to
activation of STAT3 and a 28-fold induction of a chloramphenicol
acetyltransferase reporter construct containing the
319 to +85
region of the Spi 2.2 promoter. This suggests the turpentine-induced
increase of Spi 2.2 is mediated primarily by IL-6. In contrast,
although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6
does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of
hepatocytes with IL-6 results in a 5.4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this
promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in
part to differences in the promoters of these genes at the GAS sites.
IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression
that results from turpentine-induced inflammation.
acute phase response; serpin; Janus kinase-STAT pathway; STAT3; STAT5
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INTRODUCTION |
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INFLAMMATORY STIMULI CAUSE alterations in the expression of a limited number of genes in the liver. This series of coordinated changes is known as the hepatic acute phase response (APR) and is mediated in part at the level of transcription. During the APR, there is increased expression of one subgroup and decreased expression of another subgroup of hepatic genes (19). These effects are mediated by the products of stimulated monocytes and macrophages in combination with glucocorticoids (3, 16).
We have been interested in the transcription of a subset of acute phase reactants, the rat hepatic serine protease inhibitor (Spi) genes Spi 2.1 and 2.2. The nucleic acid and deduced amino acid sequences of these genes suggest they are members of the serpin gene family. The serpin gene family encodes a large number of proteins that share significant sequence homology but perform diverse biological roles. We have characterized the physiological regulation of rat Spi 2.1 and 2.2 mRNAs. Because Spi 2.1 is dependent on growth hormone (GH) for maximal hepatic mRNA content (24, 40), it has been used to study hepatic gene regulation by GH. In contrast, Spi 2.2 mRNA is not GH dependent (32).
Despite significant promoter sequence similarity, hepatic Spi 2.1 and 2.2 mRNAs have dramatic divergence in their responses to an inflammatory stimulus. Hepatic Spi 2.2 mRNA increases 10-fold within 12 h of inflammation induced by subcutaneous turpentine injection, whereas Spi 2.1 mRNA decreases to levels ~25% of control values 48 h after stimulus (33). Spi 2.2 is thus a positive hepatic acute phase reactant, whereas Spi 2.1 is one of a small number of hepatic proteins that are negative acute phase reactants (33). Intracellular signaling by both GH and inflammatory mediators involves the actions of latent cytoplasmic signal transducer and activation of transcription (STAT) proteins. STAT proteins are tyrosine phosphorylated in response to a number of cytokine-receptor interactions. STAT3 was isolated from mouse livers following treatment with interleukin (IL)-6 and was initially called APRF, for acute phase response factor (1). STAT5, originally identified as a prolactin-induced factor, is activated in response to GH and binds to the Spi 2.1 promoter to mediate its transcriptional induction. However, GH is known to cause tyrosine phosphorylation of multiple STAT proteins, including STAT3 (5, 8, 15). Because Spi 2.1 and Spi 2.2 have divergent responses to inflammatory stimulation, we were interested in examination of potential STAT binding to the Spi 2.1 and 2.2 promoters after turpentine treatment to examine the specificity of differential gene expression in the Spi 2 gene family. As the proximal promoters of these genes differ only at putative STAT binding sites, we hypothesized that the hepatic APR following turpentine injection would activate STAT proteins and that differences in binding of STAT proteins would be critical to specificity and differential regulation of Spi 2 gene expression. To facilitate the examination of these model genes using a manipulatable in vitro system, we used hormone- and cytokine-treated primary hepatocytes to complement our previous studies in whole animals with the initial hypothesis that IL-6 would be the primary cytokine regulating the APR of the Spi 2 genes.
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METHODS |
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Induction of the hepatic APR by turpentine. Adult male rats (either hypophysectomized or normal) were sedated, injected subcutaneously with 0.5 ml turpentine/100 g body wt, and killed at intervals after injection. Use of hypophysectomized animals permitted examination of the STAT response without the effects of pituitary hormones except as supplied exogenously. For animals treated with GH, 150 µg human recombinant GH/100 g body wt were administered intraperitoneally 1 h before death. The livers were removed, and crude nuclear extracts were prepared as described (7). All animal studies were evaluated and approved by the Institutional Animal Care and Use Committee of the University of Minnesota.
Electrophoretic mobility shift assays.
Probes used in electrophoretic mobility shift assays (EMSA) included a
high-affinity sis-inducible element
(SIEa; 5'-GTCAGACATTTCCCGTAAATCGTCGA-3') (14) that binds
STAT1 and STAT3 (5) and a fragment of the Spi 2.2 gene spanning
137 to
109
(5'-GAGTCCA
ATCATCCCGTC-3'). This fragment is derived from the Spi 2.2 promoter region
corresponding (27) to the Spi 2.1 GH response element (GHRE) (39) and
contains a single
-activated sequence (GAS; underlined in the
element sequence), a feature noted in genes activated by STAT proteins. An oligonucleotide encompassing the Spi 2.1 GHRE was used for EMSA to
monitor for STAT5 binding.
Plasmid construction and hepatocyte assays.
The construction of Spi 2.2 (319 to +85) into the
Pst I sites of the parent
chloramphenicol acetyltransferase (CAT) plasmid pCAT(An)
was as described previously for other constructs (5). Spi 2.1 constructs [p(
275 to +85)-Spi 2.1-CAT (Spi-A-CAT), del (
149 to
102)-Spi 2.1-CAT (Spi-O-CAT), and a construct
containing a mutation of the 3' GAS site of the GHRE
(Spi-E-CAT)] have been described previously (5, 39). An
additional construct mutating the 5' GAS site (underlined) of the
GHRE was also prepared, containing the mutated GHRE sequence
5'-AC
TCCATGTTCTGAGAAATTCTAGAGTCTGCCCA-3' (Spi-G-CAT). This mutation completely abolishes the response to GH in
transfection assays (Bergad, Towle, and Berry, unpublished observations). All sequences were confirmed by DNA sequencing. All
experiments were repeated using independent plasmid preparations to
ensure reproducibility of the response of each construct.
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RESULTS |
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Activation of hepatic STAT3 by turpentine stimulation.
We speculated that the physiological response to turpentine stimulation
would include rapid activation of STAT proteins, particularly STAT3. To
examine the time course of STAT protein activation after turpentine
stimulation, normal male rats were injected with subcutaneous turpentine and killed at intervals after treatment. Hepatic nuclear extracts were prepared and tested for the presence of STAT protein binding using EMSA assays. Binding assays were performed using SIEa, a
high-affinity sis-inducible element
known to bind to STAT1 and STAT3, as a probe (Fig.
1A.)
EMSA of hepatic nuclear extracts from untreated animals did not display
any protein-DNA complexes. However, by 2 h after turpentine treatment,
a single complex that binds to SIEa was present. The intensity of
binding of this complex was further increased at 4 h after treatment.
EMSA using these extracts and labeled GHRE from the Spi 2.1 promoter
(which binds STAT5 and not STAT1 or STAT3) showed no changes in binding
from the low levels of basal STAT5 binding (data not shown).
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Pituitary dependence of STAT binding activity in hepatic nuclear
extracts.
To evaluate whether there were pituitary-dependent differences in the
STAT proteins activated after turpentine treatment, hepatic nuclear
extracts from hypophysectomized and normal male animals treated with
GH, with turpentine, or with both were used in EMSA with labeled SIEa
and GHRE. Figure 2 shows that
small amounts of activated STAT3 are present in hypophysectomized
animals. Injection with GH results in activation of STAT1, STAT3, and
STAT5 binding in hypophysectomized animals, as noted previously (5), but only STAT5 is activated in normal animals. Turpentine injection results in increased binding of STAT3 in both normal and
hypophysectomized animals. STAT5 binding is not seen in
hypophysectomized, turpentine-treated animals, and no increase over
basal STAT5 binding is seen in normal animals treated with turpentine.
The addition of GH treatment 4 h after turpentine treatment of a
hypophysectomized animal results in a binding pattern that is not
different from that seen after GH treatment alone. Thus there are no
substantial differences in the pattern of turpentine-stimulated STAT
protein binding attributable to GH state.
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Potential for STAT binding to the Spi 2.2 GAS element.
The activation of STAT3 after turpentine stimulation of inflammation
suggests the potential for STAT3 to function as an activating protein
for Spi 2.2 transcription. To determine whether STAT proteins can bind
to the Spi 2.2 promoter, we did EMSA using a fragment of the Spi 2.2 promoter containing a GAS element. This element is found at the same
promoter position as the GHRE in Spi 2.1 but contains only a single GAS
site, rather than the paired GAS elements of the Spi 2.1 promoter. This
was bound to hepatic nuclear extracts from hypophysectomized male rats
treated with either GH or with turpentine. Four state-specific
complexes bound to the Spi 2.2 GAS element are present in extracts from
GH-treated hypophysectomized animals. These complexes migrated at
positions expected for STAT5 dimers, STAT3 homodimers, STAT1-STAT3
heterodimers, and STAT1 homodimers
(top to
bottom positions in Fig.
3A).
Addition of appropriate antibodies to various STAT proteins confirmed
that the GH-induced protein-DNA complexes include STAT1, STAT3, and STAT5 (Fig. 3, B and
C). However, only one state-specific
complex migrating at the position of STAT3 homodimers was present after turpentine stimulation. The Spi 2.2 promoter GAS site thus can bind
three STAT proteins, although the potential for these varied binding
patterns is dependent on the pituitary state of the animal. Because Spi
2.2 mRNA can be induced in the absence of GH and because only binding of STAT3-containing complexes is found after turpentine treatment of hypophysectomized rats, STAT3 is likely to be the physiologically relevant binding factor for induction of the Spi 2.2 promoter.
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Development of an in vitro APR model: IL-6 action in induction of
Spi 2.2 and 2.1 mRNAs.
Turpentine injection results in production of IL-6 and other cytokines
(10, 11). We noted that STAT3 was specifically activated after
turpentine treatment, presumably due to stimulation of IL-6 by the
tissue injury caused by the turpentine injection. We inferred that, in
turn, Spi 2.2 transcription could be induced by IL-6-activated STAT3
binding to the Spi 2.2 GAS element. To complement these in vivo
observations and to develop a model system that would allow both
investigation of the roles of individual cytokines in the hepatic APR
and studies of the responses of individual promoter elements, we used
primary hepatocytes. To more directly evaluate the role of IL-6 in
induction of Spi 2.2 mRNA, we examined the endogenous induction of this
mRNA in primary hepatocyte cultures treated with IL-6 because our data
to this point suggested that IL-6 was the primary cytokine required for
Spi 2.2 induction. To examine the specificity of the response of the
Spi 2.2 promoter, especially as it contains a site that is capable of
binding to a variety of STAT proteins, the effect of GH treatment on
Spi 2.2 mRNA was also evaluated. As seen on a representative Northern blot (Fig. 4,
top), Spi 2.2 mRNA was
very low in RNA isolated from untreated hepatocytes but was clearly
present in RNA from cells treated with IL-6, producing increases in Spi
2.2 mRNA (4.36 ± 0.18-fold change from untreated hepatocytes, mean ± SE, n = 10). As anticipated on
the basis of previous studies, GH treatment did not result in a
significant increase in Spi 2.2 mRNA quantities (1.10 ± 0.14-fold,
n = 7), nor did it alter the effect of
IL-6 when cells were treated with both GH and IL-6 (4.30 ± 0.06-fold, n = 4). GH effectiveness
was confirmed by an observed increase in Spi 2.1 mRNA levels in these
hepatocytes (4.60 ± 0.038-fold, n = 3). In contrast to the observed induction of Spi 2.1 mRNA by GH, IL-6
did not increase Spi 2.1 mRNA (0.63 ± 0.014-fold, n = 6).
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In vitro action of IL-6 in activation of STAT3 binding.
To determine more directly whether IL-6 is an activating ligand for
STAT3 in cultured primary hepatocytes, we performed EMSA with nuclear
extracts of hepatocytes treated with IL-6 or GH. Figure
5 shows the time course of STAT3 activation
by IL-6. Within 15 min of IL-6 treatment, activated STAT3 was already
present in the nuclear extracts. Between 30 min and 2 h there was also a transient appearance of STAT1 and STAT1-STAT3 heterodimers. Under
these conditions, however, despite the activation of STAT1, STAT3, and
STAT5 by GH in vivo and the responsiveness of hepatocytes to GH
evidenced by an increase in Spi 2.1 mRNA, we did not observe activation
of STAT3 by GH.
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Action of IL-6 on the Spi 2.2 and 2.1 promoters.
To determine whether the observed increase in Spi 2.2 mRNA is due
primarily to transcriptional activation of the Spi 2.2 promoter, induction of a Spi 2.2-CAT construct was examined in hepatocytes treated similarly to those noted above. For this purpose a construct containing a Spi 2.2 promoter fragment (319 to +85; includes the
Spi 2.2 GAS element) coupled to CAT was introduced. A 28-fold induction
of CAT protein was observed after IL-6 treatment (Fig. 6A).
Again, as a control for specificity, GH failed to induce Spi 2.2-CAT
and did not augment the effects of IL-6. Thus the observed increase in
Spi 2.2 mRNA in the treated hepatocytes is due at least in part to
transcriptional activation of its proximal promoter that can be
stimulated by IL-6.
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DISCUSSION |
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As a part of the systemic response to inflammation, the rate of
synthesis of a selected group of hepatic proteins is altered. The
hepatic APR is stimulated during any tissue injury, including burns,
surgery, parturition, and sepsis (19, 22, 23). The spectrum of proteins
altered by the APR is the same regardless of the specific injury
eliciting the response, with the quantitative response resulting from
the summation of the complex signals from various cytokines and
hormones. The major mediators of change in hepatic gene response during
inflammation include IL-6, IL-1, tumor necrosis factor-
(TNF
),
interferon-
, and glucocorticoids, although other cytokines and
hormones are also involved. Many of these cytokine mediators are
derived from stimulated monocytes and macrophages. The network of
cytokines released during inflammation have both overlapping and
competing properties. The concentration of the cytokines and hormones,
the sequence in which they reach the liver, and their interactions all
affect the response of any individual protein in the APR (20).
Transcriptional regulation of the APR is dependent on DNA elements
found in cis in the promoters of acute
phase reactant genes. Studies in rat hepatoma cell lines identified two
distinct groups of hepatic acute phase reactants: group 1 proteins are
regulated by a combination of IL-1, IL-6, and glucocorticoids, and
group 2 proteins are regulated by IL-6, with or without
glucocorticoids, but without a requirement for IL-1 (4, 12).
Correspondingly, two DNA response elements were identified: group 1 genes contain an element that binds a member of the
CCAAT/enhancer-binding protein (C/EBP) family, NF-IL-6 (C/EBP). In
response to IL-6-mediated glycoprotein 130 homodimerization, the
ras-mitogen-activated protein kinase system is activated,
eventually activating NF-IL-6 (18). Group 2 genes contain an element
that binds STAT3 (17). This DNA element (type 2 IL-6 response element)
is similar to other GAS elements known to bind members of the STAT
family (41).
Injection of turpentine is a reproducible method for eliciting the APR in vivo (26, 33). In this model, hepatic mRNA levels of typical APR products are maximally changed by 24 h (26, 33, 30). Injection of IL-6 into rats results in a more rapid increase in IL-6-responding APR gene products than turpentine injection; this difference is likely due to the requirement for intermediate stimulation of IL-6 by the tissue injury associated with turpentine injection (37). Induction of the APR using turpentine in this study resulted in the rapid appearance of activated STAT3 as evidenced by binding to both the SIEa, a GAS element that recognizes only STAT1 and STAT3, and to the Spi 2.2 GAS site. The relatively rapid appearance of activated STAT3 after turpentine stimulation, and its ability to bind to the GAS site in the Spi 2.2 promoter, could result in activation of Spi 2.2 transcription and account, at least in part, for the increases observed in mRNA levels at 12 h after injection. This pathway has been recapitulated in primary hepatocyte cultures in which the rapid appearance of STAT3 and the response of Spi 2.2 mRNA and Spi 2.2-CAT promoter constructs to IL-6 stimulation have been documented. These observations further support the conclusion that STAT3 activation in response to turpentine-stimulated IL-6 is the likely mechanism for increases in Spi 2.2 mRNA after turpentine stimulation.
The Spi 2.2 GAS element is capable of binding STAT1 and STAT5 in addition to STAT3 and thus can bind all three STAT proteins activated by GH (5, 8, 25). Despite this, we do not find evidence that endogenous STAT activation from GH treatment results in stimulation of Spi 2.2 transcription or that either STAT1 or STAT5 is necessary for Spi 2.2 mRNA induction. One source of this differential response to GH could be the architecture of the GAS elements in the Spi 2.1 and 2.2 promoters. These promoters have nearly identical (2- nucleotide difference) GAS elements. The Spi 2.1 promoter also has a second 5' GAS element in addition to the primary GAS element conserved in both Spi 2.1 and Spi 2.2 promoters. Normal transcription of GH-responsive Spi 2.1 requires the presence of these paired GAS elements in its promoter, but the second, 5'-element is disrupted in the Spi 2.2 promoter. Furthermore, we have shown previously that the 3' GAS element in Spi 2.1 as an isolated GAS site binds STAT5 only weakly (5); it also fails to bind STAT1 or STAT3. The two-nucleotide difference in the otherwise conserved 3' Spi 2.1 and Spi 2.2 GAS elements appears to be sufficient to alter the affinity of the element for STAT1, STAT3, and STAT5.
The profile of STAT proteins critical to Spi 2.2 expression in the APR is succinct despite the capacity of its GAS element to bind more than one STAT protein. In experiments using overexpression of STAT proteins, Kordula et al. (21) observed activation of the Spi 2.2 promoter by STAT5b. However, under GH conditions that result in substantial induction of Spi 2.1 promoter-CAT constructs (5), the levels of STAT proteins induced by GH do not induce a Spi 2.2 promoter-CAT construct. Furthermore, hepatic nuclear extracts from hypophysectomized animals treated with turpentine do not contain factors that bind to elements that recognize STAT5 specifically, despite the observation that this treatment increases expression of Spi 2.2 mRNA (33). There is also no increase in STAT5 binding in extracts from normal animals treated with turpentine. Thus STAT3 is an essential element in the positive APR to IL-6, as exemplified by the Spi 2.2 mRNA response. The participation of other endogenous STAT proteins in the Spi 2.2 response is less likely.
One goal of these studies was generation of an in vitro model of APR
induction using primary hepatocytes. Such a model system would be
complementary to whole animal studies because it would allow
manipulation of the hormonal milieu contributing to the APR and
specifically allow study of cis
elements of gene promoters important in the APR. An additional side
benefit is that it would reduce the number of invasive treatments that
would be required to do such studies in whole animals. Although an IL-6
increase after turpentine injection can account for the positive APR of Spi 2.2, giving IL-6 as an isolated cytokine in the hepatocyte model
does not reproduce the negative APR observed after turpentine injection
in the whole animal. If IL-6 were the sole effector of the negative APR
observed for Spi 2.1 in vivo, we should have seen repression by IL-6 of
GH induction of Spi 2.1-CAT constructs in vitro. Instead, an increase
in gene expression driven by a fragment of the Spi 2.1 promoter was
observed. The paradoxical IL-6 induction of the Spi 2.1 promoter
fragment in hepatocyte culture is mediated via the paired GAS sites of
the Spi 2.1 GHRE. Wood et al. (38) showed that neither STAT1 nor STAT3
induces transcription via the Spi 2.1 promoter and suggested that this promoter is STAT5 specific. This suggests that IL-6 may activate STAT5
and in turn activate this Spi 2.1 promoter construct via the paired GAS
element. Because an induction rather than a decrease was observed,
there could be elements present in the native Spi 2.1 gene that modify
the responses we observed using a promoter fragment encompassing only
sequences from 275 to +85. However, similar results were seen
using a fragment
3200 to +85 (not shown). If IL-6-regulated
inhibitory sequences are important in the negative APR of Spi 2.1, they
are not present in these portions of the gene.
The observed induction of the reporter gene expression driven by the
Spi 2.1 promoter fragment is discordant with the decrease in Spi 2.1 mRNA expression observed after turpentine treatment and with a lack of
increase of Spi 2.1 mRNA expression after IL-6 treatment of cultured
hepatocytes. The profile of cytokines induced by turpentine treatment,
although it includes IL-6, therefore also likely includes a factor that
suppresses Spi 2.1 expression. Downregulation of the APR may be
regulated in part by TNF and IL-1
(13). We propose that the
regulation of the observed decrease of Spi 2.1 mRNA in the APR is thus
likely to be due to the interaction of more than one cytokine and
hormonal stimulus and, unlike the upregulation of Spi 2.2 mRNA, is not
dependent on induction of IL-6 alone.
Despite the demonstrable physiological regulation of the Spi 2.1 gene
by GH, its promoter can be activated by other ligands after
transfection into cultured cells. Wood et al. (38) observed a similar
induction of this promoter in COS-7 cells cotransfected with STAT5
expression plasmid and stimulated with erythropoeitin. In cultured
cells, then, other ligand-receptor activation that results in STAT5
phosphorylation can induce expression from a Spi 2.1 promoter construct
containing the paired GAS sites that mediate GH response. A similar
observation was noted for activation of the -casein promoter by IL-6
in mammary epithelial cells (36). Thus any studies to evaluate the
effects of specific hormones in vitro need to be carefully correlated
with appropriate in vivo studies if physiological relevance is at
issue. The specific effects of IL-6 as an isolated cytokine on Spi 2.1 gene expression in vivo are not known.
The mechanisms that result in differential STAT activation and, in turn, differential expression of the divergently regulated Spi 2 genes after an acute phase stimulus are complex. Quantitative or qualitative differences in the various STAT proteins available for binding may result in differential activation of Spi 2 gene promoters, and differences in DNA element architecture or the presence of other nuclear factors may modulate the responses of Spi 2 promoters after induction of the APR. Further work examining these possibilities in this provocative model system should be of value in defining these differences in regulation. It will be necessary, however, to generate a hepatocyte model that more faithfully reproduces the in vivo response to turpentine or other acute phase stimuli to establish the mechanism of the negative APR of Spi 2.1, as treatment with IL-6 alone will not faithfully reproduce the negative APR.
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ACKNOWLEDGEMENTS |
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We thank Elizabeth Kaytor for preparation of primary hepatocytes.
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FOOTNOTES |
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This work was performed with the support of National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32817 and the Viking Children's Fund.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. J. Schwarzenberg, Dept. of Pediatrics, University of Minnesota, 420 Delaware St. SE, Box 185 FUMC, Minneapolis, MN 55455 (E-mail: schwa005{at}tc.umn.edu).
Received 27 August 1998; accepted in final form 24 February 1999.
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