1 Johns Hopkins University, Baltimore, Maryland 21205; and 2 Hennepin County Medical Center, Minneapolis, Minnesota 55415
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ABSTRACT |
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Ethanol ingestion may interrupt the
proregenerative signal transduction that is initiated by injury-related
cytokines such as tumor necrosis factor (TNF)- and TNF-
-
inducible cytokines including interleukin (IL)-6. To test this theory,
liver regeneration, TNF-
and IL-6 expression, and cytokine-regulated
prereplicative events were compared in ethanol-fed rats and
isocalorically fed controls after 70% partial hepatectomy (PH).
Ethanol feeding inhibits hepatocyte replication and recovery of liver
mass after PH but generally promotes induction of both cytokines in the
liver and extrahepatic tissues (i.e., white adipose tissue).
Cytokine-regulated events that occur early in the prereplicative period
are influenced differentially. TNF-
-dependent increases in hepatic
nuclear factor-
B (NF-
B) p50 and p65 expression and DNA binding
activity are prevented, whereas IL-6-dependent inductions of hepatic
Stat-3 phosphorylation and DNA binding activity occur normally. In
contrast, events (e.g., induction of cyclin D1, cdk-1, cyclin D3, and
p53 mRNA) that occur at the end of the prereplicative period are
uniformly inhibited. These findings indicate that chronic ethanol
ingestion arrests the regenerative process during the prereplicative
period and demonstrate that increased TNF-
, IL-6 and Stat-3 are not
sufficient to assure hepatocyte proliferation after PH.
tumor necrosis factor; interleukin-6; Stat-3; transcription factors; adipose tissue
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INTRODUCTION |
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THE INJURY-RELATED, proinflammatory cytokines tumor
necrosis factor (TNF)- and interleukin (IL)-6 promote liver
regeneration. The importance of TNF-
and IL-6 as hepatotrophic
factors is demonstrated by evidence that induction of hepatocyte DNA
synthesis after 70% (partial) hepatectomy (PH) is severely inhibited
in TNF-
receptor type 1 null mice (44) and IL-6 null mice (8) and in
normal rats that have been pretreated with anti-TNF-
antibodies
(1). On the other hand, TNF-
and TNF-
-inducible
cytokines are thought to mediate the liver injury produced by several
hepatotoxins, including ethanol (reviewed in Ref. 31). Circulating
levels of TNF-
and IL-6 are increased in patients hospitalized with alcoholic liver disease and correlate with the severity of liver injury
and mortality in these patients (4, 17, 20, 23). Similarly, increased
production of TNF-
and IL-6 has been noted in rats that develop
liver damage while receiving chronic intragastric infusions of ethanol
(22). Interestingly, ethanol is one of the few causes of liver injury
that is also known to inhibit hepatocyte proliferation both in vitro
(5) and in vivo (10, 16, 18, 25, 33, 40). This suggests that ethanol
may selectively inhibit proregenerative signaling by TNF-
and/or IL-6. Recent work by several different groups is
beginning to outline the intracellular events that appear to be
involved in TNF-
- and IL-6-mediated hepatocyte proliferation. There
is good evidence that TNF-
is responsible for the induction of
NF-
B DNA binding activity that occurs within the first hour after PH
(8, 9, 44). This may help the liver to reconstitute its mass after PH,
because one of the many functions of NF-
B is to prevent apoptosis
(39, 41, 43). NF-
B is also known to activate the transcription of
TNF-
-inducible genes, including IL-6 (30, 37, 48). Indeed, antibody
neutralization studies (1) and experiments with TNF-
receptor type 1 null mice (44) indicate that TNF-
is predominately responsible for
inducing IL-6 after PH. IL-6, in turn, has been shown to play a pivotal
role in the activation of Stat-3, which occurs later during the
prereplicative period after PH (7, 8). Some have postulated that
activated Stat-3 then promotes the progression of hepatocytes through
the G1 phase of the cell cycle and
into S phase (8, 44), a process that requires induction of cyclin D1
(2, 24, 32). Thus several cytokine-inducible molecules have been
identified that, if inactivated by chronic ethanol exposure, could
abort the hepatocyte proliferative response that is normally triggered
by PH.
To test the theory that ethanol may inhibit liver regeneration by
interrupting cytokine-initiated signal transduction, we compared liver
regeneration, cytokine expression, and cytokine-regulated prereplicative events in rats that were fed an ethanol-containing diet
and control rats that were given isocaloric volumes of a similar diet
without ethanol. The results confirm previous evidence that chronic
ingestion of ethanol inhibits liver regeneration after PH and
demonstrate that this occurs despite normal or enhanced induction of
TNF-, IL-6, and Stat-3, an IL-6-regulated transcription factor. An
ethanol-related block in TNF-
-initiated signaling has also been
identified. Specifically, TNF-
-dependent, early prereplicative
events, i.e., induction of NF-
B p50 and p65 expression and DNA
binding activities, are inhibited in the ethanol-fed group. This early
inhibition of NF-
B may be related to concomitant superinduction of
the TNF-
inhibitor IL-10 and is associated with the inhibition of
many of the subsequent events (including the induction of cyclin D1,
cdk-1, cyclin D3, and p53) that normally occur as hepatocytes exit
G1 and enter S phase.
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METHODS |
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Materials
Adult Sprague-Dawley rats (mean initial wt 175 g) were purchased from Charles River (Charles River, NJ). The 1982 formulations of the Lieber-deCarli ethanol and control diets were purchased from Bio-Serv (Frenchtown, NJ). In each liter of control diet, fat contributes 350 kcal, protein 180 kcal, and carbohydrate 470 kcal. The ethanol diet is identical, except that ethanol is substituted for 355 of the carbohydrate kilocalories. Chemicals were purchased from Sigma Chemical (St. Louis, MO) with some exceptions. Phenol was purchased from Fluka (Ronkonkoma, NY). Taq polymerase was obtained from Boehringer Mannheim (Indianapolis, IN). Moloney murine leukemia virus reverse transcriptase came from GIBCO (Grand Island, NY). The enhanced chemiluminescence detection system and Hybond N+ nylon membranes came from Amersham (Arlington Heights, IL). GeneScreen nylon membranes were from NEN Research Products (Boston, MA). [Methods
Animal feeding and PH experiments. Rats were housed under a 12-h light-dark cycle and were permitted ad libitum consumption of standard rat pellet chow. After a 1-wk equilibration period, the animals were fed either a Lieber-DeCarli control diet or an isocaloric diet with ethanol providing 36% of total dietary kilocalories. Each day, the previous day's intake was measured and the control (pair-fed) rats were fed the average intake of the ethanol-fed rats (29). Food was provided daily at 6 PM. After 5 wk the animals were subjected to 70% PH while under light ether anesthesia, in the early morning (19). PH has been shown to substantially increase the proportion of hepatocytes in S phase and is a standard technique used to assess response to various effectors of regeneration. Several groups including our own (12, 16, 33, 40) showed that rats fed ethanol by this protocol have inhibited DNA synthesis and liver regeneration after PH, but to validate this response some rats were injected with the S-phase marker 5-bromo-2'-deoxyuridine (BrdU) 2 h before death (8). At various times after PH, rats were killed and serum, liver, and epididymal fat were harvested. All experiments were performed in adherence to National Institutes of Health guidelines on the use of experimental animals. Approval of the Animal Use Committee of the Johns Hopkins University was obtained before the experiments were initiated.
Evaluation of liver regeneration. As others (8, 44) have done, we used three parameters (hepatocyte incorporation of BrdU, mitotic index, and liver weight) to assess the regenerative response at 24 and 48 h after PH. Formalin-fixed liver sections from BrdU-injected rats were treated with peroxidase-conjugated antibodies to BrdU. Two different investigators counted the number of darkly stained (BrdU positive) hepatocyte nuclei in 10 different ×400 fields on coded sections from four different rats per treatment group. The average number of BrdU-labeled hepatocytes was calculated for each animal and used to derive the mean (±SE) number of BrdU-labeled hepatocytes per treatment group. The number of hepatocytes with mitototic bodies was counted on parallel hematoxylin and eosin-stained liver sections to derive the mean (±SE) mitotic index for each group. The extent to which the liver mass had been reconstituted was also evaluated. Resected livers were weighed at the time of PH. This weight was divided by 0.7 to derive the initial (pre-PH) weight of each liver. Each post-PH liver remnant was also weighed at the time of death. For each rat, the weight of the remnant liver was normalized to the weight of that rat's entire liver at the time of PH and expressed as the percentage of the rat's initial liver weight according to the following formula: (wt of liver remnant / initial liver wt) × 100. Data from four rats per treatment group were used to calculate the mean (±SE) at each time point. Results of all three parameters were evaluated by ANOVA using computer statistical software.
RNA evaluation. Total liver RNA was isolated according to the protocol of Chomczynski and Sacchi (6) and quantified by measurement of ultraviolet absorption at 260 nm. Representative aliquots of RNA from each rat (4 rats per feeding group per time point) were separated by electrophoresis on denaturing agarose gels, followed by ethidium bromide staining to confirm RNA concentration and quality. Twenty micrograms of RNA samples per lane were fractionated on denaturing agarose gels and transferred to GeneScreen membranes. Membranes were stained with 0.04% methylene blue to confirm the lane-lane equivalency of RNA loading/transfer, and then hybridized with cDNA probes for cyclin D1, cdk-1, cyclin D3, or p53 as described previously (45). After being washed under stringent conditions, membranes were exposed to Kodak X-AR film with intensifying screens. Autoradiograms were analyzed by scanning laser densitometry (Molecular Dynamics, Sunnyvale, CA).
Cytokine transcript levels of TNF-Nuclear protein isolation, immunoblot, and gel mobility/supershift
assays for NF-B.
Nuclear protein was isolated by NUN buffer (1.0 M urea, 0.33 M NaCl,
1.0% Nonidet P-40, 27.5 mM HEPES, pH 7.0, 1 mM DTT); protein
concentrations were determined and treatment-related variations in p50
and p65 NF-
B, total Stat-3, and phosphorylated Stat-3 were analyzed
by Western blot as described previously (45). In brief,
proteins (40 µg/lane) were separated on 12% SDS-polyacrylamide gels
by electrophoresis and transferred onto nylon membranes. After
Coomassie blue staining of the gels and blots to assure lane-lane
equivalency in protein loading and transfer, the blots were probed with
commercially available antibodies to the respective proteins and
visualized by enhanced chemiluminescence. As detailed in the previous
section, extracts from three different rats per feeding group per time
point were evaluated and resultant blots were assessed by scanning
laser densitometry. A representative Western blot that was probed for
Stat-3 is shown in Fig. 7.
Serum cytokine measurements.
Commercially available ELISA kits for rat TNF-, IL-6, and IL-10 were
used to determine treatment-related variations in serum cytokine
levels. Standard curves for the respective rat recombinant cytokines
were included in each assay; samples from three to four rats per
feeding group per time point were evaluated, and samples from each rat
were assayed in triplicate. Data were analyzed by ANOVA and are
graphically displayed beneath the cytokine RNA data shown in Figs. 2,
4, and 5.
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RESULTS |
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As reported by many other groups (reviewed in Refs. 27-29), rats fed ethanol in liquid diets develop a fatty liver without appreciable hepatic necrosis or inflammation. Also, as we (10-12) and others (16, 33, 40) have reported, ethanol feeding inhibits hepatocyte proliferation after PH. In the present study, this is evidenced by decreased hepatocyte incorporation of BrdU (Fig. 1A), mitoses (Fig. 1B), and restitution of liver mass (Fig. 1C) at 24-48 h after PH in ethanol-fed rats compared with controls.
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Because TNF- has been implicated in the pathogenesis of
alcohol-induced liver injury but this particular ethanol feeding protocol does not overtly injure the liver, it was initially uncertain whether TNF-
expression would be increased in these ethanol-fed rats. To resolve this question, levels of TNF-
mRNA and protein were
compared in the livers and sera of ethanol-fed rats and isocalorically maintained (pair-fed) controls before and after PH. As shown in Fig.
2, hepatic TNF-
mRNA levels increase
within 15 min after PH in pair-fed rats, remain above baseline at 60 min post-PH, and decline to pre-PH levels by 3-6 h after PH.
Compared with these pair-fed controls, ethanol-fed rats overexpress
TNF-
mRNA before PH and for the first 15 min post-PH. Although
hepatic expression of TNF-
mRNA is somewhat lower in ethanol-fed
rats from 30 to 60 min after PH, it becomes greater again in
ethanol-fed rats than controls at later time points (i.e., 3-6 h
post-PH). Ethanol-associated increases in liver TNF-
mRNA expression
are generally accompanied by increases in circulating levels of TNF-
protein. Hence, post-PH inductions of hepatic TNF-
mRNA and
circulating TNF-
proteins are somewhat enhanced by chronic ethanol
ingestion.
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In normal rats, TNF- mediates the activation of NF-
B that occurs
within an hour after PH (44). Gel retardation assays (Fig.
3, A and
B) and immunoblot analyses (data not
shown) of liver nuclear extracts were done to determine whether ethanol influences regenerative induction of NF-
B. NF-
B DNA binding activity increases transiently at 30 min after PH in pair-fed rats
(Fig. 3A). Supershift (Fig.
3B) and immunoblot analyses indicate that this increased NF-
B binding activity results, at least in part,
from increases in the nuclear accumulation of both p50 and p65.
Surprisingly, increased NF-
B DNA binding activity is not observed
after PH in ethanol-fed rats (Fig.
3A). Consistent with this finding,
no increase in p50 or p65 expression is demonstrated by immunoblot
analysis of post-PH liver nuclear extracts from ethanol-fed animals
(data not shown).
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The anti-inflammatory cytokine IL-10 is known to inhibit
lipopolysaccharide (26) and TNF- (42)-dependent activation of NF-
B in monocytic cells. To begin to evaluate the possibility that
ethanol feeding may inhibit regenerative induction of NF-
B DNA
binding activity by an IL-10-mediated mechanism, we compared IL-10
expression in ethanol- and pair-fed rats. As shown in Fig. 4, IL-10 mRNA increase transiently in the
livers of pair-fed rats from 30 to 60 min after PH. Induction of
hepatic mRNA for IL-10 is followed by increases in circulating IL-10
protein. We (34) reported similar post-PH induction of IL-10 in ad
libitum-fed rats and suggested that such increases in IL-10 may play a
role in limiting the TNF-
response after PH. Consistent with this theory, the post-PH increases in hepatic IL-10 expression temporally correlate with abrupt declines in NF-
B DNA binding activity in pair-fed animals. Regenerative induction of IL-10 mRNA and protein is
increased in ethanol-fed rats compared with controls (Fig. 4).
Superinduction of IL-10 correlates with decreased NF-
B DNA binding
activity in the ethanol-fed group. Although this correlation does not
definitively establish a cause-effect relationship between increased
IL-10 expression and decreased NF-
B activity, the results are
consistent with the possibility that overexpression of this cytokine
may contribute to ethanol-associated inhibition of NF-
B activation.
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Activation of NF-B is essential for TNF-
-dependent induction of
IL-6 (48). Thus ethanol-related inhibition of NF-
B suggests that
IL-6 expression may be decreased in ethanol-fed rats. If true, the
latter could explain the antiregenerative actions of ethanol, because
IL-6 is necessary for liver regeneration after PH (8). To evaluate the
possibility that ethanol feeding may inhibit regenerative induction of
IL-6, IL-6 expression was compared in ethanol-fed rats and pair-fed
controls. As shown in Fig. 5, in pair-fed
rats IL-6 mRNA begin to increase in the liver at ~30 min, peak at 60 min, and remain abundant for at least 6 h after PH. As we (1) and
others (38) reported, these increases in hepatic IL-6 expression are
followed by increases in circulating levels of IL-6 protein from 6 to
24 h after PH. In contrast, induction of IL-6 mRNA is delayed and
attenuated in the livers of ethanol-fed rats. However, circulating
levels of IL-6 protein are not comparably reduced. Indeed, quite the
opposite is true: serum levels of IL-6 increase earlier after PH and
are significantly greater in ethanol-fed rats than pair-fed controls at
every time point evaluated during the initial 12 h after PH. The
ethanol-related increases in serum IL-6 concentrations are apparent as
early as 3 h after PH. At that time point, no IL-6 can be detected in
control rat sera by ELISA but the mean serum IL-6 concentration in the
ethanol-fed group is 57.5 ± 2.5 pg/ml
(P < 0.027). Taken together, these
findings suggest that extrahepatic tissues may be overproducing IL-6 in the ethanol-fed animals. To evaluate this possibility, cytokine gene
expression was assessed in white adipose tissue (WAT) that was
harvested from the epididymal fat pads of both groups. WAT was selected
for scrutiny because other workers demonstrated that this tissue can
express cytokines under certain circumstances (21).
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As shown in Fig. 6, the expression of
several cytokine mRNA differs dramatically in the WAT of ethanol- and
pair-fed rats. TNF-, IL-6, and IL-10 are not expressed basally in
WAT of pair-fed animals, but WAT expression of each of these cytokines
is significantly increased by 3 h after PH. In contrast,
TNF-
mRNA are relatively abundant in WAT from ethanol-fed rats even
before PH. In addition, in the ethanol-fed group, increases in TNF-
,
IL-6, and IL-10 mRNA begin to occur as early as 30 min after PH and are
amplified and prolonged relative to those noted in the pair-fed
controls. These results suggest that extrahepatic tissues such as WAT
may be important sources of circulating cytokines after PH,
particularly in ethanol-fed rats.
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Although serum concentrations of IL-6 protein are relatively increased in the ethanol-fed rats compared with the controls after PH, hepatic expression of IL-6 appears to be attenuated by ethanol feeding. Thus it remained uncertain whether IL-6-dependent events would be induced normally in the livers of ethanol-fed animals. To explore the implications of ethanol-associated alterations in IL-6 expression, we compared Stat-3 phosphorylation and DNA binding activity in liver nuclear extracts from ethanol- and pair-fed rats. Stat-3 was selected for scrutiny because others showed that IL-6 is responsible for the inductions of Stat-3 phosphorylation and DNA binding activity that follow PH (7, 8, 38). Consistent with this fact and with the previously cited evidence that ethanol ingestion does not reduce serum concentrations of IL-6 proteins, neither the nuclear accumulation of phosphorylated Stat-3 (Fig. 7A) nor the induction of Stat-3 DNA binding activity (Fig. 7B) was inhibited in the ethanol-fed group.
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Stat-3 has been implicated as a prerequisite for hepatocyte progression through the cell cycle after PH (8, 44). Consistent with this theory, induction of cyclin D1, a gene that is important for G1-S transition in hepatocytes (2, 24, 32), is inhibited in IL-6 null mice, in which regenerative induction of Stat-3 is also aborted (8). Given this background information that links Stat-3 induction with cell cycle progression after PH, we expected that cyclin D1 mRNA would accumulate normally in the livers of ethanol-fed rats after PH. Surprisingly, this was not the case. Steady-state mRNA levels of cyclin D1 and several other genes (e.g., cdk-1, cyclin D3, and p53) increase in the livers of pair-fed rats by 24 h after PH, as was reported for ad libitum-fed rodents (2). However, little, if any, induction of these genes can be detected by Northern blot analysis of hepatic RNA isolated from ethanol-fed rats at various time points after PH (Fig. 8). Cyclin mRNA and proteins may be discoordinately regulated after PH (2). However, our preliminary observations suggest that induction of cyclin D1 protein is also inhibited by ethanol ingestion (data not shown). Thus it appears likely that ethanol-associated decreases in cyclin D1 expression contribute to the ability of ethanol to arrest cell cycle progression at the G1/S boundary. These results complement the data shown in Fig. 1 and indicate that regenerative activation of Stat-3 is not sufficient to ensure that hepatocytes will progress from G1 into S phase after PH.
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DISCUSSION |
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There is now compelling evidence that the injury-related cytokines,
TNF- and IL-6, promote liver regeneration and, therefore, are
required for recovery from liver injury (1, 8, 13, 14, 44). Habitual
consumption of ethanol is one of the most common causes of liver
disease in the world (15), and it is conceivable that this occurs
because ethanol can both injure the liver and impair regeneration. The
present study extends existing knowledge about the effects of ethanol
on liver regeneration by demonstrating that ingestion of ethanol in
subhepatotoxic amounts (27-29) permits normal-to-enhanced
induction of proregenerative cytokines, e.g., TNF-
and IL-6, after a
regenerative stimulus (e.g., PH). The results also identify the
importance of extrahepatic sites such as WAT as sources of these
cytokines after liver injury, particularly in individuals who have been
chronically exposed to ethanol.
Although chronic ethanol ingestion does not inhibit injury-related
increases in TNF- or IL-6 expression, it does selectively impede
cytokine-initiated signal transduction during the regenerative response. Induction of hepatic NF-
B, an event that requires
activation of type 1 TNF-
receptors after PH (44), is blocked in
ethanol-fed rats. It is tempting to speculate that ethanol-associated
overexpression of IL-10 may contribute to the decreased induction of
hepatic NF-
B, because IL-10 is known to block TNF-
-dependent
activation of NF-
B in other settings (26, 42). The role of NF-
B
as a regulator of hepatocyte proliferation after PH is unclear because another group showed that supplemental IL-6 can restore hepatocyte DNA
synthesis after PH in TNF receptor type 1 null mice, although these
animals exhibit decreased NF-
B DNA binding activity (44). This
finding prompted those investigators to conclude that NF-
B is not
necessary for hepatocyte DNA synthesis after PH. On the other hand,
there is recent evidence that treatment of a cultured hepatocyte cell
line with purified specific inhibitor
B promotes apoptosis (3).
Furthermore, another group has observed that pretreatment with
inhibitors of NF-
B results in massive apoptosis of hepatocytes in
the G2/M stage of the cell cycle
after PH (D. Brenner, personal communication). Our work documents
relatively decreased liver mass in the ethanol-fed group after PH, but
these tissues were not evaluated for apoptosis. However, the
possibility that ethanol-related inhibition of NF-
B may promote
hepatocyte apoptosis is provocative and merits careful evaluation in
future studies.
It is also important to emphasize that ethanol-associated TNF-
"resistance" appears to exhibit tissue selectivity, because induction of IL-6, a TNF-
- and NF-
B-regulated event, is somewhat inhibited in the liver but markedly enhanced in the WAT of ethanol-fed rats. The present study did not evaluate NF-
B activity in WAT. Thus
it is not clear whether decreased induction of this factor occurs in
the fat of ethanol-fed rats as it does in the livers of these animals.
If induction of adipose NF-
B DNA binding activity was preserved in
fat, however, this could explain the observed discrepancy between
hepatic and adipose tissue IL-6 expression in the ethanol-fed group.
Alternatively, other signals (e.g., mediated by cAMP or prostaglandins)
that regulate IL-6 by both transcriptional and posttranscriptional
mechanisms (47) may help to increase IL-6 expression when NF-
B
activity is inhibited. In any case, upregulation of adipose IL-6
expression is associated with increased circulating levels of IL-6
protein in ethanol-fed animals. The latter may function hormonally to
compensate for the delayed induction of hepatic IL-6. This is suggested
by at least two lines of evidence (i.e., Western blot and gel mobility shift analyses) that indicate that post-PH activation of Stat-3 in the
liver, an IL-6-dependent process (8), proceeds normally in the
ethanol-fed group.
Others (8, 44) suggested that IL-6-mediated activation of Stat-3 is
necessary for the induction of later events, including cyclin D1
expression, that permit hepatocytes to exit
G1 and enter replicative phases of
the cell cycle. However, the present results demonstrate that increases
in serum IL-6 and the resultant activation of hepatic Stat-3 are not
sufficient to ensure induction of many different late
G1 events after PH. These findings
complement evidence that ethanol ingestion inhibits hepatocyte
incorporation of BrdU and mitoses. Taken together, these data suggest
that additional factors are necessary for hepatocytes to escape
G1. Ethanol feeding appears to
inhibit one or more of these other factors. Thus injury-related cytokines such as TNF- and IL-6 act predominantly as
"initiation" factors, working to move quiescent
(G0) hepatocytes into the
prereplicative (G1) phase of the
cell cycle. Although initiation is critically important for eventual
hepatocyte replication, factors other than TNF-
and IL-6 appear to
be required for hepatocytes to "progress" out of
G1 and into S phase, where DNA is
actually replicated.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants R01-AA-09347, R01-AA-10154, and K-020173 to A. M. Diehl.
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FOOTNOTES |
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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: A. M. Diehl, 912 Ross Bldg., Johns Hopkins Univ. School of Medicine, 720 Rutland St., Baltimore, MD 21205.
Received 17 February 1998; accepted in final form 11 May 1998.
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