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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 9, 2001 as doi:10.1096/fj.01-0158fje. |
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,2
* Instituto de Bioquímica, Centro Mixto CSIC-UCM, Facultad de Farmacia, Universidad Complutense, 28040 Madrid;
Instituto de Biomedicina del CSIC, 46010 Valencia,
Instituto de Neurobiología Santiago Ramón y Cajal del CSIC, 28071 Madrid, Spain; and
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA
3Correspondence: Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid, Spain. E-mail: pmartin{at}eucmos.sim.ucm.es
SPECIFIC AIMS
Partial hepatectomy (PH) triggers a rapid regenerative response in liver to reinstate organ function and cell numbers. Among the molecules that change in the course of regeneration is an accumulation of prostaglandin E2 (PGE2) in the serum of animals after PH. The origin of this prostaglandin and its relevance to the events leading to liver mass recovery will be investigated.
PRINCIPAL FINDINGS
Liver regeneration is a complex physiological response to hepatic injury during which the remnant organ initiates a series of reactions in order to reestablish the hepatic-dependent homeostasis and promote cell growth. This process is accomplished through the release of hepatic growth factors, proinflammatory cytokines, and hormones that elicit a priming effect on the hepatocytes that is followed by cell cycle progression.
In the course of regeneration, an increase in serum levels of PGE2 was measured. However, adult hepatocytes fail to express cyclooxygenase 2 (COX-2) upon proinflammatory challenge. Only fetal hepatocytes, which exhibit a phenotype distinct from the adult counterpart, expressed this enzyme. The inducible isoform of COX, COX-2 accounts for the elevated production of prostaglandins in response to various proinflammatory stimuli and other kinds of cellular stress, such as endotoxemia and septic shock. In addition to inflammation, COX-2 expression has been associated with cell growth regulation and carcinogenesis.
Previous data suggest a close relationship between COX-2 induction and
decreased levels of C/EBP-
, which binds to the NF-IL-6 site of the
COX-2 promoter. C/EBPs are known to play important roles in regulating
the expression of multiple hepatocyte specific genes and to control
hepatocyte progression through the cell cycle. C/EBP- is expressed
abundantly in adult hepatocytes and induces growth arrest and
differentiation. C/EBP-ß and -
are implicated primarily in the
regulation of genes involved in inflammation and cell proliferation and
are up-regulated during the acute-phase response. Consistent with this
view, variations in the expression of C/EBP mRNAs, proteins, and DNA
binding activities have been documented during liver development,
acute-phase response, and regeneration. The expression and activities
of C/EBP isoforms fluctuate in regenerating liver and are likely to be
important targets of regulation during this process. C/EBP- is the
predominant isoform expressed by adult hepatocytes in healthy livers.
During the initial 24 h after 70% PH, the levels of C/EBP-
mRNA and protein decline, whereas levels of C/EBP-ß and - increase
in the early prereplicative period after PH and return to basal levels
in the S phase. Taking this into account, we investigated the
inducibility and potential role of COX-2 after PH. Our results show
that hepatocytes from regenerating liver express COX-2, a process that
is reinforced after treatment of these cells in culture with
proinflammatory stimuli. Using pharmacological inhibitors of COX-2 and
mice with a disrupted COX-2 gene, we observed that PGs produced by
COX-2 are important for the early steps of liver regeneration with
respect to DNA synthesis after PH.
Serum levels of PGE2 increase after PH
The levels of PGE2 were measured in the serum of control, sham,
and PH animals. As shown in Fig. 1
, a 12-fold increase in PGE2 level was observed at
16 h. At the same time, the concentration of nitrate in the serum
increased 1.8-fold. The enzyme responsible of the synthesis of
PGE2 was COX-2, as deduced from pharmacological
criteria using the selective inhibitor NS398. As shown in Fig. 1
, COX-2
protein was clearly detected at 5 h after PH and persisted for at
least 96 h. However, NOS-2 levels were transiently enhanced after
PH, with peak levels at 16 h. Immunolocalization of COX-2 in the
remnant liver revealed its presence in hepatocytes located near the
pericentral area. The staining involved particulate structures in the
cytosol, presumably the endoplasmic reticulum, and in the perinuclear
membrane.
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COX-2 activity influences early hepatocyte growth
The potential role of PGs synthesized by COX-2 in the course of
regeneration was evaluated in animals treated with NS398 and in COX-2
KO mice. Under these conditions, the rise of proliferating cell nuclear
antigen (PCNA) after PH was significantly inhibited (76% at 48 h); the same occurred with cyclins D1 and E, which showed 80% and 75%
inhibition, respectively. An accumulation of
p27kip1 was observed after PH but in the absence
of PGE2 synthesis. Moreover, COX-2 KO mice
exhibited an impaired incorporation of
[3H]thymidine into hepatocyte nuclei after PH
(Fig. 2
). However, analysis of liver mass recovery 1 wk after PH in animals
treated with NS398 or in COX-2 KO mice did not show a significant
blockage of the regenerative process.
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CONCLUSIONS AND SIGNIFICANCE
Liver regeneration after the loss of hepatic tissue is defined as
a coordinate response to specific stimuli involving sequential changes
in gene expression, growth factor production, and extracellular matrix
remodeling. Many growth factors cytokines (HGF, EGF, TGF-ß, TNF-,
and IL-6, among others) and transcription factors (c-Myc, c-Fos, c-Jun,
p53, NF-
B) have been identified as important regulators of this
process. Liver regeneration after PH is accomplished by proliferation
of all existing mature cell populations resident in the remaining
organ. These include hepatocytes, biliary epithelial cells, fenestrated
endothelial cells, Kupffer, and Ito cells. Hepatocytes are the first to
proliferate after PH; the other cells of the liver enter into DNA
synthesis later after PH, suggesting that hepatocytes provide the
mitogenic stimuli required for proliferation.
One of the genes expressed in the remnant liver after PH and required for regeneration is NOS-2, an enzyme implicated in inflammation and whose regulation is similar to COX-2. Moreover, COX-2 can be regarded as an immediate early gene producing PGs within the nucleus and influencing nuclear functions, such as replication and differentiation.
Previous results demonstrated that the presence of high levels of
C/EBP- impairs COX-2 expression in adult hepatocytes challenged with
proinflammatory stimuli. The levels of C/EBP- decrease during the
initial 24 h after PH, a period coincident with the induction of
COX-2 and an increase in the ß and isoforms. These results
suggest that COX-2 expression occurs after a loss of differentiation in
the liver and that down-regulation of C/EBP- levels after PH plays
an important role for the expression of this enzyme. Indeed, C/EBP-ß
knockout (KO) mice exhibit impaired hepatocyte proliferation and
decreased liver regeneration after partial PH. C/EBP- is known to
stabilize p21/WAF, a protein that inhibits transition from the
prereplicative (G1) period into S phase. There is
also some evidence that C/EBP-ß may regulate positively the
expression of genes that promote cell cycle progression like cyclin
D2, cyclin A, and retinoblastoma protein.
Studies of the physiological significance of COX-2 expression after PH
showed alterations in various parameters of cell cycle progression in
animals pretreated with NS398 or in COX-2 KO: PCNA levels, a marker of
S phase, increased at 24 h after PH but remained low in animals
lacking PG synthesis, suggesting that these PGs were required for
hepatocytes to enter into the S phase of the cell cycle (Fig. 3
). The expression of cyclin D1 and E showed a
reduced response under these conditions. Therefore, in the
pharmacological model of COX-2 inhibition and the genetic model of
COX-2 targeting, the lack of PG synthesis results in a delay in the
first cycle of hepatocyte proliferation. However, measurement of
overall liver mass recovery 1 wk after PH failed to show significant
differences between control and COX-2 deficient animals, which suggests
that other growth-promoting mechanisms play a more significant role in
the latter phase of the regenerating process. In conclusion, this study
suggests that COX-2 is expressed in regenerating liver after PH, in
agreement with the observation that COX-2 is a growth-associated early
gene. Analysis of cell cycle-associated proteins revealed that PGs
produced by COX-2 are important in the early steps of liver
regeneration after PH (24–48 h, depending on the species). These
results agree with previous data showing an overexpression of COX-2 in
several liver diseases, including hepatocellular carcinoma, and with
the potential usefulness of COX inhibitors in cancer
prevention.
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FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.01-0158fje ; to cite this
article, use FASEB J. (July 9, 2001)
10.1096/fj.01-0158fje 
2 M.C. and N.A.C. contributed equally to this
work.
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