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加拿大留学生医学论文-医学分子实验学论文范文-Gene expression during the priming ph

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核心提示:加拿大留学生医学论文-医学分子实验学论文范文-Gene expression during the priming phase of liver regeneration after partial hepatectomy in mice

加拿大留学生医学论文-医学分子实验学论文范文,Gene expression during the priming phase of liver regeneration after partial hepatectomy in mice
Andrew I. Su*, Luca G. Guidotti†, John Paul Pezacki*‡, Francis V. Chisari†§, and Peter G. Schultz*§¶
*Department of Chemistry and †Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037;
¶Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121; and ‡The Steacie Institute for Molecular
Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6
代写加拿大论文Contributed by Peter G. Schultz, June 14, 2002
Understanding the gene-expression patterns during liver regenerationmay help to reveal how regenerative processes are initiated andcontrolled as well as shed new light onto processes that lead to liverdisease. Using high-density oligonucleotide arrays, we have examinedthe gene-expression program in the livers of mice after partialhepatectomy. A time course was constructed for gene expressionbetween 0 and 4 h after partial hepatectomy, corresponding to thepriming phase of liver regeneration. The genomic program for liverregeneration involves transcription-factor generation, stress and inflammatoryresponses, cytoskeletal and extracellular matrix modification,and regulation of cell-cycle entry. The genome-wide changesthat are observed provide a detailed and comprehensive map of theinitial priming stage of liver regeneration.
The liver’s ability to regenerate in mammals is relatively unique.Only a few species, including certain worms, insects, reptiles,
and amphibians, can readily undergo various types of reparativeregeneration including epimorphic reconstruction of entire bodyparts. In contrast, humans and larger mammals have little regenerativecapacity (1, 2). Examples of organs and body parts that showreasonably good regenerative capacities are few and include theliver, fingertips, and peripheral nerves, and stem cells may be http://www.liuxuelw.com/jianadalunwen/asource of the regenerative capacity (2). Among these types ofreparative processes, liver regeneration stands out. The liver iscapable of modulating its mass according to functional requirements,
proliferating under conditions of functional deficiency, andundergoing apoptosis under functional excess. In both of theseprocesses, the liver undergoes remodeling of the entire organphysiology to preserve normal histological organization (3, 4). Liverregeneration does not rely on stem cells, although liver stem cellsmay contribute to the process, and each cell type has the capacityto enter into a proliferative state and also alter their differentiationso that liver cells have innate progenitor cell characteristics (5, 6).
Hepatocytes are the first cell types to enter into the cell cycle afterfunctional deficiency in the liver (4), and they show an almostlimitless capacity to proliferate (7). Also, during liver regeneration
the liver cells continue to perform crucial metabolic functions suchas glucose regulation, synthesis of many blood proteins, the secretionof bile, and biodegradation of toxic compounds required for
homeostasis (3). Understanding the molecular mechanisms andgenomic program of liver regeneration is of fundamental importanceand is the first step toward controlling these events and other
regenerative processes.Liver regeneration can be initiated in several ways. Classical
methods for initiating liver regeneration in animal models involveeither partial hepatectomy (PHx) or injection of hepatotoxic compoundssuch as CCl4. Pioneering studies by Taub and coworkers (8)as well as Fausto (3) have defined the roles of many immediate-early
genes and cytokines in liver regeneration. On a molecular level, theentry of hepatocytes into cell cycle is stimulated by various cytokinesand growth factors. Examples include IL-6, hepatocytegrowth factor (HGF), epidermal growth factor, tumor necrosisfactor (TNF)-, transforming growth factor-, insulin, and otherreceptor ligands that have been implicated in various stages of
hepatocyte proliferation (3, 4). These ligands, through complexmolecular mechanisms, activate transcription factors including NF-
B, signal transducer and activator of transcription 3 (STAT3),activator protein 1 (AP-1), and CCAATenhancer-binding protein
(CEBP) that initiate a cascade of gene expression that ultimatelyis responsible for proliferation (9).
Before cell-cycle entry, quiescent hepatocytes (G0) undergo apriming phase (G0 3 G1) during which the cells reenter the cell
cycle and prepare for proliferation. The concept of priming phasein hepatocytes, originally proposed by Fausto et al. (3, 10–12), is thefirst stage of liver regeneration, the duration of which is speciesdependent
(13). For mice this stage lasts for4 h after PHx. During
this time, immediate-early genes such as c-fos and c-jun are induced
(3, 8, 14, 15). In fact, many genes have been identified as beingdifferentially expressed during hepatocyte priming and the followingstages of the cell cycle leading up to DNA replication and
mitosis. Known exogenous priming stimuli include sham surgery,protein deprivation, and collagenase treatment as well as infusion
of TNF, epidermal growth factor, or HGF (3, 4).
Given that the cells in a regenerating liver have progenitor cellcharacter, we used functional genomic technologies to study cellularpriming in mice. We have characterized the genome-wide expressionchanges in mice after70% PHx over the course of 4 h, whichcorresponds to the priming stage of hepatocyte proliferation. Shamsurgeries were conducted to eliminate responses that are caused bythe surgery alone, and resected liver specimens functioned as abaseline for gene-expression changes in each mouse. We found thatgenes associated with transcription-factor production, stress andinflammatory responses, cytoskeletal and extracellular matrix modification,and regulation of cell-cycle entry all are involved in theearly stages of liver regeneration. It is noteworthy that the liver iscomposed of many different types of cells, and most of its functionis confined to hepatocytes (which represent 70% of the liver),
Kupffer cells (macrophages), and bile ductule epithelium. Thus, itis possible that some of the changes reported may have occurred innonparenchymal cells of the liver rather than hepatocytes.
Materials and Methods
Mice, Tissues, and RNA Preparation. Groups of three 8–10-week-oldmale C57BL6 mice were anesthetized and subjected to eithersham operation or 70% PHx as described (16). At 1, 5, 10, 30, 90,and 240 min posthepatectomy, mice were killed, and liver tissuesamples were harvested. Total RNA was isolated from thesesamples and the respective surgically resected liver sections asdescribed (16) and analyzed by high-density oligonucleotide microarray,Northern blot, and RNase protection assay (RPA).
High-Density Oligonucleotide Microarray Analysis. Double-stranded
DNA was synthesized from 5 g of total RNA by using theSuperScript Choice system (Life Technologies, Grand Island, NY)and a primer containing poly(dT) and a T7 RNA polymerase
Abbreviations: PHx, partial hepatectomy; HGF, hepatocyte growth factor; TNF, tumornecrosis factor; RPA, RNase protection assay; MAPK, mitogen-activated protein kinase;
CEBP, CCAATenhancer-binding protein; PPAR, proliferator-activated receptor.
§To whom reprint requests may be addressed. E-mail: Schultz@Scripps.edu or Fchisari@
Scripps.edu.www.pnas.orgcgidoi10.1073pnas.122359899 PNAS  August 20, 2002  vol. 99  no. 17  11181–11186
CELL BIOLOGY
Table 1. GeneChip analysis of changes in liver gene expression after partial hepatectomy
Fold change vs. resected liver specimens () for differentially expressed genes as well as the fold change vs. sham surgery (ES) are listed in rows for time points
10, 30, 90, and 240 min. Genes are listed by GenBank accession number (Acc.#) and name and are grouped according to their function. The fold change for gene
expression with values greater than 5 are highlighted; yellow corresponds to values 5; green corresponds to values 10; red corresponds to values 50. Genes
that previously had been identified in liver regeneration, particularly in the priming stage prior to entry into cell cycle, are indicated with a [Y].
11182  www.pnas.orgcgidoi10.1073pnas.122359899 Su et al.
promoter sequence (Genset, La Jolla, CA). In vitro transcription
using double-stranded cDNA as a template in the presence of
biotinylated UTP and CTP was carried out by using an Ambion
(Austin, TX) in vitro transcription kit. Biotin-labeled cRNA was
purified, fragmented, and hybridized to Mu6.5K arrays (Affymetrix,
Santa Clara, CA). The arrays were washed and stained with
streptavidin-phycoerythrin and then scanned with an Affymetrix
GeneChip scanner. Primary image analysis was performed by using
the GENECHIP 3.1 software, and images were scaled to an average
difference value of 200 as described previously. Hybridizations were
performed in duplicate, and only differential expression observed
in both replicates was analyzed further. Comparison analyses for
data at each time point were calculated by using the t0 and sham as
baselines. Gene-expression profiles were established from RNA
samples isolated from four different mice per time point (two PHx
and two sham).
Reverse Transcription–PCR, Northern Blots, and RPA. Probes for
Northern blotting were amplified from RNA in the 240-min PHx
time point by using the following primer pairs: Pip92Fwd (5-
GAGTCTGCAGCTATCCCTCG-3), Pip92Rev (5-CACGTTGAGCATATTGTCGG-
3); Tis21Fwd (5-CCTAGCCAAGGTAAAAGGGG-
3), Tis21Rev (5-GGTCCTCTCCATCTTAGCC-
3); and glvr-1Fwd (5-GGTGGGATGTGCAGTTTTCT-
3), glvr-1Rev (5-CCTTGTGCACGGTGTGATAC-3). RNA
samples were analyzed by electrophoresis and transferred to nitrocellulose
membranes. The 32P-labeled cDNA probes along with a
32P-labeled probe for the housekeeping gene glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) then were used to probe
message levels by using established protocols (16). Quantitation of
chemokines MCP-1 and GRO as well as L32 was performed also
by RPA exactly as described previously (17, 18).
Results and Discussion
We have examined the gene-expression changes in the liver of mice
after PHx over a time course spanning 4 h by using high-density
oligonucleotide arrays with probes for 6,500 genes. Liver RNA
samples were removed at 1, 5, 10, 30, 90, and 240 min after PHx.
No reproducible effects were observed at 1 and 5 min after PHx
(data not shown). The experiments showed that 185 genes had
altered gene expression for at least one of the remaining time points
(Table 1). All of these genes showed higher induction or repression
in regenerating livers than in sham controls, indicating the importance
of including these controls in our analysis. We have excluded
genes that were induced or repressed at similar levels in sham and
regenerating livers, because these gene-expression changes are
likely indicative of the effects of the surgery. It is important also to
note that a wide variety of the genes observed to be differentially
regulated according to microarray analysis have not been described
previously in more limited studies of hepatocyte priming during
liver regeneration after PHx.
Transcription Factors. During liver regeneration, liver cells are
exposed to stresses associated with functional deficiency, and these
stresses ultimately lead to cell proliferation (9, 19, 20). We have
identified 19 immediate-early transcription-factor genes that are
differentially regulated during the priming phase (Table 1), many of
which overlap with previously established immediate-early genes
implicated by Taub and coworkers (8, 15) and Fausto and coworkers
(3, 10–12) that have established the importance of inflammation
and protooncogenes in the early stages of liver regeneration. It is
Table 1. (continued)
Acc.#
Gene name
(synonyms)
Known
in LR
[Y]
10 min 30 min 90 min 240 min
Acc.#
Gene name
(synonyms)
Known
in LR
[Y]
10 min 30 min 90 min 240 min
 ES  ES  ES  ES  ES  ES  ES  ES
Su et al. PNAS  August 20, 2002  vol. 99  no. 17  11183
CELL BIOLOGY
well established that liver regeneration involves the posttranslational
activation of the transcription factors NF-B, STAT3, AP-1,
and CEBP by mechanisms connected to increased levels of
cytokines and reactive oxygen species within the liver (9, 15, 19).
Immediate-early genes such as the archetypal immediate-early
genes c-fos, c-jun, and c-myc are up-regulated as a consequence (9).
Immediate-early transcription factors generated in response to PHx
represent a critical step in controlling the proliferation of hepatocytes
within a regenerating liver. Cooperatively, they activate the
proliferative program within quiescent hepatocytes.
Four immediate-early transcription factors were activated as
early as 10 min after PHx: c-fos, c-jun, Zif268, and pip92. Of these,
c-fos and c-jun are primary immediate-early genes expressed by
many cell types in response to cellular stress. Zif268 (EGR-1)
contains three zinc fingers recognizing a GC-rich sequence, which
has been identified in the promoter regions of a number of genes
including PRL-1, a mitogenic phosphatase associated with regenerating
livers that plays an important role in cell growth in a number
of different tissue types (21). The other immediate-early transcription
factor in the 10-min time point is PIP92, the induction of which
has been confirmed by Northern blot (Fig. 1). PIP92 encodes a
short-lived proline-rich protein with no sequence homology to
other known proteins. PIP92 is known to be stimulated by cytokines
such as fibroblast growth factor and by mitogen-activated protein
kinase (MAPK) signaling pathways and has been implicated in
processes such as differentiation and stimulation of fibroblasts (22).
We observed high induction of ATF3, also known as liverregenerating
factor (LRF-1) because it is highly expressed in
regenerating livers (23). Expression of ATF3 is known to modulate
glucose homeostasis and other primary functions of the liver and
likely plays a role in altering cell function before cell-cycle entry
(24). Ets-2 is another immediate-early transcription factor that was
highly induced after PHx. Ets-2, in conjunction with CEBP, and
CEBP, rapidly increases transcription from the p21 promoter via
multiple binding elements within the enhancer region (25). Expression
of Ets-2 has direct downstream effects on both cell-cycle
progression and MAPK signaling.
We also observed that the gene encoding the peroxisome proliferator-
activated receptor  (PPAR) was down-regulated during
liver regeneration. PPAR genes mediate the proliferation of peroxisomes,
which are organelles that participate in many primary
functions of the liver including lipid metabolism, catabolism of
purines, polyamines, and amino acids, H2O2-based respiration,
cholesterol synthesis, and the production of bile acids (26). Downregulation
of PPAR linked to an immediate-early response to
functional deficiency may be a natural response to the increase in
H2O2 concentration in the remaining hepatocytes after PHx. Retinoic
acid, a natural ligand of the retinoid X receptor (RXR), has
been implicated as an important cytokine in regenerating livers
(27). Consistent with a functional role of retinoic acid in liver
regeneration, we observe the induction of Stra14 (stimulated by
retinoic acid 14), the role of which in liver regeneration is unknown.
We also detected transient expression of other transcription
factors such as hepatocyte nuclear factor 3(HNF-3) and -, which
are known to activate liver-specific genes such as albumin, and
influence expression of genes involved in bile acid and glucose
homeostasis (28, 29). HNF-3 isoforms mediate the hepatocytespecific
transcription of numerous genes important for liver function,
and homozygous knockout mice do not survive embryonic
development (28, 29). Gene expression of HNF-3 isoforms are
reduced in the liver after injury by CCl4 (30), a model system for
studying liver regeneration, suggesting an important role for these
transcription factors in response to damage and to controlling the
differentiation state and proliferation of hepatocytes.
By 240 min, we observed transcription factors associated with the
beginnings of tissue remodeling. One example is the hypoxiainduced
factor (HIF1), which is an angiogenic gene expressed
during oxygen starvation (hypoxia) that promotes vascular growth
(31). At this time point, three IFN-inducible transcription factors
also were induced: IFN-stimulated gene factor-3 (ISGF-3) and IFN
regulatory factors-1 and -2 (IRF-1 and IRF-2). ISGF-3 and IRF-1
have been identified as transcriptional activators of IFN- signaling,
whereas IRF-2 is thought to act as a repressor of such activity (32).
Given that IFN- was induced by 90 min after PHx and this
cytokine has been involved with cell-growth suppression (33, 34),
these results suggest that ISGF-3, IRF-1, and IRF-2 may differentially
modulate regeneration. Although many of the immediateearly
transcription factors induced after PHx have proliferative
functions, several genes involved in regulation and arrest of the cell
cycle were also induced. This apparent dichotomy must be linked
with the precise control of cell-cycle entry and progression.
Cell-Cycle Genes. During hepatocyte priming in mice, we observed
genes related to the cell cycle up-regulated as early as 10 min after
PHx (Table 1). Consistent with the expression of both pro- and
antiproliferative transcription factors, we observe the differential
regulation of genes that stimulate and inhibit cell-cycle entry.
Overall, we detected the differential regulation of 19 cell-cycle
control genes during the time course of hepatocyte priming, and
many are detected far earlier than reported previously. The majority
of the genes are checkpoint genes at major cell-cycle transitions
that can act to inhibit the cell cycle.
Three cell-cycle checkpoint genes induced during liver regeneration
were GADD45, TIS21, and p21, which act at different
cell-cycle transitions (Fig. 2). The levels of TIS21 mRNA, a
p53-dependent growth arrest gene that inhibits the G1S transition
(35), increased by 10 min and reached maximum levels by 90–240
min after PHx (Table 1) as also confirmed by Northern blot (Fig.
1). Immediate-early transcription factors regulate the expression of
the cell-cycle checkpoints (25), thus implying that autonomous
control of cell-cycle entry by the hepatocytes begins almost immediately.
In addition, we observe the up-regulation of cell-cycle genes
that are apoptosis inhibitors such as Bcl-2, Bcl-X, and GADD45,
previously implicated in hepatocyte priming (3).
Signal Transduction. We observed differential regulation of several
genes related to signal transduction. For example, we observe the
Fig. 1. Induction of pip92, groKC, tis21, glvr-1, MCP-1, and C10 during the
priming phase of liver regeneration. Age-matched C57BL6 male mice (three
mice per group) were subjected to 70% PHx and killed at different points
afterward. Total RNA (20 or 10 mg) extracted from their livers were analyzed by
Northern blot (NB) and RPA analyses, respectively, for the expression of pip92,
groKC, tis21, glvr-1, MCP-1, and C10. Results from a representative mouse per
group were compared with those obtained either in itsownresected liver section
(t0) or in mice that were subjected to sham operation and killed at the same time
point. The housekeeping genes glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and ribosomal protein light 32 (L32) were used to normalize either the
amount of RNA bound to the membrane (NB) or the amount of RNA loaded in
each lane (RPA).
11184  www.pnas.orgcgidoi10.1073pnas.122359899 Su et al.
differential regulation of genes related to the activation of MAPK
and related pathways. MAPK signaling is mediated by increased
expression of the cyclin-dependent kinase inhibitor protein p21,
which is induced by Ets-2 andCEBP(25), all of which are observed
in our gene-expression profile.MAPKactivation has been linked to
interleukin-6 (IL-6) pathways (8) and is an essential component of
the genetic program that leads quiescent hepatocytes into a proliferative
state. Our results with regards toMAPK-related pathways
are consistent with the findings of Taub and coworkers, which are
discussed in detail elsewhere (8).
It is noteworthy also that follistatin and inhibin, inhibitors of
activin A, have proliferative properties and were up-regulated
during hepatocyte priming. Activin A is a member of the transforming
growth factor- superfamily and is known to be involved
in kidney organogenesis and development (36). ActivinAcan block
the activity of the key inflammatory cytokine IL-6, which is known
to be critical in liver regeneration (37). Up-regulation of inhibitors
of activin A therefore are likely to play a role in hepatocyte priming
by enhancing the effects of IL-6, which leads to proliferation.
Extracellular MatrixCell Structure- and Membrane-Associated Genes.
It is well established that modification of the extracellular matrix is
integrally linked with liver regeneration (3). We have identified 28
differentially regulated genes that are associated with cell structure
and extracellular matrix modification. Cyr61, an angiogenic extracellular
matrix modifier (38, 39), appeared as early as 10 min after
PHx and was overexpressed consistently during hepatocyte priming.
Fisp12 (CTGF), another angiogenic extracellular matrix modifier
that usually is observed in conjunction with Cyr61 (40),
appeared only at the 90-min time point onward. The role of these
two genes in liver regeneration has not been established previously,
although they seem to be critical to the formation of new blood
vessels and critical to proliferation in certain cancers (41). Both
Cyr61 and Fisp12 are involved in wound healing and can stimulate
chemotaxis and promote proliferation in endothelial cells and
fibroblasts in culture and induce neovascularization in vivo (42).
They also promote adhesive signaling responses that lead to sustained
activation of p42p44 MAPKs and prolonged geneexpression
changes including up-regulation of MMP-1 (collagenase-
l) and MMP-3 (stromelysin-l) mRNAs (42). The remainder of
the genes began to be differentially expressed 90 min after PHx,
suggesting that there is a lag between the initial response to PHx and
extracellular modification. This was confirmed also by the fact that
little or no induction of matrix metalloproteinases (MMP, proteinases
known to play a role in tissue remodeling; ref. 43) was detected
by microarray (Table 1) up to the 240-min time point.
It also is worth mentioning that plasminogen activator inhibitor
1 (PAI-1), a serine protease inhibitor that specifically inhibits
plasmin activation (44) and liver regeneration, was very highly
expressed from90 min after PHx onward.PAI-1 specifically inhibits
liver regeneration (3, 4) by forming a complex with the urokinasetype
plasminogen activator (uPA) and contributes to the inactivation
of HGF. HGF is a known stimulus of liver regeneration after
priming (3, 4).
We also observed induction of RhoB, which regulates signal
transduction from plasma membrane receptors (45, 46). RhoB is
known also to regulate DNA synthesis and is expressed as a result
of genotoxic stress (47). A number of other genes associated with
cell adhesion and migration appear later in the priming phase
(Table 1). Related to genes that are involved with cell adhesion and
migration are those that are involved with cell–cell communication.
Connexins comprise a class of cell membrane proteins that allow
passive transport of small molecules between networked cells in
tissues (48). Connexin 26, which shows antiproliferative behavior
when overexpressed in human hepatoma cells (49), appeared
up-regulated during liver regeneration. We also observed clcn3
down-regulated during the priming phase of hepatocyte proliferation.
This gene is a voltage-gated chloride channel for regulation of
cell volume (50). Expression of both pro- and antiproliferative
extracellular matrix-modifying genes, exemplified by Cyr61 and
Fisp12 compared with plasminogen activator inhibitor 1 and RhoB,
is consistent with observations of expression patterns of transcription
factors and cell-cycle genes and again suggestive of tight control
of cell-cycle entry during hepatocyte priming.
Inflammatory Responses. The pioneering work by Taub and coworkers
regarding the role of IL-6 in liver regeneration in mice establishes
the importance of inflammatory cytokines during hepatocyte
priming (8). Inflammatory responses have been implicated in the
priming of liver and other types of regeneration (51). For example,
prevention of macrophage invasion impairs peripheral axonal regeneration,
whereas implantation of macrophages into central
nervous system nerves allows them to regenerate after axonal crush
(51). Inflammation also is implicated in secondary degeneration
after spinal cord injury (51). Inflammatory responses can be
triggered by cytokines such as those of the TNF family, which
activate immediate-early genes such as AP-1 (52). With the exception
of IFN- that, as mentioned before, was detected 90 min after
PHx, other cytokines such as IL-6, IL-2, IL-3, IL-4, IL-5, TNF-,
TNF-, and IFN- were not found in our samples, indicating that
if induction of these genes occurred, it was below our detection
limit. However, the messages for the chemoattractants MCP-1 and
GROwere increased in the regenerating liver by 30 and 90 min after
PHx, respectively. These chemokines can recruit monocytes
macrophages, which have the potential to exert both stimulatory
and inhibitory influences on hepatocyte proliferation (53), in the
liver after PHx (54). MCP-1 and GRO also may play a role in
angiogenesis and tissue remodeling (55).
Other inflammatory genes included CD14, the lipopolysaccharide
receptor, being highly induced after PHx, consistent with
previous observations by Taub and coworkers (8), and the gibbon
ape leukemia virus cell-surface receptor (glvr-1) that is involved in
sodiumphosphate cotransport and induced during inflammatory
responses (56). Up-regulation of the latter gene indicates an
increase in the intracellular import of inorganic phosphate, which
is required for activation of signaling pathways involving phosphorylation
and nucleotide triphosphate synthesis. Northern blot analysis
confirmed the increase in mRNA for the glvr-1 gene in the
regenerating liver (Fig. 1).
Glucose- and Metabolism-Related Genes. The liver plays an important
role in maintaining metabolic and biosynthetic homeostasis.
Glucose homeostasis is an important liver function and involves
glycogenolysis, which breaks down glycogen into glucose, and
gluconeogenesis, which involves synthesis of glucose from noncarbohydrate
precursors (57). In a regenerating liver the majority of
homeostatic responses involving metabolic functions occur after
the priming phase of hepatocytes (15). In addition to previously
mentioned genes ATF3 and PPAR, we observed the up-regulation
of phosphoenol-pyruvate carboxykinase (PEPCK) and glucose-6-
Fig. 2. The influence of observed genes on cell cycle.
Su et al. PNAS  August 20, 2002  vol. 99  no. 17  11185
CELL BIOLOGY
phosphatase (G6Pase), which are known to be involved in maintaining
glucose levels after the acute loss of liver mass posthepatectomy
(15, 57). G6Pase is involved in glycogenolysis and
associated with the endoplasmic reticulum (ER), where it hydrolyses
the G6P into glucose and phosphate. Misregulation of this gene
has been implicated in type 1 glycogen storage diseases (58).
PEPCK is a gene involved in the synthesis of glucose from noncarbohydrate
precursors. In addition, we observed up-regulation of
the insulin-like growth factor-binding protein (IGFBP-1), which is
induced by IL-6 and HGF, and is known to be up-regulated during
the course of liver regeneration (59). IGFBP-1 shares common
promoter elements with other hepatic genes associated with the
maintenance of metabolic homeostasis following large functional
deficiency after PHx such as G6Pase (20).
Heme oxygenase 1 (HMOX1) is a protein that catalyzes the
oxygen-dependent degradation of heme to biliverdin, free iron, and
carbon monoxide. Of the two isoforms, the inducible HMOX1
primarily functions in the liver and the spleen. The increased
expression of HMOX1 during liver regeneration is likely a reflection
of the increased metabolic workload of the remaining hepatocytes
after PHx and also may mediate oxidative stress by modulating
iron levels and indirectly participate in antiproliferative
proapoptotic pathways that control cell-cycle entry (60).
Uncharacterized Genes. We have identified a number of genes
corresponding to expressed sequence tags that were differentially
expressed after PHx in mice. The molecular function can be
inferred for these genes based on sequence homology. For example,
the genes AA107455 and AA105081 appear to have high homology
to elongation factor-binding proteins. The gene AA145127 appears
homologous to a serine protease inhibitor, and W08822 is homologous
to a putative GTPase-activating protein for Arf. However,
sequence homology does not reveal the cellular or physiological
role of these proteins, and their specific roles in liver regeneration
are unclear.
Conclusions. We have shown that the genome-wide expression
profile of hepatocyte priming after PHx in mice is complex and
covers different classes of proteins including transcription factors,
metabolic enzymes, proteins associated with stress and inflammatory
responses, and those involved in cytoskeletal and extracellular
matrix modification. We have adapted a diagram originally produced
by Fausto (3) to summarize these results (Fig. 3, which
is published as supporting information on the PNAS web site, www.
pnas.org). These genes are likely to have a broad effect on the liver.
Along with the concept of hepatocyte priming (3, 10–12), it is worth
mentioning that all these changes occurred well before DNA
synthesis, suggesting that the transcriptional control of liver regeneration
involves early and diverse cellular responses. It also was
somewhat surprising to find that the expression levels of genes
associated with the cell cycle over the time course of 4 h indicate
that antiproliferative genes are favored during hepatocyte priming.
These potential antiproliferative responses are in keeping with the
concept of an autonomous control of cell-cycle entry by the
hepatocytes and suggest that tight regulation of liver cell proliferation
originates very early after a regenerative stimulus. Future
experiments to determine which of the changes during hepatocyte
priming are primary-cause factors in tissue regeneration may
provide new insights into regenerative processes in mammals and
potentially may lead to new approaches to the development of
therapeutic agents for the treatment of liver diseases.
We thank Heike Mendez, Rick Koch, and Margie Chadwell for excellent
technical assistance. We also thank Ian Campbell, Valerie Asensio, and
Monte Hobbs for providing the cytokine, chemokine, and metalloproteinase
gene probes used in the RPA experiments. This work was
supported by National Institutes of Health Grants AI40696 (to L.G.G.) and
CA40489 (to F.V.C.) and funds from Novartis. This is manuscript number
15024-CH from The Scripps Research Institute.
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