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Molecular and Cellular Biology, May 2000, p. 3742-3751, Vol. 20, No. 10
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of Growth-Differentiation Factor
15, a Transforming Growth Factor 
Superfamily Member Induced
following Liver Injury
Edward C.
Hsiao,1
Leonidas G.
Koniaris,1,2
Teresa
Zimmers-Koniaris,1
Suzanne M.
Sebald,1
Thanh V.
Huynh,1 and
Se-Jin
Lee1,*
Department of Molecular Biology and
Genetics1 and Department of
Surgery,2 The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
Received 2 February 2000/Accepted 18 February 2000
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ABSTRACT |
We have identified a new murine transforming growth factor 
superfamily member, growth-differentiation factor 15 (Gdf15), that is expressed at highest levels in adult
liver. As determined by Northern analysis, the expression of
Gdf15 in liver was rapidly and dramatically up-regulated
following various surgical and chemical treatments that cause acute
liver injury and regeneration. In situ hybridization analysis revealed
distinct patterns of Gdf15 mRNA localization that appeared
to reflect the known patterns of hepatocyte injury in each experimental
treatment. In addition, treatment of two hepatocyte-like cell lines
with either carbon tetrachloride or heat shock induced
Gdf15 mRNA expression, indicating that direct cellular
injury can induce Gdf15 expression in the absence of other
cell types, such as inflammatory cells. In order to investigate the
potential functions of Gdf15, we created Gdf15 null mice by
gene targeting. Homozygous null mice were viable and fertile. Despite
the dramatic regulation of Gdf15 expression observed in the
partial-hepatectomy and carbon tetrachloride injury models, we found no
differences in the injury responses between homozygous null mutants and
wild-type mice. Our findings suggest either that Gdf15 does
not have a regulatory role in liver injury and regeneration or that
Gdf15 function within the liver is redundant with that of other
signaling molecules.
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INTRODUCTION |
The transforming growth factor (TGF-) superfamily consists of a diverse group of structurally
related proteins involved in the growth, differentiation, and repair of
many tissues (reviewed by McPherron and Lee [26]). For
mammals, more than 30 different members of this superfamily have been
reported, including the TGF-s, bone morphogenetic proteins, inhibins
and activins, Müllerian inhibiting substance, nodal, leftys,
TGF--related neurotrophic factors (GDNF, neurturin, persephin, and
artemin), and a heterogeneous group of proteins referred to as
growth-differentiation factors.
Each member of the TGF- superfamily is synthesized as a large
precursor protein that undergoes two proteolytic processing steps. The
first involves removal of the N-terminal hydrophobic signal sequence.
The second cleavage event occurs at a conserved RXXR site approximately
120 amino acids from the C terminus, generating an N-terminal proregion
and a biologically active C-terminal region. The C-terminal regions of
all superfamily members are structurally related, and the various
TGF- superfamily members can be classified into distinct subgroups
based on sequence homology in this region. C-terminal amino acid
identities within a particular subgroup generally range from 70 to
90%, although homologies between subgroups are considerably lower. For
most of the family members, the active species appears to be a
disulfide-linked homodimer of C-terminal fragments. Heterodimers of
some family members, such as the TGF-s and inhibins and activins,
have also been shown to be biologically active, although in some cases
with biological properties distinct from those of either homodimeric form.
We have been using degenerate PCR and low-stringency screening methods
to identify new TGF- family members that may play important
regulatory roles during embryonic development or adult tissue
homeostasis. Using a human sequence previously reported as human
TGF-PL (hTGF-PL) (42), hMIC-1 (2), hPDF
(32), hPLAB (16), and hPTGFB (21) (for
simplicity, we refer to this gene as hTGF-PL) as a probe, we
identified a novel sequence which we designated Gdf15. While
our studies on this clone were being completed, the murine and rat
Gdf15 sequences and their expression patterns were published
(3, 4). Here we describe the regulation of Gdf15
expression in multiple models of acute liver and bile duct injury and
the characterization of Gdf15 null mice.
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MATERIALS AND METHODS |
Library screening, hybridization, and in situ analysis.
A
murine 129/SvJ genomic library (25) and a liver cDNA library
(Stratagene, La Jolla, Calif.) were screened as described previously
(22), using the region corresponding to the entire C
terminus of hTGF-PL (2, 16, 21, 32, 42) as a probe. Low-stringency genomic Southern analysis was carried out as described before (8, 35). The blots were hybridized at 33°C below
the melting temperature of the probes. Northern analysis of RNA samples prepared using the one-step RNAzol B method (Tel-Test, Inc.,
Friendswood, Tex.) was carried out as described previously
(35). In situ hybridization using digoxigenin-labeled cRNA
probes corresponding to the 5' untranslated region and
propeptide-coding region of Gdf15 was carried out
essentially as described before (20, 36), except that slides
were washed overnight prior to detection and that BM-purple (Boehringer
Mannheim, Indianapolis, Ind.) was used as the colored precipitant.
Color development reactions were carried out for 16 to 24 h.
In vivo injury models.
All animal studies were approved by
the Animal Care and Use Committee at The Johns Hopkins University
School of Medicine. Partial hepatectomy was performed on 5- to
7-week-old male CD-1 mice (Charles River, Wilmington, Mass.) as
previously described (15), using methoxyflurane
(Mallinckrodt Veterinary, Inc., Mundelein, Ill.) as an inhaled
anesthetic. Each sham-operated animal was anesthetized, followed by
incision of the peritoneal cavity, gentle manipulation of the liver,
and closure of the abdomen with sutures. Carbon tetrachloride
(CCl4), D-galactosamine (GalN), and
methylenedianiline (DAPM) (all from Sigma Chemical Corporation, St.
Louis, Mo.) were administered to 5- to 6-week-old male C57BL/6J mice
(Jackson Laboratories, Bar Harbor, Maine) by intraperitoneal injection
in a volume of 0.1 ml. Doses for these chemicals were 20 µl of
CCl4 in soy oil (Wesson vegetable oil; Hunt-Wesson, Inc.,
Fullerton, Calif.) per mouse, 0.7 g of GalN in saline per kg of
body weight, and 50 mg of DAPM in 50% ethanol in saline per kg of body
weight. Ethanol was delivered by intraperitoneal injection of 4 g
of absolute ethanol per kg of body weight, diluted with water to a
final volume of 0.3 ml. Control samples for each chemical injury were
isolated from animals that received injection of the carrier alone.
Serum samples were collected by cardiac puncture of anesthetized
animals just prior to euthanasia and analyzed by Antech Diagnostics
(Farmingdale, N.Y.).
Dividing cells were labeled for 2 h with 5-bromo-2'-deoxyuridine
(BrdU) by injecting mice with 200 mg (per kg body weight) of a 10:1
molar ratio of BrdU (Sigma) and 5-fluoro-2'-deoxyuridine (Sigma)
resuspended in phosphate-buffered saline (11). BrdU-labeled nuclei were detected on frozen sections using the FLUOS in situ cell
proliferation kit (Boehringer Mannheim) according to the manufacturer's instructions, counterstained with
4',6'-diamidino-2-phenylindole (DAPI) (Molecular Probes, Eugene,
Oreg.), and mounted in ProLong antifade mounting medium (Molecular
Probes). Terminal deoxynucleotidyltransferase-mediated dUTP-X nick end
labeling (TUNEL)-positive cells were detected on frozen sections using
the fluorescein in situ cell death detection kit (Boehringer Mannheim)
and counterstained and mounted as described above.
Cell isolation and culture.
Peritoneal macrophages were
isolated from 5- to 6-week-old C57BL/6J female mice as described before
(6). Briefly, reactive macrophages were isolated from mice
injected with thioglycolate (Sigma) 6 days before harvest and allowed
to adhere to plastic tissue culture dishes for 3 h at 37°C in
5% CO2. The culture medium was removed and replaced with
either fresh medium or fresh medium supplemented with 160 nM
phorbol-12-myristate-13-acetate (PMA) (Sigma) for an additional 3 h.
Separation of liver cells into parenchymal and nonparenchymal fractions
was carried out as described previously (
13), using
Liver
Perfusion and Liver Digest media from Life Technologies
(Gibco BRL,
Rockville, Md.). NMuLi cells (ATCC CRL-1638) and AML12
cells (ATCC
CRL-2254) from the American Type Culture Collection
(Manassas, Va.)
were grown to near confluence in plastic flasks.
CCl
4 was
added directly to the culture medium at a final concentration
of 20 or
40 mM. The flasks were sealed and incubated on an orbital
platform at
37°C and 30 rpm. For the heat shock experiments, near-confluent
cells
were incubated in 5% CO
2 at 44°C for 30 min and then
allowed
to recover at 37°C in 5% CO
2.
Gdf15 null mice.
The genomic structure of the
murine Gdf15 gene was determined using restriction mapping
and sequence analysis of lambda phage clones isolated from a 129/SvJ
genomic library. A targeting construct was designed and transfected
into R1 embryonic stem cells (30) and selected in 2 µM
ganciclovir (Roche Discovery Welwyn, Hertfordshire, England) and 200 µg of G418 (Gibco BRL) per ml following established protocols
(34). Injection of targeted clones into C57BL/6 blastocysts and embryo transfer into pseudopregnant females were carried out by the
Johns Hopkins Transgenic Core Facility (Baltimore, Md.). The
Gdf15 null mice and their wild-type littermates were
maintained on a hybrid C57BL/6/129/SvJ background. Mice were screened
using probes both external and internal to the targeted vector (see Fig. 6).
Nucleotide sequence accession number.
The nucleotide
sequence of Gdf15 is available under GenBank accession
number AF159571.
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RESULTS |
Identification of Gdf15.
We identified
Gdf15 in a screen for novel murine TGF- superfamily
members while probing a murine genomic library with the hTGF-PL
sequence. While the experiments described here were being completed,
the cloning and expression pattern of Gdf15 in the mouse and
rat were published (3, 4).
Pairwise comparisons between the C-terminal regions of Gdf15 and the
other TGF-
superfamily members revealed that
Gdf15 is
most closely related to hTGF-
PL (67% amino acid identity and
72%
nucleotide identity [data not shown]). Because most other
TGF-
family members are more highly conserved across species,
we attempted
to identify the human ortholog of
Gdf15 and the murine
ortholog of the hTGF-
PL gene by screening both human and murine
genomic DNA libraries with these probes. Despite extensive screening
of
these libraries, we were unable to identify a murine sequence
more
closely related to the hTGF-
PL gene than
Gdf15 or a human
sequence more closely related to
Gdf15 than the hTGF-
PL
gene.
To further investigate the possibility that Gdf15 and hTGF-
PL
are orthologs, we performed genomic Southern analysis of murine
and
human DNA using probes corresponding to the C-terminal regions
of the
two proteins. As shown in Fig.
1, the
most intensely hybridizing
bands identified by the hTGF-
PL probe on
murine genomic DNA corresponded
to the
Gdf15 gene. The
results of the converse experiment (hybridization
of the
Gdf15 probe to human genomic DNA) were inconclusive.
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FIG. 1.
Genomic Southern analysis of human and mouse DNA probed
with fragments corresponding to the C-terminal regions of Gdf15 and
hTGF-PL.
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We further examined the relationship between Gdf15 and hTGF-
PL by
comparing their expression patterns. hTGF-
PL was previously
shown to
be expressed widely in human tissues, with the highest
levels being
detected in the prostate (
32) and placenta (
16,
21,
42). By Northern analysis, we detected
Gdf15 in a
variety
of different adult mouse tissues (Fig.
2a), with highest levels
in the adult
liver. In contrast to hTGF-
PL,
Gdf15 mRNA was barely
detectable in mouse prostate and placenta (data not shown). We
also
determined whether
Gdf15 expression is up-regulated in
activated
macrophages, as previously reported for hTGF-
PL
(
2). Reactive
peritoneal macrophages were isolated
from thioglycolate-injected
C57BL/6J mice and purified by
adhesion to plastic tissue culture
surfaces. As shown in Fig.
2b,
Gdf15 mRNA levels increased significantly
in macrophages
activated with PMA relative to untreated control
macrophages. Hence,
while
Gdf15 and hTGF-
PL showed distinct expression
patterns in adult tissues, their expression patterns were similar
following macrophage activation.
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FIG. 2.
(a) Northern analysis (20 µg of total RNA) of adult
mouse tissues. (b) Northern analysis (10 µg of total RNA) of
peritoneal macrophages isolated from untreated (nonreactive) or
thioglycolate-injected (reactive) mice. Macrophages were purified by
adhesion to dishes and incubated in the absence or presence of PMA.
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Induction of Gdf15 expression during liver injury and
regeneration.
In order to determine the potential roles of
Gdf15 in the liver, we examined the regulation of
Gdf15 mRNA expression in several models of acute liver
injury and regeneration. We first examined Gdf15 expression
in the liver following administration of chemicals known to injure
hepatocytes. Administration of CCl4 to animals is known to
cause acute centrilobular necrosis of hepatocytes, followed by a
regenerative response in the surviving hepatocytes (7). As
shown in Fig. 3a, Gdf15 mRNA
levels in the liver increased rapidly and dramatically following
CCl4 administration. Gdf15 mRNA levels were
highest within 1.5 h following CCl4 treatment and then
returned to baseline levels by 6 to 12 h. We also observed a rapid
and dramatic induction of Gdf15 following intraperitoneal administration of a single dose of ethanol (Fig. 3b). As in the case of
CCl4 injury, Gdf15 RNA levels were increased 1 to 3 h after ethanol administration and returned to baseline
levels by 12 h. We also examined the expression of
Gdf15 following treatment with GalN, which causes hepatocyte
injury by inhibiting RNA synthesis (9). Unlike for
CCl4 treatment, liver regeneration following GalN injury
has been shown to involve recruitment of liver stem cells, or oval
cells (1, 7, 23). As shown in Fig. 3c, Gdf15
expression was also increased following treatment with GalN, although
the time course of induction was considerably delayed compared to that
of CCl4 injury.
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FIG. 3.
Expression of Gdf15 following liver and bile
duct injury. Northern analysis was carried out using 20 µg of total
RNA, except for panel c, in which 5 µg of twice-poly(A)-selected RNA
was used. Graphs to the right of the Northern blots show
Gdf15 expression normalized to aldolase expression and
plotted relative to uninduced levels.
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In order to determine whether the induction of
Gdf15 mRNA
was restricted to chemical injury of hepatocytes, we examined the
expression of
Gdf15 following two-thirds hepatectomy.
Partial
hepatectomy initiates a regenerative response in all cell types
in the liver remnant (
5,
38), including a large hepatocyte
proliferative response similar to that seen after CCl
4
injury.
As shown in Fig.
3d,
Gdf15 expression was induced
following partial
hepatectomy, and the time course of induction was
similar to that
observed following CCl
4 treatment.
Gdf15 mRNA levels were increased
dramatically at the
earliest time point examined (30 min following
surgery) and remained
elevated for up to 12 h, after which the
Gdf15 RNA
levels decreased to baseline. In 5 of 13 sham-operated
animals,
Gdf15 mRNA levels showed a transient increase at 1 h,
but the magnitude of induction was significantly lower than that
following partial hepatectomy (data not
shown).
We next examined the expression of
Gdf15 following chemical
bile duct injury to determine whether
Gdf15 expression could
be
induced by injury to other cell types within the liver. Following
treatment with DAPM, which selectively injures bile duct epithelial
cells (
17),
Gdf15 mRNA levels were increased at
1 h and returned
to baseline levels by 6 to 12 h (Fig.
3e).
While levels of
Gdf15 mRNA were also mildly elevated at
1 h in animals receiving the
ethanol carrier only, the magnitude
of
Gdf15 induction in these
control animals was lower than
that seen with DAPM, and
Gdf15 mRNA levels returned to
baseline by 3 h (data not shown). Hence,
Gdf15
expression appears to be induced rapidly not only following
direct
injury to hepatocytes but also following injury to bile
duct epithelial
cells.
Patterns of Gdf15 mRNA localization following liver
injury.
As shown above, Gdf15 expression in the liver
was rapidly induced following various chemical and surgical treatments
that cause liver injury and regeneration. We used in situ hybridization to determine the pattern of Gdf15 expression in these
various injury models. The most dramatic results were obtained in
livers taken from animals that had been treated with CCl4.
As shown in Fig. 4b to e,
Gdf15 mRNA was selectively localized to centrilobular regions, which coincides with the known pattern of hepatocyte injury
induced by CCl4 (28, 33). Following ethanol
administration, Gdf15 was again expressed by centrilobular
hepatocytes but in a more diffuse pattern, with various levels of
expression between individual hepatocytes (Fig. 4f to i). The
centrilobular expression of Gdf15 correlates with the
centrilobular fatty change, necrosis, and fibrosis previously described
for rodent models of acute and chronic liver injury by ethanol
(14, 18). In contrast, GalN treatment caused
Gdf15 mRNA to be expressed in a punctate pattern distributed
throughout the parenchyma, with a large number of binucleate
hepatocytes expressing Gdf15 (Fig. 4j to m). A similar punctate pattern has been described for the parenchymal distribution of
necrotic cells following GalN administration (27).
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FIG. 4.
In situ localization of Gdf15 mRNA in liver.
All panels were photographed at ×40 magnification, except for panels
c, g, k, o, and s, which are ×100 magnifications of the sections shown
in panels b, f, j, n, and r, respectively.
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In order to confirm that the
Gdf15-expressing cells were
hepatocytes, we separated liver cells from GalN-treated mice into
parenchymal and nonparenchymal populations by low-speed centrifugation
(
13) and then isolated RNA from each group. As controls for
the cell separation procedure, we carried out Northern analysis
on
these RNA samples using probes for murine hepatocyte growth
factor
(
19) and albumin as markers for nonparenchymal and
parenchymal
cells, respectively. Northern analysis showed that
Gdf15 mRNA
was restricted to the parenchymal cell fraction
(Fig.
5a), consistent
with the in situ
hybridization results. Similar results were also
obtained using
separated cell populations obtained from untreated
livers (data not
shown).
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FIG. 5.
(a) Expression of Gdf15 in parenchymal cells.
Liver cells isolated from GalN-treated mice were separated into
nonparenchymal and parenchymal fractions. HGF, hepatocyte growth
factor. (b) Expression of Gdf15 in NMuLi and AML12 cells
treated with CCl4 in culture. Control flasks containing
cells were sealed and incubated at 37°C in the absence of
CCl4. Graphs to the right of the Northern blots show
Gdf15 expression normalized to aldolase expression and
plotted relative to uninduced levels. (c) Expression of
Gdf15 following heat shock treatment of NMuLi and AML12
cells in culture. Cells were incubated at 44°C for 30 min starting at
time zero and then allowed to recover at 37°C.
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Following partial hepatectomy,
Gdf15 mRNA was induced in
cells throughout the liver parenchyma, with more intense staining
around central veins (Fig.
4n to q). Unlike the CCl
4 and
GalN
injuries, the centrilobular pattern we observed following partial
hepatectomy did not correspond to the previously reported periportal
distribution of injured hepatocytes in the early period following
surgery (
5,
38). The pattern of
Gdf15 expression
at 6 h following
DAPM treatment was also centrilobular (Fig.
4r to
u). Notably,
no
Gdf15 mRNA was detected in the periportal or
bile duct regions,
which presumably represent the primary sites of
DAPM-mediated
injury.
Induction of Gdf15 mRNA in cultured liver cells.
As shown above, a variety of acute liver injuries in vivo can result in
up-regulation of Gdf15 expression in hepatocytes. Because
many complex cellular interactions can occur in vivo in the liver, we
asked whether direct injury to a uniform population of cells in tissue
culture could also induce Gdf15 expression. We examined the
response of NMuLi cells (31), which are believed to
represent early differentiated liver epithelial cells, and AML12 cells
(40), which are derived from mature hepatocytes, to
CCl4 treatment in culture. As shown in Fig. 5b,
Gdf15 mRNA levels were rapidly elevated in a
CCl4 dose-dependent manner in both cell types.
We also exposed AML12 and NMuLi cells to a transient 44°C heat shock
to determine whether
Gdf15 could be induced by a nonchemical
stressor. As seen in Fig.
5c, levels of
Gdf15 mRNA were
mildly
elevated in both cell lines at 1 h following heat shock.
These
in vitro data indicate that
Gdf15 expression can be
induced in
hepatocytes in response to direct stress and injury. In
addition,
the induction of
Gdf15 expression can occur in the
absence of
other cell types, such as inflammatory
cells.
Generation of Gdf15 null mice.
In order to address
the potential function of Gdf15 in vivo, we used gene targeting to
generate Gdf15 null mice. As shown in Fig.
6a, the Gdf15 gene is composed
of two exons spanning approximately 4 kb of DNA. The region
corresponding to the entire mature C terminus, which is carried within
exon 2, was replaced by the neomycin resistance cassette in our
targeting construct. The targeted construct was electroporated into R1
embryonic stem cells, and clones doubly resistant to ganciclovir and
G418 were isolated. Using Southern analysis with a probe from outside
the targeting construct (Fig. 6a), we identified eight targeted clones
among 143 screened colonies. All subsequent analysis was carried out on
offspring of a single male chimera derived from blastocyst injection of
one of the clones.
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FIG. 6.
Targeting construct and genomic identification of
Gdf15 null mice. (a) Restriction map of the Gdf15
genomic locus (top line) and of the targeting vector (bottom line). S,
SalI; B, BamHI; E, EcoRI; Xh,
XhoI; Xb, XbaI; TK, thymidine kinase cassette;
Neo, neomycin resistance cassette. The external and internal probes
used for Southern blot analysis are indicated. (b) Representative
Southern blots of DNA isolated from wild-type, heterozygous, and
homozygous Gdf15 null mice. The upper blot shows genomic DNA
digested with EcoRI and probed with the external probe. The
endogenous Gdf15 allele is contained within the 13.3-kb
fragment, and the null allele is contained within the 11.4-kb fragment.
The lower blot shows genomic DNA digested with BamHI and
probed with the internal probe. The endogenous Gdf15 allele
gives a 2.5-kb fragment, while the null allele produces a 10.5-kb
fragment. (c) Northern analysis of 20 µg of total RNA isolated from
livers of wild-type (WT) and null (KO) mice treated with carbon
tetrachloride and probed with a fragment corresponding to the
C-terminal region of Gdf15.
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Gdf15 homozygous null mice were obtained by crosses of
F
1 heterozygotes. The mice were genotyped using a probe
external to
the targeted region and by a second probe internal to the
targeted
site (Fig.
6b). Absence of
Gdf15 expression in the
homozygous
null mice was confirmed by Northern analysis of liver RNA
isolated
from untreated mice and mice treated with CCl
4 for
1.5 h or 1
day. No expression of
Gdf15 was detected in
the livers of null
mice (Fig.
6c). Analysis of 775 adult offspring from
heterozygous
matings showed a ratio of wild-type, heterozygous, and
homozygous
mice that approximated 1:2:1 (196:392:184). Homozygous null
mice
were viable and fertile. Analysis of serum chemistries and
examination
of various tissues by gross pathology and microscopic
techniques
revealed no obvious abnormalities in the
Gdf15
null mice (data
not
shown).
Characterization of Gdf15 null mice after liver
injury.
Based on the high level of Gdf15 expression in
the liver and the dramatic regulation of Gdf15 expression
following liver injury, we focused most of our detailed analysis on
liver function. Liver weights of homozygous null mice at 1, 3, and 6 to
7 weeks were indistinguishable from those of wild-type littermates
(Fig. 7). Therefore, we tested the
Gdf15 null mice in the CCl4 and
partial-hepatectomy injury models.
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FIG. 7.
Mean liver weight as a percent of total body weight of
wild-type (open bars) and Gdf15 null (solid bars) mice.
Numbers above each category are the numbers of mice per point. Error
bars represent 1 standard error of the mean.
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As described above, we injected the
Gdf15 homozygous null
mice and their wild-type counterparts with CCl
4 and
assessed hepatocyte
injury by measuring serum transaminase levels as
described previously
(
10). As shown in Fig.
8, wild-type mice showed peak levels
of
serum AST and ALT levels at 1 day after administration of
CCl
4,
which normalized by 4 days after injury.
Gdf15 null mice showed
elevations in AST and ALT levels
comparable to those of wild-type
mice. In addition, comparison of liver
sections from wild-type
and null mice by microscopic histology showed
no significant differences
in the size of the necrotic zones around the
central veins (data
not shown). Examination of the time course of DNA
synthesis following
CCl
4 administration by BrdU labeling
also showed no significant
differences between wild-type and null mice
(data not shown).
Finally, TUNEL analysis of the same livers showed no
significant
differences at 1 day after CCl
4 injury, which
coincided with the
peak of TUNEL labeling seen in our experiments (data
not shown).
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FIG. 8.
Mean serum aspartate transferase (AST) and alanine
transferase (ALT) levels in wild-type (open bars) and Gdf15
null (solid bars) mice following CCl4 treatment. Numbers
above each category are the numbers of mice per point. Error bars
represent 1 standard error of the mean.
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In order to examine if
Gdf15 is involved in liver
regeneration, we performed partial hepatectomies on male
Gdf15 null and
wild-type mice. As shown in Fig.
9a, the liver masses of the wild-type
mice recovered to approximately 50 and 90% of the starting liver
mass
after 2 and 6 days, respectively, as previously described
(
41). The
Gdf15 null mice regenerated their liver
mass at the
same rate as the wild-type mice. Comparison of cell
proliferation
by BrdU incorporation at these time points (Fig.
9b) also
showed
no significant difference between wild-type and knockout mice.
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FIG. 9.
Mean liver weights following partial hepatectomy in
wild-type (open bars) and Gdf15 null (solid bars) mice. (a)
Liver weights as a percent of starting body weights in untreated mice,
mice immediately following partial hepatectomy and at 2 and 6 days
after partial hepatectomy. (b) Number of BrdU-labeled nuclei in liver
sections following partial hepatectomy. The number above each category
is the number of mice per point. Error bars represent 1 standard error
of the mean.
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DISCUSSION |
In this report, we describe our initial characterization of a new
murine TGF- family member, Gdf15. Among the known family members, Gdf15 is most closely related to hTGF-PL
(42) (also known as hMIC-1, hPDF, hPLAB, and hPTGFB), which
was identified independently by several different groups searching for
novel secreted proteins (42), for genes expressed by
activated macrophages (2), for TGF--like molecules in
expressed sequence tag databases (16, 32), and for novel
genes expressed in placenta (21). Although Gdf15
and hTGF-PL are significantly more divergent than is usually
observed for other murine and human TGF- ortholog pairs, the results
of our comparative genomic Southern analysis and the fact that
Gdf15 and the hTGF-PL gene are both expressed by
activated macrophages raise the possibility that these genes may be
orthologs. Further functional studies will be required to determine
whether these two genes play analogous roles in their respective species.
In the adult mouse, Gdf15 mRNA is expressed at highest
levels in the liver, with lower levels seen in other tissues. This expression pattern is similar to that previously found using reverse transcription-PCR detection methods (3), although we did not observe strong Gdf15 signals in the brain or prostate. We
have also shown that Gdf15 expression in the liver is
rapidly and dramatically up-regulated in various models of liver injury
and regeneration. Although a number of different experimental
treatments can induce Gdf15 expression, each type of injury
induces a distinct pattern of Gdf15 expression in the liver.
Perhaps the most striking pattern is produced by CCl4,
which results in expression of Gdf15 exclusively in
centrilobular hepatocytes. Because CCl4 causes
centrilobular liver necrosis (28, 33) and because the
expression of Gdf15 mRNA extended to cells closest to the
central veins, at least some, if not all, of the hepatocytes that
express Gdf15 in response to CCl4 must be ones
that will eventually undergo necrosis.
Similarly, it seems likely that the hepatocytes expressing
Gdf15 in the other chemical and surgical models have also
been injured. GalN causes a reversible inhibition of RNA synthesis (9), leading to diffuse liver damage with individual
necrotic cells distributed within the hepatic lobules (27).
The regenerative response following GalN injury is believed to be
mediated by the proliferation of oval cells located in the periportal
regions (7, 23). Indeed, the expression pattern of
Gdf15 following GalN administration is diffuse and does not
show any obvious localization to centrilobular or periportal regions.
The centrilobular expression of Gdf15 after a single dose of
ethanol correlates with the necrosis, fibrosis, and fatty change that
is seen in centrilobular hepatocytes after chronic ethanol injury
(14, 18). Acute doses of ethanol are known to cause changes
in lipid metabolism within the injured liver, presumably contributing
to the morphologic changes that are observed in chronic injury
(29). Given the dramatic expression of Gdf15
after ethanol administration, we would hypothesize that Gdf15 is expressed by the cells in the centrilobular area
that are being injured by the ethanol.
In the case of DAPM treatment, we would speculate that the
centrilobular hepatocytes expressing Gdf15 are ones that
have been injured secondarily by cholestasis caused by the loss of bile duct epithelial cells (24, 37). Similarly, given that the pattern of Gdf15 expression following partial hepatectomy
does not coincide with the periportal distribution of proliferating hepatocytes during the initial wave of regeneration (12), it seems possible that Gdf15 expression is a response to the
acute stresses of hepatectomy on the hepatic remnant. One of our
striking findings is that Gdf15 expression can be induced in
the absence of inflammatory cells. As we have shown with
CCl4 and heat shock treatments of hepatocyte-like cells in
culture, direct cellular injury or stress appears to be sufficient to
induce Gdf15 expression.
Given the dramatic up-regulation of Gdf15 following multiple
types of liver injury, we hypothesized that Gdf15 may be
involved in the regulation of the acute injury response in hepatocytes. Two recent reports have suggested that Gdf15 and hTGF-PL could be
regulated by multiple transcription factors that are known to regulate
cell growth and injury responses (3), including p53
(39). To determine the in vivo function of Gdf15, we
generated mice in which the Gdf15 gene had been deleted.
Homozygous null mice were viable and fertile and had no gross
abnormalities in any of the major organs. Our studies also showed that
the Gdf15 null mice and wild-type mice had comparable
degrees of liver injury and recovery after CCl4
administration. Finally, the Gdf15 null mice were able to
regenerate their liver mass at the same rate as wild-type mice after
partial hepatectomy.
Our results suggest that Gdf15 is not essential for proper
regeneration of the liver following surgical or chemical injury. It is
possible that the Gdf15 mutant mice have subtle defects that
were not revealed in our analysis. It is also possible that the loss of
Gdf15 function is compensated by other secreted growth factors or by
other regulatory pathways. A full elucidation of the roles of Gdf15
during the injury response awaits additional analysis of the null mice
and the analysis of the biological activities of the Gdf15 protein
both in vitro and in vivo.
| |
ACKNOWLEDGMENTS |
We thank Paul Dunlap for his assistance in the maintenance of
mice and Tracie E. Bunton, John K. Boitnott, and Daniel Nathans for
their helpful discussions.
T.Z.-K. and L.G.K. were supported by NIH training grant 5 T32 CA09139
to the Department of Molecular Biology and Genetics, The Johns Hopkins
University School of Medicine. E.C.H. is a trainee of the Medical
Training Scientist Program (Public Health Service grant 5 T32 GM07309).
This work was supported by grants from MetaMorphix, Inc., and American
Home Products, Inc. (to S.-J.L.). Under an agreement among Johns
Hopkins University, MetaMorphix, Inc., and American Home Products,
Inc., all of the authors are entitled to a share of royalties received
by the university from sales of Gdf15. S.-J.L., T.V.H., and
the university also own MetaMorphix stock, which is subject to certain
restrictions under university policy. The terms of these arrangements
are being managed by the university in accordance with its conflict of
interest policies. S.-J.L. is a consultant to MetaMorphix.
| |
ADDENDUM IN PROOF |
Radiation hybrid mapping (Research Genetics, Inc.) linked
Gdf15 to the mouse framework marker D8Mit233. This region of
mouse chromosome 8 is syntenic to a portion of human chromosome 19p13.1 containing TGF-PL (Bottner et al., Gene 237:105-111,
1999; see also mapping data from the Human Genome Center at Lawrence Livermore Laboratory), consistent with mGdf15 and hTGF-PL
being orthologs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 N. Wolfe St./PCTB 607, Baltimore, MD 21205. Phone: (410)
614-0198. Fax: (410) 614-7079. E-mail: sjlee{at}jhmi.edu.
| |
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Abstract
Introduction
