From the Department of Hepatology, Graduate
School of Medicine, Osaka City University Medical School, Osaka,
545-8585, § Hiroshima Proteome Laboratory, Regional Science
Program of Hiroshima Industrial Technology Organization and Japan
Science and Technology Corporation, Higashihiroshima, Hiroshima,
739-0046, ¶ Hiroshima Tissue Regeneration Project, Hiroshima
Prefecture Collaboration of Regional Entities for the Advancement of
Technological Excellence, Japan Science and Technology Corporation,
Higashihiroshima, Hiroshima, 739-0046, and the
Developmental Biology Laboratory, Department of Biological
Science, Graduate School of Science, Hiroshima University,
Hiroshima, 739-8526, Japan
Received for publication, March 23, 2001, and in revised form, April 23, 2001
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ABSTRACT |
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A proteome approach for the molecular analysis of
the activation of rat stellate cell, a liver-specific pericyte, led to
the discovery of a novel protein named STAP (stellate cell
activation-associated protein). We cloned STAP
cDNA. STAP is a cytoplasmic protein with molecular weight of 21,496 and shows about 40% amino acid sequence homology with myoglobin. STAP
was dramatically induced in in vivo activated stellate
cells isolated from fibrotic liver and in stellate cells undergoing
in vitro activation during primary culture. This induction
was seen together with that of other activation-associated molecules,
such as smooth muscle The molecular mechanism underlying the activation of hepatic
stellate cells has been extensively studied during the last decade because activated stellate cells are thought to be a key player in the
development of liver fibrosis (1-5). This activation is characterized
by a transdifferentiation from a vitamin A-storing quiescent phenotype
to a myofibroblast-like cell and accompanied by the expression of
various genes of extracellular matrix matrices, cell growth
factors, inflammatory cytokines, and receptors for growth factors
(1-5). Analysis of the difference of gene expression between quiescent
and activated stellate cells has provided profound insights into the
cell activation mechanism. For instance, subtractive hybridization
revealed the expression of Zf9, a Kruppel-like transcription factor, at the initial step of the cell activation (6) and that of
cellular prion protein in fully activated stellate cells (7).
We have undertaken a proteomics approach to obtain a deeper knowledge
on the molecular mechanism of hepatic stellate cell activation at the
protein level (8). Protein populations, proteomes, expressed in
quiescent and activated stellate cells were separated by
two-dimensional polyacrylamide gel electrophoresis
(PAGE)1 and subsequently
analyzed using electrospray ionization mass spectrometry (9, 10).
Such a protein level "differential display" identified 43 proteins
that altered their expression levels during the activation process (8).
These included the up-regulation of collagen In the present study, we report a novel protein that was discovered by
proteome analysis of the activation process of rat hepatic stellate
cells. The protein was found to be heavily up-regulated both in
in vitro and in vivo activated stellate cells and
was accordingly named STAP (stellate cell
activation-associated protein). Expression of
STAP and its gene (stap) was dramatically augmented in
fibrotic liver tissues induced by thioacetamide (TAA) administration, indicating an important role of STAP in the development of liver fibrosis.
Induction of Liver Fibrosis--
Pathogen-free male Wistar rats
(SLC, Shizuoka, Japan) were administered with 50 mg/body of TAA (WAKO
Pure Chemical Co., Osaka, Japan) intraperitoneally twice a week for 8 weeks (14). The protocol of experiments was approved by the Animal
Research Committee of Osaka City University (Guide for Animal
Experiments, Osaka City University).
Preparation of Hepatic Constituent Cells--
Hepatic
constituent cells were isolated from rat livers as previously described
(8, 15). Hepatocytes, Kupffer cells, and endothelial cells were used
immediately after the isolation. Stellate cells were plated for 3 h in Dulbecco's modified Eagle's medium (Life Technologies, Inc.,
Gaithersburg, MD) and supplemented with 10% fetal bovine serum (Life
Technologies, Inc.), and the cultures were subsequently washed to
remove dead cells and cell debris. Stellate cells isolated from normal
or fibrotic livers were referred to as quiescent or in vivo
activated stellate cells, respectively, in the present study (8).
Likewise, stellate cells isolated from normal liver and cultured for 7 days were referred to as in vitro activated stellate cells
(8).
Two-dimensional PAGE--
Two-dimensional PAGE was performed as
previously described (8, 16, 17). Proteins (100 µg) from liver cells
were applied to Immobiline DryStrips (pH 4-7, 18 cm, Pharmacia Hoefer,
Upsala, Sweden) by in-gel rehydration (18, 19). After isoelectric focusing, proteins were separated by SDS-PAGE on 9-18% acrylamide gradient gels, visualized by silver staining, scanned, and analyzed using the Melanie II two-dimensional PAGE software package from Bio-Rad
(Hercules, CA).
Tryptic In-gel Digestion of Two-dimensional PAGE Resolved
Proteins and Mass Spectrometry--
Protein spots of interest were
excised from the two-dimensional gels and in-gel-digested with trypsin
(8, 17). After extraction and purification from the tryptic digests
(17), peptides were sequenced by electrospray ionization mass
spectrometry on a quadrupole-time-of-flight mass spectrometer
(8, 17). The proteins were identified by matching the obtained amino
acid sequences against the SwissProt and GenBankTM data bases.
Reverse Transcription-Polymerase Chain Reaction--
Total RNA
was extracted from stellate cells and liver tissues using Isogen
(Nippon Gene, Tokyo, Japan). Messenger RNA expression in each sample
was determined by reverse transcription-polymerase chain reaction
(RT-PCR) using GeneAmp RNA PCR Core Kit (PerkinElmer Life
Sciences). The following primers were used: STAP,
ATGGAGAAAGTGCCGGGCGAC (forward) and TGGCCCTGAAGAGGGCAGTGT (reverse);
and glyceraldehyde-3-phosphate dehydrogenase,
ACCACAGTCCATGCCATCAC (forward) and TCCACCACCCTGTTGCTGTA (reverse).
cDNA Cloning--
Degenerative PCR was performed using a
primer pair, CCXGGXGAYTTYGARATHGA and
GCXACXCCXACRTCYTC, designed from two
amino acid sequences, PGDMEIER and ANCEDVGVA, which were derived by
electrospray ionization mass spectrometric analysis of one protein spot
later named STAP from two-dimensional gels of activated stellate cells. The template used was a cDNA library that was reverse-transcribed from total RNA of activated stellate cells using oligo(dT) primer, reverse transcriptase (SUPERSCRIPT II, Life Technologies, Inc.), and
GeneAmp RNA PCR Core Kit (PerkinElmer Life Sciences). The obtained
120-bp product was ligated into pGEM-T Easy vectors (Promega), and the
sequence of the inserted DNA was determined using ABI PRISM 310. A
rat-activated stellate cell cDNA library was inserted into Lambda
Zap II vectors (Stratagene, La Jolla, CA) and screened using this
120-bp PCR product as a probe. Positive clones were in vivo
excised to pBluescript SK( Production of Polyclonal Antibodies for STAP--
A synthetic
NH2-terminal polypeptide of STAP,
NH2-MEKVPGDMEIERRERNEE+Cys-COOH, was used as an immunogen.
This peptide fragment (0.2 mg) was immunized in rabbits first with
complete Freund's adjuvant and then twice with incomplete Freund's
adjuvant. After the third immunization was finished, the rabbits were
sacrificed, and the serum was harvested. The antibody was
affinity-purified against the synthetic peptide. The antibody produced
a single band at 21 kDa in Western blot analysis of stellate cell
homogenates and recognized monospecifically recombinant rat STAP the
preparation of which is described below.
Western Blot--
Protein samples (10 µg of protein) were
subjected to SDS-PAGE and then were transferred onto Immobilon P
membranes (Millipore Corp., Bedford, MA). After blocking, the membranes
were treated with antibodies against rat STAP, PDGF receptor- Immunohistochemistry and Immunoelectron Microscopy--
Fixed
rat liver specimens (5-µm thickness) were incubated first with
anti-smooth muscle In Situ Hybridization and
Immunohistochemistry--
Digoxigenin-labeled cRNA probes were
synthesized with a digoxigenin RNA labeling kit (Roche Molecular
Biochemicals). Hybridization was carried out at 70 °C for 15 h
in a buffer consisting of 50% deionized formamide, 5× SSC (3 M NaCl and 0.3 M sodium citrate), 50 µg/ml
Escherichia coli tRNA, 1% SDS, 50 µg/ml heparin, and 1 µg/ml heat-denatured, digoxigenin-labeled probes. The detection of
hybridized cRNA probes was performed using 5-bromo-4-chloride-3-indolyl phosphate and nitroblue tetrazolium (Roche Molecular Biochemicals) as
described previously (20). Some sections were subsequently subjected to
immunohistochemistry using anti-desmin antibodies (Monosan, Uden, The
Netherlands) and fluorescein-labeled anti-rabbit IgG (Vector Laboratories).
Generation of Recombinant Rat STAP--
The open reading frame
of cloned rat STAP cDNA was ligated into pTrcHis2 TOPO vectors
(Invitrogen, Carlsbad, CA). His6-tagged STAP was generated
in TOP10 cells cultured overnight in LB medium supplemented with 1 mM isopropyl-1-thio- Identification of Recombinant Rat STAP as a Heme
Protein--
Absorption spectra of rat recombinant STAP dissolved in
PBS at pH 7.4 were obtained at 25 °C using a Hitachi
spectrophotometer U-2001 (Hitachi, Tokyo) in the presence or absence of
Na2S2O4. A peroxidase activity of
recombinant STAP was measured as follows. Purified STAP was separated
by 15% native PAGE and then transferred electrically onto Immobilon-P
membranes. After washing with PBS twice, the membranes were treated
with either 3,3'-diaminobenzidine (DAB)/hydrogen peroxide
(H2O2) solution or ECL chemiluminescence solution (Amersham Pharmacia Biotech). Peroxidase-dependent
reaction products were detected either directly on the membranes or on the Kodak XAR5 films. The presence of protoheme in STAP was determined by the pyridine hemochrome method (21). In brief, the purified STAP was
converted to a pyridine hemochrome by the addition of 0.5 ml of
pyridine and 0.5 ml of 0.5 N NaOH solution to 4.0 ml of the protein
solution. Absorption spectra of this mixture were scanned at 25 °C
using a Hitachi spectrophotometer U-2001 (Hitachi, Tokyo) in the
presence or absence of Na2S2O4.
Peroxidase Activity--
Catalase activity of STAP was
determined spectrophotometrically by measuring the decrease of
H2O2 at 240 nm in 50 mM PBS buffer in the absence or presence of STAP (22). Fatty acid hydroperoxide peroxidase activity was determined according to a modified version of
the Kharasch's method (23). Conjugated diene formation was initiated
in the presence or absence of recombinant STAP by adding 30 mM FeCl2 into a reaction mixture of 30 mM NaCl, 1 mM linoleic acid, 20 mM
linoleic acid hydroperoxide, and 0.16% Lubrol PX, pH 7.0. The reaction
was monitored at 234 nm.
Identification and cDNA Cloning of a Protein Heavily
Up-regulated in Activated Stellate Cells--
We investigated the
overall protein expression pattern of quiescent, in vitro
activated, and in vivo activated stellate cells to obtain a
general insight into changes of the activation-associated proteins and
to identify them (8). As depicted in Fig.
1A, one protein spot with a pI
value of 6 and a molecular mass of 21 kDa was found to be
remarkably up-regulated in both in vivo and in
vitro activated stellate cells. The protein was digested with
trypsin and the peptides produced were sequenced by
quadrupole-time-of-flight flight mass spectrometry. As a result
two partial sequences were obtained: PGDME(I/L)ER and ANCEDVGVA.
Neither was listed in SwissProt or GenBankTM, indicating
that this was a novel protein. Therefore, we undertook cDNA cloning
of the gene.
Degenerative PCR was performed using cDNA produced from total RNA
of activated stellate cells as a template. Primer pairs were designed
from the two obtained amino acid sequences:
CCXGGXGAYTTYGARATHGA and
GCXACXCCXACRTCYTC. The PCR amplified a
single band at 120 bp (data not shown). DNA sequencing revealed that
the deduced amino acid sequence actually contained the two
above-mentioned peptide fragments (Fig. 1B). A rat-activated
stellate cell cDNA library was screened using the 120-bp DNA as a
probe, which yielded a 2028-bp-long cDNA clone. The open reading
frame consisted of 570 bp that encoded 190 amino acids (Fig.
1C). The calculated molecular weight was 21,496. The
amino acid sequence showed about 40% homology with myoglobin and
indicated that this protein was a cytoplasmic protein according to the
Reinhardt's criterion (24). We named this novel protein STAP after
stellate cell activation-associated protein.
Expression of STAP in Stellate Cells and Other Hepatic Constituent
Cells--
Polyclonal antibodies against STAP were raised using its
synthetic NH2-terminal 18 peptides. Western blot
analysis with these antibodies revealed that STAP protein was time
dependently induced in culture-stimulated stellate cells in a manner
similar to the expression pattern of N-CAM (11), smooth muscle
Two-dimensional PAGE analysis showed that neither endothelial cells,
Kupffer cells, nor hepatocytes isolated from normal or TAA-induced
fibrotic liver expressed detectable amounts of STAP (Fig.
2C). Immunoelectron microscopy not only supported these observations (Fig. 2D, a and b), but
also revealed a negligible expression in bile duct epithelial cells and
a significant expression in myofibroblasts located in the portal area
(Fig. 2D, c).
Expression of STAP in Fibrotic Liver--
The treatment of rats
with TAA induces the liver fibrosis and finally cirrhosis (14). The
parenchymal architecture underwent distinct changes, developing thick
bundles at 8 weeks of the treatment (Fig.
3A, a and
d). Immunohistochemistry confirmed the previous report (4,
11, 12) that the expression of smooth muscle Peroxidase Activity of STAP--
His6-tagged
recombinant rat STAP was purified under native conditions using a
nickel-NTA agarose column. Aliquots of samples at each step of
the purification were run on 15% SDS-PAGE gels as depicted in Fig.
4A. The eluted protein from
the column made a single band at the predicted position of molecular
weights and the solution was brown-colored. Absorption spectra of rat
recombinant STAP under non-reducing conditions exhibited peaks at 415 and 531 nm (Fig. 4B). When the protein was suspended in a
solution containing sodium dithionite, its absorption spectra exhibited peaks at 427, 531, and 560 (Fig. 4B), indicating that STAP
was a heme protein (26). Characterization of STAP by the pyridine hemochrome method (21) indicated that protoheme was a component of STAP
protein (data not shown). The presence of heme was further supported by
the fact that the recombinant STAP showed a peroxidase activity as
revealed by the development of brown color in
DAB/H2O2 reaction and chemiluminescence in the
ECL reaction (Fig. 4C). The actual presence of peroxidase
activity was determined by a H2O2 degradation
assay wherein the recombinant STAP was tested whether it has an
H2O2-catabolizing activity. STAP was found to have 4.5 munits/mg of catalase activity. Furthermore, STAP was found to
suppress conjugated diene formation in a dose-dependent manner when the protein was added in the fatty acid peroxidation reaction, the half-maximal inhibition occurring at ~50 nM
STAP (Fig. 4D). These results conclusively demonstrated that
STAP is a novel cytosolic protein with a peroxidase activity.
The activation of hepatic stellate cells is a key step for the
development of liver fibrosis (1-5). Growth factors, such as PDGF,
TGF- These stellate cell activation-related genes have been identified
hitherto using subtractive hybridization (6, 7) and differential
display (37). In addition to these conventional methods recent
advancements of mass spectrometry have permitted us to analyze proteins
in cells and organs on a large scale (9, 10). Previously our proteome
analysis of the stellate cell activation identified a total of 43 proteins/polypeptides that consistently altered their expression levels
when the cells were activated (8). These included the up-regulated
proteins such as galectin-1, calcyclin, and calgizzarin, and the
down-regulated proteins such as liver carboxylesterase 10 and serine
protease inhibitor 3 (8). Thus, protein level "differential
display" using proteomics is evidently a powerful approach for
displaying protein expression patterns in activated stellate cells.
In addition to the known proteins mentioned above, the proteome
analysis performed in the present study revealed an unknown protein,
dubbed STAP, which was highly up-regulated during the stellate cell
activation. STAP represents a novel protein because it has little
homology with existing entries in the protein data bases. According to
the PSORT II protein analysis STAP has no endoplasmic
reticulum retention motif, membrane retention signal, peroxisomal targeting signal, RNA-binding motif, actin-binding motif,
DNA-binding motif, and ribosomal protein motif. STAP is a cytoplasmic
protein according to the Reinhardt's criterion for cytoplasmic/nuclear
discrimination (24). This is supported by the immunoelectron
microscopic observations, which showed that STAP immunoreactivity is
ubiquitously seen in the cytoplasm of stellate cells. A further proof
hereof was the fact that STAP was only detected in cellular lysates and
not in the medium wherein stellate cells had been cultured (data not shown).
During the preparation of this manuscript, a protein whose amino acid
sequence is almost identical to that of rat STAP has been listed in the
protein data base (NCBI accession number BAB31709). The
corresponding cDNA was cloned from the brain of 13-day mouse embryo. The amino acid sequence of this protein (the mouse homologue of
STAP) has a 97% identity to rat STAP. We have already cloned the cDNA of human STAP, which showed a 90% identity to rat
STAP.2
Immunohistochemistry in the present report showed that STAP expression
is restricted to stellate cells in the liver parenchyma and
myofibroblast-like cells around the portal vein in the normal liver.
These cells are known to be able to express smooth muscle Our spectrophotometric analysis of recombinant rat STAP clearly
demonstrated that STAP is a heme protein. In general, heme proteins act
as one of the following three: (i) oxygen transporters like hemoglobin
and myoglobin, (ii) electron transporters like cytochrome P450, and
(iii) enzymes with peroxidase activity like horseradish peroxidase.
Although we have not proved it yet, STAP might be an intracellular
oxygen transporter because this protein has a 40% amino acid sequence
homology with myoglobin. This speculation is reasonable because the
oxygen-dependent cellular contractility is augmented in
activated stellate cells that are enriched in contractile proteins such
as actin and myosin (1-5, 12). This hypothesis is also supported
because the induction kinetics of STAP after the treatment with a
fibrosis inducer were similar to those of smooth muscle From a functional point of view, the induction of STAP in activated
stellate cells is understandable because stellate cells are
increasingly exposed to endogenous H2O2, lipid
hydroperoxides and trans-4-hydroxy-2-nonenal during chronic
liver trauma (36, 40, 41, 43). Scavenging of radical-derived organic
peroxides by STAP could be an adaptive reaction to normalize the
cellular redox status during the cell activation. STAP as well as
glutathione (43) could compensate the redox imbalance caused by a
down-regulation of glutathione S-transferases in the
activated stellate cells, which counteracts the toxic effects of lipid
peroxidation (42).
From a clinical point of view, STAP could be a suitable marker for
liver fibrosis and activated stellate cells in the histochemical study.
However, STAP is not a secreted protein and, therefore, cannot be used
as a serum marker. Our preliminary study reveled that STAP is evidently
expressed in stellate cells and also in diseased liver tissues of human
(data not shown).
In summary, we have isolated a novel protein named STAP from hepatic
stellate cells using proteomics. STAP was expressed in stellate cells
and myofibroblasts in the normal liver and is dramatically up-regulated
in the course of the stellate cell activation at both mRNA and
protein level. This suggests a close relationship between the STAP
induction and the liver fibrosis. Functional analysis using a
recombinant rat STAP revealed that this protein is a new heme protein
with a peroxidase activity against H2O2 and
linoleic acid hydroperoxides.
-actin, PDGF receptor-
, and neural cell
adhesion molecule. The expression of STAP protein and mRNA was
augmented time dependently in thioacetamide-induced fibrotic liver.
Immunoelectron microscopy and proteome analysis detected STAP in
stellate cells but not in other hepatic constituent cells. Biochemical
characterization of recombinant rat STAP revealed that STAP is a heme
protein exhibiting peroxidase activity toward hydrogen peroxide and
linoleic acid hydroperoxide. These results indicate that STAP is a
novel endogenous peroxidase catabolizing hydrogen peroxide and lipid
hydroperoxides, both of which have been reported to trigger stellate
cell activation and consequently promote progression of liver fibrosis.
STAP could thus play a role as an antifibrotic scavenger of peroxides
in the liver.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(I), collagen
1(III),
-actin, neural cell adhesion molecule (N-CAM), and smooth
muscle
-actin in accordance with previous reports (11-13), and that
of calcyclin, calgizzarin, and galectin-1, representing new findings
(8).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) following the manufacturer's protocol.
(PDGFR-
, Santa Cruz, CA), N-CAM (Dako, Glostrup, Denmark),
smooth muscle
-actin (Dako), or glial fibrillary acidic protein
(Dako) and then were incubated with peroxidase-conjugated secondary
antibodies. Immunoreactive bands were visualized by using ECL system
(Amersham Pharmacia Biotech).
-actin antibodies (1:500) or anti-STAP antibodies
(1:500) and then with biotinylated secondary antibodies (1:500, Dako),
followed by color development using an ABC kit (Vectastain, Burlinbame,
CA). For immunoelectron microscopy, 50-µm-thick sections were
immunostained for STAP in a similar way as described above, postfixed
in 1% osmium tetraoxide, dehydrated in ethanol, and embedded in
Polybed (Polyscience, Warrington, PA). Thin sections were stained with
saturated lead citrate and observed under a JEM-1200 EX electron
microscope (JEOL, Tokyo, Japan) at 100 kV (7).
-D-galactopyranoside (Wako Pure Chemical Co., Osaka, Japan). STAP was purified under native
conditions using nickel-NTA agarose resins (Qiagen, Valencia, CA).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of STAP from activated
stellate cells and its cDNA cloning. A,
two-dimensional SDS-PAGE analysis of proteins from quiescent
(a), in vivo activated (b), and
in vitro activated (c) stellate cells. Proteins
of these cells were extracted and analyzed by two-dimensional PAGE as
detailed previously (8). One protein spot (arrow) with pI
value of 6 and molecular mass of 21 kDa was heavily up-regulated
in both in vivo and in vitro activated stellate
cells. B, the amino acid sequence deduced from 120-bp
cDNA obtained by degenerative PCR. C, STAP cDNA and
its deduced amino acid sequence. STAP consisted of 190 amino acids
producing molecular weight of 21,496.
-actin (12), and PDGF receptor-
(25) in contrast to the constant
expression of glial fibrillary acidic protein (Fig.
2A). The augmented STAP expression was also seen in in vivo activated stellate cells
isolated from fibrotic liver induced by TAA administration for 8 weeks (Fig. 2B). RT-PCR showed that the expression of STAP
mRNA was consistently augmented in the process of the stellate cell
activation (Fig. 2, A and B).
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Fig. 2.
Expression of STAP in stellate cells.
A, stellate cells were activated time dependently by
cultivating them in Dulbecco's modified Eagle's medium containing
10% fetal bovine serum on plastic culture dishes. Expression of STAP,
N-CAM, PDGFR- , smooth muscle
-actin, and glial fibrillary acidic
protein was determined by Western blotting (W.B.). STAP
mRNA expression was determined by RT-PCR using
glyceraldehyde-3-phosphate dehydrogenase as a control of quantity of
RNA analyzed (RT-PCR). B, quiescent, in
vivo, and in vitro-activated stellate cells were
prepared as detailed previously (8). The expression of STAP was
determined at the protein (W.B.) and mRNA level
(RT-PCR). Quiescent stellate cells were those freshly
prepared from normal liver. In vivo activated stellate cells
were those freshly prepared from fibrotic liver treated with TAA for 8 weeks. In vitro activated stellate cells were those prepared
from normal liver and cultured for 7 days. C,
two-dimensional PAGE analysis of STAP expression in stellate cells,
liver tissues (total liver), endothelial cells, Kupffer cells, and
hepatocytes isolated from either normal control or fibrotic liver
treated with TAA for 8 weeks. Normal stellate cells faintly expressed
STAP (arrow) and its expression was up-regulated in the
fibrotic liver. STAP was not seen in the normal total liver tissues but
detectable in the fibrotic liver tissues. Other liver cells tested did
not express STAP in both normal and fibrotic conditions.
Arrows point to the positions where STAP could be seen if
STAP is expressed. D, immunoelectron microscopic
identification of STAP-expressing cells. Normal liver tissues were
subjected to the immunoelectron microscopic observation. a,
stellate cells (S) with lipid droplets showed the
black-colored immunoreaction homogeneously in the cytoplasm.
Endothelial cells (E) forming the sinusoidal wall were
negative for STAP. b, Kupffer cells (K) in the
sinusoidal lumen attaching to endothelial cells were negative for STAP.
c, myofibroblasts (M) in a portal area were
positive for STAP, but bile duct epithelial cells (Bi) and
hepatocytes (H) were negative. Magnification, ×5,000.
-actin, a marker of
activated stellate cells, was augmented in fibrotic liver particularly
along the fibrotic septum (compare Fig. 3A, b and
e). STAP immunoreactivity was strong around the portal area
and also weakly seen sporadically in the normal parenchyma (Fig.
3A, c). These weakly positive cells were
identified as stellate cells by their locations and morphology. The
reactivity was significantly increased particularly in the fibrotic
septa in the TAA-treated animals (Fig. 3A, f).
In situ hybridization revealed that STAP mRNA expression
was augmented in fibrotic liver exclusively along the septum but not in
the albumin mRNA-expressing hepatocytes (Fig. 3A,
g-j). A section of fibrotic liver tissues was used for in situ hybridization of STAP (Fig. 3A,
k) and was subsequently subjected to immunohistochemistry of
desmin, a stellate cell marker (Fig. 3A, l).
These double staining experiments showed a specific expression of STAP
in stellate cells in in vivo. Analyses using Western blot
and RT-PCR confirmed that the expression of STAP protein and mRNA
was augmented with increased severity of liver fibrosis (Fig.
3B).
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Fig. 3.
Expression of STAP in normal and
TAA-induced fibrotic liver tissues. A, Detection of
STAP expression by immunohistochemistry and in situ
hybridization. a-f, immunohistochemistry of normal
(a-c) and TAA-induced (8 weeks) fibrotic (d-f)
liver tissues. a and d, Azan-Mallory staining;
b and e, immunostaining of smooth muscle
-actin, an activation marker for stellate cells; c and
f, immunostaining of STAP. In normal liver, STAP expression
was seen along the sinusoid and around portal areas. In A,
c, an area enclosed by a square is enlarged 10 times and is
presented in the inset, which shows positive staining of STAP in a
stellate cell in intact parenchyma. In fibrotic liver, its expression
was greatly augmented along the fibrotic septum as that of smooth
muscle
-actin. g
j, in situ hybridization of STAP
mRNA. g, hematoxylin-eosin staining;
h, in situ hybridization of albumin mRNA;
i and j, in situ hybridization of STAP
mRNA using antisense probes (i) and sense probes
(j) in serial sections. Note that STAP mRNA was
expressed exclusively along the fibrotic septum (arrows) but
not seen in the albumin mRNA expressing hepatocytes. k
and l, STAP mRNA-expressing cells (k,
arrows) were identified as stellate cells because these
cells showed the positive signals in desmin immunostains (l,
arrows). k and l were photographs
obtained from the same section. Magnification, ×200 (a-j)
and ×600 (k and l). B, expression of
STAP protein and its mRNA in normal and fibrotic liver tissues
induced by TAA administration for 4 and 8 weeks. Liver tissues obtained
from two individual animals were tested for each group of experiments.
Note that the expression of STAP protein (W.B.) and mRNA
(RT-PCR) was increased as the treatment with TAA was
prolonged.
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Fig. 4.
Biochemical characterization of recombinant
rat STAP. A, the open reading frame of cloned rat STAP
cDNA was ligated into pTrcHis2 TOPO vectors.
His6-tagged STAP was produced in TOP10 cells by culturing
them overnight in LB medium supplemented with 1 mM
isopropyl-1-thio- -D-galactopyranoside. Native STAP was
purified using a nickel-NTA agarose column and run on SDS-PAGE
gels. Lane 1, molecular weight marker (M.W.);
Lane 2, bacterial lysate; Lane
3, flow through fraction; Lane 4, wash
1; Lane 5, wash 2; Lane 6,
eluate 1; Lane 7, eluate 2; Lane
8, eluate 3; Lane 9, eluate 4. An
arrowhead points to the position of STAP. B,
absorption spectra of rat recombinant STAP. Purified STAP (200 µg) in
PBS was photometrically characterized using a Hitachi spectrophotometer
U-2001 at pH 7.4 and 25 °C in the presence (dotted line) or absence
(line) of Na2S2O4. C,
heme staining of recombinant STAP. Purified STAP (10 µg) was
separated by 15% native PAGE gels and then transferred electrically
onto Immobilon-P membranes (a, Coomassie staining). After
washing with PBS twice, the membranes were treated with either
DAB/H2O2 solution (b) or ECL
chemiluminescence solution (c).
Peroxidase-dependent reaction products were detected on the
membranes (b) or on Kodak XAR5 films (c).
D, effect of recombinant STAP on linoleic acid oxidation.
Conjugated diene formation was initiated by adding 30 mM
FeCl2 into a reaction mixture of 30 mM NaCl, pH
7.0, 1 mM linoleic acid, 20 mM linoleic acid
hydroperoxide, and 0.16% Lubrol PX in the presence or absence of
recombinant STAP. The reaction was monitored at 234 nm using a Hitachi
U-2001 spectrophotometer without (a) and with 4 nM (b), 100 nM (c), and
200 nM (d) of recombinant STAP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and TGF-
, and reactive oxygen species as well as lipid
peroxides produced by intoxicated hepatocytes are thought to trigger
the stellate cell activation (1-5). This activation is initiated by
the activation of transcription factors, such as Sp-1 (27), Zf-9/KLF6
(6), and AP-1 (28), leading to the mRNA expression of extracellular
matrix matrices and tissue inhibitor of matrix metalloproteinase-1 and
-2 (29, 30). A variety of factors are up-regulated in the activated
stellate cells and thought to contribute to the development of fibrosis in a highly orchestrated manner, including receptors of growth factors
such as PDGFR-
and insulin-like growth factor receptor type I (31),
contractility related molecules such as smooth muscle
-actin,
endothelin-1, and endothelin receptors (32, 33), cell adhesion
molecules such as intercellular adhesion molecule 1 and integlins (34),
and cytokines such as monocyte chemotactic protein-1 (35) and
interleukin-10 (36).
-actin and
to store vitamin A (38, 39). In this context, STAP might be a suitable
new marker for retinol metabolizing myofibroblasts.
-actin as
shown in the present study.
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Acknowlegments |
---|
We thank Profs. S. Imaoka and M. Inoue for their valuable comments on this work. We also thank the following co-workers for their valuable technical support: N. Uyama and H. Okuyama, Kyoto University, and T. Mishima, N. Maeda, and H. Matsui, Osaka City University.
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FOOTNOTES |
---|
* This work was supported in part by Grant-in-aid from the Ministry of Education, Science and Culture of Japan 11670525.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. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ245663.
** To whom correspondence should be addressed: Developmental Biology Lab., Dept. of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashihiroshima, Hiroshima, 739-8526, Japan. Tel.: 81-824-24-7440; Fax: 81-824-24-1492; E-mail: kyoshiz@hiroshima-u.ac.jp.
Published, JBC Papers in Press, April 24, 2001, DOI 10.1074/jbc.M102630200
2 N. Kawada, D. B. Kristensen, K. Asahina, K. Nakatani, Y. Minamiyama, S. Seki, and K. Yoshizato, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; N-CAM, neural cell adhesion molecule; STAP, stellate cell activation-associated protein; TAA, thioacetamide; RT-PCR, reverse transcription-polymerase chain reaction; bp(s), base pair(s); PBS, phosphate-buffered saline; DAB, 3,3'-diaminobenzidine; PDGF, platelet-derived growth factor; TGF, transforming growth factor.
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