From the Departments of Gastroenterology and
¶ Endocrinology and Nephrology, University of Tokyo School of
Medicine, Tokyo 113-8655 and the
Institute for Molecular and
Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
Received for publication, August 16, 2002, and in revised form, November 20, 2002
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ABSTRACT |
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Pancreatic AR42J cells have the feature of
pluripotency of the precursor cells of the gut endoderm.
Dexamethasone converts them to exocrine cells or liver cells.
Using mRNA differential display techniques, we have identified a
novel Ca2+-dependent member of the
mitochondrial solute carrier superfamily, which is expressed during the
course of differentiation, and have designated it MCSC. The
corresponding cDNA comprises an open reading frame of 1407 base
pairs encoding a polypeptide of 469 amino acids. The
carboxyl-terminal-half of MCSC has high similarity with other mitochondrial carriers, and the amino-terminal-half has three canonical
elongation factor-hand motifs and has calcium binding capacity. The
deduced amino acid sequence revealed 79.1% homology to the rabbit
peroxisomal Ca2+-dependent member of the
mitochondrial superfamily, but the subcellular localization of the
protein was exclusively mitochondrial, not peroxisomal. Northern blot
and Western blot analyses revealed its predominant expression in the
liver and the skeletal muscle. In the liver, the expression level of
MCSC was higher in the adult stage than in the fetal stage, and MCSC
was highly up-regulated in dexamethasone-treated AR42J cells before the
expression of albumin. Taken together, MCSC may play an
important role in regulating the function of hepatocytes rather than in
differentiation in vivo.
Both endocrine and exocrine cells of the pancreas arise from
epithelial cells in the pancreatic duct (1, 2). Recently embryonic stem
cells have been shown to differentiate into insulin-secreting structures similar to the pancreatic islet (3), and embryonic endodermal cells included a bipotential precursor population for pancreas and liver (4). Pancreas originates from the dorsal and ventral
regions of the foregut endoderm (5), and liver develops from the
ventral foregut endoderm adjacent to where the ventral pancreas emerges
(6). This proximity of budding sites for the liver and the ventral
pancreas has led us to understand which molecular mechanisms control
the differentiation of these organs. Recent genetic studies indicate
that pancreatic development depends on an integrated network of
distinct transcription factors operating at various levels. A mouse
homeobox protein, insulin promoter factor-1 (IPF-1/PDX-1), is required
for the development of the pancreas (7). Islet-1, BETA2/NeuroD, Pax4,
and Pax6 are necessary for the development and generation of mature
islet cells (8-11). The basic helix-loop-helix (bHLH) protein PTF1-p48 is essential for the formation of exocrine and the correct spatial organization of endocrine pancreas (12). In an endoderm explant assay,
redundant fibroblast growth factor signaling from the cardiac mesoderm
is necessary and sufficient to induce hepatogenesis within the ventral
foregut endoderm (13).
Pancreatic AR42J cells are derived from a chemically induced pancreatic
acinar cell tumor and have the feature of pluripotency of the common
precursor cells of the pancreas (14, 15). We have shown that, when
these cells were treated with activin A (Act)1 and betacellulin (BTC)
or hepatocyte growth factor, they differentiate into insulin-producing
cells (16, 17). When exposed to dexamethasone (Dx), they become more
acinar-like cells (18). It has been shown recently that when exposed to
Dx for a long period, these cells synthesize albumin and have the
character of liver cells (19). In this way, AR42J cells resemble the
precursor cells in the gut, which differentiate into the pancreas and
the liver and provide an excellent in vitro model system to
study the differentiation of these organs. To clarify the molecular
mechanisms of differentiation, we used the method of mRNA
differential display and identified several genes that were up- or
down-regulated during differentiation (20). Among them, we have focused
on one gene that was highly up-regulated by Dx and characterized it.
This gene was predominantly expressed in the liver and the skeletal
muscle and was expressed at a higher level in the adult stage compared
with the fetal stage. It is a novel member of the
Ca2+-dependent mitochondrial carrier superfamily.
Materials--
Recombinant human Act was provided by Dr. Y. Eto
of Central Research Laboratory, Ajinomoto Inc. (Kawasaki, Japan).
Recombinant human BTC (21) was generously provided by Dr. M. Seno of
Okayama University (Okayama, Japan).
Cell Culture--
Hep3B cells were provided by the Cell Resource
Center for Biomedical Research, Institute of Development, Aging, and
Cancer, Tohoku University (Sendai, Japan). Hep3B cells were cultured in Dulbecco's modified Eagle's medium containing 20 mmol/liter
HEPES/NaOH (pH 7.4), 5 mmol/liter NaHCO3, and 10% fetal
bovine serum at 37° under a humidified condition of 95% air and 5%
CO2. For staining, cells were grown on non-coated coverslips.
Northern Blotting--
20 µg of total RNA extracted from AR42J
cells were denatured and blotted onto a Hybond N+ nylon
membrane (Amersham Biosciences). Human MTN Blot and Human Digestive
System 12-Lane MTN Blot were purchased from
Clontech (Palo Alto, CA). The blots were hybridized
with 32P-labeled cDNA probe and washed for 30 min under
high stringency conditions (0.1× standard saline citrate (SSC), 0.1%
SDS) or low stringency conditions (2× SSC, 0.1% SDS) at 65° before
exposure to x-ray film.
Cloning of Mitochondrial Ca2+-dependent
Solute Carrier (MCSC) cDNA--
From mRNA differential
display, we obtained a 131-bp DNA fragment containing a putative
poly(A) tail and a polyadenylation signal (20). cDNA was amplified
using a gene-specific primer and a 5'-end-specific vector primer. Rat
lung oligo(dT)-primed Construction of MCSC Expression Vector--
To construct the
MCSC expression vector, we amplified a fragment of MCSC (nucleotides
91-1507) by PCR using the sense primer (5'-ataagaatgcggccgctctcaccagcatgctctgcc-3') and the antisense primer
(5'-ccgctcgagtcatcaccgagactgcacgcccag-3'), followed by XhoI
and NotI digestion. The fragment was subcloned into the
XhoI/NotI sites of the vector pcDNA3.1/His
(Invitrogen) and was verified by sequencing. pEGFP-Peroxi vector was
purchased from Clontech. For transfection, FuGENE 6 transfection reagent (Roche Molecular Biochemicals) was used according
to the manufacturer's instruction. Forty-eight hours after
transfection, cells were fixed and stained.
Western Blotting--
Protein extracts from rat liver, pancreas,
stomach, colon, and skeletal muscle were prepared for immunoblotting as
described previously (22). All rat experiments were conducted in
accordance with the National Institutes of Health Guidelines for the
Care and Use of Laboratory Animals and were approved by the University of Tokyo Institutional Animal Care and Use Committee. A polyclonal antibody against MCSC was raised by immunizing rabbits with peptide (C)KISEQQAEKILKSM (residues 113-126) conjugated with keyhole limpet hemocyanine. The antiserum was purified with a PD10 gel column (Amersham Biosciences). For Western blotting, membranes were blocked by
incubation for 1 h with 10% blocking ace (Snow Brand, Japan) in
phosphate-buffered saline (PBS). After blocking, the membranes were
incubated with anti-MCSC antibody (1:100 dilution) for 1 h at room
temperature, washed three times with PBS containing 0.1% Tween 20 for
10 min, and incubated for 30 min with horseradish peroxidase-conjugated
goat anti-rabbit whole immunoglobulin (Nordic, 1:500 in PBS, 0.1%
Tween 20). After washing, the enhanced chemiluminescence Western
blotting detection reagent (Amersham Biosciences) was added, and the
reaction was allowed to proceed according to the manufacturer's
recommendations. Exposure to x-ray film was applied 1-3 min at room
temperature. To remove the probe, membranes were incubated with the
stripping buffer containing 62.5 mM Tris-HCl (pH 6.7) and
2% SDS for 30 min at 50°. Anti-actin-(1-19) antibody was purchased
from Santa Cruz Biotechnology.
Immunocytochemistry--
For immunostaining, cells were grown on
non-coated glass coverslips. Cells were fixed for 30 min in 3%
paraformaldehyde in PBS, treated with 0.1% (v/v) Triton X-100 in PBS
for 5 min, and incubated sequentially with Blocking Ace (Snow Brand,
Tokyo, Japan), first antibody, and second antibody. Anti-Xpress
Antibody (Invitrogen) was used to stain the exogenous MCSC. Second
antibodies used in this study are indocarbocyanine (Cy3)-conjugated
donkey anti-rabbit IgG, FITC-conjugated donkey anti-rabbit IgG, and
FITC-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories,
West Grove, PA). MitoTracker Red CMXRos (Molecular Probes, Eugene, OR)
and 4',6-diamidino-2-phenylinodole,dihydrochloride (DAPI) (Molecular Probes, Eugene, OR) were used to stain mitochondria and nucleus respectively. The cells were examined under a light microscope (Axiophoto; Carl Zeiss, Inc., Thornwood, NY).
Analysis of mRNA by Reverse Transcriptase-PCR--
Total RNA
was extracted from rat liver by using TRIzol Reagent (Invitrogen).
One-step RT-PCR was performed using SuperscriptTM One-Step
RT-PCR with Platinum Taq (Invitrogen) according to the manufacturer's instruction. For semiquantitative RT-PCR,
first-stranded cDNA was synthesized using SuperscriptTM
first-stranded synthesis system for RT-PCR (Invitrogen) according to
the manufacturer's instruction. Oligonucleotide primers used were
5'-tgtgctagtttcccaggaacc-3' (nucleotides 10-30) and
5'-tcagcaggaccagcccagcg-3' (nucleotides 1671-1652) for the whole
coding region of MCSC (1662-bp PCR product); 5'-tgtgagcatcagctacgtgg-3'
(nucleotides 1447-1466) and 5'-ggttccaggttctagcactag-3' (nucleotides
2167-2147) for the semiquantitative RT-PCR of MCSC (721 bp PCR
product); and 5'-tgagagggaaatcgtgcgtg-3' (nucleotides 612-631) and
5'-gatccacatctgctggaaggtg-3' (nucleotides 1071-1051) for Expression and Purification of the Amino-terminal Fragment of
MCSC--
To construct the amino-terminal fragment of MCSC, we
amplified a fragment of MCSC (residues 1-178) by PCR using the sense primer (5'-ccgctcgagcatgctctgcctgtgcctgtatg-3') and the antisense primer (5'-ccgctcgagcactgtgaactcatctgggac-3'), followed by
XhoI digestion. The fragment was subcloned into the
XhoI site of the vector, pGEX-4T-3 (Amersham Biosciences)
and was verified by sequencing. Escherichia coli strain BL21
was transfected with the final construct or pGEX-4T-3. The purification
of the GST fusion protein was essentially done according to the
recommendation of the manufacturer of glutathione-Sepharose 4B
(Amersham Biosciences).
45Ca2+ Overlay--
Proteins were
resolved in SDS-PAGE (10%) and transferred to a nitrocellulose
membrane. 45Ca2+ overlay was then performed
essentially as described (23). The membrane was washed in a solution
containing 60 mmol/liter KCl, 5 mmol/liter MgCl2, and 10 mmol/liter imidazole-HCl (pH 6.8) for 1 h with three changes of
the buffer. Then the membrane was incubated in the same buffer
containing 10 µCi of 45Ca2+/ml for 10 min.
After incubation, the membrane was rinsed twice with 40% ethanol for 5 min. Excess water was absorbed between two sheets of Whatman no. 1 filter paper, and the membrane was dried at room temperature and
exposed to x-ray film at Molecular Cloning of Rat MCSC--
In mRNA differential
display, we identified several novel genes up-regulated by more than
10-fold when AR42J cells were exposed to Dx (20). We only had small
fragments of the 3'-untranslated region (UTR) with which to obtain the
full-length cDNA of these genes. During the cloning
experiments, some genes were recorded in the GenBankTM data
base as glutaredoxin, adenine nucleotide translocator, and so on. We
picked up one gene, which was highly up-regulated when exposed to Dx,
and the size of mRNA was estimated to be about 3.5 kbp according to
Northern blot analysis (Fig. 1). First we obtained the 131-bp 3'-fragment containing a putative poly(A) tail and
a polyadenylation signal. Using the technique of gene walking by PCR
and a BLAST search, we obtained the 3136-bp cDNA, which comprises
an open reading frame of 1407 bp, a 100-bp 5'-UTR, and a 1638-bp 3'-UTR
(Fig. 2A). Computer Analysis of the Primary Sequence--
Searching the
protein data base with the deduced amino acid sequence revealed
conservation with the rabbit peroxisomal
Ca2+-dependent solute carrier (Efinal,
GenBankTM accession no. T50686): 61.6% identity and 79.1%
similarity (Fig. 2B), and Homo sapiens
calcium-binding transporter (GenBankTM accession no.
AF123303): 58.2% identity and 73.0% similarity. However, the first 51 amino acids have no homology with other proteins in the data base. The
amino-terminal-half contains three well-conserved
Ca2+-elongation factor (EF)-hand binding loops (residues
60-72, 91-103, and 127-139). These EF-hand domains are also
conserved in Efinal (24). The carboxyl-terminal-half has substantial
similarity with proteins of the mitochondrial solute carrier family
(16.4-21.9% identity) (Fig. 2C). The hydropathic profile
of this region revealed six transmembrane domains, similar to other
mitochondrial carriers (data not shown). We designated this protein as
MCSC. Fig. 2D depicts the phylogenetic tree of MCSC, Efinal,
and other rat mitochondrial carriers. Recently human KIAA1896 protein
(GenBankTM accession no. AB067483) has been reported in the
data base (26). Residues 137-568 of this protein have 93.7% identity
and 95.8% similarity with residues 50-469 of MCSC. With its strong homology, the KIAA1896 protein may be a human ortholog of MCSC, but the
first 136 amino acids has no relationships with the first 49 amino
acids of MCSC (Fig. 3). There may be an
alternative splicing of MCSC but only one transcript could be seen on
the Northern blot (Figs. 4 and 5).
KIAA1896 may be an isoform encoded by
different genes. We have to exclude the possibility of the merger of
two cDNAs in MCSC cDNA. Using a rat liver total RNA as a
template, we can amplify the whole coding region by RT-PCR (Fig.
6). Recently mouse clone IMAGE: 4239441 (GenBankTM accession no. BC019978) and IMAGE: 5098924 (GenBankTM accession no. BC022114) have been recorded in
the data base. At the nucleotide level these have 93.7% identity
through nucleotides 61-2401 of MCSC and 99.5% identity at the protein
level. These genes were predicted to be the mouse gene of MCSC but are
103 amino acids shorter than MCSC. This may result from the
misidentification of the initiation codon. These are translated from
the second ATG codon of their sequences. When translated from the first
ATG codon, 103 amino acids are added, and there is a 99.1% identity over the whole region at the protein level (Fig. 3). During the preparation of this article, another version of the KIAA1896 protein was reported by NCBI Annotation Project (GenBankTM
accession no. XP 027668). This predicted KIAA1896 protein sequence was
almost identical to that of MCSC over the whole coding region: 97.0%
identity and 98.7% similarity. This gene might be the real human
ortholog of MCSC. Some other proteins with high similarity to MCSC were
reported in the data base: GenBankTM accession no.
AAH05163, 95.2% similarity (H. sapiens, 311 amino acids);
AAH37109, 88.1% similarity (mouse, 514 amino acids); BAB70825, 71.4%
similarity (H. sapiens, 384 amino acids); XP 131097, 59.4%
similarity (mouse, 475 amino acids, may be a mouse ortholog of Efinal).
These proteins have high similarities at the carboxyl terminus of MCSC
but have low similarities at the amino terminus. In particular, there
are no similarities between the first 51 amino acids of MCSC and these
proteins.
Tissue Distribution of MCSC mRNA--
Northern blot analysis
was carried out to examine the tissue distribution of MCSC. Samples of
poly(A) mRNA isolated from human tissues were analyzed with labeled
probe specific for MCSC. MCSC was expressed predominantly in the liver
and the skeletal muscle as a single transcript (Fig. 4). With a long
exposure time, weak levels of MCSC expression were detected in all the
tissues examined (data not shown). Efinal was highly expressed in the
colon, followed by small intestine and kidney. In the small intestine,
the expression of Efinal was the lowest in duodenum and increased
toward the distal part of the intestine (25). We then examined the
expression level of MCSC in digestive tract using the Human Digestive
System 12-Lane MTN BLOT. Very weak levels of expression, compared with the liver, were detected through the esophagus to the rectum (Fig. 5).
Expression of Endogenous MCSC--
Western blot analysis was then
carried out to clarify endogenous MCSC protein expression in various
rat tissues. The expression with a molecular mass of 53 kDa was
detected in skeletal muscle and liver, consistent with Northern blot
analysis (Fig. 7).
Subcellular Localization of MCSC Protein in Hep3B Cells--
To
investigate the subcellular localization of MCSC protein, we performed
immunofluorescence study using a specific antibody to MCSC. In the
cytoplasm of Hep3B cells, both punctate and filamentous patterns were
observed, consistent with MCSC protein being present in intracellular
organelles (Fig. 8A). We could
not eliminate nuclear staining, but when we expressed the MCSC protein
ectopically using the expression vector MCSC-pcDNA3.1/His, we could
see immunostaining only in the cytoplasm (Fig. 8B). So we
believe nuclear staining is nonspecific. Omission of the primary
antibody resulted in the complete absence of immunostaining (data not
shown).
To confirm that MCSC-positive cytoplasmic organelles were
mitochondrial, we treated Hep3B cells with mitochondrial-specific fluorescent dye MitoTracker Red CMXRos to label mitochondria in the first antibody incubation step. As shown in Fig. 8C, the
MCSC staining pattern overlapped with that of mitochondria,
demonstrating that MCSC is targeted to mitochondria.
Efinal is mainly localized to peroxisomes, and some are expressed in
mitochondria (25). We then examined whether MCSC is targeted to
peroxisomes. When the pEGFP-Peroxi vector is transfected to cells, it
specifically targets to peroxisomal membranes. As shown in Fig.
8D, the staining pattern of MCSC and peroxisomes are not
superimposable, indicating that MCSC is not expressed in peroxisomes.
In rat liver strong immunoreactivity against MCSC was
observed in the cytoplasm as punctate and round patterns compatible with its expression in mitochondria of hepatocytes (Fig.
8E).
Ca2+ Binding Properties of MCSC--
Since the amino
terminus of MCSC contains three canonical EF-hands (residues 60-72,
91-103, and 127-139) that are predicted to bind calcium, we expressed
a GST-tagged truncated version of MCSC (residues 1-178) in E. coli and purified it to homogeneity (Fig.
9A). The calcium binding
ability of the polypeptide was demonstrated with a
Ca2+-overlay method. As shown in Fig. 9B, a
truncated version of MCSC exhibits an affinity to calcium even in the
SDS-denatured state.
Comparison of the Expression Level of MCSC--
MCSC was
up-regulated when AR42J cells were incubated with Dx and predominantly
expressed in liver and skeletal muscle. Because MCSC might be involved
in the differentiation of hepatocytes, we compared the expression level
in the fetal stage (18.5-day postcoitum (dpc)), the neonatal stage
(2-day-old), and the adult stage (9-week-old). As shown in Fig.
10A, the expression level of
MCSC was the greatest in the adult stage. When we compared the mRNA
level, the expression level of MCSC was higher in the adult stage
(9-week-old) than in the fetal stage (18.5 dpc) (Fig. 10, B
and C).
In the present study, we have identified a novel cDNA, MCSC,
encoding a protein with 469 amino acids belonging to a
Ca2+-dependent member of the mitochondrial
transporter superfamily. Mitochondrial carriers have a tripartite
structure, made up of related sequences about 100 amino acids in length
(27). Each repetitive element contains two hydrophobic stretches
separated by an extensive hydrophilic regions (I-II, III-IV, and V-VI)
(27). The carboxyl-terminal-half of MCSC is compatible with these
criteria and has a homology with other mitochondrial carrier proteins
(Fig. 2C).
An intriguing feature of MCSC is the presence of three EF-hand-like
domains in the amino terminus (Fig. 2B). The structural similarity by itself does not prove that EF-hand like domains of MCSC
bind calcium, but the strong homology to Efinal, and the Ca2+ binding capacity of Efinal strongly suggest that
EF-hand like domains of MCSC bind calcium. Another Ca2+
binding member of mitochondrial carrier superfamily, Aralar
(GenBankTM accession no. Y14494) was reported and was
present in human muscle and brain (28). There are four EF-hand-like
domains in the amino-terminal-half and a tripartite structure in the
carboxyl-terminal-half of Aralar. There is a 16.8% identity between
MCSC and Aralar, and the amino-terminal-half of Aralar also has calcium
binding activity (28). We examined the Ca2+ binding
properties of MCSC with a truncated protein, and the amino terminus of
MCSC clearly showed Ca2+ binding activity (Fig. 9).
Mitochondrial carriers are encoded by nuclear genes and have to be
imported into the mitochondrial membranes. Like most mitochondrial carriers except phosphate and citrate carriers in mammals (29, 30),
MCSC has no obvious amino-terminal mitochondrial import sequence. The
carboxyl-terminal Ser-Lys-Leu (SKL) sequence was the first recognized
peroxisomal-targeting sequence (31). It is not present in MCSC. But
without it, Efinal is mainly targeted to the peroxisomal membrane (28).
Localization studies presented here show clearly that MCSC is expressed
in mitochondria, not in peroxisome (Fig. 8, C and
D). When expressed ectopically, MCSC was also located in
mitochondria (Fig. 8B). MCSC has three EF-hand-like domains
in the amino terminus, and intracellular Ca2+ concentration
may be related to the localization of MCSC. In rat hepatocytes,
vasopressin, endothelin, and angiotensin II elevate intracellular
Ca2+ concentration. When we add these secretagogues to the
culture medium in Hep3B cells, there are no significant changes in the MCSC staining pattern (data not shown).
AR42J cells were originally derived from a rat pancreatic acinar cell
tumor. They possess both exocrine and neuroendocrine properties (14).
Synthetic glucocorticoid, Dx, can increase the expression of amylase in
AR42J cells and enhance the differentiation of AR42J cells toward the
exocrine phenotype (18). When a subclone of AR42J cells, AR42J-B13, was
incubated for more than 9 days with dexamethasone, the expression of
amylase was almost gone, and the expression of albumin was observed,
indicating that the cells converted into hepatocytes (19). Dx also
induced the transdifferentiation of organ cultures of pancreatic buds
from mouse embryo into hepatocytes (19). The liver and the pancreas
originate from neighboring regions of the foregut endoderm (5, 6), and
some molecular and cellular mechanisms are regulating the development
of these two organs.
MCSC was highly up-regulated when AR42J cells were incubated with Dx
for 3 days (Fig. 1), and MCSC was predominantly expressed in the liver
and skeletal muscle (Fig. 4). We therefore suggest that MCSC was
up-regulated during the differentiation of AR42J cells toward
hepatocytes rather than exocrine pancreas. Glucose-6-phosphatase is
normally found in the liver as well as in the In the study of mRNA differential display, we have identified other
mitochondrial carriers up- or down-regulated over the course of
differentiation. Adenine nucleotide translocator was up-regulated when
incubated with Dx, and citrate transporter-like protein was
up-regulated when incubated with Act + BTC.2 Similar to the
importance of the ATP/ADP ratio in insulin secretion of pancreatic
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAPII cDNA library (Stratagene, La Jolla,
CA) was used as a template. We first obtained a 956-bp cDNA
fragment. A BLAST search using this fragment led to the identification
of an expressed sequence tag from rat ovary mRNA
(GenBankTM accession no. D86666), and we could extend the
cDNA to 1128 bp. We went on gene walking by polymerase chain
reaction (PCR) using a rat lung and brain cDNA library as a
template. Finally we recovered the 3136-bp cDNA, which contained
the 1407-bp open reading frame. The entire sequence of both strands was
determined using an ABI PRISM BigDye Terminator Cycle Sequencing Ready
reaction kit and an Applied Biosystems DNA sequencer 310 (Applied
Biosystems, Cambridge, MA).
-actin
(GenBankTM accession no. V01217, 460-bp PCR product). PCR
cycles for semiquantitative RT-PCR were 25 for MCSC and 20 for
-actin.
80°.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
There may be additional regions of the 5'-UTR to be cloned; however,
there is an in-frame stop codon in the 5'-UTR of our sequence. The
sequence just before the initiation codon is in good agreement with the
Kozak sequence (24). A consensus polyadenylation signal (AATAAA),
nucleotides 3115-3120, was located 15 bases before the poly(A) tail.
The open reading frame encodes a predicted polypeptide of 469 amino
acids with a calculated molecular mass of 52.7 kDa.
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Fig. 1.
Northern blot analysis of MCSC expression in
AR42J cells. Total RNA (20 µg) isolated from naive,
dexamethasone-treated, Act-treated, and ACT + BTC-treated cells was
blotted onto a nylon membrane and probed with 32P-labeled
cDNA. The bars indicate the relative positions of 28 S
and 18 S ribosomal RNA. The blot was reprobed with
32P-labeled rat glyceraldehyde-3-phosphate dehydrogenase
cDNA (lower panel).
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Fig. 2.
cDNA and protein sequences of MCSC.
A, cDNA and protein sequences of MCSC are shown. The
putative open reading frame is indicated in uppercase
letters, and 5'-UTR and 3'-UTR are indicated in lowercase
letters. The in-frame stop codon in 5'-UTR and the possible
polyadenylation signal are underlined. B,
comparison of the predicted amino acid sequence of MCSC and Efinal. An
asterisk indicates identity, a colon indicates
conservatively substituted, and a period indicates
similarity. Three well conserved EF-hands are indicated with
horizontal lines. Amino acid sequences were aligned with
ClustalW (version 1.8). C, alignment of MCSC with other rat
mitochondrial carrier sequences. The sequences shown are solute carrier
family 25, member 5 (adenine nucleotide translocator 2, fibroblast
form; Slc25a5: D12771); solute carrier family 25, member 4 (adenine
nucleotide translocator; Slc25a4: D12770); dicarboxylate carrier
(CAA11278); solute carrier family 25, member 1 (citrate
transporter: NM017307); and oxodicarboxylate carrier
(odc gene: AJ289714). Six predicted transmembrane domains
are indicated by horizontal lines; their locations are based
on human ADP/ATP translocase (AAC2 isoform; M57424) transmembrane
domain positions. Amino acid sequences were aligned with ClustalW
(version 1.8). D, phylogenetic tree of MCSC, Efinal, and the
members of rat mitochondrial transporter superfamily. The scale shows
the evolutionary distances calculated.
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Fig. 3.
Comparison of protein sequences between MCSC,
KIAA1896, and mouse IMAGE clones. Open rectangles and
dashed rectangles indicate homologous and non-homologous
regions, respectively. Dotted rectangle indicates added
amino acids when translated from the first ATG codon in the IMAGE
clone. Numbers on the rectangles indicate the number of
amino acids of the proteins.
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Fig. 4.
Northern blot analysis of MCSC expression in
various tissues. mRNA (2 µg) isolated from various tissues
was blotted onto a nylon membrane, and Northern blotting was performed
using 32P-labeled MCSC cDNA. The blot was reprobed with
32P-labeled -actin (lower panel).
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Fig. 5.
Northern blot analysis of MCSC expression in
the digestive tract. mRNA (2 µg) isolated from various
tissues was blotted onto a nylon membrane, and Northern blotting was
performed using 32P-labeled MCSC cDNA. In the
transverse colon lane, we could not remove the strong stain. The blot
was reprobed with 32P-labeled -actin (lower
panel).
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Fig. 6.
RT-PCR of the whole coding region of
MCSC. One-step RT-PCR was performed using rat liver total RNA (1 µg) as a template. Samples with (lane 1) or without
(lane 2) reverse transcriptase were loaded. The right
lane is a size marker of HindIII-digested DNA. The
band was confirmed by sequencing.
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Fig. 7.
Western blotting of endogenous MCSC protein
in various rat tissues. An aliquot of protein (100 µg) from
various rat tissues was applied to each lane and blotted with a
specific antibody for MCSC. The membrane was reprobed by using
anti-actin antibody (lower panel).
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Fig. 8.
Localization of MCSC in Hep3B cells and the
liver. A, localization of MCSC (red) and
4',6-diamidino-2-phenyindole (DAPI, blue) in Hep3B cells.
B, Hep3B cells were transfected with MCSC-pcDNA3.1/His
and stained with anti-Xpress antibody. C, localization of
MCSC (green), Mitotracker Red CMXRos (red), and
DAPI (blue) in Hep3B cells. D, localization of
MCSC (red), pEGFP-Peroxi (green), and DAPI
(blue) in Hep3B cells. Cells were transfected with
pEGFP-Peroxi vector. E, immunohistochemistry in rat
liver. Merge indicates the overlaying images using Adobe
Photoshop 5.0.
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Fig. 9.
Calcium binding activity of the amino
terminus of MCSC. A, Coomassie Brilliant Blue-stained
SDS-PAGE (10%) gel. Shown is E. coli lysate before
(lane 2) and after (lane 1) induction with
isopropyl -D-thiogalactopyranoside. In lanes
3 and 4, 2 and 5 µg of purified GST fusion protein
were loaded. In lane 5, purified GST protein from the mock
vector-transfected E. coli was loaded. B, same
amount of proteins as shown in A was resolved and
transferred onto a membrane. The membrane was labeled with
45Ca2+ as described and was exposed to x-ray
film.
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Fig. 10.
Comparison of the expression levels of MCSC
in the fetal, neonatal, and adult stages. A, an aliquot
of protein (20 µg) from rat liver in the fetal (18.5 dpc), neonatal
(2-day-old), and adult (9-week-old) stages were applied to each lane
and blotted by a specific antibody for MCSC. The membrane was reprobed
with anti-actin antibody (lower panel). B,
mRNA for MCSC was compared in the fetal (18.5 dpc) and adult
(9-week-old) stages. Samples with or without RT treatment were loaded.
The right end lane is a 100-bp ladder size marker. Primers
for -actin span two introns, and contaminated genomic DNA, if any,
should be detected at a position of 672 bp. C, mRNA for
the whole coding region of MCSC was compared as in B. The
right lane is a size marker of HindIII-digested
DNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells of pancreas and
is one of the earliest differentiation markers.
Glucose-6-phosphatase-positive cells could be seen after 3 days of
addition of Dx in AR42J-B13 cells (19) when MCSC was up-regulated. In
contrast, albumin, a liver-specific protein, started to appear after 9 days (19). Taken together, MCSC may play a role during the
differentiation toward hepatocytes. Fibroblast growth factors have been
implicated in the primary induction of the liver (6). It remains to be seen whether fibroblast growth factors regulate the expression of MCSC.
Western blotting and RT-PCR study showed that the expression level of
MCSC was higher in the adult stage compared with the fetal stage and
the neonatal stage (Fig. 10) indicating that MCSC may play an important
role in regulating the function of hepatocytes rather than the
differentiation in vivo.
-cells, the cellular environment resulting from the functions of
these mitochondrial proteins including MCSC may be important in
regulating the function and the differentiation of pancreas and liver.
MCSC may act as a calcium binding/transducing protein by the transport
of a solute. Further studies are needed to confirm our speculations.
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ACKNOWLEDGEMENT |
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We thank Dr. Yositaka Konda of the Department of Gastroenterology, Kyoto University for helpful discussions.
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FOOTNOTES |
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* This study was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Sports and Culture of Japan and grants from the Takeda Science Foundation and Pancreatic Research Foundation of Japan.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/EBI Data Bank with accession number(s) AY043169.
§ To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 37194); Fax: 81-3-5800-9738; E-mail: hmashima1-tky@umin.ac.jp.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M208398200
2 H. Mashima, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Act, activin A; BTC, betacellulin; MCSC, mitochondrial Ca2+-dependent solute carrier; PBS, phosphate-buffered saline; UTR, untranslated region; dpc, day postcoitum; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; Dx, dexamethasone.
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