From the Institute of Pathology and
Department
of Internal Medicine I, University of Regensburg, 93053 Regensburg, Germany, ** Institute of Pathology, University of
Bonn, 52127 Bonn, Germany, and the ¶ Department of Molecular
Medicine, MPI of Biochemistry, 82152 Martinsried, Germany
Received for publication, December 11, 2002, and in revised form, February 11, 2003
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ABSTRACT |
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The novel human gene MIA2
encoding a melanoma inhibitory activity (MIA) homologous protein was
identified by a GenBankTM search. MIA2,
together with MIA, OTOR, and TANGO,
belongs to the novel MIA gene family sharing important
structural features, significant homology at both the nucleotide and
protein levels, and similar genomic organization. In
situ hybridization, reverse transcriptase-PCR, and Northern blots
presented a highly tissue-specific MIA2 expression pattern in the
liver. Promoter studies analyzing transcriptional regulation of MIA2
revealed an HNF-1-binding site at position Melanoma inhibitory activity
(MIA)1 protein was identified
previously within growth-inhibiting activities purified from the tissue
culture supernatant of the human melanoma cell line HTZ-19 (1). MIA is
translated as a 131-amino acid precursor molecule and is processed into
a mature 107-amino acid protein after cleavage of a hydrophobic
secretion signal. The position within the human genome was mapped to
chromosome 19q13.32-13.33 (2). MIA mRNA was identified
independently by differential display approaches comparing melanoma
cell lines and also comparing differentiated and dedifferentiated
cartilage cells in vitro. Therefore, MIA has also been
referred to as cartilage-derived retinoic acid-sensitive protein (3).
Subsequent studies of murine embryos and murine adult tissues
demonstrated specific mRNA expression patterns in cartilage but not
in any other non-neoplastic tissue (4). Functionally, MIA was initially
purified and described to exert antitumor activity by inhibiting
proliferation of melanoma cell lines in vitro (5). However,
further studies revealed expression patterns inconsistent with a tumor
suppressor. Expression of the wild-type MIA protein gene was not
detected in normal skin and melanocytes but was associated with
progression of melanocytic tumors. More recently, it was suggested that
the MIA protein specifically inhibits attachment of melanoma cells to
fibronectin and laminin and thereby masks the binding site of integrins
to these extracellular matrix components and promotes invasion in
vitro (6). Additional in vivo studies revealed the
importance of MIA for metastasis of malignant melanomas (7, 8).
Furthermore, we and others (6, 9) have shown that MIA adopts an SH3
domain-like structure and interacts directly with fibronectin.
Recently, an MIA homologous protein, OTOR (FDP, MIAL), was
identified and mapped to chromosome 20p11. For this protein a homology of 59% to MIA was determined. OTOR expression was reported to be
highly tissue-specific and restricted to the cochlea and eye (10-12).
The detection of OTOR stimulated us to search for
further homologous proteins. Here we describe a novel MIA homologue,
designated MIA2, and we define its expression pattern and regulation in hepatocytes.
Interestingly, we found specific expression in the liver and mechanisms
of transcriptional regulation that identify MIA2 as a potential novel
acute phase protein. Furthermore, our analysis of the expression levels
of MIA2 in patients with chronic hepatitis C infection may suggest that
MIA2 can serve as a marker of hepatic disease activity and severity.
Cell Lines and Tissue Culture--
The following cell lines were
used: Mel Im, Mel Ei, Mel Wei, SK-Mel 28, Mel Ho (DSMZ ACC 62),
HTZ-19d, Mel Ju, Mel Juso (DSMZ ACC 74) (human melanoma (13)), human
primary melanocytes NHEM (14), HeLa (human cervix carcinoma, ATCC
CRL-7923), HepG2 (ATCC HB-8065), HCT 116 (ATCC CCL-247), CaCo-2 (ATCC
HTB-37), SW48 (ATCC CCL-231), LoVo (ATCC CCL-229), SW480 (ATCC
CCL-228), HT29 (ATCC HTB-38) (human colon carcinoma cell lines), U266
(B-lymphocytes, ATCC TIB-196), and Jurkat (T-lymphocytes, ATCC
TIB-152).
Cells were grown at 37 °C in 5% CO2 in Dulbecco's
modified Eagle's medium (Invitrogen) supplemented with penicillin (100 units/ml), streptomycin (10 µg/ml) (both Sigma), and 10% fetal calf
serum (Invitrogen) and split 1:2 at confluence. Cells were detached by
incubation with 0.05% trypsin, 0.04% EDTA (Sigma) in
phosphate-buffered saline for 5 min at 37 °C.
Primary human and murine hepatocytes, hepatic stellate cells (HSC), and
Kupffer cells were isolated from human liver specimens obtained during
resection of metastasis of non-hepatic tumors or rat livers,
respectively. Exclusion criteria were known liver disease or
histological evidence for liver fibrosis or inflammation in surrounding
non-tumorous liver tissue. The cell isolation and subsequent culturing
was performed as described before (15-17). HSC were activated by
culturing on plastic for 14 days (17).
Primary human fibroblasts were isolated and cultured as described
previously (18).
Stimulation of Cells--
HepG2 cells were treated with the
following cytokines: TNF-
To obtain conditioned medium of activated human HSC, the cells were
seeded in T75 culture flasks (BD Biosciences) and cultured in
serum-free medium for 24 h (12 ml/T75 flask). Some cells were additionally treated with LPS (10 µg/ml) for the 24-h incubation period. 12 ml of pure HSC medium or medium supplemented with LPS (10 µg/ml) was filled into empty T75 flasks and incubated in parallel for
24 h to serve as controls. Supernatants and controls were collected and saved at Patients and Controls--
Subjects included 11 patients (9 men
and 2 women; age, 20-52 years, median age, 35.5) with chronic
hepatitis C infection (positive for HCV-RNA and anti-HCV) and no prior
interferon treatment. Exclusion criteria were co-infections with human
immunodeficiency virus, hepatitis B virus, or other concomitant liver
disease. Liver biopsies were taken, and histological staging and
grading was performed according to the score proposed by Desmet
et al. (19). Parts of the biopsies were used for RNA isolation.
Human liver specimens obtained during resection of metastasis of
non-hepatic tumors served as controls. Exclusion criteria were known
liver disease or histological evidence for liver fibrosis or
inflammation in surrounding non-tumorous liver tissue. RNA was isolated
from surrounding non-tumorous liver tissue.
RT-PCR Analysis--
For RT-PCR total cellular RNA was isolated
from cultured cells, from multiple tissues of C57BL/6 mice, or from
human liver tissue using the RNeasy kit (Qiagen, Hilden, Germany). The
integrity of the RNA preparations was controlled on an 1%
agarose/formaldehyde gel, and subsequently cDNAs were generated by
reverse transcription. First strand cDNA was synthesized using 2 µg of the isolated total RNA, 1 µg of random primer (Amersham
Biosciences), 4 µl of 5× First Strand Buffer (Invitrogen), 2 µl of
10 mM dithiothreitol, 1 µl of 10 mM dNTPs,
and 1 µl of Superscript Plus (Invitrogen) in a total of 20 µl. To
screen for mRNA expression, semi-quantitative PCR was performed
(PTC-200, Biozym) using the primer sequences MIA2 forward, ATG GCA AAA
TTT GGC GTT C, and MIA2 reverse, CCT GCC CAC AAA TCT TCC, with
Furthermore, MIA2 mRNA expression in primary human hepatocytes was
analyzed in comparison to the mRNA expression of
To quantify precisely the expression of MIA2 the real time PCR
LightCycler system (Roche Molecular Biochemicals) was used. For PCR
1-3 µl of cDNA preparation, 2.4 µl of 25 mM
MgCl2, 0.5 µM of forward and reverse primer,
and 2 µl of SybrGreen LightCycler Mix in a total of 20 µl were
applied. The following PCR program was performed: 60 s at 95 °C
(initial denaturation); 20 °C/s temperature transition rate up to
95 °C for 15 s, 10 s at 58 °C, 22 s at
72 °C, and 10 s at 82 °C acquisition mode single, repeated
for 40 times (amplification). MgCl2 concentration and
annealing temperature were optimized for each primer set. The PCR was
evaluated by melting curve analysis following the manufacturer's
instructions and checking the PCR products on 1.8% agarose gels. Each
quantitative PCR was performed at least in duplicate for two sets of
RNA preparations.
Northern Blots--
Multiple Tissue Expression Arrays
(Clontech) were hybridized following the
manufacturer's description. As probes cDNA fragments of hMIA2 were
phospholabeled using a Klenow DNA polymerase-based random primer
labeling kit (Bio-Rad).
In Situ Hybridization--
In situ hybridization was
performed as described previously (4). A 350-bp fragment of mMIA2 was
cloned into pBluescript (Clontech) for riboprobe
synthesis. The riboprobes were synthesized by in vitro
transcription using T7 and T3 RNA polymerase, respectively, and labeled
by incorporation of [33P]UTP.
Promoter Constructs, Transfections, and Luciferase
Assays--
The 5'-flanking regions of the human MIA2 gene
from residue
2 × 105 cells (HepG2, fibroblasts) were seeded into
each well of a 6-well plate and transiently transfected with 1 µg of
plasmid DNA using the LipofectAMINE Plus method (Invitrogen) following the manufacturer's description. 24 h after transfection the cells were lysed, and the luciferase activity in the lysate was measured. To
normalize transfection efficiency, 0.5 µg of an pRL-TK plasmid (Promega) was cotransfected, and Renilla luciferase activity
was measured by a luminometric assay (Promega).
Site-directed Mutagenesis--
Mutations in the 707-bp human
MIA2 promoter region (707mut) destroying the HNF1-binding
site were introduced using a site-directed mutagenesis kit
(Clontech) as described by the manufacturer.
Gel Mobility Shift Assays--
Complementary synthetic
oligonucleotides corresponding to the HNF1 site in the MIA2
promoter region and to the HNF1 consensus binding site (Geneka,
Montreal, Canada) were hybridized and phospholabeled (MIA2-HNF1
forward, ATC CTT GTT AAT TAT TAA ACC CTT AGG, and MIA2-HNF1 reverse,
CCT AAG GGT TTA ATA ATT AAC AAG GAT; HNF1_cons forward, CCA GTT AAT GAT
TAA CCA CTG GC, and HNF1_cons reverse, GCC AGT GGT TAA TCA TTA ACT GG).
Nuclear extracts were prepared from HepG2, primary fibroblasts, Mel Im
and Mel Ju cells, and gel shifts were performed as described previously
(20). Competition experiments were performed using a 50-fold excess of
the same binding site, a mutated binding site (HNF1_mut forward, CCA
GGC GAT GAG CGA CCA CTG GC, HNF1_mut reverse, GCC AGT GGT CGC TCA TCG
CCT GG) or an unrelated binding site. Supershifting was performed using an anti-HNF1 antibody (Geneka).
Statistical Analysis--
Statistical numbers and results are
expressed as mean ± S.D. Statistical significance between two
groups was determined by using the Student's t test. A
p value <0.05 was considered statistically significant.
We identified the MIA2 gene as a homologue of MIA by a
gene search. MIA2, together with MIA,
OTOR, and TANGO, belongs to a novel
MIA gene family sharing important structural features,
including an SH3 fold domain and two intramolecular disulfide bonds,
and similar genomic organization. The four members share 34-45% amino acid identity and 47-59% cDNA sequence identity (Fig.
1A). Surprisingly, MIA2
contains an additional C-terminal region of 422 amino acids having no
homology to known proteins. MIA2 therefore encodes a mature protein of
522 amino acids in addition to the hydrophobic secretory signal
sequence (Fig. 1B). Structure modeling and protein folding
recognition studies confirm the highly conserved SH3 structure in the N
terminus present also in MIA and OTOR (which are both known to be
secreted proteins).
236 controlling
hepatocyte-specific expression. Mutation of the site led to a complete
loss of promoter activity in HepG2 cell. Further sites detected in the
MIA2 promoter were consensus binding sites for SMAD and STAT3,
Consistently, stimulation of MIA2 mRNA expression occurred by
treatment with interleukin-6, transforming growth factor-
, and
conditioned medium from activated hepatic stellate cells. In accordance
with these results, MIA2 mRNA was found to be increased in liver
tissue of patients with chronic hepatitis C infection compared with
controls. MIA2 mRNA levels were significantly higher in patients
with severe fibrosis or inflammation than in patients with less severe
fibrosis or inflammation. In summary our data indicate that MIA2
represents a potential novel acute phase protein and MIA2 expression
responds to liver damage. The increased transcription in more severe
chronic liver disease suggests that MIA2 may serve as a marker of
hepatic disease activity and severity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(10 ng/ml), IL-1 (2 ng/ml), IL-6 (30 ng/ml), TGF-
1 (2 ng/ml), LPS (10 µg/ml), PMA (10 nM),
or with alcohol (0.1% ethanol) (all obtained from Sigma) for 6, 16, and 32 h.
70 °C before stimulation of HepG2 cells. HepG2 cells were seeded in 6-well plates and incubated with 2 ml of the
conditioned media for 6, 16, and 32 h.
-actin and glyceraldehyde-3-phosphate dehydrogenase for
standardization. Two µl of the cDNA preparation were applied to
the PCR. The following PCR program was used: 5 min at 94 °C, 32 cycles of 30 s at 94 °C, 45 s at 58 °C, and 2 min at
72 °C, final extension of 5 min at 72 °C. PCR products were
separated on a 1.8% agarose gel, stained with ethidium bromide, and documented.
1-antitrypsin (
1-AT),
2-macrogobulin
(
2-MG),
1-acid glycoprotein (
1-AG), and
1-antichymotrypsin (
1-ACT) by semi-quantitative PCR.
The following pairs of primers were used:
1-AT forward, GGG AGA GAC CCT TTG AAG TCA, and
1-AT reverse, AAG AAG ATG GCG GTG GCA T;
2-MG forward, CAG TGG AGA AGG AAC AAG CG, and
2-MG reverse, TTG
GTG GCA GTT TCA GGG ATA;
1-AG forward, ACA CCA CCT ACC TGA ATG TCC,
and
1-AG reverse, ACT CTC CCA GTT GCT CCT TG; and
1-ACT forward,
GCC CAT AAT ACC ACC CTG ACA, and
1-ACT reverse, TAC AGC CTC TTG GCA
TCC TC. For comparison of these acute phase proteins and MIA-2 mRNA
expression, 1 µl of the cDNA preparation was applied to the PCR,
and the PCR program was adapted to 28 cycles.
840 to
6,
707 to
6, and
218 to
6,
respectively, were amplified by PCR, inserted into the plasmid
pGL3-basic (Promega), and resequenced.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparison of human MIA, OTOR, MIA2,
and TANGO cDNA sequences and MIA2 protein sequence. A,
homology between the MIA gene family members on the cDNA
level shown as a phylogenetic tree. The tree was constructed using the
program DNAman based on the alignment of the complete cDNA
sequence. B, sequence of MIA2 protein in comparison to MIA.
Conserved cysteine residues are labeled with a box. Residues
marked with a * are important for the hydrophobic core of the SH3
domain. The hydrophobic signal peptide is indicated by
italics.
To analyze expression patterns of MIA2, RT-PCR studies of adult human and murine tissues and in situ hybridization of murine embryo sections were performed. In general, RT-PCR studies with primers specific for MIA2 reveal a consistent expression pattern for both human and murine tissues. In contrast to MIA, which is exclusively expressed in cartilage but not in any other non-neoplastic tissue, and OTOR, which shows a highly restricted expression pattern in cochlea, eye, and cartilage, the novel MIA related gene, MIA2, is expressed specifically in liver (Table I) and at very low levels in testis. The RT-PCR analysis was verified by in situ hybridization to mouse embryo sections of different embryonic stages between E12.5 and E14.5 using radiolabeled cRNA probes. Consistent with the RT-PCR, strong MIA2 expression was found in the developing liver (Fig. 2). Comparison between adult and fetal (gestational day 20) murine livers revealed ~5-fold higher MIA2 mRNA expression levels in adult liver (data not shown).
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Next we analyzed parenchymal and non-parenchymal human liver cells, and
we found strong MIA2 expression in primary hepatocytes but not in
Kupffer cells or quiescent or activated hepatic stellate cells (Fig.
3A). Identical results were
obtained with murine hepatic cells (data not shown). To estimate MIA2
mRNA expression relative to several known acute phase proteins,
semiquantitative PCR analysis was performed using first strand cDNA
from primary human hepatocytes. MIA2 mRNA expression was revealed
to be lower than 1-antichymotrypsin mRNA expression
but similar to the mRNA expression of
1-antitrypsin,
2-macrogobulin, and
1-acid glycoprotein
(data not shown). Furthermore, several tumor cell lines were studied,
and expression was detected only in the hepatoma cell line HepG2 (Fig.
3B).
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These data indicated highly specific expression of MIA2 in hepatocytes and prompted us to further analyze the underlying gene regulatory mechanism.
Preliminary insight into putative cis-regulatory elements of the
MIA2 promoter were obtained by data bank searches of the genomic 5'-flanking region. Analysis of 1500 bp indicated several consensus binding sites for transcription factors (Fig.
4A). Most importantly, an HNF1
site was found at 253 to
240 (relative to the adenine of the start
codon). Furthermore, STAT, SMAD, GATA-1, GATA-2, cAMP-response
element-binding protein, and AP-1 binding sites were localized.
GenBankTM search revealed EST clones identical to
MIA2 with a 5'-untranslated region of
120 indicating that
these sequences are indeed being transcribed. Consistently, a consensus
TATA box sequence is located 29 bases further upstream suggesting that
transcription initiation occurs at residue
120 or very close.
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707 and 218 bp of the promoter region were subcloned into pGL3-basic, a
promoterless luciferase reporter construct. Reporter gene assays
revealed strong activity of the promoter construct 707 in HepG2 cells
but not in primary human fibroblasts or melanoma cells (Mel Im) (Fig.
4B). Activity of the promoter could be further enhanced in
HepG2 by transfection of an HNF1 expression plasmid. Interestingly,
transfection of HNF1 into Mel Im melanoma cells and into human
fibroblasts was sufficient to significantly activate the
MIA2 promoter. The 218-bp promoter construct (lacking the HNF1 site) was neither active in HepG2 cells nor inducible by HNF1
(Fig. 4B). Furthermore, mutation of the HNF1 site in the 707 reporter construct by site-directed mutagenesis entirely inactivated promoter activity in HepG2 cells.
To confirm site-specific binding of HNF1 to the putative binding site
at 253 to
240 in the MIA2 promoter, gel shift assays were performed. Binding of HNF1 present in HepG2 nuclear extracts to a
consensus HNF1 site was competed by an oligomeric binding site spanning
the HNF1 site in the MIA2 promoter (Fig. 4C).
Additionally, supershifting experiments using an anti-HNF1 antibody
confirmed binding of HNF1 to the MIA2 promoter in HepG2 but
not in Mel Im melanoma cells (Fig. 4D).
We next investigated whether cytokines, known to be elevated
during liver diseases (interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-1 (IL-1
), tumor necrosis factor-
(TNF
),
transforming growth factor-
1 (TGF-
1)), lipopolysaccharide (LPS),
alcohol, and phorbol myristyl acetate (PMA) were also involved in
regulating hepatic MIA2 mRNA expression. Only IL-6 and TGF-
increased the MIA2-specific RT-PCR signal in isolated human hepatocytes
and in HepG2 cells. As measured by quantitative RT-PCR analysis, both IL-6 and TGF-
induced MIA2-specific mRNA ~8.5-fold. Combined treatment with both cytokines even further induced MIA2 mRNA
expression by ~12.3-fold (Fig.
5A). Induction of MIA2
mRNA expression already occurred 6 h after stimulation, and
maximal stimulation was observed after 16 h (data not shown).
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Regulation of MIA2 mRNA expression by the cytokines IL-6 and
TGF-1 is in accordance with the STAT3 (IL-6 regulation) and SMAD
(TGF-
regulation) consensus binding sites in the MIA2
promoter. In chronic liver diseases both cytokines are mainly expressed by activated hepatic stellate cells or activated myofibroblasts. These
cells play a major role in the pathophysiology of chronic liver
disease. Therefore, we investigated the effect of serum-free medium
conditioned by activated human HSC. MIA2 expression in HepG2 cells was
strongly stimulated by cell culture supernatant of activated HSC
pretreated with endotoxin and weakly, but still significantly, by the
supernatant from unstimulated HSC. Quantitative RT-PCR results showed
an 8.5-fold increase (2.2-fold, respectively) (Fig. 5B).
Similarly as in cytokine experiments, maximum of MIA2mRNA expression was seen after 16 h, starting at 6 h after
stimulation with conditioned medium (data not shown).
Hepatic IL-6 and TGF- levels are known to be elevated in chronic
liver diseases. It is known that trans-differentiation of HSC into
myofibroblastic cells is occurring in chronic liver diseases, leading
to proliferation of the cells, to migration into the sites of liver
damage, and increased expression of profibrotic and proinflammatory genes, including TGF-
and IL-6. Therefore, we analyzed the
expression of MIA2 mRNA in liver tissue of patients with chronic
hepatitis C infection and various stages of fibrosis and grading of
intrahepatic inflammation. Liver tissue without histological signs of
fibrosis or inflammation from human donors without liver disease served as control. Intrahepatic MIA2 mRNA expression was found to be significantly elevated in hepatitis C patients compared with controls (0.14 ± 0.13 versus 1.62 ± 1.46, respectively;
p = 0.043) (Fig. 6).
Furthermore, hepatitis C patients with only mild, periportal fibrosis
(staging 1; n = 6) had significantly lower intrahepatic MIA2 mRNA expression than patients with more advanced fibrosis (staging >1; n = 5) (0.85 ± 0.38 versus 2.55 ± 1.78, respectively; p = 0.016), as summarized in Fig. 6A. MIA2-mRNA expression
was also lower in hepatitis C patients with little intrahepatic
inflammation (grading 1 or 2), compared with patients with severe
inflammation (grading >2); however, these differences did not reach
statistical significance (1.04 ± 0.62 versus 2.64 ± 2.04, respectively; p = 0.027) (Fig.
6B).
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DISCUSSION |
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We identified the novel human gene MIA2 as a new member of the MIA gene family. Furthermore, we analyzed the expression pattern and gained first insight into transcriptional regulatory mechanisms during liver diseases.
MIA2, together with MIA, OTOR, and TANGO, belongs to the novel MIA gene family sharing important structural features, significant homology at both the nucleotide and protein level, and similar genomic organization. MIA/OTOR and MIA2/TANGO are more closely related to each other than to the other MIA gene family members. Many residues important for structural folding are conserved between the four proteins (e.g. cysteine residues, amino acids in the hydrophobic core). Therefore, it can be speculated that all proteins belonging to the MIA gene family form an SH3 domain-like structure that was recently identified by NMR and x-ray crystallography of MIA (6, 9). The N terminus coding for the signal sequence is quite divergent, but analysis by Kyte-Doolittle blots revealed conservation of the hydrophobic character that is functionally important.
In contrast to MIA, which is exclusively expressed in cartilage but not in any other non-neoplastic tissue, and OTOR, which shows a highly restricted expression pattern in cochlea, eye, and cartilage, the novel MIA-related gene MIA2 is expressed specifically in hepatocytes. The RT-PCR analysis was verified by in situ hybridization to mouse embryo sections of different embryonic stages between E12.5 and E14.5 using radiolabeled cRNA probes. Consistent with the RT-PCR data, MIA2 expression is restricted to hepatocytes also during development. Furthermore, no mRNA expression of MIA2 was detected in cartilage indicating that the four members of the MIA gene family differ entirely with respect to their expression patterns.
To analyze transcriptional regulation, luciferase reporter gene constructs containing portions of the 5'-flanking region including an HNF1-binding site were transfected into HepG2 cells, melanoma cells, and primary fibroblasts. Our results clearly show the importance of the HNF-1-binding site for the exclusive expression of MIA2 mRNA in hepatocytes.
HNF-1 is a transcription factor that is expressed in liver, digestive
tract, pancreas, and kidney (21) and is involved in the regulation of a
large number of hepatic genes including albumin, fibrinogen, or
1-antitrypsin. It has been reported previously (22-24)
that liver injury, chronic liver disease, or conditions associated with
liver damage as elevated endotoxin levels lead to a down-regulation of
HNF-1 activity. It is further interesting to note that several genes
that contain a functional HNF-1 binding sequence are down-regulated
during host response to infection or inflammation (25-27). However,
hepatic MIA2 mRNA was found to be increased in patients with
chronic hepatitis C infection. Furthermore, MIA2 transcription was even
further elevated in liver tissue with severe fibrosis or inflammation.
Our findings indicate that HNF-1 expression is required for basal
expression but may be less important for enhanced transcriptional
regulation in liver diseases.
Here, in accordance with STAT3 and SMAD consensus binding sites in the
MIA2 promoter, IL-6 and TGF- were identified as
activators of MIA2 transcription, whereas other cytokines did not lead
to enhancement of MIA2 expression.
The cytokine IL-6 is required for liver regeneration and repair and up-regulates transcriptionally a vast array of genes during liver regeneration and repair (28-30). IL-6 induces DNA binding of STAT transcription factors on regulatory elements of target genes. Although MIA2 is expressed constitutively in hepatocytes, its transcription was significantly increased during IL-6 stimulation.
TGF- has pleiotropic functions including fibrinogenic action leading
to trans-differentiation of HSC and negative regulation of
proliferation and induction of apoptosis (31).
Interestingly, we found a synergistic effect of IL-6 and TGF-
stimulation on MIA expression in HepG2 cells. The cross-talk between
IL-6 and TGF-
signaling pathways in a human hepatoma cell line was
elucidated in a recent study (32), demonstrating that IL-6-induced
activation of STAT3 activity and STAT3-mediated gene expression was
augmented by TGF-
. These activities were due to physical
interactions between STAT3 and SMAD- and MAD-related protein-3,
bridged by p300. As the consensus binding sites for STAT3 and
SMAD are in direct proximity in the MIA2 promoter, we speculate that a similar mechanism for induction of MIA2 expression may
be involved.
Our results indicate that different classes of transcription factors, tissue-specific (HNF-1) and growth- or stress-induced (STAT3 and SMAD), may interact during acute phase reaction or as an adaptive response to liver injury to amplify expression of the MIA2 gene, as demonstrated for other genes such as insulin-like growth factor-binding protein 1 (33).
STAT and SMAD pathways are known to be involved in the pathogenesis of
liver fibrosis and inflammation, and elevated systemic and intrahepatic
levels of IL-6 and TGF- were found in acute and chronic liver
diseases (30, 31). The activation process of HSC, causing
trans-differentiation of the physiologically quiescent cells to an
activated myofibroblast-like cell type, is one of the key events of
hepatic fibrosis. In healthy liver or acute liver damage, Kupffer
cells, the resident hepatic macrophages, are the main modulators of
inflammation, secreting mainly TNF or IL-1. However, during
liver disease activation of HSC occurs early, leading to participation
in the regulation of the hepatic inflammatory response (31). Both
cytokines, IL-6, and TGF-
are expressed by activated, but not
quiescent, HSC. Consequently, we found strong induction of MIA2
expression in HepG2 cells incubated with conditioned medium of
activated HSC, particularly when HSC had been challenged with
endotoxin. These results strongly suggest that activated HSC may also
participate in vivo at least in part in the regulation of
intrahepatic MIA2 expression. It is tempting to speculate that MIA2
expression may correlate with the activation process of these cells,
allowing the use of MIA2 as a marker for fibrosis. Furthermore,
transcriptional regulation and mRNA expression data, indicating
that MIA2 is an abundantly expressed gene, render MIA2 to be
a relevant acute phase protein.
In summary, our data identify MIA2 as a potential novel acute phase
protein, secreted specifically from hepatocytes. Transcriptional regulation by IL-6, TGF-, and conditioned medium from activated HSC
and increased expression in fibrotic or inflamed liver tissue indicate
that MIA2 may play a role in the pathophysiology of liver diseases and
may serve as a marker of liver damage. Here it is tempting to speculate
that in analogy to MIA, MIA-2 regulates attachment to extracellular
matrix molecules in liver similar to MIA in cartilage. Further
experiments need to address the question of whether MIA2 interacts with
the same peptide epitopes as MIA and exerts the same functions in the
regulation of cell attachment.
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ACKNOWLEDGEMENTS |
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We are indebted to Jacqueline Schlegel, Astrid Hamm, Sandra Dahmen, Janine Muyers, Nicole Krott, and Claudia Abschlag for technical assistance. We thank Wolfgang Thasler for providing human hepatocytes and human liver tissue, Matthias Froh for providing Kupffer cells, and Gerd Kullak-Ublick for providing the HNF1 construct.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to A. B. and C. H.).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.
§ To whom correspondence should be addressed: Institut of Pathology, Molecular Pathology, University of Regensburg, D-93042 Regensburg, Germany. Tel.: 49-941-944-6705; Fax: 49-941-944-6602; E-mail: anja.bosserhoff@klinik.uni-regensburg.de.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M212639200
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ABBREVIATIONS |
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The abbreviations used are:
MIA, melanoma
inhibitory activity;
RT, reverse transcriptase;
IL, interleukin;
TGF-, transforming growth factor
;
TNF-
, tumor necrosis
factor-
;
LPS, lipopolysaccharide;
PMA, phorbol myristyl acetate;
HSC, hepatic stellate cells;
1-AT,
1-antitrypsin;
2-MG,
2-macrogobulin;
1-AG,
1-acid
glycoprotein;
1-ACT,
1-antichymotrypsin;
SH3, Src
homology 3.
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REFERENCES |
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---|
1. | Bogdahn, U., Apfel, R., Hahn, M., Gerlach, M., Behl, C., Hoppe, J., and Martin, R. (1989) Cancer Res. 49, 5358-5363[Abstract] |
2. | Koehler, M. R., Bosserhoff, A., von Beust, G., Bauer, A., Blesch, A., Buettner, R., Schlegel, J., Bogdahn, U., and Schmid, M. (1996) Genomics 35, 265-267[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Dietz, U. H.,
and Sandell, L. J.
(1996)
J. Biol. Chem.
271,
3311-3316 |
4. | Bosserhoff, A. K., Kondo, S., Moser, M., Dietz, U. H., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Buettner, R., and Sandell, L. J. (1997) Dev. Dyn. 208, 516-525[CrossRef][Medline] [Order article via Infotrieve] |
5. | Blesch, A., Bosserhoff, A. K., Apfel, R., Behl, C., Hessdoerfer, B., Schmitt, A., Jachimczak, P., Lottspeich, F., Buettner, R., and Bogdahn, U. (1994) Cancer Res. 54, 5695-5701[Abstract] |
6. |
Stoll, R.,
Renner, C.,
Zweckstetter, M.,
Bruggert, M.,
Ambrosius, D.,
Palme, S.,
Engh, R. A.,
Golob, M.,
Breibach, I.,
Buettner, R.,
Voelter, W.,
Holak, T. A.,
and Bosserhoff, A. K.
(2001)
EMBO J.
20,
340-349 |
7. | Guba, M., Bosserhoff, A. K., Steinbauer, M., Abels, C., Anthuber, M., Buettner, R., and Jauch, K. W. (2000) Br. J. Cancer 83, 1216-1222[CrossRef][Medline] [Order article via Infotrieve] |
8. | Bosserhoff, A. K., Echtenacher, B., Hein, R., and Buettner, R. (2001) Melanoma Res. 11, 417-421[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Lougheed, J. C.,
Holton, J. M.,
Alber, T.,
Bazan, J. F.,
and Handel, T. M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
5515-5520 |
10. | Rendtorff, N. D., Frodin, M., Attie-Bitach, T., Vekemans, M., and Tommerup, N. (2001) Genomics 71, 40-52[CrossRef][Medline] [Order article via Infotrieve] |
11. | Robertson, N. G., Heller, S., Lin, J. S., Resendes, B. L., Weremowicz, S., Denis, C. S., Bell, A. M., Hudspeth, A. J., and Morton, C. C. (2000) Genomics 66, 242-248[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Cohen-Salmon, M.,
Frenz, D.,
Liu, W.,
Verpy, E.,
Voegeling, S.,
and Petit, C.
(2000)
J. Biol. Chem.
275,
40036-40041 |
13. | Jacob, K., Wach, F., Holzapfel, U., Hein, R., Lengyel, E., Buettner, R., and Bosserhoff, A. K. (1998) Melanoma Res. 8, 211-219[Medline] [Order article via Infotrieve] |
14. | Jacob, K., Bosserhoff, A. K., Wach, F., Knuchel, R., Klein, E. C., Hein, R., and Buettner, R. (1995) Int. J. Cancer 60, 668-675[Medline] [Order article via Infotrieve] |
15. | Schlott, T., Thasler, W., Gorzel, C., Pahernike, S., Brinck, U., Eiffert, H., and Droese, M. (2002) Anticancer Res. 22, 1545-1551[Medline] [Order article via Infotrieve] |
16. |
Wheeler, M. D.,
Yamashina, S.,
Froh, M.,
Rusyn, I.,
and Thurman, R. G.
(2001)
J. Leukocyte Biol.
69,
622-630 |
17. | Hellerbrand, C., Wang, S. C., Tsukamoto, H., Brenner, D. A., and Rippe, R. A. (1996) Hepatology 24, 670-676[Medline] [Order article via Infotrieve] |
18. | Wach, F., Bosserhoff, A., Kurzidym, U., Nowok, K., Landthaler, M., and Hein, R. (1998) Skin Pharmacol. 11, 43-51[CrossRef] |
19. | Desmet, V. J., Gerber, M., Hoofnagle, J. H., Manns, M., and Scheuer, P. J. (1994) Hepatology 19, 1513-1520[Medline] [Order article via Infotrieve] |
20. |
Bosserhoff, A. K.,
Hein, R.,
Bogdahn, U.,
and Buettner, R.
(1996)
J. Biol. Chem.
271,
490-495 |
21. |
Cereghini, S.
(1996)
FASEB J.
10,
267-282 |
22. | Ninomiya, T., Hayashi, Y., Saijoh, K., Ohta, K., Yoon, S., Nakabayashi, H., Tamaoki, T., Kasuga, M., and Itoh, H. (1996) J. Hepatol. 25, 445-453[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Trauner, M.,
Arrese, M.,
Lee, H.,
Boyer, J. L.,
and Karpen, S. J.
(1998)
J. Clin. Invest.
101,
2092-2100 |
24. | Burke, P. A., Luo, M., Zhu, J., Yaffe, M. B., and Forse, R. A. (1996) Surgery 120, 374-380[Medline] [Order article via Infotrieve] |
25. |
Gabay, C.,
and Kushner, I.
(1999)
N. Engl. J. Med.
340,
448-454 |
26. |
Navasa, M.,
Gordon, D. A.,
Hariharan, N.,
Jamil, H.,
Shigenaga, J. K.,
Moser, A.,
Fiers, W.,
Pollock, A.,
Grunfeld, C.,
and Feingold, K. R.
(1998)
J. Lipid Res.
39,
1220-1230 |
27. | Memon, R. A., Bass, N. M., Moser, A. H., Fuller, J., Appel, R., Grunfeld, C., and Feingold, K. R. (1999) Biochim. Biophys. Acta 1440, 118-126[Medline] [Order article via Infotrieve] |
28. |
Cressman, D. E.,
Greenbaum, L. E.,
DeAngelis, R. A.,
Ciliberto, G.,
Furth, E. E.,
Poli, V.,
and Taub, R.
(1996)
Science
274,
1379-1383 |
29. | Kovalovich, K., DeAngelis, R. A., Li, W., Furth, E. E., Ciliberto, G., and Taub, R. (2000) Hepatology 31, 149-159[Medline] [Order article via Infotrieve] |
30. | Streetz, K. L., Wustefeld, T., Klein, C., Manns, M. P., and Trautwein, C. (2001) Cell. Mol. Biol. 47, 661-673[Medline] [Order article via Infotrieve] |
31. | Gressner, A. M., Weiskirchen, R., Breitkopf, K., and Dooley, S. (2002) Front. Biosci. 7, d793-d807[Medline] [Order article via Infotrieve] |
32. | Yamamoto, T., Matsuda, T., Muraguchi, A., Miyazono, K., and Kawabata, M. (2001) FEBS Lett. 492, 247-253[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Leu, J. I.,
Crissey, M. A.,
Leu, J. P.,
Ciliberto, G.,
and Taub, R.
(2001)
Mol. Cell. Biol.
21,
414-424 |
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