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Vol. 14, Issue 6, 2327-2341, June 2003
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* Duke University Liver Center and the Departments of Cell Biology and Medicine,
Duke University Medical Center, Durham, North Carolina 27710;
Liver Center Laboratory and the Department and Medicine, University of
California, San Francisco, San Francisco, California; and
Biogen, Inc, Cambridge, Massachusetts 02142
Submitted June 7, 2002;
Revised December 3, 2003;
Accepted February 26, 2003
Monitoring Editor: Carl-Henrik Heldin
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1, a cytokine
integral to wound healing, stimulated ET-1 production. This effect was due to
ECE-1 mRNA stabilization and increased ECE-1 expression in stellate cells,
which in turn was a result of de novo synthesis of the identified 56- and
62-kDa ECE-1 3' UTR mRNA binding proteins. These data indicate that
liver injury and the hepatic wound healing response lead to ECE-1 mRNA
stabilization in stellate cells via binding of 56- and 62-kDa proteins, which
in turn are regulated by transforming growth factor-. The possibility
that the same or similar regulatory events are present in other forms of wound
healing is raised. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Friedman and Arthur, 1989;
Kent et al., 1976;
Maher and McGuire, 1990).
During stellate cell activation, these cells exhibit features of
myofibroblasts, producing increased quantities of extracellular matrix
proteins as well as smooth muscle 
-actin. A further characteristic of
stellate cell activation is enhanced production of the vasoconstrictive
peptide, endothelin-1 (ET-1), which contributes to perpetuation of the
fibrogenic process (Gandhi et
al., 1998; Rockey and
Chung, 1996) as well as in control hepatic vascular tone
(Bauer et al., 1994;
Kawada et al., 1993;
Rockey and Weisiger,
1996).
Each of the three known endothelin isoforms (-1, -2, -3) arise by
proteolytic processing of large precursors (
200 amino acid residues).
Intermediates, termed big ET-1, -2, and -3 (38–41 aa) are excised from
prepropeptides at sites containing paired basic amino acids. Big endothelins,
which have little or no biological activity
(Yanagisawa, 1994), are
cleaved at Trp-21-Val/Ile-22 to produce mature 21-residue, biologically active
peptides. The enzyme responsible for the specific cleavage at Trp-21 has been
termed endothelin-converting enzyme (ECE); it is a neutral membrane-bound
metalloprotease with Mr = 120 kDa, belonging to the
endo-peptidase-24.11 family found in brain
(Ohnaka et al., 1993;
Turner and Murphy, 1996). The
production of ET-1 seems to be regulated at the level of preproET-1 as well as
by ECE. The mechanism by which ET-1 overproduction in the injured liver occurs
is linked to increased ECE-1 expression and presumed posttranscriptional
modulation of ECE-1 mRNA (Shao et
al., 1999).
The transforming growth factor- (TGF-) superfamily consists of
multiple family members, including the highly homologous isoforms of
TGF- (TGF-1–5) and the related bone morphogenetic proteins
activins, and inhibins (Massague et
al., 1992). The TGF-s have a wide variety of biological
actions, including in cell growth, differentiation, and fibrogenesis (form
review, see Massague et al.,
1992). TGF-1 in particular has emerged as a key component of
the fibrogenic response to wounding and is upregulated during many different
types of wound healing, including in the liver
(Munger et al., 1999;
Nakatsukasa et al.,
1990; Sanderson et
al., 1995) (for review, see
(Border and Noble, 1994). In
the liver, the importance of TGF-1 has been emphasized by the
demonstration of its direct effects on stellate cells, presumably at the level
of extracellular matrix gene transcription
(Armendariz-Borunda et al.,
1992).
Posttranscriptional processes, including mRNA stabilization, play an
important role in gene expression (Beelman
and Parker, 1995; Burd and
Dreyfuss, 1994; Dreyfuss
et al., 1996;
Jacobson and Peltz, 1996).
Stability of most mRNAs is determined by sequences in their
3'-untranslated regions (3' UTRs). AU-rich elements (AREs) and
poly(C) regions found in the 3' UTR are among the most common stability
determinants identified in mammalian cells
(You et al., 1992;
Levine et al., 1993;
Wang et al., 1995;
Holcik and Liebhaber, 1997;
Peng et al., 1998;
Czyzyk-Krzeska and Bendixen,
1999; Laroia et al.,
1999; Paulding and
Czyzyk-Krzeska, 1999). Functionally, AREs and poly(C) regions seem
to mediate mRNA deadenylation and cleavage of mRNA itself. mRNA stability is
generally controlled by the interaction between these specific RNA sequences
and RNA binding proteins. Several AU-specific binding proteins and poly(C)
binding proteins have been described and characterized
Wang et al., 1995;
DeMaria and Brewer, 1996;
Ma et al., 1997;
Fan and Steitz, 1998;
Czyzyk-Krzeska and Bendixen,
1999). Interaction of RNA binding proteins and 3' UTR
sequences is thought to lead to configurational changes that prevent mRNA
degradation and decrease mRNA turnover. Most RNA binding proteins seem to be
cytoplasmic proteins of 30–70 kDa
(Hamilton et al.,
1993; Zhang et al.,
1993 Gueydan et al.,
1999; Tillmann-Bogush et
al., 1999).
Given previous data demonstrating enhanced expression and stabilization of
ECE-1 mRNA in stellate cells during hepatic wound healing, we aimed to
identify regulatory regions in the ECE-1 3' UTR that might mediate this
process. In addition, the prominence of ET-1 and TGF- in wound healing
led us to hypothesize that these two factors are linked and to specifically
investigate the possibility that TGF- might regulate production of ET-1
in vivo during the wounding process. We have used a hepatic wound healing
model not only to dissect the molecular and cellular regulatory events
controlling ET-1 synthesis in vivo during wounding but also molecular
mechanisms by which TGF- controls ET-1 production.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Kountouras et al.,
1984). Bile duct ligation (BDL) resulted in periportal expansion
of biliary duct cells with concomitant periductular and lobular matrix
deposition. Repeated administration of carbon tetrachloride led to extensive
portal-portal, portal-central, and central-central bridging fibrosis, also
typical of severe hepatic wounding. In some experiments, rats undergoing bile
duct ligation received a soluble TGF- receptor (generated by fusion of
rabbit type II TGF- receptor with Fc region of human IgG1;
Smith et al., 1999;
this construct inhibits biological activity of TGF-1 and TGF-3) or
matched IgG1 control (each 5 mg/kg intraperitonally) 1 d before bile duct
ligation and 4 d later. Animal care and experimental procedures were approved
by the Duke University Medical Center Institutional Animal Care and Use
Committee as set forth in the Guide for the Care and Use of Laboratory Animals
published by the National Institutes of Health.
Cell Isolation and Culture
Nonparenchymal cells were isolated as described previously
(de Leeuw et al.,
1984). Briefly, stellate and endothelial cells were isolated by
perfusion in situ with 20 mg/100 ml pronase (Boehringer Mannheim Biochemicals,
Indianapolis, IN) and 7.3 mg/100 ml collagenase (Crescent Chemical, Hauppauge,
NY). The cell suspension was layered on a discontinuous density gradient of
8.2 and 15.6% accudenz (Accurate Chemical & Scientific, Westbury, NY). The
resulting upper layer consists of >95% stellate cells. Endothelial cells
were further purified by centrifugal elutriation (18 ml/min flow). Purity was
assessed by specific markers for each stellate cells and endothelial cells.
Cells were plated in modified medium 199, containing 20% serum (10% horse
serum and 10% calf serum; Invitrogen, Carlsbad, CA), 4 mg/100 ml streptomycin,
and 0.25 mg/100 ml amphotericin at a density of 1 x 106
cells/ml. Cultures were incubated at 37°C in a humidified incubator
(containing 95% O2 and 2.5% CO2), and the medium was
changed at every 24 h. Cell viability was >80% in all cultures used.
ET-1 Radioimmunoassay
ET-1 in conditioned medium was measured using a radioimmunoassay kit
according to manufacturer specifications (Peninsula Laboratories, Palo Alto,
CA). In brief, samples were incubated with 100 µl of rabbit anti-ET-1
antibody for 12 h at 4°C. Then 100 µl of 125I-ET-1 (10,000
cpm) was added and incubated for 12 h at 4°C. After incubation with 100
µl of goat anti-rabbit IgG and normal rabbit serum for 90 min at room
temperature, the mixture was added to buffer and centrifuged at 3000 rpm for
20 min; 125I-ET-1 in the precipitate was measured (Beckman 5500,
Beckman Coulter, Palo Alto, CA). Counts were normalized to total protein
content for each sample (Bio-Rad, Hercules, CA). The ET-1 antibody used in the
radioimmunoassay kit exhibited <5% cross-reactivity with unlabeled ET-3,
<3% cross-reactivity with unlabeled ET-2, and 3% cross-reactivity with big
ET-1. Inter- and intraassay variability of the radioimmunoassay system were
each <5%.
ECE-1 3' UTR cDNAs and Oligonucleotide Mutation
Based on the published rat ECE-1 sequence
(Shimada et al.,
1994), oligodeoxynucleotide primers encoding the proximal 675 base
pairs of the ECE-1 UTR (distal to the stop codon) were synthesized (Operon
Technologies, Alameda, CA). 5' and 3' sequences for sense and
antisense primers were 5'-TAAGGGCTGAAGCGCAGA-3' and
5'-GCCTGATTTATGAGCATG-3', respectively. Different cDNA fragments
encoding specific portions of the ECE-1 3' UTR (431, 361, 307, 190, 60,
or 46 base pairs, respectively) were generated by restriction digestion (Nsi
I, DraI, AvaII, Csp45 I, SmaI, or PstI).
Oligonucleotides and mutants were synthesized (Operon Technologies) and cloned
into pGEM3+. All cDNA fragments were sequenced by the dideoxy chain
termination method and found to have 100% homology to published sequences.
Preparation of Cell Cytoplasmic and Nuclear Extracts
Cells were resuspended in lysis buffer containing 10 mM Tris-Cl (pH 7.4),
10 mM MgCl2, 3 mM NaCl, and 0.4% Nonidet P-40 (NP-40). After
incubation on ice for 10 min, cells were centrifuged at 120,000 x
g for 10 min, and the supernatant containing the cytoplasmic fraction
was used to calculate protein concentration. Preparation of nuclear extracts
was performed by resuspending cells in phosphate-buffered saline and
sonication followed by centrifugation at 1500 x g for 5 min and
washing. The resulting cell pellet was resuspended in buffer containing 0.01 M
HEPES (pH 7.6), 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml aprotinin, 5 µg/ml
leupeptin, 5 µg/ml pepstatin, 1 mM sodium vanadate, and 2 mM KCl. After
incubation on ice for 15 min, samples were resuspended in 10% NP-40 and
centrifuged at 120,000 x g for 1 min. The pellet was dissolved
in high salt buffer containing 2 mM KCl, 0.42 M NaCl, 0.01 M HEPES (pH 7.6),
1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 2 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 1
mM sodium vanadate, 25% glycerol, and 0.2 mM EDTA. Finally, after
centrifugation at 120,000 x g for 5 min, supernatants were
collected and used for study.
RNA Isolation and RNase Protection Assay
Total RNA was isolated from cell lysates (Tri-reagent; Molecular Research,
Cincinnati, OH); RNA concentration and purity were determined
spectrophotometrically (A260/280), and the integrity of all samples was
documented by visualization of 18S and 28S ribosomal bands after
electrophoresis through a 1% agarose/formaldehyde gel. ECE-1 (270 base pairs)
cDNA was cloned as described previously. Radiolabeled probes were synthesized
by transcription of appropriate plasmid cDNA with T7 RNA polymerase in the
presence of [-32P]CTP (Amersham Biosciences, Piscataway,
NJ). Total RNA (10 µg) was incubated with 1.0 x 106 cpm of
32P-labeled cRNA, denatured at 78°C for 15 min, and hybridized
at 55°C for 12–16 h. Unhybridized RNA was digested and protected
hybrids were denatured and separated by electrophoresis through a 5%
polyacrylamide/urea sequencing gel. Dried gels were exposed to x-ray film
(X-OMat AR-5; Eastman Kodak, Rochester, NY), and scanning densitometry was
used to quantitate autoradiographic signals. All RNA samples were also probed
with a cDNA encoding 316 base pairs of glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) (Ambion, Austin, TX) to verify the integrity of mRNA in
each sample and to control internally for the amount of mRNA present in an
individual assay. tRNA and lung RNA were used as a negative and positive
control RNAs, respectively.
Mobility Shift Assay and UV Cross-Linking
32P-labeled 3' UTR RNAs encoding ECE-1 3' UTR
fragments were generated by in vitro transcription as described above.
Radiolabeled RNA transcripts (200,000 cpm/reaction) were incubated with
cytoplasmic extracts (40 µg) in 15 mM KCl, 5 mM MgCl2, 1 mM
dithiothreitol, 0.24 mM EDTA, 12 mM HEPES (pH 7.8), 0.5% NP-40, 5% glycerol,
and yeast tRNA at 30°C for 30 min. After addition of ribonuclease T1 (100
U) at 30°C for 20 min, heparin (150 µg) was added to the reaction and
the reaction was incubated on ice for 10 min. Samples were loaded on a 6%
nondenaturing acrylamide gel. For UV cross-linking, after addition of heparin,
samples were irradiated with 254-nm UV light for 10 min at a distance of 10 cm
(UVP, San Gabriel, CA). Ribonuclease T1 (1 U) was added and samples were
resolved by 10% SDS-PAGE. Dried gels were exposed to x-ray film.
Nuclear Run-On Assay
After washing with phosphate-buffered saline, cultured cells were lysed and
incubated on ice for 10 min with lysis buffer containing 10 mM Tris-Cl (pH
7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 1 mM dithiothreitol, 0.5
mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotonin. The samples were
centrifuged for 5 min at 500 x g and the pellet was resuspended
(1–2 x 107 nuclear/0.1 ml) in storage buffer containing
20 mM Tris-Cl (pH 8.0), 20% glycerol, 60 mM NaCl, 0.1 mM EDTA, and 1 mM
dithiothreitol, and followed by storage in liquid nitrogen. Transcription
assays were performed in 200 µl of reaction buffer containing 62.5 mM
Tris-Cl (pH 8.0), 10 mM MgCl2, 200 mM KCl, 50 U RNasin, 1 mM CTP, 1
mM GTP, 1 mM ATP, and 0.1 mCi of [-32P]UTP at room
temperature for 1 h. After incubation, the reaction mixture was digested with
10 U of DNase I and followed by 40 µg of proteinase K. The newly
synthesized RNA was isolated with 1 ml of Tri-reagent. RNA samples containing
1 x 106 cpm were added to 0.5 ml (final volume) of
hybridization buffer containing 50 mM PIPES (pH 6.8), 1 mM EDTA, 0.2% SDS,
2.5x Denhardt's solution, 50 µg of denatured salmon sperm DNA, and
200 µg of tRNA. The samples were then hybridized to ECE-1, GAPDH, and pGEM
3z cDNAs immobilized on nitrocellulose membranes for 3 d at 65°C.
Membranes were washed twice for 10 min at room temperature in 0.1%
SDS-2x SSC and signals were detected by exposure to X-film (X-OMat AR-5;
Eastman Kodak) for 5 d.
Reporter Construct, Transient and Stable Transfection
The proximal 675-base pair fragment of the ECE-1 3' UTR was cloned
into the C-terminal portion of a pcDNA-Luciferase plasmid construct (kindly
provided by Kevin Claffey, Center for Vascular Biology, University of
Connecticut, Storrs, CT) or the C-terminal portion of an enhanced green
fluorescent protein (GFP) vector (Clontech, Palo Alto, CA). Constructs were
transfected into stellate cells or NIH 3T3 fibroblasts by using Lipofectin
(Invitrogen) following the manufacturer's instructions. Luciferase activity
was determined 48 h after transfection as manufacturer's instructions
(Promega, Madison, WI). For stable transfection, GFP fluorescent images were
analyzed from day 1 to day 12 after cell growth in medium containing serum and
G418.
Immunoblot
Cultured cells were homogenized in 20 mM Tris-HCl buffer (pH 7.4)
containing 5 mM MgCl2 and 0.1 mM PMSF, 20 µM pepstatin A and 20
µM leupeptin. The homogenate was centrifuged at 1000 x g for
10 min, and the resulting supernatant was centrifuged at 100,000g for 60 min.
Protein content in the supernatant was quantitated (Bio-Rad) and equal
quantities of protein (30 µg) were separated by SDS-PAGE under reducing
conditions and transferred to nitrocellulose (Bio-Rad). Blots were incubated
with anti-ECE-1 primary antibody B61/104 (1:400) (kindly provided by Dr.
Thomas Sub-kowski, BASF, Ludwigshafen, Germany) and detected by
chemiluminescence (ECL kit; Amersham Biosciences).
ECE-1 Enzymatic Activity
ECE-1 enzymatic activity was determined as described previously
(Ohnaka et al.,
1993). In brief, microsomal membrane fractions (30 µg) were
preincubated with 0.1 M sodium phosphate buffer (pH 6.8) containing 0.5 M NaCl
and protease inhibitors at 37°C for 15 min before addition of 1 µM big
ET-1. The reaction was incubated at 37°C for 2 h in siliconized tubes and
terminated by adding 50 µl of 5 mM EDTA. The mixture was then processed to
detect immunoreactive ET-1.
Statistics
Data are expressed as mean ± SE and n refers to the numbers of
individual experiments performed. Differences among groups were determined
using one-way analysis of variance followed by the Newman-Keuls procedure. The
0.05 level of probability was used as the criterion of significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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)
and that the mechanism involves enhanced ECE-1 mRNA stability. We therefore
postulated that specific ECE-1 3' UTR binding proteins were responsible
for ECE-1 mRNA stabilization. To investigate this possibility, we attempted to
detect RNA binding proteins within the proximal 675 base pairs of the ECE-1
3' UTR. We focused on this fragment because it contains multiple
putative binding elements, including three AUUUA repeats, three ACCCCA
repeats, and eight CCCU repeats (Shimada
et al., 1994). Electromobility shift assays and UV
cross-linking experiments, performed using stellate and endothelial cell
cytoplasmic extracts from both normal and injured livers, reproducibly
revealed an RNA–protein complex in the cytoplasmic fraction from normal
and injured populations of endothelial cells, but only in stellate cells after
liver injury (either BDL or carbon tetrachloride)
(Figure 1). The results of UV
cross-linking revealed that RNA binding protein sizes were 56 and 62 kDa
(Figure 1). We also performed
RNA protein binding assays by using nuclear fractions, but failed to identify
RNA–protein complexes in either cell type (our unpublished data). The
results identify the proximal 675-base pair region of the ECE-1 3' UTR
as potentially important in regulation of ECE-1 mRNA stability in stellate
cells after liver injury. This result is highly relevant from a
pathophysiological standpoint, because liver injury resulting in activation of
stellate cells is typified by increases in ET-1 production
(Shao et al.,
1999).
|
We confirmed the specificity of cytoplasmic protein binding to the cloned 675 base pairs of ECE-1 3' UTR, by adding molar excess nonradiolabeled ECE-1 3' UTR as a specific competitor (Figure 1B). RNA protein binding activity was significantly inhibited by addition of nonradiolabeled 3' UTR; and additionally, binding by 56- and 62-kDa proteins was eliminated.
Identification of 56- and 62-kDa Protein Binding Regions
Because AREs and poly(C) regions mediate 3' UTR stabilization, we
aimed to identify whether these regions play a similar role in ECE-1 3'
UTR protein binding. RNA protein binding activity was effectively abolished by
excess poly(C) nucleotide but not with excess poly (A), (G), or (U)
nucleotides (Figure 2). These
data suggest that the two putative RNA binding proteins (56 and 62 kDa)
bind to C-rich regions in the 675-base pair ECE-1 3' UTR.
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To identify specific regions responsible for protein binding within the proximal ECE-1 3' UTR 675 base pairs, we isolated multiple 3' UTR fragments (Figure 3A) and examined their ability to mediate protein binding. In electromobility shift assays and UV cross-linking experiments, progressively truncated fragments containing more than the first 193 base pairs distal to the stop codon possessed both 56- and 62-kDa protein binding activity. Importantly, the sequence from 519–547 base pairs (containing three ACCCCA) repeats did not possess binding activity for either protein. The proximal 46- and 60-base pair sequences retained 56-kDa protein binding activity, but did not exhibit 62-kDa binding activity (Figure 3, A and B). A fragment containing bp 21–46 retained binding activity, but the proximal 20-base pair fragment did not (Figure 3C). Interestingly, we were unable to detect protein binding by using the 47- to 675-base pair construct (or with other constructs that lacked the proximal fragment; our unpublished data), despite multiple attempts. Protein binding in the proximal 46-base pair area was inhibited by addition of excess poly(C) (Figure 3D), consistent with poly(C) competitive inhibition of protein binding in the full 675-base pair 3' UTR fragment. The data suggest that the 56-kDa protein binds to one or more poly(C) regions in the 21- to 46-base pair segment of the ECE-1 3' UTR and that the 62-kDa protein interaction is with one or more poly(C) regions between 60 and 193 base pairs. Furthermore, the data suggest that the binding of the 62-kDa protein is dependent on binding of the 56-kDa protein.
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We next mutated the poly(C) region in the proximal ECE-1 3' UTR 46-base pair fragment (Figure 4A). Examination of the ECE-1 3' UTR revealed two sequences containing poly(C) sequences: GCGC and ACAC at 12 and 21 base pairs from the stop codon, respectively (Figure 4A). Substitution of the GCGC with GGGG (mutant 1) had no effect on protein binding activity, whereas substitution of the ACAC with AGAG (mutant 2) resulted in loss of RNA protein binding of the 56-kDa species (Figure 4B). These data, which are highly consistent with those shown in Figure 3C, indicate that the 56-kDa binding protein interacts with the ACAC poly(C) region in the proximal 46-base pair fragment.
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A Functional Role for ECE-1 3' UTR Binding Proteins
To evaluate a potential functional role for the ECE-1 3' UTR as a
stabilizing element, we introduced the 3' UTR sequence into reporter
constructs in stellate cells as well as in NIH 3T3 fibroblasts. We
additionally chose to examine NIH 3T3 fibroblasts because of their great
transduction efficiency and because in preliminary experiments, we verified
that NIH 3T3 fibroblasts contain abundant quantities of each 56-and 62-kDa UTR
binding proteins (our unpublished data). Cells were transiently transfected
with a chimeric luciferase-ECE-1 3' UTR construct in which the
full-length 675-base pair 3' UTR was cloned downstream of a
cytomegalovirus-Luciferase construct. As shown in
Figure 5A, luciferase activity
in stellate cells transfected with the vector containing the 3' UTR was
significantly higher than that in cells transfected with vector alone.
Identical results were found in NIH 3T3 cells transfected in a similar manner
(our unpublished data). To further demonstrate a functional role for the
3' UTR, we exposed stably transfected NIH 3T3 fibroblasts containing a
chimeric ECE-1 3' UTR-GFP construct to TGF-1 (which we have shown
to mediate ECE-1 mRNA stability; see below)
(Figure 5B). GFP degradation in
control cells began 2 d after transduction and continued throughout the course
of the experiment. In contrast, GFP expression in cells containing the
GFP-3' UTR construct was substantially prolonged. Furthermore, exposure
of cells containing the GFP-3' UTR construct to TGF-1 resulted in
more intense and longer GFP expression than those not exposed to TGF-1.
Further controls consisted of constructs lacking putative protein binding
regions (i.e., the 47- to 675-base pair, 60- to 193-base pair, and 193- to
675-base pair constructs); these revealed luciferase activity similar to the
vector control (our unpublished data). The data indicate that the ECE-1
3' UTR plays an important role in mRNA stabilization and moreover
suggests that TGF-1 could play a direct role in regulation of proteins
that bind to the ECE-1 3' UTR.
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Effect of TGF- on ET-1 Production in Liver Wound Healing
Potential sources of ET-1 in the liver include each sinusoidal endothelial
and stellate cells (Shao et al.,
1999), the latter, a key effector in the wounding response to
liver injury. Furthermore, of factors that seem to be important in the
fibrogenic response to injury, TGF-1 is prominent. Therefore, we
investigated the effect of TGF-1 on ET-1 production in hepatic
endothelial and stellate cells. TGF-1 had no effect on ET-1 release in
quiescent (i.e., those isolated from normal liver) stellate cells but caused
significant increases in the release of ET-1 in activated (i.e., those from
livers after injury induced by BDL) stellate cells
(Figure 6A). Interestingly,
higher TGF-1 concentrations did not stimulate stellate cell release of
ET-1. The effect of TGF-1 on ET-1 release in normal and activated
stellate cells contrasted sharply with that in endothelial cells in which
TGF-1 induced an increase in ET-1 release in normal endothelial cells
but not in injured endothelial cells
(Figure 6A). Similar results
were elicited with each sinusoidal endothelial and stellate cells isolated in
other models of injury and wounding (i.e., eight consecutive doses carbon
tetrachloride injury; our unpublished data).
|
To further investigate whether ET-1 production in vivo could be mediated by
TGF-1, we induced liver injury in rats (by bile duct ligation for 8 d)
and simultaneously inhibited TGF- binding (TGF-1 and -3)
with a soluble TGF- receptor (STR) as described in MATERIALS AND
METHODS. ET-1 production in freshly isolated endothelial cells from normal
liver was 26-fold higher than that from normal stellate cells
(Figure 6B). However, after
liver injury induced by BDL, ET-1 production in freshly isolated stellate
cells increased markedly, whereas it was significantly decreased in
endothelial cells. Inhibition of TGF- in vivo in rats with liver injury
led to a significant decrease in ET-1 production in stellate cells
(Figure 6B). We also
investigated the effect of TGF-1 on ET-1 production in vivo at earlier
time points, including 4 d after liver injury and likewise found that
suppression of the effect of TGF- with the soluble TGF- receptor
inhibited ET-1 production in stellate cells (n = 4; our unpublished data).
Importantly, exposure of rats to isotype matched (i.e., to the soluble
TGF- receptor) control IgG had no effect on ET-1 production.
TGF- Controls ET-1 Production during Wounding by Regulation of
ECE-1
To explore the mechanism by which TGF- modulates ET-1 production in
liver injury, we examined both precursor ET-1 and ECE-1. Liver injury led to a
fourfold increase in preproET-1 mRNA expression in stellate cells but had no
significant effect on endothelial cell expression of preproET-1 mRNA
(Figure 7, A and B). Inhibition
of TGF- with the soluble TGF- receptor
(Figure 7, A and B) had no
effect on stellate or sinusoidal endothelial cell expression of preproET-1
mRNA. These data suggest that TGF- does not play a direct role in
regulation of precursor preproET-1 mRNA in stellate and endothelial cells
after liver injury, but raise the possibility that other factors such as
extracellular matrix, ET-1 itself and/or other cytokines lead to increases in
preproET-1 mRNA in activated stellate cells.
|
Because TGF- had prominent effects on stellate (or endothelial) cell
production of ET-1, but did not seem to affect precursor preproET-1 mRNA, we
examined the effect of TGF-1 on ECE-1. After liver injury, ECE-1 mRNA
levels were reduced by 20% in stellate (and endothelial) cells
(Figure 7, C and D). However,
inhibition of TGF- binding with soluble TGF- receptor resulted in
normalization of ECE-1 mRNA in stellate cells but not in endothelial cells. At
the protein level, after liver injury, ECE-1 levels were increased by fourfold
in stellate cells but decreased by 48% in endothelial cells
(Figure 7, E and F).
Furthermore, inhibition of TGF- in vivo during liver injury led to a
significant reduction in stellate cell ECE-1
(Figure 7, E and F), whereas it
had little effect on endothelial cell ECE-1 expression. Finally, we found
there was no change in ECE-1 enzymatic catalytic activity (conversion of big
ET-1 to ET-1) in stellate cells after BDL-induced liver injury or after
exposure of injured liver to the soluble TGF- receptor (our unpublished
data). These data suggest that TGF- regulates ECE-1 protein expression
(rather than preproET-1 levels or ECE-1 enzymatic activity), which in turn
contributes to the observed production of ET-1 in each stellate and
endothelial cells after liver injury
(Figure 6).
Given that ECE-1 mRNA became reduced while ECE-1 and protein expression was
increased (Figure 7, C and D, and E and
F), we hypothesized that ECE-1 mRNA must be stabilized after its
synthesis, and moreover, that TGF- plays a role in this process.
Therefore, we examined posttranscriptional regulation of ECE-1 in vivo by
inhibiting TGF- signaling with the soluble TGF- receptor. When
TGF- was inhibited in vivo, we found that stellate cell ECE-1 mRNA
stability was shifted toward normal (Figure
8A). ECE-1 mRNA decay in quiescent stellate cells was more rapid
than that in activated cells (half-life t1/2 16 vs. 35 h),
and moreover, inhibition of TGF- reduced ECE-1 mRNA
t1/2, consistent with a role for TGF-1 in ECE-1 mRNA
stabilization. To further evaluate the transcriptional and/or
posttranscriptional effect of TGF- on ECE-1 mRNA, we performed nuclear
run-on assays in isolated stellate cells (we used cells at 3 d in culture to
mimic the early activation process). As shown in
Figure 8B, TGF-1
significantly inhibited, rather than stimulated, ECE-1 mRNA transcription. To
further explore the role of TGF-1 in control of ECE-1 mRNA
stabilization, we performed experiments in which we exposed isolated stellate
cells (again early in the activation process) to TGF-1 and measured
ECE-1 mRNA stability; ECE-1 mRNA stabilization was more pronounced
(t1/2 = 35 h) in stellate cells stimulated with
TGF-1 than that (t1/2 = 20 h) of control cells
(Figure 8, C and D).
|
Because TGF- seemed to stabilize ECE-1 mRNA in vivo, we explored the
possibility that this was a result of stimulation of putative ECE-1 mRNA
3' UTR binding proteins by TGF-. Thus, we examined
RNA–protein complexes in stellate cell lysates (as in
Figure 1) and the effect of
TGF- inhibition with the soluble TGF- receptor. After induction of
liver injury by bile duct ligation, RNA–protein complexes were
identified in stellate cell lysates (as in
Figure 1) and inhibition of
TGF-1 abrogated this protein expression
(Figure 9). These data
demonstrate that in vivo, TGF- modulates expression of ECE-1 UTR binding
proteins and suggest that the mechanism by which TGF- modulates
expression of ECE-1 is via modulation of the expression of these binding
proteins.
|
Mechanisms Underlying TGF-–induced ECE-1 Stabilization
and ET-1 Production in Stellate Cells
To further explore the mechanisms by which TGF-1 exerts its effects
on stellate cell ECE-1 expression, we used a culture based in which stellate
cells are isolated from normal livers and allowed to undergo spontaneous
activation (induced by serum and plastic substratum). Cells from normal livers
grown in very early (24–48 h) culture mimic the normal quiescent
phenotype, whereas those after more prolonged culture mimic the injured
activated phenotype (Friedman,
1993). This model was chosen specifically to allow study of the
requirement for de novo protein synthesis. TGF-1 had no effects on
preproET-1 mRNA or ECE-1 mRNA expression in quiescent stellate cells,
consistent with data presented in Figures
6 and
7 (our unpublished data). In
contrast, analogous to the situation after in vivo activation
(Figure 7, C and D), exogenous
TGF-1 led to a reduction in ECE-1 expression, and as predicted,
inhibition of TGF- with the soluble TGF- receptor inhibited this
effect (Figure 10, A and B). Furthermore, exposure of activated cells to the soluble TGF- receptor
reduced ECE-1 levels to those found in quiescent stellate cells, a finding
consistent with known endogenous production of TGF-1 in activated
stellate cells (Bissell et al.,
1995). Also analogous to the situation after in vivo activation,
ECE-1 protein levels in culture-activated stellate cells were elevated
(Figure 10B). Furthermore,
TGF-1 stimulated ECE-1 protein expression, whereas the soluble
TGF- receptor inhibited this effect
(Figure 10B). Finally,
inhibition of protein synthesis with cycloheximide abolished the effects of
TGF-1 on ECE-1 mRNA (Figure
10A) as well as protein expression
(Figure 10B), suggesting that
the effect of TGF-1 on ECE-1 in activated stellate cells is dependent on
new protein synthesis.
|
We further evaluated TGF-1's ability to modulate expression of ECE-1
3' UTR binding proteins. In isolated stellate cells, normal cells did
not express ECE-1 3' UTR binding proteins; however, binding proteins
were expressed after activation. Notably, TGF-1 significantly increased
expression of both 56- and 62-kDa complexes
(Figure 10C) and the formation
of RNA–protein complexes was inhibited by addition of cycloheximide
(CHX), an inhibitor of protein synthesis. These data suggest that TGF-
leads to increased ECE-1 mRNA stability through new synthesis of RNA binding
proteins.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
;
McGary et al., 1997;
Zehner et al., 1997).
Among the most common elements found in mRNAs that mediate protein binding are
AREs and poly(C) regions found in the 3' UTRs of many mRNAs
(Wang et al., 1995;
Holcik and Liebhaber, 1997;
Xu et al., 1997;
Czyzyk-Krzeska and Bendixen,
1999; Paulding and
Czyzyk-Krzeska, 1999;
Tillmann-Bogush et al.,
1999); indeed, in the system described in this study, poly(C)
regions found in the ECE-1 3' UTR are the target of mRNA binding
proteins.
Our findings, in the context of other recent work in stellate cells,
emphasizes the concept that wound healing as a general biological phenomenon,
may alter gene and protein expression via unique regulatory processes. For
example, stellate cell type collagen I mRNA is stabilized after wound healing
and contributes to enhanced type I collagen mRNA and protein expression
(Stefanovic et al.,
1997,
1999). In these experiments,
increased mRNA stability was a result of CP binding to poly(C)-rich
areas of the type I collagen 3' UTR mRNA
(Focht and Adams, 1984;
Herget et al., 1989;
Stefanovic et al.,
1997,
1999). Thus, our work has
extended the concept that hepatic wound healing is associated with changes in
mRNA stability by identifying TGF-, a critical cytokine involved in
diverse forms of wounding (Border and
Noble, 1994; Sanderson et
al., 1995), as a regulator of 3' UTR binding proteins
that contribute to mRNA stability during this process.
An important finding of this work was that poly(C) elements, rather than
AREs, mediated protein binding to the ECE-1 3' UTR. AREs, in particular,
those containing AUUUA pentamers, have been studied extensively
(Levine et al., 1993;
Chen and Shyu, 1994;
Chen et al., 1995;
Chung et al., 1996;
Fan and Steitz, 1998;
Peng et al., 1998).
For example, a number of binding proteins (such as HuC, HuD, HuR, and Hel-N1
expressed in differentiated neurons) have been shown to interact with ARE of
the c-fos 3' UTR mRNA and to stabilize c-fos mRNA; likewise, a variety
of other AU-rich binding proteins have been identified in different cell types
(Zhang et al., 1993;
DeMaria and Brewer, 1996;
Ma et al., 1997;
Laroia et al., 1999).
It is notable that the proximal portion of the ECE-1 3' UTR mRNA
includes a classic ARE, containing three AUUUA pentamers. However, we were
unable to demonstrate binding of stabilizing proteins to this area, suggesting
that ECE-1 mRNA stabilization in this system is ARE independent. Because no
other AREs (i.e., other than those identified in the proximal sequence
examined in our study) are found in the ECE-1 3' UTR mRNA, it is
unlikely that AREs are involved in ECE mRNA stabilization.
The differential effects of TGF- on the central cellular sources of
ET-1, endothelial and stellate cells, in the hepatic wound healing model were
notable. In normal liver endothelial cells, TGF-1 stimulated ET-1
production in a "linear" dose-response manner, yet had little
effect on endothelial cells from injured liver
(Figure 6A). This finding
suggests that TGF- could be an important regulator of ET-1 in the normal
liver. Interestingly, TGF-1 did not have an effect on stellate cells
from normal livers, but rather had prominent effects on stellate cells from
injured livers. Interestingly, stellate cell synthesis of ET-1 was stimulated
in a nonlinear manner. The mechanism underlying this phenomenon is not
understood at present. However, because TGF-1 acts locally, it may not
be necessary for very high concentrations of TGF-1 to be present in
situ. Not only was ET-1 production by sinusoidal endothelial cells markedly
decreased after liver injury but also it did not seem to be affected
TGF-1. The decrease in ET-1 synthesis in sinusoidal endothelial cells
was presumably due to decreased ECE-1 production
(Figure 7E); further evidence
supporting the important role of ECE-1 was that neither liver injury nor
TGF-1 had significant effects on the precursor ET-1 synthetic pathway in
sinusoidal endothelial cells (Figure
7A).
The mechanism for the differential effect of TGF- in endothelial and
stellate cells from normal and injured livers is unknown. We have considered
the possibility that changes in receptor (i.e., the TGF- type II
receptor complex) density or affinity for TGF-1 on stellate or
endothelial cells account for the differences. For example, increased binding
of TGF-1 has been documented during stellate cell activation in vivo and
in culture (Friedman et al.,
1994). This could explain increased responsiveness of injured
stellate cells to TGF-1, but does not explain lack of a response to
normal cells, because these cells clearly possess TGF- receptor.
Alternatively, signal transduction pathways may be altered after injury.
Current evidence indicates that TGF- signals through each the p38
mitogen-activated protein kinase or SMAD pathways
(Derynck et al.,
1998; Datto et al.,
1999; Hanafusa et
al., 1999). In preliminary studies (Shao and Rockey,
unpublished observation), divergent TGF- signaling pathways after
hepatic injury and wound healing have been identified in each stellate and
sinusoidal endothelial cells, making it attractive to speculate that divergent
signaling pathways account for the differences in responses by sinusoidal
endothelial and stellate cells.
The importance of TGF- was further assessed in vivo by using a
soluble TGF- receptor that binds to TGF- and inhibits its
function. Although these experiments (Figures
6B and
7) clearly verified the
important regulatory effect of TGF- on stellate cells in vivo during
liver injury, they further emphasized the complexity of the regulatory
pathways. For example, we found that precursor preproET-1 mRNA was stimulated
in injured stellate cells, but that this effect was not linked to TGF-
(Figure 7, A and B). However,
consistent with an important role for TGF- in regulation of ECE-1
expression, inhibition of TGF- reduced ECE-1 levels. Notably, TGF-
does not seem to be the only factor involved, because inhibition of TGF-
only partially abrogated the up-regulation of ECE-1 after injury in stellate
cells (Figure 7, E and F). In
contrast to the effect of TGF- on stellate cells, its effect on
sinusoidal endothelial cells was less prominent. Indeed, the soluble
TGF- receptor had little effect on either sinusoidal endothelial cell
precursor preproET-1 or ECE-1, suggesting that factors other than TGF-
are important inhibitors of ECE-1 expression in injured sinusoidal endothelial
cells (Figure 7F). In
aggregate, these data emphasize the importance of TGF- in the regulation
of stellate cell ET-1 synthesis in the injured liver, and likewise support the
contention that other putative factors in the wounding system also affect both
stellate and endothelial cell ET-1 synthesis.
Our data suggest that factors other than TGF- are likely to be
important in regulation of ET-1 in the injured liver. For example, the
wounding milieu is complex and the wounding response is regulated by interplay
among the various systems. Indeed, other cytokines and the extracellular
matrix are prominent in many forms of wounding and are likely to be important
regulators of ET-1 in this environment. Nonetheless, TGF- has been
reported to lead to increased production of ET-1 in other systems
(Kurihara et al.,
1989; Kanse et al.,
1991; Schnermann et
al., 1996Eakes and Olson,
1998), consistent with its effect in liver wounding. In these
reports and in systems in which other factors stimulate ET-1 synthesis, the
effect has been largely via modulation of precursor ET-1 (i.e., preproET-1
mRNA expression), primarily at the level of preproET-1 mRNA transcription. To
our knowledge, a system in which TGF- plays a major role in regulation
of ET-1 via effects on ECE-1 has not described.
The current data suggest that ET-1 synthesis may be compartmentalized
(i.e., in endothelial and stellate cells). Classic ET biology assigns ET-1
production to endothelial cells; endothelial derived ET-1 in turn has
important paracrine effects on smooth muscle cells
(Yanagisawa et al.,
1988; Goto and Warner,
1995). Furthermore, ET-1 is thought to act in a paracrine, or
autocrine manner, physically its source of synthesis. In the current report,
we have used an in vivo model to explore cell systems important in ET
production in the intact organ and have demonstrated an important deviation
from the canonical synthetic paradigm. In this model, ET-1 production shifted
from the sinusoidal endothelial cell in the normal liver to the hepatic
stellate cell after injury (Figure
6A). Although relative ET-1 production by stellate and endothelial
cells seemed to be similar after injury
(Figure 6A), from a
quantitative standpoint, the bulk of ET-1 produced in the healing wound is
likely to be stellate cell derived. Striking proliferation of stellate cells
after liver injury results in a dramatic increase in the total number of
stellate cells (Geerts et al.,
1991); therefore, the overall increase in production of ET-1 in
the injured liver (Pinzani et
al., 1996) is likely to be a result of stellate rather than
endothelial cell synthesis and release of ET-1. Furthermore, localization of
ET-1 in the injured liver further seems to assign its production to stellate
cells (Pinzani et al.,
1996). Inhibition of soluble TGF- inhibited total hepatic
ET-1 production, is highly consistent with this conclusion. The transition of
ET-1 production from the endothelial cell to a nonendothelial compartment
after injury is consistent with emerging data in other forms of wound healing
in which nonendothelial elements such as fibroblasts and smooth muscle cells
can produce endothelins (Sung et
al., 1994). This "switch" in the cellular source
of ET-1, from endothelial cells in normal tissue to mesenchyme-derived cells
after injury seems to be characteristic in particular of pathological
situations.
We found that new protein synthesis is required for the up-regulation of
ECE-1 by TGF-. The requirement for new protein synthesis and the
induction of 56- and 62-kDa RNA binding proteins
(Figure 10, A–C) by
TGF- strongly supports the contention that TGF- helps control the
ECE-1 synthetic pathway after liver injury (and perhaps in other forms of
injury in which ET-1 is stimulated). Further investigation in which the 56-
and 62-kDa RNA binding proteins are identified and the mechanism by which
TGF- controls their synthesis will therefore be required.
The findings reported herein have important therapeutic implications for
wound healing. Most studies involving TGF- and wound healing have
presumed that TGF- is directly responsible for reduced fibrogenesis in
effector cells such as stellate cells. Although TGF- clearly has direct
effects on stellate cell fibrogenesis in cell culture systems, its effect is
unlikely to be this simple in vivo; indeed, we have clearly shown important
indirect effects of TGF- on the endothelin system. Moreover, because
enhanced production of ET-1 is common to many forms of wound healing,
including pulmonary fibrosis, heart failure, and renal fibrosis
(Forbes et al., 1996;
Kakugawa et al.,
1996; Karam et al.,
1996; Kohan, 1997;
Park et al., 1997;
Cho et al., 2000),
all of which are also characterized by elevated production of TGF-, we
speculate that in vivo, TGF- modulates the wound healing response
additionally by regulating the production of ET-1. Given the emerging
importance that ET-1 itself plays in the wound healing response (i.e., as a
stimulator of each cellular proliferation, fibrogenesis, and wound
contraction), our data emphasize a novel relationship between TGF- and
ET-1 in wound healing.
|
ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
receptor; TGF-1, transforming growth
factor-1; 3' UTR, 3' untranslated region.
Present address: Storr Liver Unit, Department of Medicine, University of
Sydney at Westmead Hospital, Westmead, New South Wales 2145, Australia. 
|| Corresponding author. E-mail address: don.rockey{at}duke.edu.
|
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