 |
INTRODUCTION |
The family of endothelins is comprised of three 21-residue
peptides, endothelin-1, 2, and 3 (1, 2). Endothelin-1
(ET-1),1 the most prevalent
member of the endothelin family, is produced by a sequence of cleavage
steps. The translational product of preproET-1 mRNA (preproET-1) is
cleaved by as yet poorly identified furin-like enzymes to yield a
peptide of 39 amino acid residues, which has been termed big ET-1 (1,
3). This enzymatic step has been felt to be relatively nonspecific and
not rate-limiting (1). Big ET-1 is subsequently cleaved at the Trp-21
residue by a specific membrane-bound endothelin-converting enzyme-1
(ECE-1) (4). Recently, two ECE-1 isoforms have been identified and termed ECE-1
and -1
, which are alternatively spliced products of
the same gene and share the same C-terminal domain (5, 6). ECE-1 has
been localized predominantly to endothelial cells, the apparent primary
source of ET-1 (4). Thus, ECE-1 has been regarded as an integral
component of ET-1 processing in endothelial cells.
Persistent hepatic injury leads to a wound healing response that
ultimately results in the entity known as cirrhosis. This scarring
process is analogous to injury in other organs and includes components
of both increased fibrogenesis and wound contraction. Recent evidence
indicates that a key component of the hepatic wounding response is the
hepatic stellate cell (also Ito cell or lipocyte), a pericyte-like
perisinusoidal cell of mesenchymal lineage that lies in close proximity
to sinusoidal endothelial cells (7). After injury, stellate cells
undergo a programmed cascade of events, including enhanced matrix
synthesis, cellular proliferation, and remarkable de novo
expression of smooth muscle
-actin, the latter event suggesting a
stellate cell to myofibroblast transition (8-11). This process, termed
"activation," may be stimulated by ET-1 and is mediated by
endothelin A and B receptors on stellate cells (12, 13). Further study
has demonstrated that ET-1 causes stellate cell proliferation and
contraction and collagen production during liver injury (13-15). Thus,
the data suggest that ET-1 has important effects in hepatic wound
healing via its actions on stellate cells.
Although ET-1 production during hepatic wounding is likely to be
regulated (at least in part) at the level of preproET-1 mRNA production in liver endothelial and stellate cells (13), we hypothesized that ECE-1 could also be an important regulator of ET-1
production. To test this hypothesis, we have investigated ET-1 release
as well as ECE-1 mRNA and protein regulation in cells from normal
and injured rat livers. We have taken advantage of our ability to
reproducibly create a wounding healing environment in the liver and,
moreover, the ability to isolate specific cellular components that are
the sources of ET-1. The data are consistent with a unique combination
of regulatory events in specific cellular compartments during hepatic
wound healing and have important implications for endothelin biology in
parenchymal organ injury.
 |
MATERIALS AND METHODS |
Liver Injury and Hepatic Fibrosis--
Male Sprague-Dawley rats
(450-500 gram, Harlan Sprague-Dawley, Indianapolis, IN) were
maintained on standard chow and water ad libitum. Hepatic
fibrogenesis was induced by gavage with carbon tetrachloride (1.0 ml/kg
mixed with corn oil) on consecutive weeks or by ligation of the common
bile duct (16, 17).
Cell Isolation and Culture--
Nonparenchymal cells were
isolated as described previously (18). Briefly, stellate and
endothelial cells were isolated by perfusion in situ with 20 mg/100 ml Pronase and 7.5 mg/100 ml collagenase (both from Boehringer
Mannheim). The cell suspension was layered on a discontinuous density
gradient of 8.2 and 15.6% nycodenz (Accurate Chemical and Scientific,
Westbury, NY). The resulting upper layer consists of more than 95%
stellate cells. Endothelial cells in the lower layer were further
purified by centrifugal elutriation (18 ml/min flow). Purity was
assessed by the use of specific markers for each stellate cells and
endothelial cells (19). Cells were suspended in modified medium 199, containing 20% serum (10% horse serum and 10% calf serum, Life
Technologies, Inc.), 4 mg/100 ml streptomycin, and 0.25 mg/100 ml
amphotericin at a density of approximately 1 × 106
cells/ml. Cultures were incubated at 37 °C in a humidified incubator (containing 95% O2 and 5% CO2), and the
medium was changed at every 24 h. Cell viability was greater than
80% in all cultures utilized for study.
ET-1 Radioimmunoassay--
Cells were cultured in
serum-containing medium for 48 h, after which time serum-free
conditions were introduced. ET-1 in supernatants was measured using a
radioimmunoassay kit according to manufacturer specifications
(Peninsula Labs, Palo Alto, CA). Immunoreactive ET-1 was normalized to
total protein content for each sample (Bio-Rad, Richmond, CA). The ET-1
antibody used in the radioimmunoassay kit exhibited <5%
cross-reactivity with unlabeled ET-3 and <3% cross-reactivity with
unlabeled ET-2. Inter- and intra-assay variability of the
radioimmunoassay system were 3.8 and 1.5%, respectively.
ECE-1 cDNAs--
Based on the reported sequence of rat ECE-1
(5, 20), oligodeoxynucleotide primers that could detect each ECE,
ECE-1
, and ECE-1
mRNA were synthesized (Fig. 1) (Operon
Technologies, Alameda, CA). 5' and 3' sequences used to isolate ECE-1
were as follows: CGG CTG GTG GTT CTG GTG (154-171bp) and CTT GGT TGT
GCT CCC AGA GGT (404-424bp). 5' and 3' sequences encoding
alternatively spliced ECE-1
and ECE-1
mRNA variants (5, 20)
were the following: ATG ATG TCA TCC TAC AAG (1-18 bp) and CTT GGT TGT
GCT CCC AGA GGT (404-424bp) were used to clone ECE-1
cDNA, and
ATG GGC AGC CTG AGG CCT (1-18 bp) and CAT GGA GTT TAG GAT GGA
(304-321 bp) were used to obtain ECE-1
cDNA
(Fig. 1). cDNA fragments were amplified and cloned into pGEM7+ (Promega, Madison, WI). cDNAs, resulting in 270 (ECE-1), 424 (ECE-1
), and 321 (ECE-1
) base pair
fragments, respectively, were sequenced by the dideoxy chain termination method and found to have 99-100% homology to published cDNAs (5, 20).

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 1.
ECE-1 constructs. ECE-1, -1 , and
-1 cDNA fragments encoding the areas depicted by the
closed bars were synthesized. ECE-1 and -1 are
alternatively spliced (6) and diverge only at the initial 84 and 108 bp, respectively (hatched boxes). The conserved
transmembrane domain region and zinc-binding and catalytic sites are
highlighted by the closed and stippled
boxes, respectively.
|
|
RNA Isolation and RNase Protection Assay--
Total RNA was
extracted (Tri-reagent, Molecular Research Inc, Cincinnati, OH) from
cell lysates; RNA concentration and purity were determined
spectrophotometrically (A260/280). The integrity of all samples was documented by visualization of 18 and 28 S ribosomal
bands after electrophoresis through a 1% agarose/formaldehyde gel.
Radiolabeled probes were synthesized by transcription of appropriate
plasmid cDNA with T7 or T3 RNA polymerase in the presence of
[
-32P]CTP (Amersham Pharmacia Biotech). Total RNA (20 µg) was incubated with 1.0 × 106 cpm of
32P-labeled cRNA, denatured at 78 °C for 15 min, and
hybridized in hybridization buffer 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 Co., Rochester, NY), and scanning densitometry was used to quantitate autoradiographic signals. All RNA samples were also probed with either a cDNA encoding 585 bp of ribosomal protein S-14
(21) or a cDNA encoding 316 bp of glyceraldehyde-3-phosphate dehydrogenase (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. Transfer RNA and lung RNA were used as
negative and positive RNAs, respectively, for all experiments.
Immunoblot--
Cultured cells were homogenized in 20 mM Tris-HCl buffer (pH 7.4) containing 5 mM
MgCl2 and protease inhibitors, 0.1 mM
phenylmethylsulfonyl fluoride, 20 µM pepstatin A, and 20 µM leupeptin. The homogenate was centrifuged at 1000 × g for 10 min, and the resulting supernatant was
centrifuged at 100,000 × g for 60 min. The microsomal
membrane fraction was resuspended, and samples (30 µg) were incubated
with ECE-1 antibody (10 µg/sample) at 4 °C overnight. The mixture
was subsequently incubated with protein A-Sepharose beads (Sigma) precoupled with rabbit anti-mouse IgG for 1 h at 4 °C. After
centrifugation at 12,000 × g for 40 s, the beads
were washed three times, and immune complexes were disassociated by
heating to 100 °C for 3 min. After centrifugation at 12,000 × g for 40 s, supernatants were subjected to
immunoblotting. For immunoblot, microsomal membrane proteins (30 µg)
or the sample proteins (30 µg) from immunoprecipitation were
separated by SDS-polyacrylamide gel electrophoresis under reducing
conditions (5% mercaptoethanol) and transferred to the nitrocellulose
(Bio-Rad). Blots were incubated with ECE-1 primary antibody B61/104
(1:400) (kindly provided by Dr. Thomas Subkowski, BASF,
Aktiengesellschaft, Germany) followed by horseradish
peroxidase-conjugated rabbit anti-mouse IgG (1:1000) (ECL kit, Amersham
Pharmacia Biotech). Immunoreactive protein was detected after addition
of substrates and exposed to x-ray film. Bands corresponding to ECE-1
were quantitated by scanning densitometry.
ECE-1 Enzymatic Activity--
ECE-1 enzymatic activity was
determined as described previously(4). 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 prior to 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 as above to detect
immunoreactive ET-1.
Statistics--
Data are expressed as mean ± standard
errors (S.E.) 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 test. The 0.05 level of probability was used as the
criterion of significance.
 |
RESULTS |
Liver Injury--
We utilized two models of hepatic wounding to
examine ET-1 regulation in stellate and endothelial cells. Histological
assessment of livers after injury revealed that administration of
CCl4 for 14 doses resulted in extensive portal-central,
central-central, and portal-portal bridging fibrosis with portal
hypertension documented in all cases; bile duct ligation for 8 days
resulted in bile duct proliferation with extensive periportal expansion
of biliary radicals and extension of fibrous bands from portal tracts
into the lobule.
ET-1 Production in Stellate and Endothelial Cells from Injured
Liver--
Previous studies have demonstrated that preproET-1 mRNA
is up-regulated in both stellate and endothelial cells after bile duct
ligation, but to a much greater degree in stellate than endothelial cells (13). Although these data elucidate a potential site for ET-1
regulation after wounding, we postulated that because ECE-1 converts
big ET-1 to biologically active ET-1, production of the final 21 amino
acid product might be regulated not only at the level of precursor
preproET-1/big ET-1, but also by the quantity and/or activity of ECE-1.
Initial experiments, therefore, examined ET-1 release by normal and
injured stellate and endothelial cells. Experiments were designed such
that ET-1 levels were measured in culture media soon after cell
isolation, and thus would reflect in vivo production and
release of ET-1. We found that ET-1 production in normal endothelial
cells was 25-fold higher than that from normal stellate cells
(Fig. 2). However, after liver injury
(both BDL and CCl4-treated rats), ET-1 release from
stellate cells was increased, whereas it was decreased in sinusoidal
endothelial cells. These data provide strong evidence that endothelial
cells are the main source of ET-1 production in normal liver but that, after liver injury, the cellular source of released ET-1 shifts to
stellate cells.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Immunoreactive ET-1 production by stellate
and endothelial cells from injured liver. Cells were isolated from
normal (Nor) rats, 8 days after bile duct ligation or after
14 doses of CCl4. After isolation, cells were grown for
48 h in culture with standard culture medium. Serum-free
conditions were introduced, and immunoreactive ET-1 in culture medium
supernatants was measured 24 h later by radioimmunoassay as
described under "Materials and Methods." *, p < 0.05 compared with normal for each cell type (n = 8).
|
|
ECE-1 and ECE-1
mRNA Regulation in Stellate and Endothelial
Cells after Liver Injury--
During initial examination of ECE in
liver injury, we explored ECE-1 mRNA and protein expression in
stellate and endothelial cells from normal and injured liver. As under
"Materials and Methods" and Fig. 1, ECE-1 has two isoforms,
ECE-1
and ECE-1
. ECE-1
appears to be localized to microsomal
membranes, whereas ECE-1
is found on the plasma membrane (6, 22).
Therefore, these isoforms may be responsible for endogenous and
exogenous conversion of big ET-1 to ET-1, respectively. We found that
in each stellate and endothelial cells, ECE-1
mRNA represents
the predominant form, making up 80% of total ECE-1 (e.g.
ECE-1
constitutes 20% of total ECE-1)
(Fig. 3).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
ECE-1 isoform mRNA expression in normal
stellate and endothelial cells. Normal stellate and endothelial
cells were isolated and cultured as in Fig. 2. Total cellular RNA was
isolated and hybridized (20 µg/sample) with 32P-labeled
cRNAs (S14 and ECE-1 and -1 ) as described under "Materials and
Methods." In panel A, a representative RNase protection
assay is shown. Bars represent cRNA probe positions, and
arrowheads represent specific mRNA bands. In panel
B, specific bands were scanned, quantitated, normalized to S14
signal, and expressed as the percentage of normal stellate cells
(n = 2).
|
|
During liver injury, we unexpectedly found that ECE-1 mRNA levels
were reduced in stellate cells (approximately 30%) in both models of
liver injury (Fig. 4). ECE-1 mRNA in
endothelial cells was also decreased after each bile duct ligation and
carbon tetrachloride administration, but to a lesser extent than for
stellate cells. Additionally, mRNA expression of the predominant
isoform of ECE-1, ECE-1
was significantly decreased in both stellate
and endothelial cells after liver wounding
(Fig. 5), whereas ECE-1
mRNA
levels were not altered (data not shown).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 4.
ECE-1 mRNA expression in stellate and
endothelial cells from injured liver. Cells were isolated after
bile duct ligation or CCl4 administration and cultured as
in Fig. 2. Forty-eight h after plating, total cellular RNA was isolated
and hybridized (20 µg/sample) with 32P-labeled cRNAs
(ECE-1 and S14) as described under "Materials and Methods." The
ECE-1 cDNA was cloned from the amino-terminal, nonspliced
transmembrane domain region and therefore represents total ECE-1 and
-1 . In panel A, an RNase protection assay containing
three individual cell specimens probed for ECE-1 and S14 is shown. In
panel B, data were scanned, quantitated, normalized to S14
and expressed graphically. *, p < 0.05 compared with
normal (n = 3).
|
|

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
ECE-1 mRNA
expression is decreased in stellate and endothelial cells from injured
liver. Stellate and endothelial cells were isolated after liver
injury and cultured as in Fig. 2. Forty-eight h after plating, total
cellular RNA (20 µg/sample) was isolated and hybridized with
32P-labeled cRNAs (ECE-1 and S14) as described under
"Materials and Methods." In panel A, a representative
RNase protection assay is shown. In panel B, specific
ECE-1 bands were scanned, quantitated, and normalized to the signal
for S14 and the data presented graphically. *, p < 0.05 compared with normal (n = 4).
|
|
ECE-1 Expression in Stellate and Endothelial Cells from Injured
Liver--
To determine whether the decreased ECE-1 mRNA
expression was associated with a similar change in protein expression
in stellate and endothelial cells after liver injury, we measured
intracellular expression of ECE-1 by immunoblotting of microsomal
fractions. In contrast to the reduction in ECE mRNA levels, we
found that ECE-1 protein levels in stellate cells from injured liver
were 43.9% higher than in normal cells, whereas ECE-1 levels in
endothelial cells were decreased by 27.6%
(Fig. 6). ECE-1 protein levels in normal
sinusoidal endothelial cells were 1.3-fold greater than that in normal
stellate cells, whereas ECE-1 levels in stellate cells from injured
liver were 1.8-fold greater than that from sinusoidal endothelial
cells.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
ECE-1 regulation in stellate and endothelial
cells from injured liver. Stellate and endothelial cells were
isolated after liver injury, and grown in culture as in Fig. 2. In
panel A, 48 h after culture, microsomal membrane
fractions were prepared and subjected to SDS-polyacrylamide gel
electrophoresis. Proteins were transferred to a nitrocellulose
membrane, and immunoreactive ECE-1 was detected using monoclonal
antibody directed against ECE-1. In panel B, specific bands
were quantitated by scanning densitometry and expressed as the
percentage of normal stellate cells. *, p < 0.05 compared with normal (n = 4).
|
|
To determine whether liver injury had an effect on ECE-1 activity
itself, we examined conversion of big ET-1 to ET-1 by ECE in stellate
and endothelial cells from normal and injured livers. After normalizing
ECE-1 activity (i.e. conversion of big ET-1 to ET-1) to
immunoprecipitated ECE-1, we found no difference in ECE-1 activity in
stellate or endothelial cells after liver injury (data not shown).
Thus, the quantitative ECE-1 data parallel the results of
immunoreactive ET-1 production from stellate and endothelial cells
(Fig. 2), suggesting that altered expression of ECE-1 without alteration of its activity in stellate and endothelial cells after wounding determines release of mature ET-1.
ECE-1
mRNA Stability Is Increased in Stellate Cells from
CCl4-treated Rats--
The divergent results identified
for ECE mRNA and protein expression in stellate cells from normal
and injured liver imply a form of post-transcriptional regulation for
ECE-1. To investigate this possibility, we determined ECE-1
mRNA
stability by using the inhibitor of RNA polymerase, actinomycin D. We
found that ECE-1
mRNA decay in stellate cells from
CCl4-treated rats was much slower than that in normal rats
(Fig. 7). mRNA in normal stellate
cells was decreased after 6 h and dramatically decreased between
12 and 36 h after exposure of cells with actinomycin D. In
contrast, ECE-1
mRNA in stellate cells from
CCl4-treated rats was only slightly decreased over a 36-h
time span (Fig. 7). By interpolation (23), half-life time
(t1/2) of ECE-1
mRNA in normal stellate cells
was found to be 13 h but was 38 h in stellate cells from
CCl4-treated rats. Therefore, the 3-fold longer mRNA
stability in stellate cells from injured compared with cells from
normal liver is likely to be responsible for comparatively greater
ECE-1 protein expression in stellate cells.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
ECE-1 mRNA
stability is increased in stellate cells from
CCl4-treated rats. Stellate cells were
isolated from normal and CCl4-treated (14 doses) rats.
After cells were allowed to adhere for 48 h, actinomycin D (15 µg/ml) was added to the medium, and total cellular RNA was isolated
at the indicated times. RNA (15 µg/sample) was hybridized with
32P-labeled cRNAs (ECE-1 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH)) as described under "Materials and
Methods." In panel A, an RNase protection assay is shown.
In panel B, specific ECE-1 bands were scanned,
quantitated, and normalized to the signal for
glyceraldehyde-3-phosphate dehydrogenase, and the data presented
graphically. ECE-1 mRNA half-life computations (23) revealed
t1/2 = 13 h for normal stellate cells
(open symbols) and 38 h for stellate cells from
CCl4-treated rats (closed symbols)
(n = 3).
|
|
 |
DISCUSSION |
The wounding response to injury is carried out by cells which
contract the wound and/or produce increased amounts of extracellular matrix. For example, the mediator of the wounding healing response of
the liver is the perisinusoidal stellate cell, a cell type which
undergoes a cascade of events that confers on them the ability to
contract and to produce large amounts of extracellular matrix proteins.
Additionally, stellate cells have been shown to express abundant
endothelin and cytokine (e.g. platelet-derived growth factor
and transforming growth factor-
) receptors, the ligands for which
have been implicated in their contractility and fibrogenesis, respectively. ET-1, the predominant ET-receptor ligand, appears to
be involved in the pathogenesis of a number of pathologic wound healing
diseases such as renal, cardiac, and pulmonary fibrosis as well as
hepatic cirrhosis (24-27). In cirrhosis, plasma ET-1 levels are
elevated (24, 28), and it has been demonstrated that liver sinusoidal
endothelial and stellate cells are sources of ET-1 (13, 29). Further,
ET-1 has been shown to have potent effects on stellate cells, inducing
their contraction and resulting in their activation (12, 14, 30-33).
For this reason, an important issue with regard to liver wounding is
the regulation of ET-1.
Our data demonstrate a shift in the cellular source of ET-1 after
hepatic wounding. In normal liver, sinusoidal endothelial cells
released significantly more ET-1 than stellate cells. In contrast,
after injury, stellate cells became a more prominent source of ET-1 and
are likely to be responsible for elevated circulating levels of this
peptide in patients with cirrhosis. Moreover, the mechanism for the
change in ET-1 release appeared to be that ECE was differentially
regulated (i.e. up in stellate and down in endothelial
cells) in the two cell types. These data raise the possibility that
nonendothelial structures might be an important source of ET-1 in other
forms of wound healing.
We have previously shown that preproET-1 mRNA becomes elevated in
both stellate and endothelial cells after bile duct ligation, although
to a much greater degree in stellate than endothelial cells (13). These
data are consistent with enhanced production of ET-1 and suggest that
ET-1 production could be regulated at the level of preproET-1 mRNA
transcription and preproET-1/big ET-1 synthesis. However, because the
level of biologically active ET-1 also depends on ECE-1, the enzyme
that converts big ET-1 to ET-1, we postulated that ECE-1 could serve as
an important regulatory element. Surprisingly, while ECE-1 mRNA
decreased during liver wounding in both stellate and endothelial cells,
the level of proteolytically active ECE-1 protein increased in stellate cells but decreased in endothelial cells (a picture mirrored by cellular ET-1 release). Not only is such a differential regulatory event unique, but, because released ET-1 peptide levels closely parallel ECE-1 protein expression, this leads us to conclude that ECE-1
plays a major role in the regulation of ET-1 release into the wounding environment.
Other work is consistent with our own and suggests that ECE-1 is
important in regulation of ET-1 production in pathological states. For
example, in a rat vascular balloon injury model and human coronary
atherosclerotic lesions, ECE-1 mRNA and enzyme activity were
increased (34). The mechanism by which ECE is regulated in these models
is open to speculation but has been postulated to be related to
vascular endothelial growth factor (VEGF) as this compound has been
shown to directly stimulate ECE-1 expression (35). Additionally, in
human bronchial epithelial cells, interleukin-1 (IL-1) and tumor
necrosis factor
(TNF-
) were shown to increase not only ET-1
mRNA expression and ET-1 release but also ECE-1 mRNA expression
(26). We postulate, however, that ET-1 regulation in liver injury and
wounding is likely to be more complex than simple cytokine or growth
factor stimulation of ECE-1 mRNA and protein (or preproET-1
mRNA) because we have demonstrated divergent regulation of ECE-1
mRNA and protein in two different cell types, each of which is
prominent in liver wounding.
Phosphoramidon-sensitive ECEs are type II integral membrane proteins;
two ECEs, ECE-1 (5, 20, 36, 37) and ECE-2 (38, 39), have been cloned.
Sequence analysis of ECE-1 and ECE-2 reveals 59% amino acid homology;
the two isoforms are distinguished largely by their sensitivity to
phosphoramidon, their pH optimum, and possibly by their subcellular
localization (38). Two isoforms of ECE-1, ECE-1
and ECE-1
, have
been identified in various rat tissues, but the expression level of
ECE-1
is 5-10-fold higher than that of ECE-1
(5). In the present
study, we found that ECE-1
mRNA was 4-fold higher than ECE-1
in normal stellate and endothelial cells and that mRNA levels of
ECE-1 and ECE-1
were decreased in stellate and endothelial cells
from injured liver but ECE-1
levels were unchanged. These results
are important for two reasons. First, ECE-1
appears to be regulable,
whereas ECE-1
apparently is not. Secondly, because ECE-1
is the
major isoform and is regulable, the data imply that ECE-1
is the
major biologically isoform of ECE-1 (5).
Previous studies have demonstrated that ECE-1 is localized primarily to
endothelial cells (40-42). In our study, we have not only definitively
confirmed ECE-1 expression by liver endothelial cells but also
demonstrated ECE-1 production by stellate cells, mesenchymal cells of
an entirely different lineage than endothelial cells. Other data on
expression of ECE-1 in nonendothelial cells are limited but indicate
that ECE-1 mRNA or protein expression is predominantly a feature of
endothelial cells; few other cell types express ECE-1 (40, 43). In
contrast, we found that stellate cells were a major source of ECE.
Thus, it is likely that paracrine effects of endothelial cell-derived
ET-1 on stellate cells are important; however, our data also highlight
an important potential autocrine loop of ET-1 synthesis and binding in
nonendothelial cells.
The intracellular distribution of ECE-1 is important because different
ECE-1 isoforms localized to different subcellular fractions may behave
differently in function. The majority of ECE-1 activity has been
identified in microsomal membrane fractions (22). Currently available
data suggest that ECE-1
is most likely localized at intracellular
compartment whereas ECE-1
is on plasma membrane (6, 44).
Localization studies have demonstrated that, in Chinese hamster ovary
and/or COS cells transfected with ECE-1, ECE-1
or ECE-1
, all
three isoforms localize to the plasma and intracellular membrane (5,
41). However, other data conflict and indicate that ECE-1 and ECE-1
are found intracellularly, whereas ECE-1
is present on the plasma
membrane (22). Most of the currently available studies demonstrating
ECE-1
in intracellular locations have been performed in endothelial
cells or human umbilical vein endothelial cells, whereas studies
revealing ECE-1
and ECE-1
localization to the plasma membrane
have been done in transfected cells that normally do not express ECE-1.
Our study allows careful examination of the cellular compartmentation
of ECE-1. We have demonstrated by both subcellular fractionation and
immunohistochemistry (data not shown) that in both primary endothelial
and stellate cells, the vast majority of ECE-1 is intracellular. This
finding is consistent with the postulate that the predominant active
isoform is microsomal-bound ECE-1
rather than ECE-1
.
Our results indicate that the half-life of ECE-1 mRNA was
dramatically increased in stellate cells after liver injury and their
activation. This was associated with a striking decrease in
steady-state ECE-1 mRNA levels and an increase in ECE-1 protein; in
aggregate, these data intimate that ECE-1 mRNA transcription is
reduced in injured stellate cells, but also suggest that ECE-1 protein
levels are controlled by post-transcriptional mechanisms. In contrast,
the half-life of ECE-1 mRNA in endothelial cells is unchanged after
injury whereas ECE-1 mRNA and protein levels are reduced,
suggesting transcriptional or pre-translational control. Although we
are unaware of previous data demonstrating altered ECE-1 mRNA
stability and protein expression in a biologically important situation
such as wound healing, these data have several important implications.
For example, the data raise the possibility that the wounding response
results in production or recruitment of cellular machinery that could
stabilize ECE-1 mRNA and imply that wounding may modify cellular
protein synthesis by complex molecular mechanisms.