Regulation of Endothelin-1 Synthesis by Endothelin-converting Enzyme-1 during Wound Healing*

Rong Shao, Wei Yan, and Don C. RockeyDagger

From the Duke University Liver Center and the Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
Top
Abstract
Introduction
References

Endothelin-1 (ET-1) is involved in the pathogenesis of a number of diseases, including wound healing. In cirrhosis, the wounding response of the liver, circulating ET-1 levels are elevated; moreover, ET-1 has potent effects on hepatic stellate cells, the key effectors of cirrhosis. In this study, we have examined the regulatory role of ECE-1, a critical enzyme involved in ET-1 synthesis, in the two major cellular sources of hepatic ET-1. ET-1 release from normal hepatic endothelial cells was 25-fold higher than that from normal stellate cells. However, after liver injury, ET-1 release was increased in stellate cells but markedly decreased in endothelial cells. The two major isoforms of ECE-1, ECE-1alpha /1beta , made up 80% and 20%, respectively, of total ECE-1 in both stellate and endothelial cells. Following liver injury, ECE-1alpha mRNA was decreased by 44.2% in stellate cells, and by 16.1% in endothelial cells. ECE-1beta mRNA expression remained unchanged after injury. In contrast to ECE-1 mRNA, ECE-1 protein expression was increased by 43.9% in stellate cells but decreased in endothelial cells, while relative ECE-1 enzymatic activity was unchanged. In mRNA stability experiments, the half-life of ECE-1alpha mRNA in normal stellate cells was 13 h compared with 38 h in cells from injured livers. Thus, during hepatic wound healing, differential regulation of ECE-1 mRNA and protein appears to be critical in controlling ET-1 production.

    INTRODUCTION
Top
Abstract
Introduction
References

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-1alpha and -1beta , 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 alpha -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-1alpha , and ECE-1beta 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-1alpha and ECE-1beta 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-1alpha 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-1beta cDNA (Fig. 1). cDNA fragments were amplified and cloned into pGEM7+ (Promega, Madison, WI). cDNAs, resulting in 270 (ECE-1), 424 (ECE-1alpha ), and 321 (ECE-1beta ) 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, -1alpha , and -1beta cDNA fragments encoding the areas depicted by the closed bars were synthesized. ECE-1alpha and -1beta 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 [alpha -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-1alpha 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-1alpha and ECE-1beta . ECE-1alpha appears to be localized to microsomal membranes, whereas ECE-1beta 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-1alpha mRNA represents the predominant form, making up 80% of total ECE-1 (e.g. ECE-1beta 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-1alpha and -1beta ) 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-1alpha was significantly decreased in both stellate and endothelial cells after liver wounding (Fig. 5), whereas ECE-1beta 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-1alpha and -1beta . 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-1alpha 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-1alpha and S14) as described under "Materials and Methods." In panel A, a representative RNase protection assay is shown. In panel B, specific ECE-1alpha 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-1alpha 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-1alpha mRNA stability by using the inhibitor of RNA polymerase, actinomycin D. We found that ECE-1alpha 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-1alpha 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-1alpha 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-1alpha 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-1alpha 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-1alpha bands were scanned, quantitated, and normalized to the signal for glyceraldehyde-3-phosphate dehydrogenase, and the data presented graphically. ECE-1alpha 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-beta ) 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 alpha  (TNF-alpha ) 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-1alpha and ECE-1beta , have been identified in various rat tissues, but the expression level of ECE-1alpha is 5-10-fold higher than that of ECE-1beta (5). In the present study, we found that ECE-1alpha mRNA was 4-fold higher than ECE-1beta in normal stellate and endothelial cells and that mRNA levels of ECE-1 and ECE-1alpha were decreased in stellate and endothelial cells from injured liver but ECE-1beta levels were unchanged. These results are important for two reasons. First, ECE-1alpha appears to be regulable, whereas ECE-1beta apparently is not. Secondly, because ECE-1alpha is the major isoform and is regulable, the data imply that ECE-1alpha 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-1alpha is most likely localized at intracellular compartment whereas ECE-1beta is on plasma membrane (6, 44). Localization studies have demonstrated that, in Chinese hamster ovary and/or COS cells transfected with ECE-1, ECE-1alpha or ECE-1beta , all three isoforms localize to the plasma and intracellular membrane (5, 41). However, other data conflict and indicate that ECE-1 and ECE-1alpha are found intracellularly, whereas ECE-1beta is present on the plasma membrane (22). Most of the currently available studies demonstrating ECE-1alpha in intracellular locations have been performed in endothelial cells or human umbilical vein endothelial cells, whereas studies revealing ECE-1alpha and ECE-1beta 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-1alpha rather than ECE-1beta .

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.

    FOOTNOTES

* This work was presented in part at the 49th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), November 8-10, 1998 and was supported by grants from the National Institutes of Health (DK 02124 and DK 50574) and the American Digestive Health Foundation (The Fiterman Basic Research Award).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.

Dagger To whom correspondence should be addressed: Rm. 334, Sands Bldg., Box 3083, Liver Center, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-8727; Fax: 919-684-4983; E-mail: dcrockey{at}acpub.duke.edu.

The abbreviations used are: ET, endothelin; ECE, endothelin-converting enzyme; BDL, bile duct ligation; CCl4, carbon tetrachloride; t1/2, half-life; bp, base pair(s).
    REFERENCES
Top
Abstract
Introduction
References

  1. Rubanyi, G. M., and Botelho, L. H. (1991) FASEB J. 5, 2713-2720[Abstract/Free Full Text]
  2. Masaki, T., Kimura, S., Yanagisawa, M., and Goto, K. (1991) Circulation 84, 1457-1468[Medline] [Order article via Infotrieve]
  3. Simonson, M. S., and Dunn, M. J. (1990) FASEB J. 4, 2989-3000[Abstract]
  4. Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWit, D., and Yanagisawa, M. (1994) Cell 78, 473-485[Medline] [Order article via Infotrieve]
  5. Shimada, K., Takahashi, M., Ikeda, M., and Tanzawa, K. (1995) FEBS Lett. 371, 140-144[CrossRef][Medline] [Order article via Infotrieve]
  6. Valdenaire, O., Rohrbacher, E., and Mattei, M. G. (1995) J. Biol. Chem. 270, 29794-29798[Abstract/Free Full Text]
  7. Friedman, S. L. (1993) N. Engl. J. Med. 328, 1828-1835[Free Full Text]
  8. Kent, G., Gay, S., Inouye, T., Bahu, R., Minick, O. T., and Popper, H. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3719-3722[Abstract]
  9. Maher, J. J., and McGuire, R. F. (1990) J. Clin. Invest. 86, 1641-1648[Medline] [Order article via Infotrieve]
  10. Friedman, S. L., and Arthur, M. J. (1989) J. Clin. Invest. 84, 1780-1785[Medline] [Order article via Infotrieve]
  11. Ballardini, G., Fallani, M., Biagini, G., Bianchi, F. B., and Pisi, E. (1988) Virchows Arch. B Cell Pathol. 56, 45-49[Medline] [Order article via Infotrieve]
  12. Housset, C., Rockey, D. C., and Bissell, D. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9266-9270[Abstract]
  13. Rockey, D. C., Fouassier, L., Chung, J. J., Carayon, A., Vallee, P., Rey, C., and Housset, C. (1998) Hepatology 27, 472-480[Medline] [Order article via Infotrieve]
  14. Kawada, N., Tran-Thi, T. A., Klein, H., and Decker, K. (1993) Eur. J. Biochem. 213, 815-823[Abstract]
  15. Bauer, M., Zhang, J. X., Bauer, I., and Clemens, M. G. (1994) Am. J. Physiol. 267, G143-G149[Abstract/Free Full Text]
  16. Kountouras, J., Billing, B. H., and Scheuer, P. J. (1984) Br. J. Exp. Pathol. 65, 305-311[Medline] [Order article via Infotrieve]
  17. Proctor, E., Chatamra, K., Friedman, S. L., and Roll, F. J. (1982) Gastroenterology 83, 1183-1190[Medline] [Order article via Infotrieve]
  18. de Leeuw, A. M., McCarthy, S. P., Geerts, A., and Knook, D. L. (1984) Hepatology 4, 392-403[Medline] [Order article via Infotrieve]
  19. Friedman, S. L., and Roll, F. J. (1987) Anal. Biochem. 161, 207-218[Medline] [Order article via Infotrieve]
  20. Shimada, K., Takahashi, M., and Tanzawa, K. (1994) J. Biol. Chem. 269, 18275-18278[Abstract/Free Full Text]
  21. Rhoads, D. D., Dixit, A., and Roufa, D. J. (1986) Mol. Cell. Biol. 6, 2774-2783[Medline] [Order article via Infotrieve]
  22. Gui, G., Xu, D., Emoto, N., Yanagisawa, M., Parnot, C., Le Moullec, J. M., Cousin, M. A., Guedin, D., Corvol, P., and Pinet, F. (1993) J. Cardiovasc. Pharmacol. 22 (suppl.), 53-56
  23. Ross, J. (1995) Microbiol. Rev. 59, 423-450[Abstract]
  24. Moore, K., Wendon, J., Frazer, M., Karani, J., Williams, R., and Badr, K. (1992) N. Engl. J. Med. 327, 1774-1778[Abstract]
  25. Gandhi, C. R., Sproat, L. A., and Subbotin, V. M. (1996) Life Sci. 58, 55-62[CrossRef][Medline] [Order article via Infotrieve]
  26. Saleh, D., Furukawa, K., Tsao, M. S., Maghazachi, A., Corrin, B., Yanagisawa, M., Barnes, P. J., and Giaid, A. (1997) Am. J. Respir. Cell Mol. Biol. 16, 187-193[Abstract]
  27. Stewart, D. J., Kubac, G., Costello, K. B., and Cernacek, P. (1991) J. Am. Coll. 18, 38-43
  28. Moller, S., Gulberg, V., Henriksen, J. H., and Gerbes, A. L. (1995) J. Hepatol. 23, 135-144[CrossRef][Medline] [Order article via Infotrieve]
  29. Pinzani, M., Milani, S., De Franco, R., Grappone, C., Caligiuri, A., Gentilini, A., Tosti-Guerra, C., Maggi, M., Failli, P., Ruocco, C., and Gentilini, P. (1996) Gastroenterology 110, 534-548[Medline] [Order article via Infotrieve]
  30. Rockey, D. C., and Chung, J. J. (1996) J. Clin. Invest. 98, 1381-1388[Abstract/Free Full Text]
  31. Okumura, S., Takei, Y., Kawano, S., Nagano, K., Masuda, E., Goto, M., Tsuji, S., Michida, T., Chen, S. S., and Kashiwagi, T. (1994) Hepatology 19, 155-161[Medline] [Order article via Infotrieve]
  32. Zhang, J. X., Pegoli, W. J., and Clemens, M. G. (1994) Am. J. Physiol. 266, G624-G632[Abstract/Free Full Text]
  33. Zhang, J. X., Bauer, M., and Clemens, M. G. (1995) Am. J. Physiol. 269, G269-G277[Abstract/Free Full Text]
  34. Minamino, T., Kurihara, H., Takahashi, M., Shimada, K., Maemura, K., Oda, H., Ishikawa, T., Uchiyama, T., Tanzawa, K., Yazaki, Y., Yorimitsu, K., Moroi, K., Inagaki, N., Saito, T., Masuda, Y., Masaki, T., Seino, S., and Kimura, S. (1997) Circulation 95, 221-230[Abstract/Free Full Text]
  35. Matsuura, A., Kawashima, S., Yamochi, W., Hirata, K., Yamaguchi, T., Emoto, N., and Yokoyama, M. (1997) Biochem. Biophys. Res. Commun. 235, 713-716[CrossRef][Medline] [Order article via Infotrieve]
  36. Ikura, T., Sawamura, T., Shiraki, T., Hosokawa, H., Kido, T., Hoshikawa, H., Shimada, K., Tanzawa, K., Kobayashi, S., and Miwa, S. (1994) Biochem. Biophys. Res. Commun. 203, 1417-1422[CrossRef][Medline] [Order article via Infotrieve]
  37. Schmidt, M., Kroger, B., Jacob, E., Seulberger, H., Subkowski, T., Otter, R., Meyer, T., Schmalzing, G., Hillen, H., Matsuura, A., Kawashima, S., Yamochi, W., Hirata, K., Yamaguchi, T., Emoto, N., and Yokoyama, M. (1994) FEBS Lett. 356, 238-243[CrossRef][Medline] [Order article via Infotrieve]
  38. Emoto, N., and Yanagisawa, M. (1995) J. Biol. Chem. 270, 15262-15268[Abstract/Free Full Text]
  39. Yorimitsu, K., Moroi, K., Inagaki, N., Saito, T., Masuda, Y., Masaki, T., Seino, S., and Kimura, S. (1995) Biochem. Biophys. Res. Commun. 208, 721-727[CrossRef][Medline] [Order article via Infotrieve]
  40. Barnes, K., Walkden, B. J., Wilkinson, T. C., and Turner, A. J. (1997) J. Neurochem. 68, 570-577[Medline] [Order article via Infotrieve]
  41. Takahashi, M., Fukuda, K., Shimada, K., Barnes, K., Turner, A. J., Ikeda, M., Koike, H., Yamamoto, Y., and Tanzawa, K. (1995) Biochem. J. 311, 657-665[Medline] [Order article via Infotrieve]
  42. Yoshimura, H., Nishimura, J., Sakihara, C., Kobayashi, S., Takahashi, S., and Kanaide, H. (1997) Am. J. Respir. Cell Mol. Biol. 17, 471-480[Abstract/Free Full Text]
  43. Takada, J., Hata, M., Okada, K., Matsuyama, K., and Yano, M. (1992) Biochem. Biophys. Res. Commun. 182, 1383-1388[Medline] [Order article via Infotrieve]
  44. Schweizer, A., Valdenaire, O., Nelbock, P., Deuschle, U., Dumas Milne Edwards, J. B., Stumpf, J. G., and Loffler, B. M. (1997) Biochem. J. 328, 871-877[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.