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Volume 270, Number 12, Issue of March 24, 1995 pp. 6997-7003
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endothelin Receptor Subtype B Mediates Autoinduction of Endothelin-1 in Rat Mesangial Cells (*)

(Received for publication, November 29, 1994; and in revised form, January 18, 1995)

Shigeki Iwasaki ,  Toshio Homma ,  Yuzuru Matsuda (1),  Valentina Kon (§)

From the Division of Pediatric Nephrology, Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2584 Kyowa Hakko Kogyo Co., Tokyo 194, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Autoinduction of endothelin-1 (ET-1) has been suggested to be involved in the profound and long-lasting effects of ET-1. We examined mechanisms that underlie autoinduction of ET-1 in cultured rat glomerular mesangial cells. Incubation of mesangial cells with ET-1 resulted in an immediate and dose-dependent stimulation of preproET-1 mRNA expression as assessed by polymerase chain reaction coupled with reverse transcription. Within 1 h of exposure to ET-1 (10M), preproET-1 mRNA expression was increased to a maximal level of 465 ± 43% of the control value (p < 0.01), which was accompanied by significant stimulation of production of the immunoreactive ET-1 peptide. Nuclear run-off analysis revealed increases in the transcriptional rate of preproET-1 mRNA to 239 and 175% above the control values at 1 and 3 h of ET-1 stimulation, respectively. ET-1 also increased the stability of preproET-1 mRNA, resulting in an mRNA half-life of 60 min from 20 min seen in non-stimulated cells. Addition of an ET(B)-specific antagonist, RES701-1, at >10M abolished ET-1 stimulation of preproET-1 mRNA (p < 0.001), whereas an ET(A)specific antagonist, BQ123, was without effects (up to 10M). The ET(B) agonist, sarafotoxin S6c (10M), significantly stimulated preproET-1 mRNA expression to 201 ± 14% above controls (p < 0.01), an effect that was lessened significantly by RES701-1 (p < 0.05). RES701-1 abolished the ET-1-induced production of the ET-1 peptide (p < 0.001). Taken together, we demonstrates that in mesangial cells, autoinduction of ET-1 occurs through the ET(B) receptor subtype via increases in both preproET-1 transcription and mRNA stability.

INTRODUCTION

Endothelins are a family of peptides which include endothelin-1 (ET(^1)-1), ET-2, and ET-3. These isopeptides have a variety of biological effects, including unparalleled vasoconstriction, induction of cell hyperplasia and/or hypertrophy, regulation of renal tubule reabsorption, as well as modulation of other hormone and cytokine production(1, 2, 3, 4, 5, 6, 7) . These effects are mediated through at least two receptor subtypes, ET(A) and ET(B), both of which are extensively distributed(8, 9, 10) . The wide distribution of ET receptors is paralleled by a similarly extensive and overlapping localization of ET production and mRNA expression(10) , emphasizing the potential importance of paracrine and/or autocrine mechanisms for the actions of ET. When vascular endothelial cells are cultured on permeable support, a major fraction of produced ET-1 is secreted in the basolateral direction(11) , where it can interact in a paracrine fashion with receptors on the underlying smooth muscles and modulate contractile tone and proliferative responses. Autocrine mechanisms have also been implicated by the fact that cells capable of producing ET-1 also express ET receptors (12, 13, 14, 15) . Thus, both HeLa and HEp-2 cells produce ET-1 and proliferation of these cells is inhibited by polyclonal antibody to ET-1(12) . Angiotensin II-induced hypertrophy of cultured cardiomyocytes is associated with increases in preproET-1 mRNA expression and ET-1 peptide production and the hypertrophic response is inhibited by an ET(A) receptor antagonist, without affecting preproET-1 mRNA expression(13) . Similarly, platelet-derived growth factor-induced proliferation of glomerular mesangial cells is inhibited by an ET(A) receptor antagonist(14) . More recently, transfection and overexpression of ET-1 in cultured smooth muscle cells has been shown to promote proliferation of these cells in a manner inhibitable by an ET(A) receptor antagonist(15) . However, while autocrine mechanisms may be important for regulation of cell function by ET produced by that cell, this mechanism does not readily account for the persistence of ET's actions.

In this connection, increasing evidence indicates the possible autoinduction of ET-1. In human endothelial cells, production of ET-1 is augmented by ET-3 (16) and ET-1(17) . Enhanced expression of preproET-1 mRNA has been observed in cardiomyocytes upon exposure to ET(13) . Autoinduction has been documented for TGF-alpha(18) , B-chain of platelet-derived growth factor(19) , TGF-beta(20, 21) , and interleukin 1 (22) and has been suggested to be a mechanism for signal amplification that underlies the enduring cellular proliferation characteristic of these growth factors. Similarly, the persistent actions of ET may be accounted for by its autoinduction, although the mechanisms underlying autoinduction of ET-1 are yet to be elucidated. In vascular endothelial cells, ET receptors expressed are predominantly the ET(B) subtype, and ET-1-induced synthesis of nitric oxide is mediated by the ET(B) receptors(23) , suggesting that the autoinduction of ET-1 may also be channeled through the ET(B) receptor subtype. However, autoinduction of ET-1 also occurs in vascular smooth muscle cells (24) in which the ET(A) receptor subtype is predominant and accounts for ET-1-induced contraction and proliferation(5) . It is not known whether autoinduction of ET-1 occurs through transcriptional or post-transcriptional regulation.

Mesangial cells are smooth muscle cell-like cells of the glomerulus and have been recognized to be key in pathogenesis of both acute and progressive renal damage(6) . Various lines of evidence suggest that ET mediates alterations of mesangial cell function seen under these pathophysiologic settings (6) and that glomerular mesangial cells possess all the components necessary for autoinduction of ET-1. In response to ET-1, mesangial cells contract(3, 25) , undergo proliferation(3) , and increase collagen production and extracellular matrix deposition(26) . Both expression preproET-1 mRNA and the ET-1 peptide production in mesangial cells are regulated in response to a variety of stimuli both in vivo and in vitro(27, 28, 29, 30) . Furthermore, the presence of both ET(A) and ET(B) receptor subtypes has been demonstrated in mesangial cells(30, 31) . ET-1-induced proliferation and contraction occur through the ET(A) receptor subtype(25) , whereas the ET(B) receptor is suggested to mediate cGMP generation through production of nitric oxide(32) . Thus, glomerular mesangial cells provide a system suitable for elucidating mechanisms involved in autoinduction of ET-1. In the present study, we examined mechanisms that underlie autoinduction of ET-1 in mesangial cells. Results indicate that autoinduction of ET-1 is mediated by the ET(B) receptor subtype and involves both transcriptional and post-transcriptional mechanisms.

EXPERIMENTAL PROCEDURES

Methods

BQ123 was provided by Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd. (Tsukuba, Japan). Random primer, ribonuclease H (RNase H), dithiothreitol (DTT), SuperScript reverse transcriptase with reaction buffer (5times) (20 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01% Nonidet P-40, and 50% glycerol), RPMI 1640, Dulbecco's modified Eagle's medium, Hank's balanced salt solution, and HEPES were purchased from Life Technologies, Inc. RNasin (RNase inhibitor), Taq DNA polymerase, with reaction buffer (10times) (50 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 50% glycerol, and 1.0% Triton X-100), deoxynucleotide mixture (dNTP), RNase-free DNase I and MgCl(2) were from Promega Corporation (Madison, WI). ET-1 and ET-3 were from Peptide Institute (Louisville, KY). Sarafotoxin S6c, Nonidet P-40, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole were from Sigma. I-ET-1, I-ET-3, [-P]ATP, and [alpha-P]UTP were from DuPont NEN. RES701-1 was prepared at Kyowa Hakko Kogyo Co. (Tokyo, Japan)(33, 34) .

Cell Culture

Mesangial cells were cultured from glomeruli of rat kidneys and maintained in RPMI 1640 supplemented with 16% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 95% air, 5% CO(2), as described previously(25, 30) . Cells between the 5th and 12th passage were used. Cells were grown to subconfluence and, 24 h prior to experiments, medium was changed to RPMI 1640 containing 0.4% fetal bovine serum.

Polymerase Chain Reaction with Reverse Transcription (RT-PCR)

Total RNA was extracted by the acid-guanidinium-phenol-chloroform method (35) as described previously (25, 35) . Briefly, mesangial cells were lysed in 10 ml of RNA zol B® (TEL-TEST Inc., Friendswood, TX). 1.0 ml of chloroform/isoamyl alcohol mixture (49:1) was added, and the lysates were incubated on ice for 15 min. After centrifugation at 12,000 times g for 30 min, RNA in an aqueous phase was precipitated with isopropyl alcohol. Total RNA was pelleted by centrifugation at 12,000 times g, washed with 75% ethanol, and dissolved in diethylpyrocarbonate-treated H(2)O. Samples were stored at -70 °C.

RT was performed as we have previously described(36) . Briefly, total RNA (3 µg) was used in an RT reaction. The samples were heated to 70 °C for 10 min, cooled on ice, and mixed with 19 µl of the RT reaction mixture containing 50 ng of random primer, 40 units of RNasin, 10 mM DTT, 1 mM dNTP, 5 times reaction buffer, and 200 units of reverse transcriptase. The reaction mixture was incubated at 42 °C for 60 min. At the end of the incubation, the reaction mixture was heated to 95 °C for 5 min to inactivate the reverse transcriptase activity and to denature RNA-cDNA hybrids. The samples were treated with RNase H at 37 °C for 30 min. For each PCR reaction, 2 µl of cDNA templates were used.

PCR was performed with oligonucleotide primers specific for rat preproET-1, ET(A), or ET(B) prepared by a DNA synthesizer (Applied Molecular Physiology in Vanderbilt University, Nashville, TN). PreproET-1 primer 1 (antisense) was defined by bases 210-229, sequence 5`-CTCTGCTGTTTGTGGCTTTC-3`; primer 2 (sense), bases 700-719, sequence 5`-TCTTGATGCTGTTGCTGATG-3`(37) . The cDNA amplification product was predicted to be 510 bp in length. ET(A) primer 1 (antisense) was defined by bases 538-557, sequence 5`-GAAGTCGTCCGTGGGCATCA-3`; primer 2 (sense), bases 734-753, sequence 5`-CTGTGCTGCTCGCCCTTGTA-3`(38) . The cDNA amplification product was predicted to be 216 bp in length. ET(B) primer 1 (antisense) was defined by bases 1004-1023, sequence 5`-TTACAAGACAGCCAAAGACT-3`; primer 2 (sense), bases 1549-1568, sequence 5`-CACGATAGAGGACAATGAAGAT-3`(9) . The PCR product was predicted to be 565 bp in length. We performed RT-PCR of beta-actin as an internal standard(39) . beta-Actin primer 1 (antisense), bases 1670-1687, sequence 5`-GCCAACCGTGAAAAGATG-3`; primer 2 (sense), bases 2531-2548, sequence 5`-TGCCGATAGTGATGACCT-3`. beta-Actin primers spanned one intron and resulted in a 465-bp product. For each PCR reaction, 25 pmol of each of primers 1 and 2 and 5 units of Taq DNA polymerase were used. The reaction mixture (100 µl) was overlaid with 50 µl of mineral oil. The tubes were placed in a Programmable Thermal Controller, PTC-100 (MJ Research Inc., Watertown, MA) programmed as follows. First, incubation at 94 °C for 3 min 15 s (initial melt); then, (a) preproET-1, ET(B): 30 cycles (preproET-1) or 35 cycles (ET(B)) of the following sequential steps: 94 °C for 45 s (melt); 60 °C for 1 min (anneal); 72 °C for 90 s (extension); (b) ET(A) and beta-actin: 28 cycles (ET(A)) or 22 cycles (beta-actin) of the following sequential steps: 94 °C for 45 s; 60 °C for 1 min; 72 °C for 1 min. Final incubation was performed at 72 °C for 5 min. The optimum number of amplification cycles used for quantitative RT-PCR was chosen on the basis of previously published data to be within the linear range of the reaction(36) .

Southern Blot Analysis of PCR Products

PCR products were identified and quantitated by Southern hybridization. The PCR products were size-fractionated by electrophoresis in 2% agarose gel. PCR products were blotted to GeneScreen (DuPont NEN) in 10 times SSC. The DNA was fixed to the membrane by baking at 80 °C for 2 h. Oligoprobes were synthesized to hybridize PCR products. The sequence of oligonucleotide was 5`-CAAAGAACTCCGAGCCCAAA-3` (preproET-1), 5`-CCCCTTGATTACCGCCATTTG-3` (ET(A)), 5`-TGTGCTGCTGGTGCCAAACG-3` (ET(B)), and 5`-CTGCGTCTGGACCTGGCTGGCCGGG-3` (beta-actin), respectively(37, 38, 39) . The synthetic oligonucleotide was end-labeled with [-P]ATP (6,000 Ci/mmol) using a 5`-end oligonucleotide-labeling method. Prehybridization (3 h) and hybridization (overnight) were carried out at 50 °C for preproET-1 and beta-actin or at 65 °C for ET(A) and ET(B). After washing once with 2 times SSC, 0.5% SDS for 20 min, autoradiography was performed at room temperature for 30 min, 50 min, 4 h, and overnight for beta-actin, ET(B), preproET-1, and ET(A), respectively. Experimental OD values were determined by Videodensitometry (Bio-Rad).

Determination of mRNA Stability and Nuclear Run-off Assay

Cells were preincubated for 30 min with 60 µM 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole, the specific RNA polymerase II inhibitor, prior to incubation with 100 nM ET-1 or vehicle. RNA was extracted at the time points indicated in the figure legends and assayed by RT-PCR as described above.

Nuclear run-off transcription assays were performed as described previously(40) . Three 100-mm dishes of confluent cells were incubated with ET-1 (100 nM) or vehicle for 1 or 3 h. Cells were lysed for 15 min on ice in Nonidet P-40 buffer consisting of 0.5% Nonidet P-40, 150 mM KCl, 5 mM MgCl(2), 250 mM sucrose, 50 mM Tris-HCl at pH 7.4). Nuclei were collected by centrifugation at 500 times g for 5 min, resuspended in glycerol storage buffer (50 mM Tris, 5 mM MgCl(2), 0.1 mM EDTA, 30% glycerol), and stored at -70 °C. For in vitro transcription, nuclei were thawed and incubated for 30 min at 30 °C with 10 mM Tris buffer, 150 µCi of [alpha-P]UTP (6,000 Ci/mmol), 1 mM ATP, 1 mM GTP, 1 mM CTP, 5 mM MgCl(2), 300 mM KCl, and 2 units/µl RNasin. The reaction products were treated with 2 units of RNase-free DNase I for 15 min at 16 °C and incubated with 200 µg/ml proteinase K in the presence of 0.1% SDS for 20 min at 37 °C. The P-labeled RNA was extracted with phenol-chloroform extraction in the presence of 50 µg of yeast tRNA and denatured by heating at 65 °C. The labeled RNA was hybridized for 48 h at 50 °C to cDNAs immobilized on Nylon membrane. The membranes were washed with 0.1 times SSC, 0.1% SDS at 50 °C, and exposed to Kodak X-OMAT AR x-ray film at -70 °C for 2 days.

Binding Studies

Cells were grown in 24-well tissue culture clusters and washed twice with the binding buffer consisting of Hank's balanced salt solution buffered with 40 mM HEPES, pH 7.4, and supplemented with 0.2% bovine serum albumin. Binding was performed by incubating cells in the binding buffer supplemented with 20 pMI-labeled ET-1 or ET-3 in the absence or presence of varying concentrations of unlabeled ligand as we have previously described(41) . After 3 h of incubation at room temperature, cells were washed three times with ice-cold PBS supplemented with 0.2% bovine serum albumin, and dissolved in 0.25 M NaOH/0.1% SDS. Radioactivity was determined by a LKB-Wallac Gamma Counter (Turku, Finland).

Determination of Endothelin-1

ET-1 was measured as described previously(30) . Culture medium was extracted by Amprep C2 cartridge (Amersham Corp.). Samples were assayed by using the endothelin 1-21-specific radioimmunoassay system (Amersham). I was counted by an LKB-Wallac Gamma Counter (Turku, Finland). In preliminary studies, we determined by using I-ET-1 that, under the present conditions, washing cells five times with ice-cold phosphate-buffered saline effectively removed exogenously added ET-1, with 2-5 fmol/ml remaining bound to cells.

Statistics

Data are presented as mean ± S.E. Data were analyzed by Wilcoxon signed rank test or analysis of variance, followed by unpaired Student's t test, as appropriate. p < 0.05 was considered to be significant.

RESULTS

Effect of Exogenously Added ET-1 on the Steady-state Levels of mRNA for PreproET-1 in Mesangial Cells

Fig. 1A shows the ethidium bromide staining of agarose gels and corresponding Southern blots for the PCR products for preproET-1 mRNA and beta-actin mRNA in mesangial cells. The expected size of the PCR product is 510 bp and 465 bp for preproET-1 and beta-actin, respectively. Southern hybridization using the specific oligoprobes confirmed the identity of these PCR products. When the PCR was carried out in the absence of reverse transcription, these bands were not seen, indicating that each band is derived from mRNA, and not from the genomic DNA (data not shown). The PCR products were quantitated by densitometric analysis of the Southern blots, and preproET-1 mRNA levels were normalized to that of beta-actin, an internal standard for the RT-PCR reaction.

Figure 1: RT-PCR analysis of steady-state mRNA levels for preproET-1 in mesangial cells after addition of ET-1. Mesangial cells were incubated with ET-1 (10M) and, at the indicated times, total RNA was extracted and subjected to RT-PCR analysis as described under ``Experimental Procedures.'' A, the upper panel of each set shows the ethidium bromide-stained PCR products in 2% agarose gel. Arrows indicate the expected size for each PCR product. The lower panel shows the corresponding Southern blot probed with P-labeled oligonucleotides that localized each PCR primer. Southern blot of PCR product for beta-actin are presented as an internal standard. Lane 1, control; lane 2, 1 h; lane 3, 3 h; lane 4, 6 h; lane 5, 12 h; and lane 6, 24 h of incubation with ET-1. B, products of RT-PCR were quantitated by densitometry of Southern analysis, normalized to beta-actin mRNA, and expressed as percentage of values observed in control cells. Each data point represents mean ± S.E. (n = 6). *p < 0.01 versus control.


Fig. 1B shows the time course of changes in the steady-state preproET-1 mRNA levels that occurred in mesangial cells in response to exogenous ET-1. Exposure of mesangial cells to ET-1 (10M) resulted in a prompt increase in preproET-1 mRNA levels, reaching maximum at 1 h (466 ± 43% of that observed in nonstimulated control cells; p < 0.01). PreproET-1 mRNA levels gradually declined; 156 ± 26%, 163 ± 40%, 145 ± 31% and 125 ± 33% of control values, respectively, at 3, 6, 12, and 24 h. The observed stimulation was concentration-dependent. After 1 h of incubation, ET-1 induced a significant increase in preproET-1 mRNA expression to 164 ± 19% above base line at the concentrations of 10M (p < 0.05). Stimulation observed at 10M was more variable and did not reach statistical significance.

Effects of ET-1 on PreproET-1 Gene Transcription and mRNA Stability

In order to determine whether the ET-1-induced stimulation of preproET-1 mRNA expression occurs through transcriptional or post-transcriptional mechanisms, we performed nuclear run-off transcriptional assay (Fig. 2A). The rate of preproET-1 gene transcription was significantly increased by ET-1 to 239% and 175% above base-line levels at 1 and 3 h of incubation, corresponding to the time-dependent increase in the steady-state levels of preproET-1 mRNA. ET-1 did not affect transcription of beta-actin.

Figure 2: Effects of ET-1 on preproET-1 transcription and mRNA stability. A, nuclear run-off transcription analysis of ET-1 effects on preproET-1 gene transcription. Cells were incubated with ET-1 (10M) or vehicle for 1 or 3 h. Nuclei were isolated, subjected to in vitro transcription, and aliquots of the P-labeled transcripts were hybridized at 50 °C for 2 days with the indicated cDNAs immobilized to membranes. Representative autoradiographs are presented (n = 3). B, effects of ET-1 on the stability of preproET-1 mRNA in mesangial cells. Cells were preincubated with 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (60 µM) for 30 min, and incubated for the indicated times in the absence (control) or presence of ET-1 (10M). Total RNA was extracted, and expression of each mRNA was analyzed by RT-PCR and Southern blotting (top panel) and quantitated (bottom panel) as described in Fig. 1. (n = 2-3).


Effect of ET-1 on the stability of preproET-1 mRNA was examined by determining decay of mRNA in the presence of the RNA polymerase II inhibitor, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (Fig. 2B). In cells treated with ET-1 (10M), decay of preproET-1 mRNA was slower than that in non-treated control cells. Densitometric measurements of preproET-1 mRNA showed that, in control cells, 50% of preproET-1 mRNA decayed within approx20 min, a finding in agreement with the previously estimated half-life of 15 min(42, 43, 44) . By contrast, 50% mRNA decay in cells treated with ET-1 occurred at approx60 min, indicating an increase in mRNA stability.

Profile of ET-1 and ET-3 Binding to Mesangial Cells

Competitive binding assay showed that ET-1 inhibited binding of I-ET-1 to mesangial cells more potently than ET-3, with 50% displacement seen at 5.4 times 10M and 2 times 10M for ET-1 and ET-3, respectively (Fig. 3A). In contrast, ET-1 and ET-3 were equally effective in inhibiting binding of I-ET-3 (Fig. 3B); 50% displacement occurred at 1.3 times 10M and 1 times 10M for ET-1 and ET-3, respectively. In agreement with previous studies(31) , these findings provide evidence for the presence of pharmacologically defined ET(A) and ET(B) receptor subtypes in glomerular mesangial cells.

Figure 3: Competitive inhibition of I-ET-1 and I-ET-3 binding by ET isopeptides and ET receptor antagonists. Cells were incubated with 20 pMI-ET-1 (A) or I-ET-3 (B) in the presence or absence of varying concentrations of ET-1 (up triangle) or ET-3 (bullet) for 3 h at room temperature. Results are expressed as percentage of specific binding. Each point represents mean ± S.E. (n = 6).


Effects of ET(A) and ET(B) Receptor Antagonists and ET(B) Agonist on Autoinduction of ET-1

To determine the receptor subtype that mediates the ET-1-induced increase in preproET-1 mRNA expression in mesangial cells, we utilized the ET(A)-specific antagonist, BQ123(45) , and the ET(B)-specific antagonist, RES701-1(33, 34) . RES701-1 is a cyclic peptide recently identified in the culture broth of Streptomyces sp. RE-701(33) . Subsequent studies have demonstrated that RES701-1 is an ET(B)-specific receptor antagonist, inhibiting the ET-1-induced increase in intracellular Ca concentration in COS-7 cells expressing ET(B) receptors, but not in ET(A) receptor-expressing COS-7 cells(34) .

Stimulation of preproET-1 mRNA expression induced by ET-1 was not affected by the ET(A)-specific antagonist, BQ123, at concentrations up to 10M (Fig. 4A). Compared with ET-1 alone at 10M, which increased preproET-1 mRNA to 496 ± 77% of the control values, the increases were 431 ± 83, 562 ± 121, and 504 ± 90% in the presence of BQ123 at 10, 10, and 10M, respectively. In contrast, the ET(B)-specific antagonist, RES701-1, effectively reduced the ET-1-induced stimulation of preproET-1 mRNA expression to 118 ± 23, 106 ± 19, and 124 ± 33% at 10, 10, and 10M of RES701-1, respectively (Fig. 4B). Exposure of mesangial cells to the ET(B) agonist, sarafotoxin S6c(46) , increased preproET-1 mRNA levels to 201 ± 14 and 178 ± 24% of the control values at 10 and 10M, respectively (Fig. 4C). This stimulation by sarafotoxin S6C (10M) was significantly inhibited by RES701-1 (10M) to 160 ± 5% of the control values (p < 0.05). These data indicate that the ET(B), but not the ET(A), receptor subtype is involved in the stimulation of preproET-1 mRNA expression induced by ET-1.

Figure 4: The effects of BQ123 and RES701-1 on stimulation of preproET-1 mRNA expression induced in mesangial cells by ET-1 or sarafotoxin S6c (STX). Mesangial cells were incubated for 1 h with vehicle (control) or ET-1 (10M) in the presence or absence of either BQ123 (A) or RES701-1 (B). C, cells were incubated with vehicle (control), STX, or STX+RES701-1 for 1 h. Total RNA was extracted and subjected to RT-PCR analysis for expression of preproET-1 mRNA. The upper panel of each set demonstrates the ethidium bromide-stained PCR products in 2% agarose gel. The graph shows the results of corresponding Southern blot probed with P-labeled oligonucleotides that localized each PCR primer. Values are expressed as percent of value observed in controls. Each point represents the mean ± S.E. (n = 6). *p < 0.01 versus control.


Fig. 5shows the effects of ET-1 on secretion of immunoreactive ET-1 by mesangial cells. In agreement with previous reports(30) , non-stimulated mesangial cells produced ET-1. After 1 h of incubation, the level of the immunoreactive ET-1 peptide in the medium was 8.0 ± 0.5 fmol/ml, followed by a further increase to 19.5 ± 2.1 fmol/ml at 3 h (n = 6). When cells were incubated with ET-1 (10M) for 1 h, extensively washed to remove bound ET-1, and further incubated in fresh media, a significant increase was observed in secretion of immunoreactive ET-1 into media such that at 3 h the ET-1 in media was 50.7 ± 4.1 fmol/ml (Fig. 5A, p < 0.001 versus time controls). Addition of RES701-1 (10M) during incubation with ET-1 suppressed the ET-1-induced secretion of ET-1 to 11.8 ± 0.7 fmol/ml at 3 h of incubation (p < 0.001). These findings further indicate that in mesangial cells, the ET(B) receptor subtype mediates autoinduction of ET-1.

Figure 5: Effects of RES701-1 on ET-1 secretion stimulated by ET-1 in mesangial cells. A, the effects of ET-1 on ET-1 secretion from mesangial cells. Cells were incubated either without (control, open bars) or with ET-1 (10M, closed bars) for 1 h at 37 °C, washed five times with ice-cold PBS, then incubated for 1 or 3 h at 37 °C in fresh media, and ET-1 in media was determined by radioimmumoassay. Each point represents the mean ± S.E. (n = 6). *p < 0.05,**p < 0.01,***p < 0.001 versus control. B, effects of RES701-1 on ET-1 secretion stimulated by ET-1 in mesangial cells. Cells were incubated either without (control, closed bar) or with RES701-1 (10M, hatched bar) for 30 min, washed five times with ice-cold PBS, then incubated for 3 h at 37 °C in fresh media. Each point represents the mean ± S.E. (n = 6) p < 0.001 versus ET-1 stimulation.


DISCUSSION

Regulation of peptide production occurs at transcriptional and/or post-transcriptional levels. Lack of prominent secretory granules in endothelial cells and the finding that little ET-1 or its precursor, big ET-1, is found intracellularly(47) , have led to the idea that synthesized ET-1 is constitutively secreted and that ET-1 production is regulated primarily through changes in preproET-1 mRNA levels. Previous studies have shown that preproET-1 mRNA levels are modulated by a variety of stimuli, including thrombin(27) , TGF-beta(43) , angiotensin II(48) , calcium ionophores(49) , hypoxia/anoxia(50) , and cyclosporine (28, 30, 36) . The current study showed that stimulation of mesangial cells with ET-1 causes a rapid increase in preproET-1 mRNA expression, in association with > 6-fold increase in production of the mature ET-1 peptide. Possible autoinduction of ET-1 has also been suggested in other cell types. Exogenous ET-1 increases preproET-1 mRNA expression in neonatal cardiomyocytes (13) and production of the ET-1 peptide in human umbilical vein endothelial cells(16, 17) . While the phenomenon of autoinduction is rare in regulation of cytokine synthesis, previous studies have shown that autoinduction occurs for several growth factors, including TGF-alpha(18) , B-chain of platelet-derived growth factor(19) , TGF-beta(20, 21) , as well as interleukin 1(22) , leading to the hypothesis that autoinduction underlies both the signal amplification and the enduring effects that characterize these growth factors. Similarly, the current demonstration of autoinduction of ET-1 underscores the possibility that this phenomenon is involved in the potent and long-lasting effects of ET, resulting in augmentation of potentially destructive processes occurring within the glomerulus.

To investigate the mechanisms by which ET-1 increases the steady-state preproET-1 mRNA expression, we examined the rate of transcription and the stability of preproET-1 mRNA. Nuclear transcription run-off analyses revealed that ET-1 increased transcription of preproET-1 gene to 239 and 175% above base line at 1 and 3 h after exposure to ET-1, respectively (Fig. 2A). Regulation of the transcriptional rate has been viewed as an overriding control of gene expression for a variety of genes. Analyses of 5`-flanking region of preproET-1 gene suggest the presence of response elements that are linked to constitutive endothelial cell-specific transcription of preproET-1 mRNA (51) , down-regulation with retinoic acid(52) , and binding of the fos/jun complex(53) . In addition, more recent reports indicate that cis-acting elements upstream to, and distinct from the AP-1 and GATA sites are involved in down-regulation of preproET-1 gene in response to shear stress(54) . Of interest in this regard are the observations that phosphoinositide breakdown is induced through the ET(B) receptor not only in endothelial cells(22, 23, 26) , but also in Chinese hamster ovary cells transfected with the ET(B) receptor cDNA(55) . This coupling of the ET(B) receptor and phospholipase C indicates that the ET(B) receptor-mediated ET-1 autoinduction in mesangial cells may also occur through activation of phospholipase C and subsequent activation of protein kinase C- and Ca-dependent pathways. Whether the observed auto-stimulation of preproET-1 mRNA expression involves these or other regulatory elements remains to be determined.

Steady-state mRNA levels are also determined by the rate of mRNA degradation. We found that the half-life of preproET-1 mRNA in quiescent mesangial cells was approx20 min, consistent with previous reports in bovine aortic endothelial cells(42, 43, 44) . This short life span of preproET-1 mRNA has been attributed to the presence of three AUUUA sequences in the 3`-untranslated region that are thought to mediate selective mRNA degradation(56) . Exposure of mesangial cells to ET-1 significantly increased stability of preproET-1 mRNA to a half-life of approx60 min (Fig. 2B). Stabilization of preproET-1 mRNA in response to other cytokines has been noted previously. TGF-beta increases the half-life of preproET-1 mRNA in Madin-Darby canine kidney cells from 15 to 30-45 min(43) . In bovine aortic endothelial cells, atrial natriuretic peptide increases the half-life of preproET-1 mRNA from 25 to approx70 min(44) . The present findings indicate that in mesangial cells, ET-1 increases preproET-1 mRNA expression by increasing both the rate of transcription and the stability of mRNA. Since the increase in transcriptional rate was maximal at 1 h while prolonged stability of the preproET-1 mRNA remained significant beyond 1 h (Fig. 2), it is suggested that rapid and transient stimulation of preproET-1 gene transcription primarily accounts for the increased steady-state preproET-1 mRNA levels. It is interesting to speculate that increased transcription rate is a primary mechanism responsible for the early surge in the preproET-1 mRNA expression, while increased mRNA stability contributes more to a sustained elevation in preproET-1 gene expression observed in some pathophysiologic settings(57, 58, 59) .

Competitive binding studies showed that in mesangial cells, ET-1 inhibited binding of I-ET-1 >100 times more potently than ET-3, whereas I-ET-3 binding was inhibited equally potently by ET-1 and ET-3 (Fig. 3), demonstrating the presence of both ET(A) and ET(B) receptor subtypes. Therefore, in subsequent studies, we investigated which ET receptor subtype is involved in autoinduction of ET-1 (Fig. 4). Addition of the ET(A)-specific receptor antagonist, BQ123, during incubation of cells with ET-1 had no effect on the increase in preproET-1 mRNA expression, up to 10M of BQ123 (Fig. 4A), indicating that the ET(A) receptor subtype is not involved in autoinduction of ET-1. In contrast, the ET(B)-specific receptor antagonist, RES701-1, abrogated the increase in preproET-1 mRNA expression that occurred in response to ET-1 (Fig. 4B). Consistent with these observations, exposure to the ET(B) agonist, sarafotoxin S6c, increased preproET-1 mRNA expression, and this stimulation was inhibited by the ET(B) receptor antagonist (Fig. 4C), further solidifying the notion that autoinduction of ET-1 production is channeled through the ET(B) receptor. By determining immunoreactive ET-1 levels in the media, we further investigated the effects of the ET(B)-specific receptor antagonist on the ET-1-induced stimulation of ET-1 peptide production (Fig. 5). Compared with non-treated control cells, stimulation with ET-1 resulted in significant increases in ET-1 production occurring within 1 h and a further increase was observed after 3 h of incubation (Fig. 5A). Addition of RES701-1 suppressed the ET-1-induced stimulation of ET-1 peptide production to the level not distinguishable from that in control cells (Fig. 5B). Thus, the squelching effect of the ET(B) receptor antagonist was observed both at the levels of preproET-1 mRNA expression and production of ET-1, further demonstrating that in mesangial cells, the ET(B) receptor subtype mediates autoinduction of ET-1. Endothelial cells, which possess only the ET(B) receptor, have been shown to increase production of ET-1 in response to ET-1 and ET-3, thereby implicating this receptor subtype in the autoinduction process(16, 17) . While an ET(A) receptor antagonist, BQ123, lessened ET-3 autoinduction in cardiomyocytes(13) , a considerable fraction of the autoinduction was unremitted, raising the possibility that, in cardiomyocytes, autoinduction of preproET-1 mRNA also occurs, at least in part, through ET(B). Overall, these observations not only emphasize the occurrence of the ET(B) receptor-mediated ET-1 autoinduction, but also underscore the possibility that the mechanisms mediating ET-1 autoinduction may vary among different cell types.

It was noted that the ET-1-induced increase in preproET-1 mRNA expression was effectively inhibited by RES701-1, whereas RES701-1 was less effective in inhibiting stimulation of preproET-1 mRNA expression induced by sarafotoxin S6c (Fig. 4, B and C). Accumulating evidence suggests the possibility of subpopulations within the ET(B) receptor subtype. In membranes prepared from cerebellum, caudate putamem, and hypothalamus, a subpopulation was identified within the ET(B) receptor subtype with features distinct from known receptors, including the picomolar range of binding affinity, resistance to deglycosylation, and the lack of phosphoinositide hydrolysis(60) . More recently, a gene coding for an ET receptor has been cloned from Xenopus laevis dermal melanophores which is distinct from the known ET(A) or ET(B) receptors(61) . This new receptor appears to preferentially bind and respond to ET-3 rather than ET-1. Thus, it is possible that a minor subpopulation of the ET(B) receptors mediates autoinduction of ET-1.

Previously, the ET(B) receptor subtype has been recognized as the mediator of the production of nitric oxide (NO) and prostacyclin in vascular endothelial cells(16, 23) . The ET(B) receptor subtype in mesangial cells has also been linked to NO production(32) . Since both NO and prostacyclin are anti-proliferative and dilatory for vascular smooth muscle cells through production of cGMP(62) , the ET(B) receptor subtype in the vasculature has been proposed to offer counterregulation to proliferative and vasoconstrictor actions of ET-1. In addition, NO can suppress the production of ET(63) , possibly by blunting the increase in intracellular calcium. More recently, Goligorsky et al.(64) showed that NO reduces the affinity of the receptor to its ligand, possibly by reacting with the thiol group of the ET receptors. The current study underscores an additional role for the ET(B) receptor, that of autoinduction of ET-1 production. The present findings have the additional implications that, once stimulated to produce ET-1, the cells expressing the ET(B) receptors would amplify and propagate ET-1 actions without requiring an external source for ET-1. This may have particular relevance for glomerular mesangial cells which have a central role in regulating glomerular filtration, chemoattraction, hypercellularity, and matrix deposition(6, 7) . Therefore, the current demonstration of autoinduction of ET-1 in mesangial cells suggests that continued local production of ET-1 and resulting amplification and propagation of ET-1 actions may occur without continued presence of external ET-1. Indeed, persistent increase of ET gene expression has been documented in a variety of pathophysiologic settings(6, 7, 57, 58, 59) , suggesting that stimuli (ischemia, toxins) can induce synthesis of endogenous ET-1, which in turn perpetuates its own production, even after the initiating stimulus is resolved.

In summary, we showed that in mesangial cells, ET-1 stimulates its own gene expression as well as peptide production through increased transcription and stability of the preproET-1 mRNA. We also demonstrated that the observed autoinduction of ET-1 occurs through the ET(B), but not ET(A), receptor subtype.

FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK42159 and DK44757. Preliminary accounts of this work were presented at the 27th Annual Meeting of the American Society of Nephrology, Orlando, FL, October 26-29, 1994, and were published in abstract form ((1994) J. Am. Soc. Nephrol.5, 717). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: C-4204 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232-2584. Tel.: 615-322-7931; Fax: 615-322-7929.

(^1)
The abbreviations used are: ET, endothelin; ET(A) and ET(B), endothelin receptor subtype A and B; TGF-alpha and -beta, transforming growth factor-alpha and -beta; RT-PCR, reverse transcription coupled with polymerase chain reaction; PBS, phosphate-buffered saline; NO, nitric oxide; DTT, dithiothreitol; bp, base pair(s).

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