(Received for publication, November 29, 1994; and in revised form, January 18, 1995)
From the
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
-specific
antagonist, RES701-1, at >10
M abolished ET-1 stimulation of preproET-1 mRNA (p <
0.001), whereas an ET
specific antagonist, BQ123, was
without effects (up to 10
M). The ET
agonist, sarafotoxin S6c (10
M),
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
receptor
subtype via increases in both preproET-1 transcription and mRNA
stability.
Endothelins are a family of peptides which include endothelin-1
(ET()-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
and ET
, 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
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
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
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-(18) , B-chain of platelet-derived growth
factor(19) , TGF-
(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
subtype, and ET-1-induced synthesis of nitric oxide
is mediated by the ET
receptors(23) , suggesting
that the autoinduction of ET-1 may also be channeled through the
ET
receptor subtype. However, autoinduction of ET-1 also
occurs in vascular smooth muscle cells (24) in which the
ET
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 and ET
receptor subtypes has been demonstrated in mesangial
cells(30, 31) . ET-1-induced proliferation and
contraction occur through the ET
receptor
subtype(25) , whereas the ET
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
receptor
subtype and involves both transcriptional and post-transcriptional
mechanisms.
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 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, or ET
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
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
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
-actin as an
internal standard(39) .
-Actin primer 1 (antisense), bases
1670-1687, sequence 5`-GCCAACCGTGAAAAGATG-3`; primer 2 (sense),
bases 2531-2548, sequence 5`-TGCCGATAGTGATGACCT-3`.
-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
: 30
cycles (preproET-1) or 35 cycles (ET
) 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
and
-actin: 28 cycles (ET
) or 22 cycles
(
-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) .
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, 250 mM sucrose, 50 mM Tris-HCl
at pH 7.4). Nuclei were collected by centrifugation at 500
g for 5 min, resuspended in glycerol storage buffer (50 mM Tris, 5 mM MgCl
, 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
[
-
P]UTP (6,000 Ci/mmol), 1 mM ATP,
1 mM GTP, 1 mM CTP, 5 mM MgCl
,
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
SSC, 0.1% SDS at
50 °C, and exposed to Kodak X-OMAT AR x-ray film at -70
°C for 2 days.
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
-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
-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 10
M (p < 0.05). Stimulation observed at 10
M was more variable and did not reach statistical
significance.
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-
-D-ribofuranosyl benzimidazole (60
µM) for 30 min, and incubated for the indicated times in
the absence (control) or presence of ET-1 (10
M). 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--D-ribofuranosyl benzimidazole (Fig. 2B). In cells treated with ET-1 (10
M), 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
20 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
60
min, indicating an increase in mRNA stability.
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 pM
I-ET-1 (A) or
I-ET-3 (B) in the presence or absence of varying concentrations of
ET-1 (
) or ET-3 (
) for 3 h at room temperature. Results are
expressed as percentage of specific binding. Each point represents mean
± S.E. (n = 6).
Stimulation of preproET-1 mRNA expression induced by ET-1 was not
affected by the ET-specific antagonist, BQ123, at
concentrations up to 10
M (Fig. 4A).
Compared with ET-1 alone at 10
M, 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 10
M,
respectively. In contrast, the ET
-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
10
M of RES701-1, respectively (Fig. 4B). Exposure of mesangial cells to the ET
agonist, sarafotoxin S6c(46) , increased preproET-1 mRNA
levels to 201 ± 14 and 178 ± 24% of the control values at
10
and 10
M,
respectively (Fig. 4C). This stimulation by sarafotoxin
S6C (10
M) was significantly inhibited by
RES701-1 (10
M) to 160 ± 5% of
the control values (p < 0.05). These data indicate that the
ET
, but not the ET
, 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
(10
M) 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
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 (10
M, 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.
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-(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-
(18) , B-chain of platelet-derived growth
factor(19) , TGF-
(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 receptor not only in endothelial
cells(22, 23, 26) , but also in Chinese
hamster ovary cells transfected with the ET
receptor
cDNA(55) . This coupling of the ET
receptor and
phospholipase C indicates that the ET
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
20 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
60 min (Fig. 2B). Stabilization of
preproET-1 mRNA in response to other cytokines has been noted
previously. TGF-
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
70 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
and ET
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
-specific receptor antagonist, BQ123, during incubation
of cells with ET-1 had no effect on the increase in preproET-1 mRNA
expression, up to 10
M of BQ123 (Fig. 4A), indicating that the ET
receptor
subtype is not involved in autoinduction of ET-1. In contrast, the
ET
-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
agonist, sarafotoxin S6c, increased
preproET-1 mRNA expression, and this stimulation was inhibited by the
ET
receptor antagonist (Fig. 4C), further
solidifying the notion that autoinduction of ET-1 production is
channeled through the ET
receptor. By determining
immunoreactive ET-1 levels in the media, we further investigated the
effects of the ET
-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
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
receptor subtype mediates autoinduction of ET-1.
Endothelial cells, which possess only the ET
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
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
. Overall, these observations not only
emphasize the occurrence of the ET
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 receptor subtype. In membranes prepared from
cerebellum, caudate putamem, and hypothalamus, a subpopulation was
identified within the ET
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
or
ET
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
receptors
mediates autoinduction of ET-1.
Previously, the ET 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
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
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
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
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, but
not ET
, receptor subtype.