© 1995 American Heart Association, Inc.
Articles |
Lack of Role for Nitric Oxide (NO) in the Selective Destabilization of Endothelial NO Synthase mRNA by Tumor Necrosis Factor–
Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993, and published in Circulation. 1993;88(suppl I, pt 2): I-273. Abstract.
From the McGill Vascular Biology Group, Divisions of Cardiology (J.C.M., D.J.S.) and Medical Biochemistry (P.C.), Department of Medicine, Royal Victoria Hospital, and McGill University (F.M., A.G., D.B.), Montreal, Quebec, Canada.
Correspondence to Dr Duncan J. Stewart, Royal Victoria Hospital, 687 Pine Ave W, Rm M4.76, Montreal, Quebec, Canada H3A 1A1.
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Abstract |
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Abstract The constitutive expression of endothelial nitric oxide (NO) synthase (cNOS) is essential for the physiological regulation of vascular tone and structure. The mechanism of downregulation of steady state cNOS mRNA in human umbilical vein endothelial cells exposed to tumor necrosis factor–
(TNF-) was
investigated by using Northern blot analysis of total cellular RNA.
TNF- produced a dose- and time-dependent decrease in cNOS mRNA
expression that was near maximal at 10 U/mL and 6 hours of exposure,
respectively. In contrast, steady state expression of endothelin-1 and
plasminogen activator inhibitor–1 (PAI-1) mRNA was upregulated by
TNF-. The pharmacological generation of NO using sodium
nitroprusside (10 µmol/L) and
S-nitroso-acetylpenicillamine (100 to 400 µmol/L) had no
effect on cNOS mRNA levels, and TNF-–induced downregulation of cNOS
was not prevented by coincubation with the inhibitors of NO synthesis
N
-nitro-L-arginine methyl ester
(1 mmol/L) and NG-monomethyl
L-arginine (10 mmol/L). Under control conditions, cNOS
and PAI-1 mRNA were stable after treatment with actinomycin D for
periods greater than 24 hours, whereas endothelin-1 message was rapidly
degraded (half-life, <1 hour). Pretreatment with TNF- (30 U/mL)
selectively reduced the half-life of cNOS mRNA to less than 12 hours
without altering the stability of PAI-1 message. TNF-–induced
destabilization of cNOS mRNA could be partially prevented by
coincubation with cycloheximide (1 µmol/L) but was not reproduced by
addition of sodium nitroprusside. These findings indicate that TNF-
downregulation of cNOS expression in human endothelial cells results
predominantly from the selective destabilization of the mRNA by a
mechanism involving the synthesis of new protein. However, NO
production by a TNF-–inducible isoform of NOS did not appear to
contribute either to the decrease in steady state cNOS mRNA levels or
the shortening of its half-life.
Key Words: cytokines • endothelin-1 • plasminogen activator inhibitor–1 • endothelium-derived relaxing factor • regulation of gene expression
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Introduction |
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Endothelial cells (ECs) express a "constitutive" isoform of nitric oxide (NO) synthase (cNOS) that catalyzes the oxidation of L-arginine to produce citrulline and NO.1 The activity of cNOS is tightly regulated by a calmodulin-dependent mechanism.2 3 A wide variety of receptor agonists, including acetylcholine, bradykinin, and serotonin, have the ability to elevate intracellular calcium concentrations, thereby increasing NO release from ECs.4 As a result of intimal shear stress associated with blood flow in the vascular lumen, high basal levels of NO production are also maintained under physiological conditions.5 NO acts by stimulating soluble guanylate cyclase and the resulting increases in cGMP concentration mediate its many important physiological actions,4 5 including vasodilation, prevention of smooth muscle cell proliferation, and inhibition of platelet and monocyte adhesion.1
Loss of endothelium-derived NO might therefore
lead to abnormalities in smooth muscle tone or growth and allow
adhesion of blood elements to the vessel wall.6 Recently,
several groups have demonstrated2 7 8 a potent
downregulation of steady state cNOS mRNA expression in ECs exposed to
cytokines, in particular tumor necrosis factor– (TNF-), by a
mechanism involving destabilization of cNOS mRNA.8 Thus,
decreased expression of endothelial cNOS could contribute to the
development of vascular disorders, such as atherosclerosis, in which
increased production of TNF-9 and impaired
endothelium-dependent dilator responses have been
reported.6 10
However, cytokines may also cause the expression of an inducible NOS isoform (iNOS)11 12 and increased NO production. Although identified in macrophages,13 14 15 it is now recognized that many cell types, including some EC lines,16 17 18 may have the potential to express iNOS. The functional relevance of iNOS expression in endothelium is unknown. Unlike the endothelial cNOS, the inducible enzyme is not regulated by calcium; rather, it produces large amounts of NO in a continuous manner. Such high levels of NO generation may have direct effects on cellular function, eg, by promoting ADP ribosylation,12 and by inhibiting mitochondrial respiration.19 NO has also been shown to alter gene expression, downregulating monocyte chemoattractant protein–120 and increasing levels of transferrin receptor mRNA.21 Interestingly, this latter effect was mediated by an action of NO on the stability of the mRNA.21 However, whether excessive endothelial production of NO by iNOS might contribute directly to the cytokine-induced downregulation in cNOS expression, perhaps also mediated by an effect on mRNA stability, is not known.
Therefore, the aim of the present study was to determine the
mechanism of TNF-–induced decreases in cNOS expression in human
ECs. We now report that TNF- reduced the steady state levels of cNOS
mRNA, in large part due to a posttranscriptional mechanism involving
destabilization of the mRNA and requiring de novo protein synthesis.
However, this process was independent of endogenous NO production and
was not reproduced by the pharmacological generation of exogenous
NO.
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Methods |
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EC Culture
Human umbilical vein ECs (HUVECs) were obtained from the American Type Culture Collection (ATCC) or isolated from fresh umbilical veins by 0.2% collagenase digestion.22 HUVECs were grown to confluence in T75 flasks (Fisher Scientific) equilibrated with 95% air and 5% CO2 at 37°C in Ham's/F12 medium (GIBCO) and supplemented with 10% fetal calf serum, penicillin (500 U/mL), streptomycin (50 µg/mL), and heparin (100 µg/mL) (all from GIBCO) and EC growth factor (20 µg/mL, Boehringer Mannheim). Confluent ECs between the fifth and 18th passages were washed with Hank's buffered salt solution and preincubated in 10 mL medium supplemented with 10% fetal calf serum, 500 U/mL penicillin, and 50 µg/mL streptomycin for 2 hours. For experiments on steady state mRNA expression, fresh medium was added containing the following agents singly or in combination: human recombinant TNF-
(Sigma Chemical
Co), N-nitro-L-arginine methyl
ester (L-NAME, 1 mmol/L, Sigma),
NG-monomethyl
L-arginine (L-NMMA, 10 mmol/L, Sigma), dexamethasone
(1µmol/L, Sigma), sodium nitroprusside (SNP, 10 µmol/L, Roche
Pharmaceuticals), and S-nitroso-acetylpenicillamine (SNAP,
Biomol). Unless otherwise specified, cells were incubated under the
above conditions for 24 hours. For experiments on the stability of
mRNA, actinomycin D (Sigma) was added to the cells after a 3-hour
preincubation period in the presence or absence of TNF- (30 U/mL),
cycloheximide (1 µmol/L), or SNP (10 µmol/L) singly or in
combination.
RNA Extraction and Northern Blot Analysis
RNA was isolated by a modified guanidinium
thiocyanate–phenol-chloroform method using RNAzol B (Tel-Test)
according to the manufacturer's recommendations. For the Northern
blots, total cellular RNA (20 µg) samples were separated by
electrophoresis on a 1.2% agarose gel containing 2 mol/L formaldehyde,
20 mmol/L 3-(N-morpho)propane sulfonic acid, 8 mmol/L sodium
acetate, 1 mmol/L EDTA, and 5 mmol/L NaOH and transferred by capillary
blotting to Hybond-N membranes (Amersham Corp) in 20x saline–sodium
citrate (SSC; 3 mol/L sodium chloride and 0.3 mol/L sodium citrate).
The membrane was optimally cross-linked (120 mJ/cm2) with
UV light (UVXL-1000, Fisher Scientific). Membranes were prehybridized
for 4 hours at 42°C in 50% formamide, 5x SSPE (0.75 mol/L NaCl and
0.05 mol/L NaH2PO4), 5x Denhardt's solution
(0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 0.1% bovine serum
albumin), 0.5% sodium dodecyl sulfate (SDS), and 100 µg/mL herring
sperm DNA and hybridized overnight (42°C) with specific cDNA probes
that had been radiolabeled with [-32P]dCTP
(Amersham) by the random primer technique to a specific activity of at
least 1x109 dpm/µg.23 Membranes were then
washed twice in 2x SSC and 0.1% SDS for 15 minutes at room
temperature and then once in 1x SSC and 0.1% SDS for 15 minutes at
65°C. Autoradiography was performed by using double intensifying
screens (Cronex) and Kodak XAR film at -80°C. Signal intensity was
quantified as integrated areas by using scanning densitometry.
Preparation of the cDNA Probes
A cDNA probe for human cNOS was prepared by polymerase
chain reaction (PCR) of a 10-µL aliquot of a HUVEC lambda gt-11 cDNA
library (provided by Dr Morag Park, McGill University). The primers
were designed on the basis of the sequence retrieved from the Genbank
database (accession No. M93718)24 and were as follows:
sense 5'-TTCCGGGGATTCTGGCAGGAG-3', antisense
5'-GCCATGGTAACATCGCCGCAG-3'. Amplification was as
described25 with some modifications. Briefly, PCR was
performed for 30 cycles with denaturation at 94°C for 1 minute and 20
seconds, annealing at 55°C for 2 minutes, and extension at 72°C for
2 minutes. The PCR product was analyzed in a 2% agarose gel, revealing
a band of the predicted size (299 bp). This product was then ligated
directly into the PCR II vector (Invitrogen), and the NOS sequence was
confirmed by sequencing with T7 DNA polymerase.
A full-length human prepro–endothelin-1 (ET-1) cDNA probe was prepared
by screening a HUVEC cDNA library in lambda gt-11 with a synthetic
33-base oligonucleotide complementary to the mRNA region encoding for
residues Met-59 to Leu-69 of human prepro–ET-1
(5'-CAGGTGGCAGAAGTAGACACACTCTTTATCCAT-3').26 The
oligonucleotide was 5' end labeled with T4 polynucleotide kinase and
[
-32P]dATP. Bacteriophages were plated and
transferred to nylon membranes (Hybond N, Amersham Canada, Ltd).
Filters were hybridized with radiolabeled oligonucleotide, washed, and
autoradiographed as described27 ; hybridization and washing
were performed at 50°C. DNA from hybridizing recombinant
bacteriophages was prepared by the method of QIAGEN, Inc. Inserts from
the recombinant bacteriophages were recovered by EcoRI
digestion, ligated into the plasmid pGEM-3Z, and sequenced with T7 DNA
polymerase.
GAPDH cDNA, a constitutively expressed gene, was obtained (ATCC No. 57091),28 and a 0.78-kb Pst 1/Xba I fragment was used as a cDNA probe. A full-length cDNA probe for plasminogen activator inhibitor–1 (PAI-1) was a generous gift from Dr David Ginsburg, University of Michigan, Ann Arbor29 , and a 1.1-kb fragment was used that had been generated by digestion with EcoRI and Ava I.
Nitrite Measurement
Nitrite was determined by incubating 0.25 mL of the
EC-conditioned medium with the Griess reagent (0.025 mL of 6.5 mol/L
HCl and 0.025 mL of 37.5 mmol/L sulfanilic acid) for 10 minutes at room
temperature.30 Ethylenediamine (0.025 mL) was added, and
the absorbance at 540 nmol/L was determined 30 minutes later by using a
spectrophotometer (Milton Roy). The concentrations were calculated from
a standard curve derived from prepared solutions of NaNO2
(0 to 25 µmol/L).
Western Blot Analysis
Western blot analysis was performed by using the ECL
system (Amersham). Crude cytoplasmic extracts were prepared from HUVECs
that were either incubated under control conditions or exposed to
TNF- (30 U/mL) for 24 hours, by lysis in hypotonic buffer containing
0.2% Nonidet P-40, 40 mmol/L KCl, 10 mmol/L HEPES, pH 7.9, 3 mmol/L
MgCL2, 1 mmol/L DTT, 5% glycerol, 8 ng/mL
aprotinin, 2 ng/mL leupeptin, and 0.5 mmol/L phenylmethylsulfonyl
fluoride (Sigma). The nuclei were removed by centrifugation at
14 000g for 2 minutes at 4°C. Cytoplasmic extracts were
immediately frozen on dry ice and stored at -80°C. Sample protein
(25 µg) was then loaded on a 10% to 20% gradient SDS-tricine gel
(Novex), and electrophoresis was performed at 125 V for 90 minutes. The
gel was then electrotransferred to a polyvinylidene difluoride membrane
(Novex) using 30 V for 90 minutes. After overnight blocking in 5%
nonfat milk in Tris-buffered saline at 4°C, the filter was incubated
for 1 hour at room temperature with a primary antibody (diluted
1:1000), either a polyclonal raised against human endothelial cNOS (Dr
David Harrison, Emory University, Atlanta, Ga) or a monoclonal raised
against mouse iNOS (Transduction Laboratories, Lexington, Ky). The
membrane was then incubated with the secondary antibody (anti-mouse or
anti-rabbit immunoglobulin G conjugated with horse radish peroxidase,
1:1000) following the manufacturer's recommendations and
autoradiographed for 10 to 15 seconds.
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Results |
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Incubation of human ECs with TNF-
markedly downregulated
the expression of cNOS. As shown in Fig 1
, steady state mRNA
levels of cNOS were profoundly reduced relative to GAPDH, even at
concentrations as low as 10 U/mL and after an incubation period of less
than 6 hours. In contrast, the expression of PAI-1 and ET-1 was
upregulated by TNF- (Fig 2). The decrease in levels of
cNOS mRNA induced by TNF- could not be prevented by L-NAME, an
inhibitor of NO synthesis, or dexamethasone, which inhibits the
induction of iNOS by cytokines.17
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Fig 3 shows the effect of L-NMMA, another inhibitor of NOS,
on TNF-–induced downregulation of cNOS expression. Even at a
relatively high concentration (10 mmol/L), L-NMMA did not prevent the
downregulation of cNOS mRNA by TNF-. To further exclude a role for
NO in this action of TNF-, the effect of pharmacological generation
of NO was examined by using SNP (1 µmol/L, Fig 2
) or SNAP (100 and
400 µmol/L; Fig 4A). These NO donor compounds failed to
substantially reduce the expression of cNOS in HUVECs.
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) and absence
(
) of L-NMMA. In lane 3 of the L-NMMA–treated
cells (TNF-
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The effect of TNF- on the expression of cNOS and iNOS proteins in
HUVECs was studied by Western blot analysis. As shown in Fig 4B,
pretreatment of HUVECs for 24 hours with TNF- markedly reduced the
amount of cNOS protein, while iNOS protein was not detected in HUVEC
cytoplasmic extracts either under control conditions or following
pretreatment with TNF-. In addition, nitrite levels were not
different in control (0.38±0.05 µmol/L) and TNF-–treated
(0.38±0.03, 0.46±0.06, and 0.44±0.04 µmol/L for 10, 30, and 100
U/mL, respectively; n=3) cells. In contrast, substantial increases in
nitrite could be demonstrated following incubation of cells with 100
and 400 µmol/L SNAP (8.04 and 66.55 µmol/L, respectively).
Fig 5 compares the stability of various EC mRNAs following
the complete inhibition of transcription by the administration of
actinomycin D. There was no loss of the hybridization signal for cNOS
over 24 hours; indeed there was an apparent increase in cNOS mRNA.
However, when corrected by normalization with GAPDH mRNA, the relative
density of the cNOS hybridization signal was constant (Fig 6), as was that for PAI-1. In contrast, ET-1 mRNA was
rapidly degraded with a half-life of less than 1 hour.
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The effect of TNF- on the stability of cNOS mRNA in the presence and
absence of cycloheximide is presented in Fig 7. TNF-
alone destabilized cNOS message, while that of PAI-1 remained stable.
Cycloheximide had no effect on cNOS mRNA levels prior to the addition
of actinomycin D (0 hours) but increased the amount of message at each
time point thereafter. In contrast, cycloheximide increased the
expression of ET-1 (less so for PAI-1) in the presence of TNF- but
had little effect on the half-life, while message for GAPDH was not
altered.
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The relative density of the cNOS mRNA hybridization signal
normalized for GAPDH is presented in Fig 8. TNF-
produced a shortening of the half-life to less than 12 hours, while
coincubation with TNF- and cycloheximide partially restored cNOS
mRNA stability to control levels (ie, half-life >24 hours). In the
presence of SNP, a pharmacological generator of NO, the stability of
cNOS mRNA was identical to that under control conditions.
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) or in the presence of
tumor necrosis factor–
), TNF and cycloheximide
(CHX; 
). The density
of the hybridization signal relative to GAPDH is presented on the
ordinate as a percentage of the control values immediately before the
addition of actinomycin D (Act D, 2 µg/mL). Time of incubation
following addition of actinomycin D is shown on the abscissa. Each
point represents a mean value for two or three experiments. |
Discussion |
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TNF-
produced a marked reduction in steady state mRNA levels
for cNOS in human ECs in a time- and concentration-dependent manner,
with near maximal downregulation at concentrations between 10 and 30
U/mL and as rapidly as within 6 hours of its addition to the culture
medium. Yet cNOS mRNA was remarkably stable under control conditions,
with no detectable loss of signal even 24 hours after treatment with
actinomycin D, which effectively prevents further gene
transcription.31 Thus, it is unlikely that, without a
change in the stability of the message, even the total suppression of
transcription could account for the rapid and complete disappearance of
steady state cNOS message after exposure to TNF-. Indeed, after a
3-hour preincubation with TNF-, the half-life of cNOS message was
found to have decreased to less than 12 hours, with nearly no
detectable message 24 hours after treatment with actinomycin D. This
finding agrees with the earlier report of Yoshizumi et al8
and strongly suggests that a change in stability of cNOS message is the
predominant mechanism for the downregulation of its steady state
expression. Moreover, the destabilization of cNOS message by TNF-
could be largely prevented by coincubation with cycloheximide,
indicating that synthesis of new protein(s) was required for this
effect.
The degradation of mRNA is a regulated process that is a potentially
important contributor to the level of gene
expression.32 33 Certain motifs can confer stability, such
as the stem-loop structures in the 3' untranslated regions (3'UTRs) of
some bacterial mRNAs. In contrast, UA-rich regions in the 3'UTR of
protooncogenes promote the rapid degradation of their messages.
Interestingly, the mRNA for both endothelial cNOS and ET-1 possesses
AUUUA repeats in the 3'UTR, which promote rapid degradation, yet only
ET-1 exhibits a short half-life.34 Destabilization of cNOS
mRNA in response to TNF- could result from a protein-mRNA
interaction, which would allow the rapid degradation elements in the
3'UTR to become active. An example of such a mechanism is the
destabilization of transferrin receptor mRNA in the presence of ferrous
iron.33 35 Importantly, NO also alters the affinity of the
iron regulatory factor for its mRNA binding site21 32 and
thus regulates the stability of the transferrin receptor message.
The effect of NO on the stability of the transferrin receptor mRNA
raises the possibility that, as a product of the NOS pathway, NO could
regulate the expression of cNOS mRNA in a "feedback" manner, much
as it may regulate the activity of the enzyme.36 However,
TNF-–induced downregulation of steady-state mRNA levels for cNOS
was not prevented by inhibition of the synthesis of NO using L-NAME or
L-NMMA. As well, pretreatment of ECs with dexamethasone at a
concentration that prevents the induction of iNOS in response to
cytokine stimulation17 also failed to modify the effect of
TNF- on the expression of cNOS, while the pharmacological generation
of exogenous NO by addition of SNP or SNAP had no effect on
steady-state levels or stability of cNOS mRNA. Finally, the expression
of cNOS protein was downregulated by TNF- in a manner similar to its
message, whereas no induction of iNOS was observed in HUVECs by Western
blot analysis (Fig 4B) or as shown by the measurement of nitrite, a
stable breakdown product of NO. Therefore, the results of the
present experiments provide strong arguments against an autocrine
role of NO in the cytokine-induced downregulation of cNOS mRNA in human
ECs.
Surprisingly, the message for PAI-1 was stable in human ECs, comparable
to those for cNOS and GAPDH. This finding is in disagreement with the
short half-life for PAI-1 mRNA reported for bovine ECs37
and a human hepatoma cell line.38 The reason for this
discrepancy is not clear, but it may reflect differences in
posttranscriptional regulation of PAI-1 mRNA between different cell
types. In contrast, the half-life of ET-1 message was very short in our
cells (<1 hour), comparable to other reports.34 Of note,
the stability of PAI-1 mRNA was not reduced by pretreatment with
TNF-, indicating that the destabilization of cNOS message occurred
by a selective mechanism rather than by a nonspecific increase in
degradation of mRNA.
The present findings may have important implications
regarding the role of the endothelium in the initiation and progression
of vascular disorders. The loss of cNOS expression and the resulting
decrease in the capacity to produce NO in response to physiological
stimuli could compromise the ability of the endothelium to protect
against thrombosis, vasoconstriction, and subintimal proliferation. At
the same time, TNF-–induced upregulation of the expression of ET-1
and PAI-1 mRNA would serve to actively promote these pathological
processes. Increased levels of cytokines have been demonstrated in a
variety of cardiovascular disorders, from atherosclerosis9
to heart failure.39 Thus, a better understanding of the
mechanisms of TNF-–induced endothelial dysfunction may lead to the
development of new strategies in the prevention and treatment of
vascular disease.
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Acknowledgments |
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This work was supported by a grant from the Medical Research Council of Canada. Juan Carlos Monge and Duncan John Stewart are supported by Les Fonds de Recherche en Santé du Québec.
Received December 8, 1993; accepted September 26, 1994.
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