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*
Cardiovascular Research and Hypertension Laboratory,
Fundación
Jiménez Díaz,
Madrid, Spain.
Department of Pathology, Fundación,
Jiménez Díaz,
Madrid, Spain.
Correspondence to Dr. Antonio López-Farré, Cardiovascular Research and Hypertension Laboratory, Fundación Jiménez Díaz, Av Reyes Católicos, 2, Madrid 28040, Spain. Phone: 91-5504821; Fax: 91-5494764; E-mail: ALopeza{at}fjd.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Regulation of mRNA stability has emerged as an important control mechanism for the regulation of mRNA levels. Although the mechanism for regulating the cellular stability of different genes has unique features, it seems that in each case, specific RNA sequences are required for the recognition of protein factors (11, 12). Some of these sequences have been identified within the 3'-untranslated region (3'-UTR) of mRNA. The 3'-UTR interacts with transacting factors (regulatory proteins), which may affect the half-life of the mRNA. In this regard, we recently obtained new evidence that bovine aortic endothelial cells contain cytosolic proteins that form complexes with in vitro transcribed 3'-UTR eNOS mRNA (13). These cytosolic proteins seem to be involved in the stabilization of eNOS mRNA (13).
The mesothelium is a flat epithelial lining of the peritoneal, pleural, and pericardial cavities. Like the endothelium in the vessel wall, mesothelial cells provide a nonadhesive surface and gate the traffic of molecules and cells between the circulation and these body compartments. Moreover, mesothelial cells share a number of morphologic and functional properties with the endothelium, including the expression of several adhesive molecules and the production of cytokines, fibrinolytic agents, and vasodilator substances such as prostacyclin (14,15,16). However, whether eNOS protein is expressed in human mesothelial cells is not evident. Here we have shown that in human peritoneum, both capillary endothelial and mesothelial cells expressed endothelial-type NOS.
Peritoneal inflammation has been implicated as a major cause of morphologic and functional alterations of the peritoneum (17, 18). Loss of eNOS expression in mesothelial and capillary endothelial cells may contribute to altering their functional properties. Therefore, we analyzed the effect of bacterial lipopolysaccharide (LPS) on the expression of eNOS protein in human peritoneal samples, determining the presence of cytosolic proteins that specifically bind to the 3'-UTR eNOS mRNA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All patients gave oral informed consent. The peritoneum was carefully approached from the antemesothelial side. Routine pathologic examination of the visceral peritoneum disclosed signs of inflammation linked to neoplasia extension or diffuse ischemic lesions. The samples were quickly isolated with a curved surgical clamp and cut. We tried to avoid undue manipulation, particularly extended exposition and surgical handling, which usually cause tissue disruption. Peritoneal samples were washed with isotonic saline to remove the remaining blood and cut into similar portions. The peritoneal portions were preincubated in RPMI medium containing 10% fetal calf serum, 5 mM glutamine, 2 x 10-5 U/L penicillin, and 2 x 10-5 µg/L streptomycin during 1 h. Afterward, the medium was removed and replaced by fresh RPMI medium containing 10% fetal calf serum. LPS (10 µg/ml) was added for different periods of time. Peritoneal samples were then frozen in liquid nitrogen for molecular biology determinations or embedded in paraffin wax for immunohistochemistry analysis.
Western Blot Analysis
eNOS protein was analyzed in human peritoneum by Western blotting as
described previously (19).
Human peritoneal samples were homogenated and lysed in Laemmli buffer
containing 2-mercaptoethanol
(20). Equal amounts of protein
(20 µg/lane) estimated by bicinchoninic acid reagent (Pierce, Rockford, IL)
were loaded. To verify that equal amounts of proteins had been loaded in the
gel, we ran and stained a parallel gel with identical samples with Coomassie
to compare the intensities of protein bands. Proteins were separated on
denaturing sodium dodecyl sulfate (SDS)-10% polyacrylamide gels. The separated
proteins were then blotted into nitrocellulose (Immobilon-P, Millipore Corp.,
Iberica, Madrid, Spain). Blots were blocked overnight at 4°C with 5%
nonfat dry milk in Tris-buffered saline-Tween (20 mM Tris-HCl, 137 mM NaCl,
0.1% Tween-20). Western blot analysis was performed with a monoclonal antibody
against eNOS (Transduction Laboratories, Lexington, UK). Blots were incubated
with the first antibody (1:2500) for 1 h at room temperature and, after
extensive washing, with the second antibody (horseradish peroxidase-conjugated
anti-mouse Ig antibody) at a dilution of 1:1500 for 1 h. Specific eNOS protein
was detected by enhanced chemiluminescence (ECL; Amersham Iberica, Madrid,
Spain). Prestained protein markers were used for molecular mass
determinations.
Immunohistochemistry
eNOS expression was also detected by immunohistochemistry. Human peritoneal
pieces were incubated in the presence and in the absence of LPS (10 µg/ml)
for 24 h. Then they were dehydrated with sequentially increasing
concentrations of ethanol followed by xylene and embedded in paraffin wax.
Paraffin blocks were sectioned at 4 µm on a standard rotary microtome
(Leitz 1512, MICROM GmbH, Walldorf, Germany), and the sections were recovered
from a water bath on acid-alcohol—cleaned slides. Sections were stained
with hematoxylin and eosin using an automated staining system.
The sections were incubated further with phosphate-buffered saline containing 3% normal goat serum (ICN Biochemicals Ltd, Barcelona, Spain) and 0.1% Triton X-100 for 30 min and subsequently with a monoclonal antibody against eNOS protein (Transduction Laboratories) at the dilution of 1:20 for 1 h or monoclonal antibodies to identify mesothelial (HBME-1) and endothelial (factor VIII) cells. The sections were washed in phosphate-buffered saline containing 0.05% bovine serum albumin, then incubated with biotinylated antiserum to goat IgG (1:300 dilution, Vector Laboratories Ltd, Madrid, Spain) for 1 h and in a solution of peroxidase-linked avidin-biotin complex (ABC kit, Vector Laboratories Ltd) for 1 h as previously reported (19, 21). To reveal the peroxidase activity, the nickel-enhanced diaminobenzidine procedure was used.
Northern Blot Analysis
To test the stability of mRNA, human peritoneal samples were preincubated
with actinomycin D (10 µg/ml) for 1 h. Then, human peritoneal samples were
incubated in the presence and in the absence of LPS (10 µg/ml) during
different periods of time. Each experiment was performed four times. Total RNA
was isolated according to the method of Chomazynski and Sacchi
(22). Twenty micrograms of RNA
were fractionated in 1.3% agarose-formaldehyde gels and transferred by
capillarity to Genescreen nylon membranes (Dupont, Boston, MA). The membranes
were prehybridized at 42°C for 4 h in a solution containing 50% formamide,
1% SDS, 5x SSC (1x SSC is 150 mM NaCl plus 150 mM sodium citrate),
1x Denhardt's solution, 100 µg/ml denatured herring sperm DNA, and 50
mM phosphates (pH 6.5) and hybridized for 16 to 18 h in the same solution
supplemented with dextran sulfate (10% final concentration) containing 500,000
cpm of radiolabeled eNOS probe per milliliter. The cDNA probe used was the
HindIII-BamHI fragment of pNOS UTR-L (see below). The equal
loading of RNA was confirmed by the ethidium bromide staining of 28S and 18S
rRNA.
Plasmids and In Vitro Transcription
Oligonucleotides complementary to eNOS cDNA (GenBank accession number,
BTNIOXSY) were purchased from Bio-synthesis, Inc. (Lewisville, TX). pNOS-UTR
plasmids were prepared as previously reported
(13). In brief,
oligonucleotide 1 (5'-GGATCTAGAACGCTATCACGAGGACATT-3') and
oligonucleotide 2 (5'-AGGAAGCTTAGTAGGTCTCCTAACTTCTG-3') were used
to produce by reverse transcriptase PCR (from BAEC total RNA) a fragment
covering 166 bases of the coding region and 393 bases of the 3'-UTR of
eNOS cDNA (from 3485 to 4012). Amplification products were purified after
agarose gel electrophoresis, subjected to restriction endonuclease digestion
with XbaI and HindIII and ligated to pGEM4Z (Promega,
Madison, WI) to create plasmid pNOS-UTR-L. To produce single-stranded RNA,
plasmids were linearized with the corresponding restriction enzyme and
transcribed with SP6 or T7 RNA polymerase. Radiolabeled RNA was produced
according to the manufacturer's recommendations (Promega) with
32P-CTP (Amersham Iberica).
Band-Shift Assays
After incubation with LPS (10 µg/ml) for different periods of time, the
human peritoneal samples were frozen in liquid nitrogen. Afterward, the
samples were pulverized and resuspended in hypotonic buffer (25 mM Tris-HCl
[pH 7.9], 0.5 mM ethylenediaminetetraacetate, and 1 mM phenylmethylsulfonyl
fluoride) followed by four freezing and thawing cycles and by centrifugation
at 12,000 g at 4°C for 15 min. The supernatant was removed, supplemented
with glycerol (10% final concentration), and frozen at -70°C until use.
The protein content of the cytosolic extracts was determined by the
bicinchoninic acid reagent (Pierce) as described by the manufacturer.
Cytoplasmic lysates (10 µg) were incubated with 5 to 10 x
104 cpm of radiolabeled UTR-L in 15 mM HEPES (pH 7.9), 10 mM KCl, 5
mM MgCl2, 1 mM dithiothreitol, 1 µg of yeast tRNA per
microliter, 40 U of RNAsin (Promega), and 10% glycerol in a total volume of 15
µl for 10 min at 25°C. Twenty units of RNase T1 per reaction (Life
Technologies-BRL, Eggenstein, Germany) were then added, and the reaction
mixtures were incubated for 30 min at 37°C. Samples were electrophoresed
on 4% native polyacrylamide gel in 0.25 x TBE
(Tris-Borate-ethylenediaminetetraacetate) as running buffer, dried, and
autoradiographed with Kodak X-OMAT-S film (Madrid, Spain).
UV Cross-Linking of RNA-Protein Complex
Ten micrograms of the cytoplasmic lysates were incubated with
105 cpm of RNA in the same buffer described above in a total volume
of 20 µl for 10 min at 25°C. The samples were transferred to 20-well
U-bottom plates and UV irradiated in ice in a Stratalinker (Stratagene LTD,
Cambridge, UK) for 20 min on the automatic setting, followed by RNase
digestion (20 µg of RNase A and 20 U of RNase T1 for 30 min at 37°C).
The samples were then heated for 10 min at 70°C in Laemmli buffer
(20) without 2-mercaptoethanol
and electrophoresed on denaturing SDS-polyacrylamide gel. After drying, the
gels were exposed for 3 to 5 d to Kodak film with two intensifying
screens.
Statistical Analyses
Results are expressed as means ± SEM. Unless otherwise stated, each
value corresponds to a minimum of six different experiments. To determine the
statistical significance of our results, we performed an ANOVA with
Bonferroni's correction for multiple comparisons or a t test (paired
or unpaired). A P value < 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS (10 µg/ml) reduced eNOS protein expression in human peritoneum (Figure 1). A marked decrease in eNOS protein expression was found 6 h after exposure to LPS, and the maximal decrease in eNOS protein expression was observed after 24 h (Figure 1).
Northern blot analysis of total RNA obtained from human peritoneal samples demonstrated the presence of eNOS mRNA (Figure 3). The eNOS mRNA half-life was determined in the presence of actinomycin D (10 µg/ml), an inhibitor of transcription. Under basal conditions, the eNOS mRNA half-life was higher than 24 h. Stimulation of human peritoneum with LPS (10 µg/ml) markedly shortened eNOS mRNA half-life to less than 6 h (Figure 3). A marked decrease in eNOS mRNA expression was in fact observed 6 h after LPS incubation with nearly no detectable message 24 h after LPS incubation (Figure 3).
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Immunolocalization of eNOS Protein
The distribution of eNOS protein was studied before and after 24 h of
exposure to LPS (10 µg/ml). Positive immunostaining for eNOS protein was
observed in peritoneal capillary endothelium and in the mesothelium
(Figure 4, A and B). This was
confirmed by the fact that eNOS protein was co-localized in cells positively
stained by HBME-1, an anti-human mesothelial cell antibody (Dako A/S,
Glostrup, Denmark) which reacts with an incompletely characterized membrane
antigen present on the microvillous surface of mesothelial cells
(23). These cells were
negatively stained by factor VIII (Dako A/S), suggesting the presence of
mesothelial cells (Figure 4, C and
D). Furthermore, eNOS protein was also expressed in cells
positively stained by factor VIII and negatively by HBME-1, indicating the
presence of endothelial cells (Figure 4, C
and D). The histologic examination also revealed that LPS reduced
eNOS protein expression in both peritoneal endothelial and mesothelial cells
(Figure 5). In all experiments,
control immunostaining in which the primary antibody was replaced with normal
rabbit serum produced no positive signal (data not shown).
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Importance of Tumor Necrosis Factor-
in the Effect of LPS on
eNOS Expression
Resident peritoneal macrophages, although low in number, were present in
human peritoneum, as demonstrated by the positive stain for CD68 antigen
(Figure 6), a
monocyte/macrophage marker
(24). LPS-activated
macrophages are a recognized source of cytokines such as tumor necrosis
factor- (TNF-). Moreover, it has been recently demonstrated that
TNF- reduces eNOS expression in cultured bovine endothelial cells
(9,
10,
13). Therefore, we analyzed
the involvement of TNF- in the above-mentioned effects of LPS on eNOS
protein expression. We first analyzed the ability of the peritoneal samples to
produce TNF-. TNF- was measured in the RPMI medium obtained
after the incubation of the peritoneal samples for 24 h at 37°C. As
previously reported (25),
TNF- was determined by an enzyme-linked immunosorbent assay kit
(Chromogenix, Mölndal, Sweden) in which the
intra- and interassay variabilities were 1.1 and 4.7%, respectively. Under
basal conditions, the production of TNF- by human peritoneum was
undetectable and it was significantly stimulated after incubation with LPS. We
then determined whether an anti—TNF- antibody could modify the
decrease in eNOS expression mediated by LPS on human peritoneum. Addition of a
polyclonal anti—TNF- antibody (1:400) to 24-h LPS-incubated
peritoneum slightly prevented the reduction of eNOS protein expression
(Figure 7). This effect was not
observed when a nonspecific IgG was used
(Figure 7). The efficacy of the
polyclonal antibody to inhibit TNF- activity was previously
demonstrated by its ability to prevent the reduction of eNOS protein
expression produced by the addition of TNF- (<500 pg/ml) to cultured
endothelial cells (25).
Moreover, an increased concentration of anti—TNF- antibody
(1:200) failed to demonstrate a greater level of prevention of eNOS expression
than that obtained with 1:400 anti—TNF- antibody (data not
shown).
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Peritoneal Cytosolic Proteins and 3' UTR of eNOS mRNA
In a previous work, we demonstrated that bovine aortic endothelial cells
contain cytosolic proteins that form complexes with 3'-UTR eNOS mRNA,
which could be involved in destabilization of eNOS mRNA
(13). Addition of peritoneal
cytoplasmic extracts to a labeled probe containing the entire 3'-UTR
eNOS mRNA, the UTR-L probe, resulted in a gelshifted band
(Figure 8A, lane 1).
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Cytosolic extracts from human peritoneum were isolated further after in vitro exposure to LPS for different periods of time. These cytosolic extracts showed an increased time-dependent binding to the labeled UTR-L probe (Figure 8A, lanes 2, 3, and 4). Complex formation showed a significant increase after 6 h of LPS incubation and demonstrated the highest binding activity at 24 h (Figure 8A, lanes 2, 3, and 4).
The complex between UTR-L and the cytosolic proteins obtained from LPS-incubated human peritoneal samples was prevented by an excess (1000 ng) of unlabeled UTR-L (Figure 8B, lane 2). Treatment of peritoneal cytosolic extracts with proteinase K (87 µg/ml) before their incubation with the UTR-L abolished the complex formation, thus indicating the involvement of cytosolic proteins in the eNOS mRNA peritoneal cytosolic extract interactions (Figure 8B, lane 3).
To characterize the peritoneal cytosolic proteins that interacted with 3'-UTR eNOS mRNA, we performed UV cross-linking experiments followed by SDS-polyacrylamide gel electrophoresis analysis. A band with an apparent molecular weight of 60 kD was observed in the mixtures of labeled 3-UTR-L and peritoneal cytosolic extracts exposed to UV light (Figure 9, lane 1). The intensity of the 60-kD band was markedly enhanced after 24 h in LPS-incubated human peritoneal samples (Figure 9, lane 2). The 24-h incubation time was chosen because the maximal binding activity induced by LPS was observed at this time (Figure 8A, lane 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various studies have demonstrated a potent destabilization of eNOS mRNA in
TNF-—stimulated endothelial cells,
(9,
10,
13). In the present work, LPS
reduced eNOS mRNA in human peritoneal tissue. This effect was observed in the
presence of actinomycin D, suggesting that a change in stability of the eNOS
message may be the predominant mechanism for the LPS-induced downregulation of
its expression. In addition, a correlative decrease in eNOS protein expression
was observed in the LPS-treated peritoneal samples. It is of note that under
basal conditions, eNOS mRNA was very stable. A similar observation was
previously reported for cultured endothelial cells
(10). However, the present
experiments will not allow us to rule out a possible effect of TNF- on
eNOS gene transcription in human peritoneum until formal experimental runoff
studies are performed.
It has been demonstrated that LPS stimulated TNF- production by
macrophages (26). In this
regard, we recently demonstrated that TNF- reduces eNOS mRNA stability
in cultured endothelial cells
(13). Therefore, TNF-
could be involved in the observed reduction of eNOS expression elicited by LPS
in the human peritoneal samples. Despite that immunohistochemistry examination
demonstrated the presence of macrophages in the peritoneal tissue, the
addition of an anti—TNF- antibody to LPS-incubated peritoneal
samples slightly prevented the decrease in eNOS protein expression. These
results suggest that TNF- has a modest participation in the effect of
LPS on eNOS expression.
Several proteins that bind to specific sequences in the 3'-UTR of many mRNAs have been implicated in the regulation of their half-life (11, 12). In a previous study, we demonstrated that the cytosol of bovine endothelial cells contains proteins that interact with 3'-UTR of eNOS mRNA (13). In the present study, we demonstrated that human peritoneal cytosolic lysates form complexes with the complete in vitro transcribed 3'-UTR eNOS mRNA. Furthermore, cytosolic lysates obtained from LPS-incubated human peritoneal samples showed an increased binding activity to the 3'-UTR eNOS mRNA. The enhanced binding activity of the cytosol obtained from LPS-stimulated human peritoneal samples was associated with a decreased level of eNOS protein and eNOS mRNA expression.
The cytosolic components of human peritoneum that bind to the 3'-UTR of eNOS mRNA were proteins. This was suggested by the fact that preincubation of the human peritoneal cytosolic extracts with proteinase K fully inhibited complex formation with the 3'-UTR of eNOS mRNA. The UV crosslinking and SDS-polyacrylamide gel electrophoresis experiments using the labeled 3'-UTR eNOS mRNA showed a band with an apparent molecular weight of 60 kD. These results suggested that a 60-kD protein binds specifically to 3'-UTR eNOS mRNA.
Functional evidence demonstrating the involvement of the 60-kD protein in eNOS mRNA destabilization is not shown in the present work. Therefore, we may establish only an association between the presence of the 60-kD protein induced by LPS and the eNOS mRNA destabilization. In this regard, the activity of other reported 3'-UTR binding proteins correlated inversely with the destabilization of mRNA (27, 28). Moreover, the molecular weight of the 60-kD eNOS mRNA binding protein was within the range of other previously reported 3'-UTR binding proteins. In this regard, proteins of apparent molecular weights between 30 kD and 141 kD have been reported to bind to 3'-UTR of different mRNA reducing their half-life (29, 30).
The data presented here could not elucidate whether the LPS-dependent increase in the binding activity of the 60-kD cytosolic protein was due to a greater binding of the protein to 3'-UTR eNOS mRNA or to the synthesis of new protein or both. In this regard, Mohamed et al. (9) and Yoshizumi et al. (10) showed that destabilization of the eNOS message by cytokines was largely prevented by coincubation with cycloheximide, suggesting that synthesis of new proteins was required for this effect. It is also noteworthy that although the immunohistochemical study showed that LPS reduced eNOS protein expression in both mesothelial and peritoneal endothelial cells, our present experimental design did not allow us to establish whether the 60-kD binding protein is expressed in endothelial, mesothelial, or both cell types. Additional studies with isolated cultured cells are needed to elucidate this question.
Loss of eNOS expression in human peritoneal cells could compromise different cellular functions, favoring the presence of peritoneal dysfunction. In this regard, peritoneal inflammation results in the expression of adhesion molecules in endothelial and mesothelial cells, and it has been demonstrated that NO limits the expression of several adhesion molecules (31,32,33,34). Moreover, recent evidence demonstrated that NO contributes to the regulation of human fibrinolytic activity (35, 36). In this sense, the blockade of NO synthesis by L-arginine competitive inhibitors stimulated the generation of the plasminogen activator inhibitor (36). It is interesting that peritoneal inflammation causes a reduction in abdominal fibrinolytic activity, resulting in the persistence of intra-abdominal fibrin with subsequent adhesion and abscess formation (37, 38). Therefore, a situation of mesothelial and endothelial dysfunction, in terms of a reduced ability to express eNOS, could favor changes in the activity of the peritoneal fibrinolytic system.
In summary, in the present study, we describe that eNOS protein is expressed in peritoneal capillary endothelial and mesothelial cells. The expression of eNOS protein was down-regulated by LPS. In addition, we observed that a 60-kD peritoneal cytosolic protein binds to the 3'-UTR of eNOS mRNA, which increased after LPS stimulation, suggesting an association between the presence of the cytosolic protein and eNOS protein expression in human peritoneum. A better understanding of the regulatory mechanism of eNOS expression in peritoneal cells may lead to new strategies in the prevention of peritoneal dysfunction during peritonitis.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Am J Physiol277
: H1317-H1325,1999 in RAW 264.7 and peritoneal cells but not in Kupffer
cells. Am J Physiol 275:G39
-G46, 1998This article has been cited by other articles:
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P < 0.05 with respect to LPS-incubated peritoneal
samples.
