From the Faculty of Pharmacy, University of Catanzaro
"Magna Graecia" and the §§ Institute of Biotechnology
Applied to Pharmacology, Consiglio Nazionale delle Ricerche, 88021 Roccelletta di Borgia, the ¶ Department of Neuroscience, DIBIT-H
San Raffaele Institute, 20132 Milan, the
Centre of Cellular and
Molecular Pharmacology, Consiglio Nazionale delle Ricerche, 20129 Milan, and the ** Department of Pharmaco-Biology, University of
Calabria, 87036 Rende, Italy
Received for publication, July 21, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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Cell death via apoptosis induced by tumor
necrosis factor- Tumor necrosis factor- The gaseous messenger nitric oxide (NO) plays a role in the modulation
of TNF- To address this question, we have investigated cell death in response
to TNF- Cell Culture, Transfection, and Selection of Stable
Clones--
Bovine eNOS cDNA, subcloned in pBluescript SK, was a
kind gift of Dr. William C. Sessa (Yale University School of Medicine, New Haven, CT). The cDNA was excised with EcoRI and
ligated in the EcoRI site of plasmid pTRE
(CLONTECH, Palo Alto, CA). The orientation of the
insert was checked by restriction mapping.
HeLa Tet-off cells (CLONTECH) were grown in
Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.,
Paisley, United Kingdom (UK)), supplemented with 10% Tet system
approved fetal bovine serum (CLONTECH), 50 UI/ml
penicillin, 50 µg/ml streptomycin, 2 mM
L-glutamine, 100 µg/ml G418 (Roche Diagnostics, Mannheim, Germany), and maintained at 37 °C under a 5% CO2 atmosphere.
Cells grown on 35-mm Petri dishes to ~40% confluence were
cotransfected by the calcium phosphate method (23) using 0.5 µg of
pTRE/eNOS plasmid and 25 ng of pTK-Hygr plasmid
(CLONTECH) per cm2 of culture dish. 3 days after transfection, the cells were detached with trypsin and
plated in the presence of doxycycline hydrochloride (0.01-1 µg/ml)
(Sigma) and hygromycin B (200 µg/ml) (Roche Diagnostics). After 3 weeks, hygromycin-resistant colonies were expanded in 24-well Petri dishes.
Stable transfectants were maintained in medium supplemented with 100 µg/ml G418, 100 µg/ml hygromycin, and 10 ng/ml doxycycline. To
induce expression of eNOS, cells were plated in the absence of
doxycycline. Unless otherwise specified, experiments were carried out
on cells cultured for 72 h with or without the antibiotic.
A mouse endothelial cell line immortalized with the middle T antigen
(E2 cells), a kind gift of Elisabetta Dejana (Mario Negri Institute,
Milan, Italy) was maintained in culture as described (24).
Protein Extraction and Immunoblot Analysis--
Cell monolayers
were washed free of medium, solubilized by direct addition of a
pre-heated (to 80 °C) denaturing buffer, containing 50 mM Tris-Cl, pH 6.8, 2% SDS, and a protease inhibitor
mixture (Complete, Roche Diagnostics), and immediately boiled for 2 min. After addition of 0.05% bromphenol blue, 10% glycerol, and 2% Immunofluorescence and Diaphorase Cytochemistry--
Cells
plated on glass coverslips, fixed with 4% paraformaldehyde (Fluka) in
120 mM sodium phosphate, pH 7.4, for 30 min at 37 °C,
were permeabilized with Triton X-100 and processed for immunofluorescence as described previously (25). Cells were doubly
immunostained with monoclonal anti-eNOS and anti-giantin polyclonal
antibodies, the latter a kind gift of Dr. M. Renz (Institute of
Immunology and Molecular Genetics, Karlsuhe, Germany; see Ref. 26).
Bound monoclonal and polyclonal antibodies were revealed by
fluorescein-labeled anti-mouse IgG and rhodamine-conjugated anti-rabbit
IgG (Jackson Immunoresearch Laboratories Inc., West Grove, PA).
Preparations were observed under a Nikon Optiphot 2 microscope equipped
for epifluorescence or with a Bio-Rad MRC 1024 laser confocal microscope.
For the diaphorase reaction, cells, fixed as above, were incubated for
30 min at 37 °C in a buffer containing 100 mM Tris-Cl, pH 7.4, 0.2% Triton X-100, 1 mM Assay of NOS Activity--
NOS activity was assayed in both
homogenized and intact cells by measuring the conversion of
L-[3H]arginine (Amersham Pharmacia Biotech)
into L-[3H]citrulline. In the first approach,
HeLa or E2 cells were detached by trypsinization, pelleted, and
resuspended (2 × 107 cells/ml) in an homogenization
buffer containing 320 mM sucrose, 1 mM
dithiothreitol, 1 mM EDTA, 20 mM HEPES, pH 7.2, supplemented with a protease inhibitor mixture (Complete, Roche
Diagnostic). After sonication (30 s at 4 °C), 50 µl of cell
extract were assayed in a total volume of 150 µl as described (28).
In some samples 500 µM
N Measurement of cGMP Formation--
NO-dependent
guanosine 3',5'-cyclic monophosphate (cGMP) generation by the
transfected HeLa clone A Induction and Detection of Cell Death--
HeLa cells seeded in
six-well plates (105 cells/cm2) were incubated
in the presence or absence of TNF-
Apoptosis was measured by flow cytometry using two different protocols
as described (31). In the first approach, phosphatidylserine exposure
was monitored by staining for 15 min at room temperature with
fluorescein isothiocyanate-labeled annexin V (0.5 µg/ml in phosphate-buffered saline). In the second approach, the hypodiploid DNA
peak in single parameter DNA histograms typical of apoptotic cells was
identified. To this end, DNA was stained in unfixed cells incubated for
60 min at 37 °C in 0.1% sodium citrate, 50 mg/ml propidium iodide,
100 µg/ml RNase A (Sigma), and 0.01% Nonidet P-40. Cells were
analyzed for either DNA content or annexin V staining using a
fluorescence-activated cell sorter (FACStar Plus, Becton Dickinson,
Sunnyvale, CA).
Measurement of Ceramide Concentration--
Samples containing
2 × 106 HeLa cells were incubated at 37 °C with or
without PDMP (50 µM) and Fum (10 µM) for
3 h, then treated with TNF- Statistical Analysis--
The results are expressed as
means ± S.E. of the mean (S.E.); n represents the
number of individual experiments. Statistical analysis was carried out
using Student's t test for unpaired variables (two-tailed).
*, **, and *** or +, ++, and +++ in the figure panels and tables refer
to statistical probabilities (P) of <0.05, <0.01, and
<0.001, respectively, as detailed in the legends to figures and tables.
Characterization of HeLa Tet-off Clones Inducibly Expressing
eNOS--
For our experiments we chose the Tet-off system (32), which
allows repression of a single gene by tetracycline or a tetracycline derivative, such as doxycycline. After transfection of HeLa Tet-off cells with eNOS cDNA together with a plasmid conferring resistance to hygromycin, we expanded 30 resistant clones of which 6 expressed eNOS when doxycycline was removed from the medium as established by
Western blotting and by immunofluorescence. Since the Tet-off system,
by allowing comparison between the "on" and "off" states of the
transfected cDNA, permits direct assessment of the consequences of
the expression of a single gene in an internally controlled system, we
carried out a detailed analysis on only one of the selected clones,
clone A
We first analyzed the time course of eNOS induction by removal of
doxycycline from the medium (Fig.
1A). As shown in Fig. 1A, the removal of doxycycline led to a rapid induction of
the expression of eNOS, detectable as a 140,000 Mr band (arrowhead in Fig.
1A), which was visible already at 4 h and continued to increase in intensity during the following 3 days. At the later time
points, lower Mr bands appeared (see 24-, 48-, and 72-h time points of Fig. 1A); these were presumably
products of eNOS proteolysis, since they were not present in noninduced
cells (0- and 2-h time points).
Next, we investigated the dependence of eNOS levels on doxycycline
concentration. As can be seen from Fig. 1B, induction was detectable at doxycyline concentrations
To obtain a semiquantitative evaluation of the degree of induction of
eNOS expression by doxycyline removal, we compared by Western blotting
30 µg of total protein extract from cells grown in the presence of
doxycycline with different quantities of extract from cells grown in
the absence of the antibiotic (Fig. 1C). By loading a large
amount of protein and by increasing exposure time of the blot, it was
possible to detect a faint eNOS band in noninduced cells, due to basal
expression of the cloned cDNA (lane 1). This band was much less prominent than that obtained with 1/10 the amount of
lysate from induced cells, whereas it was of intensity comparable to
that of the band from 1/100 this amount of lysate (0.3 µg of protein,
lane 3). This result indicates that, in response to doxycycline removal from the medium, this clone exhibits an ~100-fold induction of eNOS expression.
We also compared the level of eNOS expression in induced cells with
that of endogenous eNOS in a murine endothelial cell line (E2 cells).
As shown in Fig. 1D, the level of eNOS in induced clone A
To investigate whether eNOS localization in induced cells corresponds
to that of the endogenous enzyme in endothelial cells (33), we carried
out immunofluorescence analysis. As shown in Fig.
2A, eNOS appeared most
concentrated on the surface and in a perinuclear structure in the Golgi
region of the cells, as shown by staining for the Golgi marker giantin
in the same field of cells (Fig. 2B). Confocal analysis
(panels D-F) confirmed the Golgi localization of
eNOS, as is apparent by the yellow color in the
merged image of the double-stained cells (F).
We then analyzed the Ca2+-dependent activity of
eNOS in homogenized and intact cells, by measuring the conversion of
its substrate, L-arginine, into L-citrulline.
As shown in Fig. 3A,
citrulline formation in eNOS-expressing (dox
In intact cells (Fig. 3B), NOS activity was stimulated using
ATP, an agonist of P2y receptors known to increase
[Ca2+]i in HeLa cells. ATP
increased L-citrulline formation with respect to untreated
controls only in eNOS-expressing (dox
We also carried out a morphological analysis of the diaphorase activity
of eNOS. As shown in Fig. 3C, the
NADPH-dependent reduction of nitro blue tetrazolium
generated a dark precipitate in eNOS-expressing cells grown in the
absence of doxycyline (panel b), whereas cells
cultured in the presence of the antibiotic remained colorless
(panel a). The localization of the precipitate in
induced cells was reminiscent of that of the enzyme itself detected by immunofluorescence (Fig. 2).
To investigate whether eNOS-expressing cells are able to generate
bioactive NO, we measured formation of cGMP, a good proxy for NO since
soluble guanylate cyclase is activated by nanomolar concentrations of
the gas (36). Exposure to ATP (15 min) of cells grown in the presence
of doxycycline did not result in any increase in cGMP over non
stimulated controls (0.36 ± 0.02 and 0.35 ± 0.03 pmol/mg
min eNOS Expression Inhibits TNF-
To further characterize the protective effect of eNOS expression, we
measured, by flow cytometry, an early feature of apoptosis, i.e. the appearance of phosphatidylserine on the outer
leaflet of the plasma membrane, as well as another apoptosis
hallmark, the formation of hypodiploid DNA. Phosphatidylserine,
measured by annexin V staining, was not detected in cells either in the presence or absence of doxycycline (Fig.
4 left, a and
c, respectively; Table II).
Incubation with TNF- Stimulation with TNF-
We considered the mechanism by which TNF-
The data of Table III show that ceramide
levels in A
We then proceeded to investigate the effects of Fum and PDMP on
TNF- The aim of this study was to investigate the interaction between
eNOS and TNF- The protective effect of NO on TNF- To elucidate the pathway responsible for the TNF- Several lines of evidence suggest that generation of ceramide by
TNF- (TNF-
) plays an important role in many
physiological and pathological conditions. The signal transduction
pathway activated by this cytokine is known to be regulated by several
intracellular messengers. In particular, in many systems nitric oxide
(NO) has been shown to protect cells from TNF-
-induced apoptosis.
However, whether NO can be generated by the cytokine to down-regulate
its own apoptotic program has never been studied. We have addressed
this question in HeLa Tet-off cell clones stably transfected with the
endothelial NO synthase under a tetracycline-responsive promoter.
Endothelial NO synthase, induced about 100-fold in these cells by
removal of the antibiotic, retained the characteristics of the native enzyme of endothelial cells, both in terms of intracellular
localization and functional activity. Expression of the endothelial NO
synthase was sufficient to protect from TNF-
-induced apoptosis.
This protection was mediated by the generation of NO. TNF-
itself
stimulated endothelial NO synthase activity to generate NO through a
pathway involving its lipid messenger, ceramide. Our results identify a
novel mechanism of regulation of a signal transduction pathway activated by death receptors and suggest that NO may constitute a
built-in mechanism by which TNF-
controls its own apoptotic program.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF-
)1 is a pleiotropic
cytokine involved in the regulation of important physiological
functions, including the development of tissues and the coordinate
activation of immune responses, as well as in the onset and progress of
pathological conditions (1-4). Most of these actions are exerted by
TNF-
via its ability to induce cell death via apoptosis. This
process is activated by the cytokine through the stimulation of its
55-kDa, type I receptor (TNF-RI), whose signal transduction pathway has been characterized in quite some detail. Binding of TNF-
results in
receptor trimerization with clustering of the cytosolic death domains,
interaction with the complementary domains present in specific
cytosolic proteins, including TRADD and FADD, and formation of the
transduction complex, referred to as the DISC (5). This leads to the
recruitment and proteolytic activation of a specific pro-protease,
procaspase-8 (and possibly also -10) (5), which initiates a cascade of
signaling events, including generation of the lipid metabolite
ceramide, cytochrome c release from mitochondria, apoptosome
formation as well as activation of downstream caspases, all of them
integrated in an operational network leading to cell death (6-8).
signaling. When generated after cytokine-stimulated expression of the inducible isoform of its synthesizing enzymes, NO
synthase (NOS), NO may contribute to later stages of apoptosis (see,
e.g., Refs. 9-12; but see also Ref. 13). By contrast, administration of NO prior to, or together with, TNF-
has been shown
to inhibit apoptosis stimulated by the cytokine (13-19). Two lines of
evidence suggest that this early protective effect of NO might be of
physiological relevance. First, NO acts by inhibiting key apoptogenic
signal transduction events triggered by TNF-
, including ceramide
accumulation and TRADD recruitment to the DISC complex, cytochrome
c release, as well as the activity of initiator and effector
caspases, both before and after their enzymatic cleavage (13-15,
17-19). Second, inhibition of endogenous NOS activity during stimulation with TNF-
increases apoptosis induction by the cytokine (18, 20, 21). A direct link between NOS activity and protection from
TNF-
-induced apoptosis was demonstrated by infection experiments with the inducible isoform of NOS, an enzyme that generates NO continuously (16). These experiments, however, left open the question
of whether endothelial and neuronal NOS, whose activity is instead
regulated by intracellular messengers (22), would be stimulated to
counteract the apoptogenic effect of TNF-
, and whether this would be
sufficient to protect cells from death.
of a HeLa Tet-off cell line expressing bovine endothelial
NOS (eNOS) in an inducible fashion. By comparing the degree of
TNF-
-induced apoptosis in cells expressing or not expressing the
enzyme, we were able to show that expression of eNOS is sufficient to
induce a partially resistant phenotype, and that this effect is due to
generation of NO. We have also investigated the mechanism of this
resistance, and found that eNOS activity is stimulated by TNF-
itself through a pathway involving ceramide accumulation. Our study
shows that eNOS activity is regulated by, and itself regulates, the
apoptotic process triggered by TNF-
in a complex regulatory circuit.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, samples were boiled again and loaded onto 10% SDS-polyacrylamide gels. After electrophoresis, polypeptides were electrophoretically transferred to nitrocellulose filters (Schleicher & Schuell, Dassel, Germany). Monoclonal anti-eNOS (Transduction Laboratories, Lexington, KY) and monoclonal anti-
-actin (Sigma) antibodies were used to reveal the respective antigens. After incubation with secondary reagent (polyclonal anti-mouse horseradish peroxidase conjugate; Transduction Laboratories), blots were developed with the enhanced chemiluminescence procedure (ECL-Plus; Amersham Pharmacia, Little Chalfont, UK).
-NADPH (Sigma), and 0.2 mM nitro blue tetrazolium (Sigma), and observed by bright
field light microscopy (27).
-monomethyl-L-arginine
(L-NMMA) or 1 mM EGTA were included. NOS activity in intact cells was measured on monolayers washed and then
incubated for 20 min at 37 °C in a reaction buffer containing: 145 mM NaCl, 5 mM KCl, 1 mM
MgSO4, 10 mM glucose, 1 mM
CaCl2 and 10 mM HEPES, pH 7.4, with or without
N
-nitro-L-arginine methyl ester
(L-NAME; 500 µM) (Sigma). In the experiments
with fumonisin B1 (Fum) (10 µM; Sigma) or
DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) (50 µM; Calbiochem, Bad Soden, Germany) cell were
pre-incubated for 3 h in culture medium containing the compounds,
which were also added during the subsequent incubation in the reaction
buffer. At the end of the pre-incubation, 2.5 µCi/ml
L-[3H]arginine was added 5 min before cell
stimulation with ATP (100 µM) or TNF-
(100 ng/ml;
Alexis Italia, Florence, Italy). Nonstimulated cells were run in
parallel. 15 min later the monolayers were washed with 2 ml of ice-cold
phosphate-buffered saline, pH 7.4, supplemented with
L-arginine (5 mM) and EDTA (4 mM).
0.5 ml of 100% cold ethanol was added to the dishes and left to
evaporate before a final addition of 2 ml of 20 mM HEPES,
pH 6.0. Separation of L-[3H]citrulline from
L-[3H]arginine was obtained by Dowex 50X8-400
chromatography (Sigma) as described (29). In the assay carried out with
homogenized cells, values obtained from samples incubated without cell
extracts were subtracted. Data in the intact cells are presented
without background correction.
L-[3H]Citrulline formed was normalized to
protein content (bicinchoninic acid assay; Pierce).
was measured in both the cells themselves
and in a coincubation system using PC12 cells as a reporter system.
PC12 cells were grown at 37 °C, 5% CO2 in DMEM
supplemented with 10% heat-inactivated horse serum and 5% Fetal Clone
III, then detached by trypsinization for the experiment. Samples of
suspended 2 × 106 HeLa and 0.5 × 106 PC12 cells, either separate or together, were treated
for 15 min at 37 °C in the presence or absence of either ATP (100 µM), TNF-
(100 ng/ml), or
S-nitrosoacetylpenicillamine (100 µM;
Calbiochem) in 1 ml of a solution containing 125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2 mM
CaCl2, 6 mM glucose, 1 mM
L-arginine, 0.6 mM isobuthylmethylxanthine, and
25 mM HEPES, pH 7.4, with or without L-NAME
(500 µM). In the experiments with Fum (10 µM) and PDMP (50 µM), pre-incubations were
for 3 h as described above. The reaction was terminated by
addition of ice-cold trichloroacetic acid (final concentration 6%).
After ether extraction, cGMP levels were measured using a
radioimmunoassay kit (Perkin Elmer Life Sciences) and normalized
to cellular proteins as described above.
(100 ng/ml) and cycloheximide (CHX; 1 µg/ml). In some samples L-NAME (500 µM) was also present. Cell death was measured using the
colorimetric assay with
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT;
Sigma) (30). At the end of the treatments, cell plates were washed with
RPMI 1640 (without phenol red) and incubated for 3 h at 37 °C
with 2.4 mM MTT in the same medium, the untransformed MTT
carefully removed and the dye crystals solubilized in 1 ml of
2-propanol. Absorbance was read immediately in a Uvikon 941 spectrophotometer (test wavelength of 570 nm; reference wavelength of
690 nm).
(100 ng/ml) for another 15 min.
Conversion of ceramide to the 32P-labeled ceramide
phosphate was carried out by diacylglycerol kinase treatment of the
extracted lipids in the presence of 10 µCi of
[
-32P]ATP as described previously (19). The ceramide
phosphates produced were separated by one-dimensional thin layer
chromatography using chloroform/methanol/acetic acid (65:15:5; v/v/v)
as solvent. The relevant spots were identified by autoradiography and
their radioactivity measured by liquid scintillation counting. The
concentration of ceramide per sample was determined versus a
standard curve encompassing the range of ceramide expected in the samples.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.
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Fig. 1.
Immunoblots of proteins extracted from clone
A . A, time course of eNOS
induction by removal of doxycycline from the medium. A
cells were
grown in 100-mm dishes to 80% confluence in the presence of
doxycycline (10 ng/ml). At time 0, cells were washed with
phosphate-buffered saline, split into smaller culture dishes (1/20 of
the initial cultures/35-mm dish), and incubated further in the absence
of the antibiotic. At the indicated time points, cells were harvested
and analyzed by 9% SDS-polyacrylamide gel electrophoresis followed by
electroblotting. ~10 µg of total cellular protein were loaded onto
each lane. Protein load was checked by
-actin immunostaining, shown
in the lower part of the panel.
B, dependence of eNOS expression on doxycyline
concentration. Cells grown in the presence of doxycycline were split as
described for panel A and then cultured in the
presence of 1, 0.5, 0.1, 0.05, 0.01, 0.001, and 0 ng/ml doxycycline
(lanes 1-7, respectively) for 72 h. Shown
is a Western blot, in which ~10 µg of total protein for each
condition were analyzed. The lower part of the
panel shows
-actin immunostaining of the same samples.
C, semiquantitative evaluation of induction of eNOS
expression by doxycycline depletion. Lysates were prepared from cells
grown in the presence or absence of the antibiotic for 72 h.
Lane 1 contained 30 µg of protein total extract
from cells grown in the presence of doxycycline, whereas
lanes 2 and 3 contained 3 and 0.3 µg, respectively. Removal of doxycycline results in a ~100-fold
induction of eNOS expression. D, comparison of eNOS levels
in induced A
and E2 cells. The indicated amounts of proteins from
A
cells (lane 1) and E2 cells
(lanes 2 and 3) were loaded. In
panels A-D, the arrowhead indicates eNOS
(Mr 140,000), whereas the asterisks
in panels A, B, and D
indicate
-actin immunostaining for the same samples.
Numbers on the left represent
Mr × 10
3 of molecular
size standards from Bio-Rad.
0.1 ng/ml
(lanes 3-7). With decreasing doxycyline
concentrations, the expression of the enzyme gradually increased, to
reach a maximum at an antibiotic concentration of 0.001 ng/ml.
cells (lane 1) was only slightly higher compared
with the endothelial cell line (lanes 2 and
3).
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Fig. 2.
Double-staining immunofluorescence of clone
A cells. Cells expressing eNOS were
doubly stained with monoclonal anti-eNOS and polyclonal anti-giantin
antibodies, followed by fluorescein-labeled anti-mouse IgG
(A and D) and rhodamine-conjugated anti-rabbit
IgG (B and E), respectively. Panels
A-C show the same field of cells viewed under the
fluorescein filter for eNOS (A), the rhodamine filter for
giantin (B), and in phase contrast (C). eNOS
(A) appears concentrated on the surface and in a perinuclear
structure corresponding to the Golgi region, as shown by the giantin
staining (B). Panels D-F show a
single field of cells analyzed by confocal microscopy; fluorescein
(eNOS; D), and rhodamine (giantin; E)
fluorescence are superimposed on the transmitted light image, and
colocalization of the two is demonstrated by the yellow
color in the merged image (F).
Bar in panels A-C, 15 µm;
panels D-F, 10 µm.
) cells was stimulated by
Ca2+ (363 ± 4.1%, n = 4 over the
activity in the presence of EGTA). The
Ca2+-dependent activity was ~2.5 pmol/mg
min
1, a value approximately 3-fold higher
than those reported in the literature for endothelial cells (see,
e.g., Ref. 34). We also obtained similar activity values in
the E2 cell extracts (data not shown). The
Ca2+-dependent NOS activity was nearly
completely abolished by the NOS inhibitor L-NMMA. In cells
grown in the presence of doxycycline, instead, neither EGTA nor
L-NMMA induced significant changes in citrulline
formation.
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Fig. 3.
Characterization of eNOS activity in clone
A . A, calcium dependence of
eNOS activity in cell lysates. Lysates, prepared from cells grown in
the presence (dox+) or absence (dox
) of
doxycycline for 72 h, were resuspended in a buffer containing
L-[3H]arginine and either 0.45 mM
CaCl2 (Ca2+), 0.45 mM
CaCl2 and 1 mM EGTA (EGTA), or 0.45 mM CaCl2 and 500 µM
L-NMMA (L-NMMA) for 10 min at
37 °C. eNOS activity was estimated as pmol/min
L-[3H]citrulline formed in the reaction,
normalized to protein content. B, Ca2+
dependence of eNOS activity in intact cells. Cell monolayers, grown in
the presence or absence of doxycycline, were incubated for 15 min at
37 °C in a medium containing
L-[3H]arginine without other additions
(control), with 100 µM ATP (ATP),
or with 100 µM ATP and 500 µM
L-NAME (ATP + L-NAME).
eNOS activity was estimated as in A. C,
cytochemical analysis of NADPH tetrazolium blue reductase activity of
eNOS in cells grown in the presence (a) or absence
(b) of doxycycline in the culture medium. Preparations were
examined in bright field. A dark precipitate is visible in cells
induced to express eNOS (b), whereas noninduced cells are
barely visible (a). Photography, digital acquisition, and
printing were exactly the same for the two panels. Bar, 15 µm. The results shown are from one experiment representative of four
consistent ones. D, cGMP generation induced by activation of
eNOS. 2 × 106 A
cells, grown in the presence or
absence of doxycycline, were suspended together with 0.5 × 106 PC12 cells and incubated for 15 min at 37 °C under
the conditions described in B. cGMP accumulation was
estimated by a radioimmunoassay and normalized to protein content.
Statistical probability in panels A,
B, and D is indicated by the asterisks
and calculated versus EGTA-containing lysates (A)
or untreated control cells (B and D), whereas the
crosses refer to the statistical probability in samples
treated in the presence versus absence of either
L-NMMA or L-NAME (n = 4) (see
"Experimental Procedures" for details).
) cells, and this effect was
prevented by the NOS inhibitor L-NAME. In contrast, no
changes were observed in cells cultured in the presence of doxycycline
(Fig. 3B). The amplitude of the
[Ca2+]i increase induced by ATP
(measured with fura-2; see Ref. 35) was similar in dox
and dox+ cells
(data not shown).
1, respectively; n = 4).
In eNOS-expressing cells, administration of ATP resulted in a slight
increase in cGMP formation over controls (0.44 ± 0.03 and
0.33 ± 0.01 pmol/mg min
1, respectively;
n = 3), which was not observed in the presence of
L-NAME (0.28 ± 0.02 pmol/mg min
1),
suggesting that these cells express low levels of guanylate cyclase.
Therefore, to detect generation of cGMP, we used another cell type, the
pheochromocytoma PC12, as a reporter system. These cells were chosen
because they respond to NO with generation of cGMP (0.33 ± 0.04 and 9.45 ± 0.1 pmol/mg min
1 in controls and cells
incubated with 100 µM of the NO donor
S-nitrosoacetylpenicillamine, n = 3, p < 0.001), whereas they do not generate cGMP when
treated with ATP (data not shown). As shown in Fig. 3D,
coincubation of eNOS-expressing (dox
) HeLa cells together with PC12
cells resulted in an increased generation of cGMP after administration
of ATP, which was inhibited by the presence of L-NAME. No
changes in cGMP formation were observed with cells grown in the
presence of doxycycline (Fig. 3D).
-induced Cell Death--
To
investigate the effects of the expression of eNOS on TNF-
-induced
cell death, cells grown in the presence or absence of doxycyline were
treated with TNF-
together with the protein synthesis inhibitor CHX
for 6 h. In cells grown in the presence of doxycyline, this
treatment resulted in death as measured by the decreased conversion of
MTT into formazan (Table I).
eNOS-expressing (dox
) cells were less sensitive to death induction by
TNF-
/CHX. However, when these cells were exposed to TNF-
/CHX in
the presence of L-NAME, cell sensitivity to the treatment
was similar to that observed in cells not expressing eNOS (Table I).
The effect of L-NAME on eNOS-expressing cells was due to
its inhibition of eNOS activity since the compound was without any
appreciable effect in dox+ cells (Table I).
Effects of eNOS expression on the TNF--induced conversion of MTT
into formazan
cells, seeded in six-well plates (10 × 104
cells/cm2), were cultured in the presence (dox+) or absence
(dox
) of doxycycline and then treated with TNF-
(100 ng/ml) and
CHX (1 µM) for 6 h or left untreated (control). When
indicated, this treatment was carried out in the presence of
L-NAME (500 µM). Cell were then washed and
incubated with MTT (2.4 mM) for another 3 h. Conversion of MTT
into its derivative formazan by mitochondrial dehydrogenases was
measured as described under "Experimental Procedures." Statistical
probability vs. controls is indicated by the asterisks,
calculated as described under "Experimental Procedures"
(n = 4). The differences between values observed after
TNF-
/CHX treatment in dox+ vs. dox
and
dox
/L-NAME vs. dox
(indicated by the
crosses) are highly significant (p < 0.001 in both
cases).
/CHX (12 h) resulted, in both cell preparations,
in the appearance of phosphatidylserine (Fig. 4 left,
panels b and d, respectively),
although to a significantly lower extent in eNOS-expressing cells (Fig.
4 left, panel d; Table II). Similarly,
the increase of hypodiploid DNA after treatment with TNF-
/CHX
observed in cells grown in the presence of doxycycline was
significantly higher than that in dox
cells (Fig. 4 right, panels g and h, and i and
l, respectively; Table II). When eNOS-expressing cells were
exposed to TNF-
/CHX in the presence of L-NAME, the appearance of both phosphatidylserine (Fig. 4 left,
f) and hypodiploid DNA (Fig. 4 right,
n) was significantly enhanced and not
significantly different from that observed in noninduced (dox+) cells.
The effect of L-NAME was specific because incubation with this compound had no appreciable action in dox+ cells (Table II).
View larger version (20K):
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Fig. 4.
Effects of eNOS expression on
TNF- -induced apoptosis. Cell monolayers,
grown in the presence (dox+) or absence (dox
)
of doxycycline were incubated without (control) or with TNF-
(100 ng/ml) and CHX (1 µg/ml) for an additional 12 h.
L-NAME (500 µM) was added during the
incubation where indicated. Apoptosis was analyzed measuring, by flow
cytometry, phosphatidylserine exposure and DNA staining with propidium
iodide. The left-hand panel shows the exposure of
phosphatidylserine, assessed using fluorescein isothiocyanate
labeled-annexin V (x axis, arbitrary units). The increase in
annexin V-positive cells after treatment with TNF-
/CHX is lower in
eNOS-expressing cells (d) than in noninduced cells
(b) or eNOS-expressing cells treated with L-NAME
(f). No significant annexin V staining is present in control
cells not exposed to the cytokine and the protein synthesis inhibitor
(a, c, and e). The
right-hand panel shows the DNA content analyzed
by flow cytometry measuring the binding of propidium iodide
(x axis, arbitrary units). In these experiments HeLa cells
were not synchronized; accordingly, G0/G1, S,
and G2/M phases were present as already described for these
cells (55). The sub-G1, hypodiploid DNA peak,
characteristic of apoptosis, is clearly detectable in cells treated
with TNF-
/CHX (h, l, and n) with
respect to untreated controls (g, i, and
m) and is lower in eNOS-expressing cells (l).
Incubation with L-NAME resulted in a sub-G1
peak similar to the one observed in noninduced cells (n).
The results shown are from one out of four representative experiments.
M1 indicates the region considered for the statistical
analyses of Table II.
Effects of eNOS expression on the TNF--induced exposure of
phosphatidylserine and formation of hypodiploid DNA
cells, cultured in the presence (dox+) or absence (dox
) of
doxycycline were treated with TNF-
(100 ng/ml) and CHX (1 µM) for 12 h, or left untreated (control), before
analysis of annexin V and propidium iodide staining by flow cytometry
as described under "Experimental Procedures." Values represent the
percentage of cells measured in the M1 regions, defined as shown in
Fig. 4. Statistical probability vs. controls is indicated by
the asterisks, that of dox+ vs. dox
and dox
/L-NAME
vs. dox
cells after TNF-
/CHX treatment by the crosses,
both calculated as described under "Experimental Procedures"
(n = 4).
Increases eNOS Activity and NO
Generation--
To investigate whether generation of NO from eNOS was
induced as a consequence of cell stimulation with TNF-
, we measured the effect of a 15-min stimulation with the cytokine (in the absence of
CHX) on NOS activity in intact HeLa cells and the generation of cGMP in
the PC12 cell reporter system. As shown in Fig.
5A, NOS activity was
stimulated by TNF-
in eNOS-expressing (dox
) HeLa cells.
Consistently, these cells, when stimulated by the cytokine, caused
increased cGMP generation in coincubated PC12 cells (Fig.
5B). Both these effects were prevented by
L-NAME. In addition, TNF-
did not have any effect on NOS
activity and cGMP generation when HeLa cells grown in the presence of
doxycycline were used.
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Fig. 5.
Effect of TNF- on
eNOS activity and cGMP generation. A, dependence of NOS
activity on TNF-
and drugs interfering with sphingolipid metabolism.
Cell monolayers, grown in the presence (dox+) or absence
(dox
) of doxycycline, were incubated for 3 h at
37 °C in culture medium with 10 µM Fum
(fourth bar), 50 µM PDMP
(fifth bar), or without other additions
(first three bars). Cells were then
treated for an additional 15 min at 37 °C in a medium containing
L-[3H]arginine with or without either TNF-
(100 ng/ml), L-NAME (500 µM), Fum, or PDMP as
indicated. NOS activity was estimated as described in Fig.
3B. B, effects of TNF-
and drugs interfering
with sphingolipid metabolism on cGMP generation. 2 × 106 A
cells, grown in the presence or absence of
doxycycline, and incubated with Fum or PDMP as in A, were
suspended together with 0.5 × 106 PC12 cells and
treated for 15 min at 37 °C with or without either TNF-
,
L-NAME, Fum, and PDMP as described in panel
A. cGMP accumulation was estimated as described in Fig.
3D. Statistical probability versus untreated
control cells is indicated by the asterisks, that of samples
incubated with TNF-
in the presence versus absence of
either L-NAME, PDMP or Fum by crosses, both
calculated as described under "Experimental Procedures"
(n = 4).
stimulates eNOS activity.
TNF-
is not known to increase
[Ca2+]i, the best known activator
of eNOS (22). In accordance, we did not detect any increases in
[Ca2+]i in HeLa cells during a
15-min stimulation with the cytokine (data not shown). Generation of
ceramide is among the intracellular events involved in induction of
apoptosis by TNF-
(6). Since short chain analogues of this lipid
messenger have been reported recently to activate eNOS (37), we
investigated whether ceramide could be involved in the activation of
eNOS by TNF-
. To this end we preincubated the cells with the
ceramide synthase inhibitor, Fum, and the glycosylceramide synthase
inhibitor, PDMP, which are known to decrease and increase,
respectively, the generation of ceramide by TNF-
, presumably by
altering the levels of substrate for sphingomyelinases (19, 38,
39).
cells were affected by TNF-
, Fum, and PDMP in the
same way as described for other cells (19, 38, 39). TNF-
caused an
early increase in ceramide concentration, which was augmented by
preincubation with PDMP and diminished by Fum. These compounds had no
significant effect on basal levels of ceramide.
Effects of TNF-, PDMP, and fumonisin B1 on ceramide accumulation in
HeLa dox+ cells
cells, cultured in the presence of doxycycline, were
incubated for 3 h with or without PDMP (50 µM) or
Fum (10 µM) prior to a 15 min treatment in the absence
(control) or presence of TNF-
(100 ng/ml). Ceramide was measured as
described under "Experimental Procedures." Values are expressed as
pmol/mg proteins. Statistical probability vs. controls is
indicated by the asterisks, and that of cells incubated with PDMP or
Fum vs. non-incubated cells after TNF-
/CHX treatment by
the crosses, both calculated as described under "Experimental
Procedures" (n = 3).
-induced activation of eNOS. In the absence of TNF-
, Fum and
PDMP did not modify NOS activity and cGMP formation in either
eNOS-expressing or -nonexpressing cells (data not shown). These
compounds had no effect also when given in combination to TNF-
to
HeLa cells not expressing eNOS (Fig. 5, dox+). However, similarly to the effects on ceramide generation, PDMP enhanced, and Fum
reduced, the stimulatory effect of TNF-
on NOS activity in the
induced cells (Fig. 5, dox
), suggesting that eNOS
activation by TNF-
is mediated through a pathway involving ceramide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and the effect of this interaction on TNF-
-induced cell death via apoptosis. To do this we have chosen HeLa cells because
they express TNF-RI and die via apoptosis when treated with TNF-
(40, 41). We generated clones stably transfected with the eNOS cDNA
under the control of a doxycycline-responsive promoter, in which
transcription of the mRNA and expression of the protein, suppressed
in the presence of the antibiotic, is initiated by its removal, at
levels inversely proportional to the concentration of antibiotic. The
induced eNOS, localized mainly to the plasma membrane and to the Golgi
complex, was positive for NADPH diaphorase staining and showed a
Ca2+-dependent activity in HeLa cell extracts,
at levels comparable to those of the endogenous enzyme in endothelial
cells. In addition, ATP, which increases
[Ca2+]i, stimulated eNOS activity
with generation of bioactive NO, the latter assessed by formation of
cGMP in a PC12 cell reporter system. These observations indicate that
eNOS, when induced in HeLa cells, retains the intracellular
distribution and functional characteristics of the native enzyme (33,
34, 42). Thus, eNOS Tet-off HeLa clones represent a clean, well
controlled system to study the interaction between eNOS and TNF-
,
and the effect of this interaction on apoptosis, because all
differences observed between cells cultured with or without doxycycline
can be attributed to the presence/absence of the enzyme.
-induced apoptosis, when the gas
was administered together with or prior to the cytokine, had already
been shown in several studies. This effect of NO, however, had been
demonstrated using either NO donors or continuous fluxes of endogenous
NO, generated as a consequence of constitutive expression of inducible
NOS after adenoviral gene transfer (13-19). These studies, therefore,
did not establish whether generation of NO could be a built-in,
physiological mechanism triggered by TNF-
to modulate its own
apoptotic effect. To elucidate this, we have studied cell death induced
by the cytokine, in the presence of the protein synthesis inhibitor
CHX, in cells either expressing or devoid of eNOS. In particular, we
measured the activity of mitochondrial dehydrogenases and two markers
of apoptosis, i.e. the cell surface appearance of
phosphatidylserine and the formation of hypodiploid DNA. Our results
show that induction of eNOS is sufficient per se to reduce
significantly, yet not to abolish, the degree of cell death induced by
TNF-
. This partial protection was eliminated by the NOS inhibitor
L-NAME, indicating that the action of eNOS was due to
generation of NO. This generation of NO was due to stimulation of eNOS
by TNF-
, and was already observed in the first minutes after its
administration. Taken together, these results demonstrate that, in
cells endowed with eNOS, the apoptotic program triggered by TNF-
is
modulated through the autocrine generation of NO induced by the cytokine.
-induced generation
of NO, we have taken into consideration intracellular events known to
stimulate eNOS. TNF-
did not induce any changes in
[Ca2+]i, thus excluding the cation
as the intracellular mediator. We concentrated on ceramide as a
possible messenger since it is generated by TNF-
(6) and its short
chain analogues can stimulate eNOS (37). To investigate the role of
ceramide, we used PDMP and Fum, inhibitors, respectively, of
glycosylceramide synthase and ceramide synthase, which increased and
decreased, respectively, the early wave of ceramide generation induced
by TNF-
in our Tet-off HeLa cells. PDMP increased, while Fum
inhibited the stimulation by TNF-
of eNOS activity and cGMP
generation, suggesting that ceramide is involved in the activation of
eNOS by TNF-
. Other mechanisms, however, may also play a role. In
particular, Akt kinase has been shown recently to phosphorylate, and
thus activate, eNOS (42, 43), and in some, although not in all cell
systems, TNF-
may stimulate this kinase (44, 45). It should be
mentioned, however, that ceramide inhibits Akt kinase (46, 47) so that it is unlikely that this enzyme plays a major role in the activation of
eNOS by TNF-
reported here.
plays an important role in the induction of apoptosis by the
cytokine. In particular, impairment of the activity of sphingomyelinases, which generate ceramide following TNF-
stimulation, reduces the ability of the cytokine to induce
apoptosis (48-52). In addition, short chain ceramide analogues
increase cell sensitivity to TNF-
(19, 53). Recently, we have
demonstrated that this is due at least in part to stimulation, by
ceramide, of the recruitment of TRADD to TNF-RI with subsequent
increased activation of caspase-8, a process that occurs shortly (a few
minutes) after receptor activation (19). Of importance, exogenous NO
was able to prevent these effects of ceramide by inhibiting the
TNF-
-induced accumulation of the lipid messenger. Now we show that,
in cells endowed with eNOS, TNF-
induces an early stimulation of the
enzyme activity in a ceramide-dependent way. Taken
together, these results suggest that ceramide and NO constitute a
two-messenger system, triggered by TNF-
to regulate bidirectionally
the initial steps of its own apoptotic signaling pathway. A model for
this mechanism of regulation is described in Fig.
6.
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Fig. 6.
Schematic model for modulation of the
TNF- -triggered apoptosis by NO and
ceramide. Activation by TNF-
of TNF-RI results in receptor
trimerization and recruitment of TRADD and then FADD, which leads to
activation of caspase-8. The ensuing ceramide generation (56, 57), acts
as an amplifying factor of the response to TNF-
, by increasing TRADD
recruitment (19). Moreover, it may contribute to apoptosis by
triggering other signaling events (58). Ceramide, however, stimulates
also eNOS activity and the generated NO down-regulates the accumulation
of the lipid messenger. In addition, NO may counterbalance the
apoptogenic effect of ceramide also because of its action on additional
events involved in TNF-
-induced apoptosis (see text for details).
The functional coupling between eNOS and TNF-RI might be facilitated by
the subcellular localization of the enzyme and the receptor, both known
to concentrate in the caveolae at the plasma membrane (34, 59,
60).
In the present study the mechanisms by which endogenously generated NO
protects cells from TNF--stimulated apoptosis have not been
investigated. Although the model of Fig. 6 focuses on the NO-ceramide
feedback loop, it is likely that more than one of the effects described
for exogenous NO, such as inhibition of caspase activity, Bcl-2
cleavage, and cytochrome c release (13-15, 17-19), in
addition to inhibition of ceramide formation (19), are relevant also to
the action of endogenous NO. Among the substrates of NO, particularly
relevant is caspase-3 because of its role as a central effector in many
apoptotic pathways (7). Recent evidence with CD95 indicates that the
inhibition of caspase-3 cleavage by S-nitrosylation, and the
removal of this inhibition by activation of the death receptor, act as
a switch to turn the apoptotic program on (54). Furthermore,
NO-dependent inhibition of caspase-3 cleavage, observed in
endothelial cells when eNOS activity was increased by shear stress, was
found to protect from apoptosis induced by various stimuli (20). Thus,
in situations in which other apoptogens are present together with
TNF-
, such as during inflammation, the functional coupling of the
cytokine with eNOS we have shown, might act as a broad regulator of apoptosis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jacopo Meldolesi for critical revision of the manuscript; Rosaria Arcone, Clara Sciorati, and Vittorio Colantuoni for help during the initial stages of this work; William Sessa and Elisabetta Dejana for gifts of eNOS cDNA and E2 cells, respectively; and Teresa Sprocati for assistance with the preparation of the illustrations.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Italian Association for Cancer Research (to E. C.), Consiglio Nazionale delle Ricerche, Target Project Biotechnology (to E. C.), Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Cofinanziamento 99 (to E. C.), Schering-Plough Italia (to E. C.), Armenise-Harvard Foundation (to E. C.), and Programma Operativo Plurifondo 1994/99, Regione Calabria (to D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: DIBIT-H San
Raffaele Inst., via Olgettina 58, 20129 Milan, Italy. Tel.:
39-02-2643-4807; Fax: 39-02-2643-4813.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M006535200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF-, tumor
necrosis factor-
;
TNF-RI, p55 receptor for tumor necrosis
factor-
;
[Ca2+]i, intracellular
concentration of calcium;
CHX, cycloheximide;
DMEM, Dulbecco's
modified Eagle's medium;
Fum, fumonisin B1;
MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5,-diphenyltetrazolium bromide;
NOS
and eNOS, nitric-oxide synthase and its endothelial isoform;
L-NAME, N
-nitro-L-arginine methyl ester;
L-NMMA, N
-monomethyl-L-arginine;
PDMP, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol;
dox, doxycyline;
FADD, Fas-associated death domain;
TRADD, TNF-receptor-associated death domain.
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