From the Research Group Immunobiology and
§ Department of Dermatology, MED-Heinrich-Heine-University
of Düsseldorf, Postfach 10 10 07, D-40001 Düsseldorf,
Germany
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ABSTRACT |
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A variety of toxic and modulating events induced
by UVA exposure are described to cause cell death via apoptosis.
Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and
endothelial cells (ECs). We have now searched for the role of iNOS
expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as
detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation
with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the
onset of apoptosis. This protection was completely abolished in the
presence of the iNOS inhibitor
L-N5-(1-iminoethyl)-ornithine
(0.25 mM). Additionally, preincubation of cells with the NO
donor
(Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate at concentrations from 10 to 1000 µM as an exogenous
NO-generating source before UVA irradiation led to a
dose-dependent inhibition of both DNA strand breaks and
apoptosis. In search of the molecular mechanism responsible for the
protective effect, we find that protection from UVA-induced apoptosis
is tightly correlated with NO-mediated increases in Bcl-2 expression
and a concomitant inhibition of UVA-induced overexpression of Bax
protein. In conclusion, we present evidence for a protective role of
iNOS-derived NO in skin biology, because NO either endogenously
produced or exogenously applied fully protects against UVA-induced cell
damage and death. We also show that the NO-mediated expression
modulation of proteins of the Bcl-2 family, an event upstream of
caspase activation, appears to be the molecular mechanism underlying
this protection.
Exposure of eukaryotic cells to UVA radiation results in cellular
inactivation and death (1, 2). UVA-induced oxidative damage has been
reported to occur in lipids (3), coenzymes (4), and DNA (5). On the
level of organelles, nucleated mammalian cells exposed to UVA radiation
were reported to bear damaged cellular structures, notably the
microtubuli (6), the plasma membrane, the nuclear membranes, and the
rough endoplasmic reticulum (7, 8). Besides causing membrane damage,
UVA radiation is also known to result in DNA damage, mostly in the form
of single-strand breaks and protein-DNA cross-links. The amount of UVA
radiation-mediated damage to membranes and DNA was found to correlate
with the onset of cell death via apoptosis (5, 9).
Apoptosis is a distinct form of cell death controlled by an internally
encoded suicide program. The morphological changes associated with
apoptosis include the condensation of nucleoplasm, blebbing of
cytoplasmic membranes, and fragmentation of the cell into apoptotic
bodies that are rapidly phagocytosed by neighboring cells. The
biochemical markers of apoptosis include activation of a caspase
cascade, DNA fragmentation into nucleosomal fragments, and cleavage of
various caspase substrates (10, 11), including gelsolin, leading to
rounding and detachment of cells (12), and nuclear lamin, allowing for
nuclear fragmentation (13, 14).
A plethora of anti-oxidant and antiapoptotic defense mechanisms exist
in mammalian cells. These can be broadly classified into the expression
of enzymes, including superoxide dismutase, catalase, and several
peroxidases, compounds to quench reactive oxygen intermediates like
ascorbate, ureic acid, carotenoids, and glutathione (15), or
apoptosis-protective proteins such as Bcl-2 as critical factors
controlling the apoptosis program (14). Recently, it has also been
shown that the radical nitric oxide
(NO),1 which is known to
exert cytotoxic effects and mediate onset of apoptosis in a variety of
mammalian cells (16), may also protect against the injurious actions of
superoxide, hydrogen peroxide, and alkyl peroxides (17) as putative
mediators of UV radiation-induced cytotoxicity.
NO and equal amounts of citrulline are synthesized from the guanidino
nitrogen of L-arginine by nitric-oxide synthases (NOSs) found in endothelial cells and neurons and upon activation in macrophages and many other cell types. This enzyme family consists of
three isoenzymes: the endothelial ecNOS and the neuronal ncNOS are
constitutively expressed and calcium/calmodulin-regulated and produce
regulated and low amounts of NO for short pulses, whereas the
cytokine-inducible and calcium-independent isoenzyme (iNOS) synthesizes
large amounts of NO for long periods of time (18, 19).
During inflammatory processes cytokines are known modulators of
endothelial cell functions (20). One prominent effect that cytokines
can exert in endothelial cells is the induction of iNOS, followed by
high output NO synthesis (21, 22). The iNOS-generated NO is thought to
serve mainly as nonspecific immune protection (23), and prolonged iNOS
activity was described as dangerous for the host, as evidenced by its
role in the pathogenesis of septic shock and cytokine-induced
hypotension (24), the suppression of various cellular functions
(25-27), inflammatory tissue destruction (28), and the induction of
apoptosis (18).
Recently, UV irradiation has been shown to modulate local NO production
in human skin (29). Additionally, our observation of a strong epidermal
as well as endothelial iNOS expression in UV-irradiated normal skin
(30) led us to search for a possible role of cutaneous UV-induced NO
formation. Here, we tested the role of iNOS-generated as well as
exogenously added NO in the UVA-induced apoptosis of resting or
cytokine-activated ECs, respectively. The experiments presented here
demonstrate for the first time a NO-mediated modulation of the Bcl
family of proteins as a strong protective mechanism rescuing ECs from
UVA-induced cell death.
Reagents and Materials--
Recombinant human interleukin 1 Endothelial Cells--
ECs were isolated by outgrowth from male
Wistar rat aortic rings exactly as described previously (31). Aortic
segments were placed on top of a collagen gel (1.8 mg collagen/ml) in
24-well tissue culture plates and incubated in RPMI 1640 medium with
20% FCS and 100 µg endothelial cell growth supplement/ml in a
humidified incubator at 37 °C in a 95% air/5% CO2
atmosphere for 5 days. Aortic explants were then removed, and cells
were detached with 0.25% collagenase in Hanks' balanced salt solution
and replated onto plastic culture dishes in RPMI 1640 medium with 20%
FCS. Cells were subcultured for up to eight passages, and removal from culture dishes for each passage was performed by treatment with trypsin/EDTA.
Cellular Characterization of Cultured Endothelial
Cells--
Cells were passaged from tissue culture dishes onto sterile
glass coverslips and allowed to grow as subconfluent monolayers. Cells
were washed with PBS and fixed with acetone at Experimental Design--
All measurements were performed with
cells from passages two to eight. Endothelial cells (2 × 105) were cultured in 12-well tissue culture plates or on
8-well chamber Tec glass slides in a humidified incubator at 37 °C
in a 95% air/5% CO2 atmosphere in RPMI 1640 medium with
20% FCS. For iNOS induction and endogenous NO production, endothelial
cells were activated 24 h before UVA irradiation (2, 4, 6, 8, and
10 J/cm2) by the addition of IL-1 Nitrite Determination--
After 24 h of incubation,
nitrite was determined in these control culture supernatants using the
diazotization reaction as modified by Wood et al. (35) and
using NaNO2 as a standard.
Detection and Quantification of Nuclear DNA
Fragmentation--
DNA strand breaks of cells grown on 8-well chamber
Tec slides were visualized by the in situ nick translation
method (36) 1-24 h after UVA irradiation (2-10 J/cm2). In
acetone-fixed cells, endogenous peroxidase activity was blocked with
methanol plus 0.3% H202 for 30 min. The nick
translation mixture contained 3 µM biotin-dUTP, 5 units/100 µl Kornberg polymerase, 3 µM each of dGTP,
dATP, and dCTP, 50 mM Tris-HCL, pH 7.5, 5 mM MgC12, and 0.1 mM dithiothreitol, and the
reaction was performed at room temperature for 20 min. Slides were
washed in PBS and processed for immunocytochemical detection of
biotin-labeled UTP by peroxidase-labeled avidin, followed by an enzyme
reaction using 3,3'-diaminobenzidine as the substrate. In each sample,
a minimum of 500 cells were counted, and labeled nuclei were expressed
as a percentage of the total number of nuclei.
Detection of Nuclear Chromatin Condensation and Nuclear
Fragmentation--
1-24 h after UVA irradiation (2-10
J/cm2), endothelial cells grown in 12-well culture plates
were washed with PBS and stained with Hoechst dye H33342 (8 µg/ml)
for 5 min, and nuclei were visualized using a Zeiss fluorescence
microscope. In each sample, a minimum of 400 cells were counted, and
condensed or fragmented nuclei were expressed as a percentage of the
total number of nuclei.
Polymerase Chain Reaction (PCR)--
Total cellular RNA (1 µg
each), prepared 16 h after irradiation, from adherent growing
nonirradiated and UVA-irradiated (6 J/cm2) ECs that were
resting or cytokine-activated (addition of 200 units/ml IL-1 Western Blot Analysis of Bcl-2 and Bax Protein
Expression--
After a 16-h incubation after UVA irradiation (6 J/cm2), resting, cytokine-activated (200 units/ml IL-1 Statistical Analysis--
Data are given as arithmetical
means ± S.D. Values were calculated using Student's t
test (two-tailed for independent samples).
UVA irradiation of endothelial cells in a
dose-dependent manner leads to cell death via apoptosis.
Resting ECs were exposed to UVA radiation with increasing intensities
as indicated. The relative number of viable or dead cells was
determined using neutral red staining or trypan blue exclusion,
respectively. Nuclear fragmentation or DNA strand breaks were
visualized using Hoechst DNA dye H33342 or the in situ nick
translation method, respectively.
Irradiation of ECs with UVA in a dose-dependent manner led
to endothelial cell death (Fig. 1). We
find that 24 h after maximal UVA irradiation with 10 J/cm2, 37 ± 7% of the initial cell number are still
adherent; of these, about half were trypan blue-positive (Fig.
1A). Thus, maximal UVA irradiation leads to the cell death
of 80% of cells within 24 h. About 60% of cells detach from the
dish as a first indication for ongoing apoptosis, in which cellular
detachment and cytoskeletal disruption occur. Half-maximal cytotoxicity
was found at a UVA dose of 6 ± 1 J/cm2 (Fig.
1A). Indeed, at and above 6 J/cm2, staining with
Hoechst DNA dye or in situ nick translation revealed nuclear
alterations such as chromatin condensation and fragmentation, as well
as DNA strand breaks in 35-80% of adherent cells (Figs. 1B
and 2, C and
D).
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
(IL-1
), recombinant human tumor necrosis factor
(TNF-
), and
recombinant murine IFN-
were obtained from HBT (Leiden,
Netherlands). Endothelial cell growth supplement, Neutral Red (3%
solution), type I collagen, collagenase (from Clostridium
histolyticum), rabbit anti-human von Willebrand factor antiserum,
and Hoechst dye H33342 were from Sigma. Monoclonal antibody Ox43 was
from Serotec (Camon, Wiesbaden, Germany). The rabbit anti-rat Bcl-2
antibody and Bax antisera were from PharMingen (San Diego, CA).
Peroxidase-conjugated porcine anti-rabbit IgG was from DAKO (Hamburg,
Germany), and peroxidase-conjugated goat anti-mouse IgG was from
Zymed Laboratories Inc. Trypsin, EDTA, fetal calf
serum (FCS, endotoxin-free), RPMI 1640 medium (endotoxin-free),
biotin-dUTP, Kornberg polymerase, dGTP, dATP, dCTP, dithiothreitol,
oligo(dT)16 primer, reverse transcriptase, and
Taq polymerase were purchased from Boehringer Mannheim or Life Technologies, Inc. 3,3'-Diaminobenzidine was from Serva GmbH (Heidelberg, Germany). The interleukin-converting enzyme inhibitor Z-VAD was obtained from Enzyme Systems (Livermore, CA). The NO donor
(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) was kindly provided by Prof. H. Weber
(Heinrich-Heine-University of Düsseldorf, Düsseldorf,
Germany). Denitrosilated DETA/NO (DETA) was obtained by incubating
stock solutions (50 mM) of DETA/NO for 10 days at 37 °C.
The iNOS inhibitor
L-N5-(1-iminoethyl)-ornithine (NIO) was a kind
gift from Boehringer Mannheim. We used a Sellas-2000 lamp (Sellas
Medizinische Geräte GmbH, Geveisberg, Germany) emitting
the UVA1 spectrum (340-390 nm) as a UVA source.
20 °C for 10 min,
followed by inhibition of endothelial peroxidase activity with 0.3%
H2O2 in ethanol and three washing steps in
Tris-buffered saline. After blocking unspecific binding with 0.5%
bovine serum albumin in Tris-buffered saline for 30 min and rinsing,
specimens were incubated with a 1:50 dilution of rabbit anti-human-von
Willebrand factor antiserum, which was previously shown to cross-react
with the rat antigen (32), in a moist chamber for 45 min. After an additional wash, slides were incubated in a 1:50 dilution of
peroxidase-conjugated porcine anti-rabbit IgG for 45 min at room
temperature. After washing, peroxidase activity was visualized with
0.05% diaminobenzidine plus 0.015% H2O2 for
10 min at room temperature. Control cultures were incubated with a
nonrelevant rabbit hyperimmune serum instead of the first antiserum.
Positive controls (human platelets) and negative controls (rat alveolar
macrophages and the fibroblastoma cell line L929) were also tested with
this anti-von Willebrand factor antiserum. The rat vascular
endothelium-specific monoclonal antibody Ox43 (33) was used in a 1:50
dilution. A peroxidase-conjugated goat anti-mouse IgG was diluted 1:50
before use. Otherwise, conditions were as described above.
(200 units/ml),
IL-1
plus TNF-
(500 units/ml), or IL-1
plus TNF-
plus
IFN-
(100 units/ml). Inhibition of the cytokine-induced iNOS
activity was reached by adding the iNOS inhibitor NIO (0.25 or 0.5 mM) to the activated endothelial cell cultures. NIO was
present in the culture medium during cytokine activation as well as
during and 24 h after UVA irradiation. Additionally, 24 h
before UVA irradiation, resident endothelial cells were incubated with
the NO donor DETA/NO or DETA alone at the concentrations indicated. 10 min before UVA exposure, cells were washed extensively with PBS and
covered with 1 ml of Hanks' balanced salt solution/HEPES or RPMI 1640 medium without phenol red; after irradiation, cells were washed in PBS,
and RPMI 1640/FCS medium was added. 24 h after irradiation, the
relative number of living ECs was detected by neutral red staining
(34). Cells were incubated for 90 min with neutral red (1:100 dilution
of the 3% solution) and then washed twice with PBS. Cells were then
dried and lysed by isopropanol containing 0.5% of 1 N HCl.
Extinctions of the supernatants were then measured at 530 nm.
Additionally, the viability of endothelial cells was routinely
controlled at the beginning and end of every experiment using the
trypan blue exclusion assay.
+ 500 units/ml TNF-
+ 100 units/ml IFN-
24 h before UVA
irradiation), or preincubated with DETA/NO or DETA (1 mM
each for 24 h before UVA irradiation) (37) was used for cDNA
synthesis (38). Reverse transcription was carried out at 42 °C for
60 min, using the oligo(dT)16 primer. The cDNA
was then used as a template for PCR, primed by either the
oligonucleotides TATGATAACCGGGAGATCGTG (sense; bases 26-45 of rat
Bcl-2 cDNA) and CAGATGCCGGTTCAGGTACTC (antisense; bases 526-546 of
rat Bcl-2 cDNA) for specific Bcl-2 amplification (39) or the
oligonucleotides CAAGAAGCTGAGCGAGTGTCT (sense; bases 62-82 of rat
Bax cDNA) and GGTTCTGATCAGCTCGGGCAC (antisense; bases 279-299 of
rat Bax cDNA) for specific Bax amplification (39). For specific rat
glyceraldehyde-3-phosphate dehydrogenase cDNA amplification,
the oligonucleotides CAACTACATGGTTTACATGTTCC (sense; rat
glyceraldehyde-3-phosphate dehydrogenase cDNA bases 153-175) and
GGACTGTGGTCATGAGTCCT (antisense; rat glyceraldehyde-3-phosphate dehydro- genase cDNA bases 549-568) were used (40). Additionally, to exclude unspecific amplification by mRNA or DNA contamination, control PCR was performed with all additives but without cDNA or
with the RNA probes, respectively. PCR was carried out following standard protocols (41) with the following cycle profile: 33 cycles
with 30 s at 94 °C, 30 s at 58 °C, and 30 s at
72 °C for Bcl-2 or Bax mRNA amplification and 25 cycles with
30 s at 94 °C, 30 s at 62 °C, and 30 s at 72 °C
for glyceraldehyde-3-phosphate dehydrogenase mRNA amplification. A
final incubation step was performed at 72 °C for 10 min. An aliquot
of each reaction was subjected to electrophoresis on 1.5% agarose
gels. Bands were visualized by ethidium bromide staining.
+ 500 units/ml TNF-
+ 100 units/ml IFN-
), EC cultures preincubated
with DETA/NO (1 mM for 24 h before UVA irradiation) or
DETA (1 mM for 24 h before UVA irradiation) were
washed, and adherent growing cells were lysed, scraped from the dishes,
transferred to a microcentrifuge tube, and boiled for 5 min exactly as
described previously (42). Proteins (50 µg/lane) were separated by
electrophoresis in a 12% SDS-polyacrylamide gel and transferred to
nitrocellulose membranes. To control equal loading of total protein in
all lanes, blots were stained with Ponceau S solution immediately after
transfer. Blots were incubated for 2 h with blocking buffer (2%
bovine serum albumin, 5% non-fat milk powder, and 0.1% Tween 20 in
PBS buffer), incubated for 1 h with a 1:1000 dilution of the
anti-Bcl-2 or anti-Bax antiserum, washed, incubated for 1 h with a
1:1000 dilution of the secondary horseradish peroxidase-conjugated
porcine anti-rabbit IgG antibody, incubated for 5 min in ECL reagent
(Pierce, Rockford, IL), placed into a plastic bag, and exposed to an
enhanced autoradiographic film.
RESULTS
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Fig. 1.
UVA irradiation of endothelial cells leads to
cell death. Resting endothelial cells were exposed to UVA
radiation with increasing intensities, as indicated. The relative
number of viable ( ) or dead adherent cells (
) was determined by
neutral red staining or trypan blue exclusion, respectively. The
relative number of all adherent cells (viable plus dead) is also
indicated (
). Furthermore, apoptotic nuclei and nuclear DNA
strand breaks were visualized using Hoechst DNA dye H33342 (
) or the
in situ nick translation method (
) and calculated as a
percentage of all adherent cells, respectively. A,
irradiation of ECs with UVA in a dose-dependent manner led
to endothelial cell death as detected 24 h after irradiation.
B, increasing UVA intensities led to an increasing number of
cells with pygnotic nuclei, nuclear chromatin condensation, and nuclear
fragmentation as well as DNA strand breaks as detected 24 h after
irradiation. C-F, time course of UVA-induced cell death and
apoptotic events; 4 or 8 h after irradiation with 6 (D)
or 10 J/cm2 (F), a low but significant increase
in the number of fragmented nuclei and nuclei with DNA strand breaks
was found. At
16 h after UVA irradiation, the number of apoptotic
nuclei is significantly increased. Values are the mean ± S.D. of
3-12 individual experiments.
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Fig. 2.
UVA irradiation of endothelial cells leads to
apoptosis and DNA strand breaks. Resident or Z-VAD-treated (30 µM) ECs were exposed to UVA radiation (6 J/cm2), and after 16 h, apoptotic nuclei or nuclear
DNA strand breaks were visualized using Hoechst DNA dye H33342
(A, C, and E) or the in situ nick
translation method (B, D, and F), respectively.
UVA irradiation leads to nuclear chromatin condensation and nuclear
fragmentation (C) as well as DNA strand breaks in most of
the nuclei (D) as indicators of ongoing apoptosis, whereas
Z-VAD (30 µM) inhibits the development of these apoptosis
symptoms (E and F). A and
B, nonirradiated control cultures. Magnification: ×650 in
A and E, ×700 in C, ×300 in B,
D, and F.
Next we examined the time course of UVA-induced cell death, nuclear
damage, and DNA damage; between 8 and 16 h after irradiation with
6 (Fig. 1C) or 10 J/cm2 (Fig. 1F), we
found a highly significant increase in the number of nuclei with DNA
strand breaks, indicating that DNA damage is not an immediate
consequence of UVA irradiation. At 16 h after UVA irradiation, the
number of altered nuclei increased to 35-65% of adherent cells at 6 J/cm2 or 40-90% of adherent cells at 10 J/cm2
.
Three lines of evidence argue in favor of apoptosis as the main course of cell death. As shown in Fig. 2, Hoechst staining of UVA-irradiated cells led to the formation of shrunken pygnotic nuclei and nuclear fragmentation (Fig. 2C). Furthermore, DNA strand breaks occur (Fig. 2D) in focal areas of unbroken nuclei 16 h after half-maximal UVA irradiation (6 J/cm2). Finally, incubation of ECs with the caspase inhibitor Z-VAD (30 µM) for 6 h before and for 24 h after UVA irradiation completely inhibits all morphological alterations of nuclei (Fig. 2, E and F).
Cytokine-induced Activation and Concomitant Endothelial iNOS
Expression Fully Protects against Cell Damage--
ECs were activated
by proinflammatory cytokines (200 units/ml IL-1, 500 units/ml
TNF-
, and 100 units/ml IFN-
). Activation led to the production of
NO as evidenced by increased nitrite concentrations in culture
supernatants. Incubation of endothelial cell cultures with different
combinations of cytokines (200 units/ml IL-1
, IL-1
plus 500 units/ml TNF-
, and IL-1
plus TNF-
plus 100 units/ml IFN-
)
shows a specific pattern of iNOS activity (Fig.
3A), exactly as described
previously (32, 43). The cultures of resident or activated cells were
exposed to UVA radiation, and the relative number of living cells was
determined after an additional 24 h.
|
A highly significant protection against UVA-induced cell death in close
correlation to the amount of NO produced was observed (Fig. 3,
B-D). EC cultures incubated with the single cytokine IL-1 show a half-maximal iNOS activity, leading to 3.5 ± 0.6 nmol of nitrite in culture supernatants. This leads to significantly protective effects against UVA-induced cell death (40 ± 5%
surviving cells at 8 J/cm2; 33 ± 5% surviving cells
at 10 J/cm2). Maximal endothelial iNOS activity was
achieved after incubation with the cytokine combinations IL-1
plus
TNF-
(10.7 ± 1.0 nmol of nitrite) or Il-1
plus TNF-
plus
IFN-
(11.8 ± 1.7 nmol of nitrite) and resulted in a highly
significant protection (90 ± 6% of surviving cells at 6 J/cm2; 82 ± 7% of surviving cells at 10 J/cm2). When activation of ECs was performed in the
presence of the NOS inhibitor NIO (0.25 mM), complete
abrogation of the protective effects further indicated that iNOS
activity does indeed mediate the protective mechanism (Fig.
3C). Cell survival after UVA irradiation shows a linear
correlation with iNOS activity as measured by nitrite concentrations in
culture supernatants (Fig. 3D).
Additionally, parallel to these experiments, apoptotic nuclei and DNA
strand breaks were visualized 16 h after half-maximal UVA
irradiation (6 J/cm2). In cytokine-activated cultures
(IL-1 plus TNF-
plus IFN-
), cells are fully protected from
UVA-induced apoptosis (Fig. 4, A and B) as seen in comparison to resident and
UVA-irradiated cells (Fig. 2, C and D) where
apoptosis is frequent. Inhibition of endothelial NO production by the
addition of NIO (0.25 mM) abrogates this protection (Fig.
4, C and D).
|
Exogenous NO Is Sufficient to Protect against UVA Irradiation-induced Cell Damage-- To test whether NO alone is sufficient to mediate the above described protection, resident ECs were incubated 24 h before UVA irradiation with various concentrations of the chemical NO donor DETA/NO.
Neutral red staining of living cells performed 24 h after UVA irradiation revealed that at and above concentrations of 50 µM DETA/NO, protection was always significant (Fig. 5A). With 1 mM DETA/NO, full protection was achieved (86 ± 9% of surviving cells at 6 J/cm2 UVA; 78 ± 7% of surviving cells at 10 J/cm2 UVA). In contrast, DETA without NO had no protective effect at all (Fig. 5B), proving the NO specificity. A linear correlation between protection and the concentration of the exogenous NO donor was found (Fig. 5C). Neither DETA/NO nor any of the other compounds used showed any cytotoxic effects toward the ECs within the incubation times.
|
Again, visualization of apoptotic nuclei and DNA strand breaks (Fig. 6) exactly as described above demonstrated that EC cultures preincubated with the NO donor before irradiation were exactly like the untreated and nonirradiated controls (Fig. 2), whereas DETA alone had no protective action against UVA-induced nucleus aberrations.
|
NO or UVA Modulates the Expression of Endothelial Bcl-2 or Bax mRNA as well as Bcl-2 or Bax Protein-- In search of the molecular mechanism(s) responsible for the protective effects, we examined the mRNA expression and the relative amounts of the two proteins Bax and Bcl-2 after the various treatments.
As described above, 16 h after UVA irradiation (6 J/cm2), 72 ± 7% of ECs were still adhering to the plate, and only these could be used for PCR or Western blot analysis. Of the adherent population, 79% were live cells positively stained by neutral red and excluding trypan blue.
Both endogenously produced NO after cytokine challenge (Fig. 7A) and exogenously applied NO (Fig. 7C) result in a 2.5-3-fold increase in Bcl-2 mRNA expression that persists for 24 h, whereas activation of cells in the presence of the NOS inhibitor NIO (0.25 mM) or incubation with DETA alone had no effects on Bcl-2 mRNA expression (Fig. 7, B and D), indicating the NO specificity of these effects. Moreover, the time course also reveals that UVA will not counteract the up-regulated Bcl-2 gene expression.
|
Furthermore, 1 h after UVA irradiation (6 J/cm2) of resting cells, we found an increased Bax mRNA expression that was augmented after 24 h by a factor of 2.5-3.5 compared with the controls (Fig. 8, A and B), whereas in cytokine-preactivated ECs (Fig. 8A) or cells pretreated for 24 h with DETA/NO (Fig. 8B), UVA had no influence on endothelial Bax mRNA expression.
|
Protein expression as studied by Western blots showed analogous effects of NO or UVA (Fig. 9). Untreated resident ECs express both proteins (lanes 1 and 4). Cytokine activation and concomitant high output NO production (as seen in Fig. 3A) resulted in a significant increase in Bcl-2 levels (Fig. 9B, lanes 2 and 5), and this Bcl-2 overexpression was absent when cells were activated in the presence of NIO (0.25 mM). UVA irradiation of resting cells significantly increased endothelial Bax levels (Fig. 9A). This UVA-induced Bax overexpression was blocked by the preactivation of cells; instead, a simultaneous overexpression of Bcl-2 was found. Again, both effects were absent when activation was performed in the presence of the NOS inhibitor. Controls with resident cells and NIO addition showed no differences from untreated controls (data not shown). Parallel experiments with or without exogenous NO (24 h preincubation with 1 mM DETA/NO) and with or without UVA irradiation resulted in identical patterns of NO-mediated Bcl-2 increase and abrogation of UVA-induced Bax overexpression (Fig. 9B).
|
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DISCUSSION |
---|
A consequence of excessive UV exposure of human skin is the occurrence of programmed cell death or sunburn cells, which appears to contribute to aging and tumor development (1). We recently observed that UV challenge of normal human skin leads to the induction of iNOS expression in keratinocytes and capillary endothelial cells.
Higher amounts of NO as produced by iNOS are known inducers of cell death that often follows the route of apoptosis (44), and it was generally accepted that apoptosis induction represents one mode of action for the cytotoxic effects of NO (16). Thus, it was tempting to speculate that iNOS expression after UV challenge may contribute to cell death in skin.
However, very recent reports indicate that NO can also exert antiapoptotic activity (45, 46) by inhibiting caspase 3 via S-nitrosation (47, 48) or by modulating the expression of the antiapoptotic protein Bcl-2 (49), respectively. Indeed, with the results of our experiments, we provide evidence that NO, either endogenously produced or exogenously generated, is a powerful tool to prevent UVA-induced apoptosis.
In search of the intracellular events that help to explain this protective effect, we find that events upstream of caspase 3 activation appear to be responsible, namely, the modulation of proteins from the Bcl family at their relative expression levels by both UVA and NO, albeit in opposing patterns. Proteins of the Bcl family regulate apoptosis either positively (as, for instance, Bax) or negatively (as, for instance, Bcl-2 (11, 50)). Both proteins are found as intracellular membrane-associated proteins of the outer mitochondrial membrane, endoplasmatic reticulum, and nuclear envelope (51). Bax was shown to form channels in lipid membranes, and the proapoptotic effect of Bax appears to be elicited through an intrinsic pore-forming activity (52), thus leading to leakage of cytochrome c from the mitochondrial intermembrane compartment into the cytosol. Cytosolic cytochrome c initiates the apoptotic program by activating specific caspases, leading to the cleavage of fodrin and lamin (53) and apoptotic nuclear morphology (54). In contrast, Bcl-2 overexpression prevents the release of cytochrome c and ensuing events and thus mediates antiapoptotic effects (42, 55), and an inhibitory role of Bcl-2 on Bax channel-forming activity appears likely (52). Thus, onset of apoptosis is controlled by the ratio of death agonists (like Bax) to antagonist (like Bcl-2). This death-life rheostat is mediated, at least in part, by competitive dimerization: with excess Bax, Bax homodimer formation will dominate, and apoptosis occurs; with excess Bcl-2, Bax/Bcl-2 heterodimers will dominate, and cells are protected (56, 57).
As mentioned above, the antiapoptotic activity of NO has been mainly attributed to NO-mediated S-nitrosation and the partial inhibition of caspase 3 or activation of Bcl-2. With the results presented here, we now show that NO can also act upstream of caspase 3, mainly by regulating the expression levels of Bcl-2. The proapoptotic effects of UVA lead to significant increases in relative Bax expression. In contrast, cytokine-activated cells exhibit a significant increase in Bcl-2 levels and also an apparent suppression of UVA-induced Bax overexpression. Endogenous NO synthesis and exogenously applied NO thus very effectively counteract the proapoptotic UVA activity. Because NO-mediated protection tightly correlates with Bcl-2 overexpression at the time of UVA challenge, data presented are in full agreement with several publications demonstrating that Bcl-2 protects against diverse cytotoxic insults including UV irradiation (for a review, see Ref. 58).
The concentration of exogenously added NO necessary to achieve full
protection lies well within physiological levels as deduced from
published data. It has been shown (59, 60) that DETA/NO spontaneously
releases NO with a predictable first-order kinetic and a half-life of
7.7 h, thus providing a constant NO supply over a period of hours.
According to the first-order kinetic laws (t1/2 = ln2/k and c = c0 × ekt), 1 mM DETA/NO generates 2.7 µM NO/min. Recently, a steady-state concentration of
about 4-5 µM NO has been calculated to be present in the
immediate vicinity of a cell monolayer that enzymatically generates NO
(61). In consideration of these calculations and measured data, the
amount of NO needed for full protection is readily available under
physiological conditions.
In summary, we show that NO protects against UVA-induced cell death and
that endogenously produced or exogenously generated NO is sufficient to
achieve full protection. Thus, iNOS expression in human skin after UV
exposure appears to represent a beneficial response helping to minimize
sunburn-associated skin damage. This concept is further backed by our
recent finding in cutaneous Lupus erythematosus patients (30). These
patients are known to exhibit photosensitivity leading to skin lesion
formation after UV exposure. We could demonstrate that in these
patients, a significantly delayed UV-induced iNOS expression correlates
with lesion formation as further evidence for the beneficial activity
of iNOS expression in UV exposure.
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ACKNOWLEDGEMENTS |
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We thank Prof. H. Weber for kindly providing DETA/NO, Christa-Maria Wilkens, Marija Lenzen, and Ulla Lammersen for technical assistance, and Martha Turken for photographic assistance.
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FOOTNOTES |
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* This study was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 503, A3, to V. K.-B.).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.
¶ To whom correspondence should be addressed: Institute of Immunobiology Geb. 14.80, MED-Heinrich-Heine-University, P. O. Box 10 10 07, D-40001 Düsseldorf, Germany. Tel.: 49-211-81-19184; Fax: 49-211-81-19532; E-mail: Bachofen{at}uni-duesseldorf.de.
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ABBREVIATIONS |
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The abbreviations used are:
EC, endothelial
cell;
NO, nitric oxide;
NOS, nitric-oxide synthase;
iNOS, inducible
NOS;
IL-1, interleukin 1
;
TNF-
, tumor necrosis factor
;
DETA/NO, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate;
DETA, denitrosilated DETA/NO;
NIO, L-N5-(1-iminoethyl)-ornithine;
PBS, phosphate-buffered saline;
FCS, fetal calf serum;
PCR, polymerase
chain reaction.
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REFERENCES |
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