Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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Nitric oxide (NO) can either prevent or promote apoptosis, depending on cell type. In the present study, we tested the hypothesis that NO suppresses ultraviolet B radiation (UVB)-induced keratinocyte apoptosis both in vitro and in vivo. Irradiation with UVB or addition of the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) increased apoptosis in the human keratinocyte cell line CCD 1106 KERTr, and apoptosis was greater when the two agents were given in combination. Addition of the chemical NO donor S-nitroso-N-acetyl-penicillamine (SNAP) immediately after UVB completely abrogated the rise in apoptosis induced by L-NAME. An adenoviral vector expressing human inducible NOS (AdiNOS) also reduced keratinocyte death after UVB. Caspase-3 activity, an indicator of apoptosis, doubled in keratinocytes incubated with L-NAME compared with the inactive isomer, D-NAME, and was reduced by SNAP. Apoptosis was also increased on addition of 1,H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. Mice null for endothelial NOS (eNOS) exhibited significantly higher apoptosis than wild-type mice both in the dermis and epidermis, whereas mice null for inducible NOS (iNOS) exhibited more apoptosis than wild-type mice only in the dermis. These results demonstrate an antiapoptotic role for NO in keratinocytes, mediated by cGMP, and indicate an antiapoptotic role for both eNOS and iNOS in skin damage induced by UVB.
ultraviolet radiation; skin; dermis; epidermis
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INTRODUCTION |
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KERATINOCYTES ARE EXPOSED to many environmental insults, including ultraviolet radiation, against which they form the body's first line of defense. Ultraviolet B radiation (UVB) induces apoptosis in all cell types. However, keratinocytes are particularly resistant to UVB, an essential feature in a cell type continually exposed to solar irradiation. Under appropriate conditions, keratinocytes can be stimulated to undergo apoptosis. Death receptor ligand-dependent and -independent pathways act via the FADD/caspase 8 cascade(23, 30, 36). DNA damage upregulates p53 expression, accompanied by G1 arrest and either DNA repair or apoptosis (8). Finally, UVB also activates p38 mitogen-activated protein kinase with resultant mitochondrial cytochrome c release (2).
Nitric oxide (NO) is produced at low levels from the amino acid L-arginine in a calcium-dependent fashion by the constitutive neuronal and endothelial nitric oxide synthases (nNOS and eNOS, respectively) and at higher levels in a calcium-independent manner by the inducible NOS (iNOS). All three NOS isozymes are present in human skin (48). NO release has been described within 1 min of UVB irradiation in a calcium-dependent manner, leading to the production of cGMP produced by the soluble guanylate cyclase (sGC) (9, 32), both features of constitutive NOS. Additionally, iNOS mRNA and protein expression peak 24 h after two minimal erythema doses of UVB in healthy human skin (21).
NO exerts both pro- and antiapoptotic effects (17). In macrophages (1), thymocytes (12), human neutrophils (45), and mesangial cells (11) NO induces apoptosis, often in combination with reactive oxygen intermediates or at high concentrations. In contrast, NO inhibits apoptosis in endothelial cells (42) and hepatocytes (44). This process occurs both directly by nitrosative inactivation of caspases (24) and indirectly via the cGMP pathway (18) and interference with activator protein-1 (AP-1)-mediated upregulation of the CD95L promoter (27). The presence of elevated concentrations of NO in psoriatic (3, 28) and irradiated skin, and the changes in keratinocyte proliferation and apoptosis in these states, suggest that NO can play a pathophysiological role in certain skin disorders.
Ultraviolet radiation both induces apoptosis and causes NO release. We wanted to determine whether the pro- or antiapoptotic effects of NO predominate after exposure of skin to UVB and to examine the mechanisms for this action. Our findings suggest that NO derived from both eNOS and iNOS is antiapoptotic for keratinocytes challenged with UVB and, through isoform-specific patterns of NO release, serves as both an immediate and a sustained mechanism to protect keratinocytes from the toxicity of exposure to UVB.
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MATERIALS AND METHODS |
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Tissue culture. All chemicals were obtained from Sigma (St. Louis, MO), unless otherwise stated. The E6/E7-transformed keratinocyte line CCD 1106 KERTr (American Type Culture Collection, Manassas, VA) was cultured in 175-cm2 culture flasks in an equal-parts mixture of medium 154 (Cascade Biologics, Seattle, WA) and keratinocyte-SFM, to each 500 ml of which 2.5 µg of human recombinant EGF and 25 mg of bovine pituitary extract had been added (GIBCO BRL, Grand Island, NY). Penicillin and streptomycin were added to the culture medium (GIBCO BRL), and the cells were maintained in a humidified incubator at 37°C and 5% CO2.
Ultraviolet irradiation. For cell culture experiments, two Philips FS20 lamps (Bulbtronics, Farmingdale, NY) were screened with Kodacel filter K6808 (International Polarizer, Marlborough, MA) to remove ultraviolet C wavelengths (14). Lamp output was measured with a UVP model UVX digital radiometer and UVX-31 probe (UVP, San Gabriel, CA) after the lamps were allowed to warm up for 15 min. At 20 cm from the cells, an irradiance of ~0.5 mW/cm2 was measured. Cells were plated onto a six-well tissue culture dish 24 h before irradiation. The relevant treatment was added 18 h later, and after 6 h the culture medium was replaced with 600 µl/well Hanks' balanced salt solution (containing Ca2+ and Mg2+; GIBCO BRL) and the cells were irradiated. The original media were returned to each well, the cells were returned to the incubator, and apoptosis was measured 18 h later. For irradiation of mice, four FS40 lamps, similarly screened with Kodacel and calibrated, were used.
Cell treatments.
L-NAME and
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ) were obtained from Alexis (San Diego, CA), prepared in
keratinocyte growth medium or DMSO, and added to the cells 6 h
before radiation. 8-Bromo-cGMP (8-BrcGMP) was obtained from Sigma.
N-propyl-1,3-propanediamine-NO (PAPA-NO) was the kind gift
of Dr. Klaus Kroncke (Research Group Immunobiology, Heinrich Heine
University, Düsseldorf, Germany). For infection with adenoviral
vectors, the cells were washed with Hanks' balanced salt solution
containing both Ca2+ and Mg2+ and were infected
with adenoviral vectors carrying the genes for bacterial
-galactosidase (Ad-LacZ) or human iNOS (Ad-iNOS), prepared as
described previously (35). Cells were incubated at a
multiplicity of infection (MOI) of 10 pfu/ml in a volume of 600 µl of
Opti-MEM (Life Technology). After a 4-h infection, the medium was
changed to fresh keratinocyte culture medium.
Cell survival assay. Cell viability was determined by the crystal violet method. Cells were stained with 0.5% crystal violet in 30% ethanol and 3% formaldehyde for 5 min at room temperature. Plates were then washed four times with tap water. Cells were solubilized with 1% sodium dodecyl sulfate solution, and dye uptake was measured at 550 nm with a microplate reader.
Nitrite assay. Nitrite concentration was measured by a standard Griess reagent assay with a multiplate spectrophotometer at 550 nm. The Griess reagent consisted of one part 1% sulfanilamide in 5% orthophosphoric acid.
Caspase activity assay. Six hours after irradiation of 70% confluent cells in 10-cm dishes, cells were harvested at 4°C, rinsed in PBS, and resuspended in 5× volume of hypotonic buffer (in mM: 20 HEPES-KOH, pH 7.5, 10 KCl, 1.5 MgCl2, 1 EDTA, 1 EGTA) to which fresh protease inhibitors (2 µg/ml leupeptin, 2 µg/ml aprotinin, 2 µg/ml pepstatin A, and 1 mM PMSF) had been added. Cells were lysed with three freeze-thaw cycles in liquid nitrogen and centrifuged at 4°C at 16,000 g for 15 min to separate cell fragments from supernatant. The protein concentration of the supernatant was corrected to 100 µg of protein per 100 µl of buffer. To each 100 µl of lysate, in a 96-well plate, DEVD-pNA caspase-3 substrate (Promega, Madison, WI) was added to a final concentration of 200 µM. The plate was incubated at 37 °C, and optical density (OD) was measured every 5 min for 1 h at 405 nm in a 96-well optical plate reader (Versamax tunable reader with Softmax Pro software; Molecular Devices, Sunnyvale, CA).
Flow cytometry. Apoptosis was assessed by flow cytometric analysis of annexin V with a Becton Dickinson FACSort (San Jose, CA) as described by Schindl et al. (34). Supernatants and cells harvested with 0.05% trypsin were rinsed in PBS and suspended in 100 µl of annexin binding buffer (10 mM HEPES-NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), 5 µl/100 µl of annexin V, and 5 µg/ml of propidium iodide (PI; BD PharMingen, San Diego, CA). Emission filters used were BP 530/30 nm [fluorescein isothiocyanate (FITC)] and BP 585/42 nm (PI). A minimum of 10,000 cells per sample were recorded, and cell debris was excluded by appropriate forward light scatter threshold setting. Data analysis with CELLQuest software (Becton Dickinson, St. Louis, MO) was performed, and numbers of cells positive for annexin V, PI, or combinations thereof were calculated.
UVB irradiation of mice.
All animal studies were approved by the Institutional Animal Care and
Use Committee of the University of Pittsburgh. Wild-type C57Bl/6 mice
were from Charles River (Wilmington, MA). eNOS/
mice
were SV129 × C57BL/6J and were a kind gift from Drs. Victor Laubach and Edward Shesley (University of Virginia, Charlottesville, VA). eNOS
/
mice were used at the N6
generation and thus were 98.4% homogeneous to the C57Bl/6 mice.
iNOS
/
mice were a kind gift from Dr. John Mudgett
(Merck Research Laboratories, Rahway, NJ); they were received as
N4 and we backcrossed them to N10 (C57BL/6) 7- to 10-wk-old wild-type females. Accordingly, iNOS
/
mice were 99.91% homogeneous to the C57Bl/6
mice. iNOS
/
and eNOS
/
mice were housed
in cages in a room with controlled temperature and humidity and
alternating 12:12-h light-dark cycles. Mice were fed with a commercial
diet and had water ad libitum. They were anesthetized with isofluorane
(Abbott, North Chicago, IL) for shaving of their backs before
irradiation and were unrestrained in uncovered cages during
irradiation. Mice were euthanized by CO2 inhalation 24 h after irradiation, the time point at which apoptosis has been
shown to be maximal (29). The dorsal skin was removed and
placed in 2% paraformaldehyde overnight and then in 30% sucrose for
4 h before being frozen in optimum cutting temperature (OCT)
compound (Tissue-Tek, Torrance, CA) and stored at
80°C until
analyzed. Single mice were initially irradiated at UV doses ranging
from 50 to 1,600 mJ/cm2 to determine the dose at which the
greatest differences in apoptosis could be discerned (data not
shown). Three or four mice per group were then irradiated at 400 and
1,000 mJ/cm2, the doses determined to be optimal.
Apoptosis assay. Sections (7 µm) were cut and incubated at 37°C for 1 h with a FITC conjugate of the cell-permeant caspase inhibitor VAD-FMK (CaspASE FITC-VAD-FMK, 10 µM; Promega, Madison, WI), which binds to activated caspase sites. The sections were counterstained for 3 min at room temperature with 10 µg/ml of Hoechst 33258 dye, followed by mounting in Gelvatol [23 g poly(vinyl alcohol 2000), 50 ml glycerol, 0.1% sodium azide to 100 ml PBS]. TdT-mediated dUTP nick end labeling (TUNEL), which measures DNA strand breaks, was also performed with a kit (in situ cell death detection kit; Roche, Indianapolis, IN). This analysis produced results analogous to caspase staining, but with a higher number of falsely positive stained cells (data not shown). TUNEL in irradiated skin is not specific for apoptosis (13). The caspase labeling technique measures a biochemical intermediate of the apoptotic pathway and is more specific in UV-induced apoptosis.
eNOS immunohistochemistry. Sections (7 µm) were blocked with 5% normal goat serum in bovine serum albumin for 1 h at room temperature and then probed with a 1:2,000 dilution of anti-eNOS polyclonal antibody (BD Transduction Laboratories, Franklin Lakes, NJ) overnight at 4°C, followed with a 1:3,000 dilution of Cy3-labeled goat anti-rabbit secondary antibody (Jackson, West Grove, PA) for 1 h at 37°C. Omission of primary and secondary antibodies in control wild-type sections confirmed the specificity of eNOS staining.
Sections were photographed with an Olympus Provis epifluorescence microscope connected to an Olympus digital camera (Melville, NY) with 480/535 and 550/570 filters for fluorescein and Cy3, respectively. Total and apoptotic cells were counted with Metamorph software (Universal Imaging, Dowmington, PA), with the epidermis and dermis being differentiated morphologically. Between 6 and 12 high-power fields were counted for each dose of UV, with an average of 800 cells per high-power field.Statistics. All values are means ± SD unless otherwise indicated. Comparisons of multiple groups were done by one-way analysis of variance followed by the Tukey post hoc test, using a 95% confidence interval (P < 0.05) to denote statistical significance.
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RESULTS |
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Effect of NO on UVB-induced keratinocyte apoptosis and
survival.
We first determined the effects of NO on UVB-induced apoptosis
in human keratinocytes in vitro by treating the cells with UVB in the
presence or absence of the nonspecific NOS inhibitor L-NAME. Addition of L-NAME and irradiation with
UVB both increased apoptosis, and the combination of
L-NAME and UVB produced a higher amount of
apoptosis than control (P < 0.001),
L-NAME alone (P = 0.009), or UVB alone
(P = 0.021). We showed previously (49) that other NOS antagonists also increase apoptosis after UVB. Addition of S-nitroso-N-acetyl-penicillamine
(SNAP) immediately after UVB significantly abrogated the rise in
apoptosis due to L-NAME (P = 0.012)
but not that due to UVB (Fig.
1A). The reduction in
keratinocyte cell death is not solely a feature of addition of
nitrosothiols such as SNAP. The NONOate PAPA-NO also markedly reduced
cell death after UVB irradiation (Fig. 1B). We next infected keratinocytes with AdiNOS at a MOI of 10 pfu/cell. AdiNOS-transduced iNOS protein was functional, because the stable end product of NO,
NO-galactosidase gene (AdLacZ; Fig.
2A). AdiNOS-infected cultures
also exhibited significantly reduced cell death after irradiation
(P < 0.001 vs. UVB + AdLacZ or UVB only; Fig.
2B). Interestingly, AdiNOS also increased the viability of
keratinocytes not exposed to UVB (P < 0.001 vs. AdLacZ or UVB only).
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Caspase-3 activity after UVB irradiation.
We next wished to determine whether the apparent increase in
apoptosis assessed by annexin V staining correlated with
increased caspase activity. Caspase-3 activity after irradiation
doubled in cells incubated with 1 mM L-NAME compared with
caspase-3 activity in cells treated with the inactive isomer,
D-NAME (P = 0.021). Addition of 100 µM
SNAP partially reduced the rise in caspase-3 activity in both
D-NAME- and L-NAME-treated cells
(P < 0.001), so that caspase-3 activity in cultures
treated with L-NAME + SNAP did not differ
significantly from cultures treated with D-NAME (Fig.
3). Caspase activity was not
significantly different in control cells, to which neither NAME isomer
had been added, and D-NAME-treated cells (control
nonirradiated 28.6 ± 1.9 × 104,
D-NAME nonirradiated 32.1 ± 1.21 × 10
4, control irradiated 68.2 ± 9.41 × 10
4, D-NAME irradiated 68 ± 2.27 × 10
4 OD · mg
protein
1 · min
1).
|
cGMP activity and apoptosis after UVB.
Keratinocytes become increasingly resistant to UVB-induced
apoptosis as they become more confluent. Total annexin-positive cells, when cultured at a density of 0.6 × 105
cells/cm2 in a six-well plate and allowed to grow to 70%
confluence, increased from 5% to 47% after irradiation with 100 mJ/cm2 UVB. This number was reduced to 21% after addition
of the nonhydrolyzable cGMP analog 8-BrcGMP (800 µM; Fig.
4A). The annexin-positive
fraction of cells that had grown to 90% confluence increased from 4%
to 9% after UVB, and this was increased to 26% after addition of the
sGC inhibitor ODQ (Fig. 4B).
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Dermal and epidermal apoptosis in wild-type,
iNOS/
,
and
eNOS
/
mice.
Given the potent effects of NO on UVB-induced keratinocyte
apoptosis, we next examined whether NO had a similar effect in vivo by examining the effects of UVB on wild-type,
iNOS
/
, and eNOS
/
mice. We carried out
initial UVB dose-finding experiments in wild-type mice, which were
shaved and irradiated with 0, 50, 100, 150, 400, 800, and 1,600 mJ/cm2 UVB to determine the doses of UVB required to obtain
apoptosis. In these studies, apoptosis was assessed by
immunostaining with an antibody to the active site of caspase-3. A
clear dose response was seen, with dermal and epidermal
apoptosis increasing in proportion to UV dose after 24 h
and eNOS
/
mice being most prone to apoptosis
(data not shown). On the basis of these results, triplicate mice (wild
type, eNOS
/
, or iNOS
/
) were irradiated
with 0, 400, and 1,000 mJ/cm2, respectively; representative
immunostaining for caspase-3 active site is shown in Fig.
5 and quantified in Fig. 6. In the
dermis, increasing UVB dose produced
increasing apoptosis, with both iNOS
/
(P < 0.005) and eNOS
/
(P < 0.001) mice exhibiting significantly higher
apoptosis at 400 mJ/cm2. In the dermis, the
apoptosis rate of eNOS
/
mice was double that of
either wild-type or iNOS
/
mice at a UVB dose of 1,000 mJ/cm2 (P < 0.001; Fig. 6A). In
the epidermis, eNOS
/
mice underwent significantly more
apoptosis than wild-type mice (P < 0.001).
iNOS
/
mice showed a tendency to increased
apoptosis, but this did not reach statistical significance.
There were no differences among groups at 1,000 mJ/cm2
(Fig. 6B).
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Localization of eNOS.
Our studies suggested a profound antiapoptotic activity of eNOS in
the context of UVB irradiation, but it was necessary to localize eNOS
in the skin of wild-type mice. Immunohistochemistry was performed with
a polyclonal anti-eNOS antibody. Staining for eNOS protein was strong
in keratinocyte cytoplasm and in endothelial cells and weaker in dermal
fibroblasts. There was no difference in eNOS expression between
iNOS/
and wild-type mice or in irradiated and
nonirradiated skin. As expected, no staining was observed in
eNOS
/
mice (Fig. 7).
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DISCUSSION |
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One of the primary functions of the skin is to protect the body from the external environment. UVB radiation is a potent carcinogen, causing DNA damage, immunosuppression, and apoptosis (7). Keratinocytes are relatively resistant to the apoptogenic properties of UVB, and they form an effective barrier to the transmission of UVB to the underlying tissues. Here, we demonstrate that the resistance of keratinocytes to UVB-induced apoptosis is a consequence of NO release by eNOS, acting through the stimulation of cGMP production.
Previous reports demonstrated the release of NO in normal skin but did
not ascribe a clear function to this NO. Romero-Graillet et al.
(32) described the release of NO and activation of cGMP within 1 min of UVB irradiation of cultured human keratinocytes, implicating an eNOS-like enzyme. Additionally, iNOS in the skin releases NO with a peak 18-24 h after UVB irradiation
(47). Our experiments in gene knockout mice extend these
prior observations, demonstrating that keratinocyte apoptosis
is greater in both eNOS/
and iNOS
/
mice
compared with wild-type mice after irradiation with 400 mJ/cm2 UVB. We found that eNOS
/
mice are
markedly more sensitive to UVB than wild-type or iNOS
/
mice. They undergo a greater degree of apoptosis in both the dermis and the epidermis, as well as exhibiting a higher degree of
apoptosis at both 400 and 1,000 mJ/cm2 UVB. This
effect produced by eNOS is consistent with the rapid release of NO from
this constitutive NOS in the skin. Together, these data raise the
possibility that the coordinated release of NO from eNOS and iNOS
serves both as an immediate and a sustained mechanism to protect
keratinocytes from the toxicity of exposure to UVB.
Keratinocyte apoptosis occurs by several partially independent
pathways: activation of the TNF-/Fas death receptor, release of
reactive oxygen species, mitochondrial damage with subsequent release
of cytochrome c, and DNA damage with resultant upregulation of p53 (22). Relative resistance to UV-induced
apoptosis has been demonstrated in stem cell-enriched
keratinocytes via an integrin signaling pathway (43), and
in normal keratinocytes stabilization of wild-type p53 after UV
irradiation occurs by p38-mediated phosphorylation (5). NO
can inhibit apoptosis directly by S-nitrosylation of cysteine groups on a number of caspases in hepatocytes
(25), indirectly via cGMP-dependent pathways (18,
37), and by quenching of reactive oxygen species released after
UV irradiation (40). The antiapoptotic effect of NO in
keratinocytes was consistently reduced by the sGC inhibitor ODQ and
replicated by addition of the nonhydrolyzable cGMP analog 8-BrcGMP.
Caspase-3 activity after irradiation was increased by blocking NOS
activity and reduced on addition of an NO donor. These observations
suggest that the protective role of NO in keratinocytes involves, at
least in part, cGMP.
The prototypic target for cGMP is protein kinase G (PKG), whereas protein kinase A (PKA) is one of the main targets of cAMP. Both cGMP and cAMP can cross talk through the activation of PKG or PKA (15, 19). Previous studies from our laboratory suggest that both cyclic nucleotides prevent hepatocyte apoptosis through the activation of PKA (26). Similar to NO, cell-permeant cGMP analogs suppressed caspase-8 activation, loss of mitochondrial membrane potential, cytochrome c release, and caspase-3 activation. Nonhydrolyzable analogs of cAMP were even more potent in the inhibition of hepatocyte cell death. The protective effects of both cGMP and cAMP were dependent, in part, on PKA. Furthermore, NO, cGMP, and cAMP all activated the Akt/protein kinase B (PKB) pathway in cultured hepatocytes (26). Future studies will be aimed at elucidating whether this same paradigm holds in keratinocytes. Future studies will also determine whether the mechanisms outlined here in a transformed keratinocyte cell line are still operative in primary cells. This is especially important because Jackson et al. (16) have shown that human papillomavirus reduces post-UV apoptosis by reducing Bak expression, whereas Simbulan-Rosenthal et al. (39) have shown that E6/7 increases sensitivity to UV-induced apoptosis.
At higher doses of UVB (1,000 mJ/cm2), the
antiapoptotic role of eNOS in the dermis is striking, whereas
neither iNOS nor eNOS appears to be able to protect the epidermis from
apoptosis. This blurring of the effect of NO in the epidermis
may be due to changes in the redox environment at these higher
radiation doses. For example, very small differences in flux of
reactive oxygen species and NO can markedly alter the balance between
oxidative and nitrosative effects (33, 52). We previously
described (49) how the presence of superoxide dismutase
enhances the antiapoptotic effects of NO in irradiated
keratinocytes. In that study, we suggested that limiting formation of
proapoptotic reactive nitrogen intermediates such as peroxynitrite,
or quenching of NO by formation of superoxide, shifts the balance
toward the antiapoptotic effects of NO (49). The loss
of the antiapoptotic effect of eNOS-derived NO at 1,000 mJ/cm2 could be due either to the inability of these levels
of NO to overcome the proapoptotic effects induced by UVB through
the mechanisms outlined above or to the presence of proapoptotic
reactive nitrogen intermediates formed at higher doses of UVB
(10).
In the dermis, we observed a direct linear relation between UVB dose
and apoptosis, with eNOS/
mice exhibiting
significantly increased apoptosis at all doses of UVB.
Apoptosis was not counted in adnexal structures and hair follicles because apoptosis in these structures is dependent on hair cycle rather than purely on UV radiation. The cells in which apoptosis was measured are thus predominantly fibroblasts. The persistently high apoptosis in eNOS
/
mice, even
at 1,000 mJ/cm2, might be due to differences in cell
type-specific responses to NO and reactive nitrogen intermediates or to
the reduced levels of UVB reaching the dermis compared with the
epidermis. We demonstrated eNOS staining intensely in the cytoplasm of
keratinocytes and diffusely in fibroblasts of iNOS
/
and
wild-type mice, in accord with the findings of other authors studying
isolated cells from human skin (20, 38). There was no
change in distribution or intensity of eNOS protein observed after
irradiation, and eNOS was not seen in eNOS
/
mice.
Besides eNOS, we define a novel role for iNOS in the protection of keratinocytes from UVB-induced apoptosis. Higher levels of NO output would be expected from iNOS, which peaks in human skin 24 h after irradiation in both keratinocytes (21) and dermal endothelial cells (41) . The time course of this iNOS upregulation correlates with marked skin erythema (46, 47). A lower-grade but more persistent erythema is seen for up to 3 wk after irradiation (51). Because erythema is a good marker for NO and cGMP production (6), we believe that a prolonged production of lower concentrations of NO after irradiation may continue to exercise an antiapoptotic effect.
The aberrant regulation of NO may have important pathophysiological sequelae. In psoriasis, plaque NO levels are persistently elevated (50), which may at least partially account for the excess keratinocyte numbers and reduced apoptosis (4) characteristic of this disease. In systemic and localized scleroderma, reduced dermal eNOS protein and NO production correlates with reduced endothelial cell growth (31). In conclusion, our findings extend to keratinocytes the antiapoptotic role of NO and raise the clinical possibility of NOS inhibition for the treatment of hyperproliferative skin disorders such as psoriasis.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of General Medical Sciences Grants R01-GM-44100 and R01-GM-50441 (T. R. Billiar) and the Gunning Research Fellowship of the University of Edinburgh, Department of Medicine (R. Weller).
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. R. Billiar, Dept. of Surgery, Univ. of Pittsburgh, F-1281 Presbyterian Univ. Hospital, Pittsburgh, PA 15213 (E-mail: billiartr{at}msx.upmc.edu).
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.
10.1152/ajpcell.00462.2002
Received 3 October 2002; accepted in final form 24 December 2002.
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