Photodermatology, Photoimmunology & Photomedicine Volume 26 issue 4 2010 [doi 10.1111%2Fj.1600 0781.2010.00522.x] Haoxiang Xu; Yan Yan; Li Li; Shiguang Peng; Tao Qu; Baoxi Wang Ultraviol

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O R I G I N A L A R T I C L E
Ultraviolet B-induced apoptosis of human skin fibroblasts involves
activation of caspase-8 and -3 with increased expression of vimentin
Haoxiang Xu, Yan Yan, Li Li, Shiguang Peng, Tao Qu & Baoxi Wang
Department of Dermatology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing, China
Key words:
apoptosis; caspase; fibroblast; UVB;
vimentin
Correspondence:
Wang Baoxi, Department of Dermatology, Peking
Union Medical College Hospital, Chinese Academy of
Medical Sciences and Peking Union Medical College,
Beijing 100730, China.
e-mail: wangbx2009@gmail.com
Accepted for publication:
28 April 2010
Conflicts of interest:
None declared.
Summary
Background: After irradiation with a high dose of ultraviolet B (UVB), cells undergo
apoptosis. Caspase-8 and -3 are key mediators of apoptosis in many cells. Vimentin, an
important cytoskeleton component, can be cleaved by caspase-3, -6, -7 and -8. Cell apoptosis
is promoted via caspase-triggered proteolysis of vimentin. In this study, we explored the
roles of caspase-8 and -3 and the changes in vimentin expression in UVB-induced apoptosis
of human dermal fibroblasts.
Methods: Skin fibroblasts were irradiated with 150mJ/cm2 UVB and cell death was
monitored by the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay
and Hoechst staining. Caspase-8 and -3 activities were detected by the caspase activity assay.
Vimentin expression was assessed by immunofluorescence and Western blot.
Results: Caspase-8 and -3 were activated by 150mJ/cm2 UVB irradiation. Caspase-8 and -3
activities changed in a time-dependent way after UVB irradiation to induce apoptosis of
fibroblasts, and caspase-8 and -3 interacted with each other in this process. However, their
substrate, vimentin, showed an enhanced expression over time after UVB irradiation.
Conclusions: UVB-triggered apoptosis of fibroblasts was dependent on the activation of
caspase-8 and -3 with an increased expression of vimentin.
S olar ultraviolet (UV) radiation is mainly divided intoultraviolet A (UVA, 315–400 nm), ultraviolet B (UVB,
280–315 nm) and ultraviolet C (UVC, 200–280 nm). The UV
light reaching the earth includes about 90% UVA and 10% UVB,
whereas, UVC is absorbed by the earth’s atmosphere. UVB rays
can penetrate the epidermis and reach the upper part of the
dermis. UVB irradiation can directly induce DNA damage, such
as cyclobutane pyrimidine dimer formation (1). In addition
to DNA damage, exposure to UVB irradiation can also lead to
inflammatory factor generation and gene expression modulation,
as well as cell apoptosis (2, 3).
Caspase-8 and -3, key members in the caspase family, play
crucial roles in apoptosis (4, 5). When factor-associated suicide
ligand, tumor necrosis factor, tumor necrosis factor-related
apoptosis-inducing ligand and other extracellular ligands bind
to their respective receptors, caspase-8 dimerizes and then
cleaves other pro-apoptotic proteins including caspase-6, -7 and
-3 (6–8). Caspase-3 is an important effector caspase because it
selectively destroys structural and functional proteins (9).
Caspase-3 has also been shown to control the Dcm collapse
and amplify the release of cytochrome c from the mitochondria
(10).
Vimentin is an important component of the cytoskeleton. In
the skin, vimentin specifically exists in fibroblasts. In in vitro
studies, it has been shown to be cleaved by caspase-3, -6, -7 and
-8 (11, 12). In another study, cleaved vimentin promoted
apoptosis by amplifying the cell death signal (13).
However, the roles of caspase-8 and -3 and the changes of
vimentin are still unknown in UVB-induced apoptosis of human
dermal fibroblasts. In this study, we examined the changes of
caspase-8 and -3 activities after UVB irradiation. To understand
how vimentin was involved in UVB-induced apoptosis of
fibroblasts, we measured levels of vimentin expression
following UVB irradiation exposure.
Materials and methods
Fibroblast culture
Normal human dermal broblasts were obtained from the
foreskins of healthy donors (18–30 years old) with informed
consent. The cells were cultured in dishes containing Dulbecco’s
modified Eagle’s media (DMEM) supplemented with 10% fetal
bovine serum (FBS), penicillin (100U/ml) and streptomycin
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(100 mg/ml). The cells were maintained in a humidified 5%
carbon dioxide, 95% air incubator at 37 1C. Experiments were
carried out at three to six passages of fibroblasts. The study
protocol was reviewed and approved by the ethics committee of
Peking Union Medical College Hospital.
UVB irradiation
The UVB source was a Waldmann UV800K cabin (Herbert
Waldmann GmbH & Co., Villingen-Schwenningen, Germany)
equiped with six TL20/12RS fluorescent tubes (Philips,
Eindhoven, the Netherlands). The spectral outputs were 70%
UVB (280–315 nm) with a peak emission at 313 nm and 30%
UVA (315–400 nm) with a peak emission at 365 nm.
Wavelengths below 280 nm were screened using a Kodacel filter
(Kodak, Rochester, NY, USA). The UVB output was 1.10mW/
cm2 measured using a UV meter (Type 595100, Herbert
Waldmann GmbH & Co.). The UVB irradiation dose was
controlled by adjusting the exposure time. The majority of the
energy was in the UVB range, and cells were exposed to radiation
for 136 s (136 s = 150mJ/cm2/1.10mW/cm2). In addition, the
dishes were left covered during exposure to radiation and the
output of UVA transmitted through the lids was 0.02mW/cm2.
Therefore, the accumulated UVA dose was 2.72mJ/cm2
(2.72mJ/cm2=136 s� 0.02mW/cm2), which was too low to
have an effect on cells. Thus, we referred to this UV irradiation as
UVB.
Cell exposure to UVB irradiation was performed in Petri
dishes containing prewarmed phosphate-buffered saline (PBS)
without sodium bicarbonate, which was immediately replaced
by fresh DMEM containing 10% FBS after UV irradiation.
Temperature was kept constant during UVB irradiation. Mock-
irradiated controls followed the same schedule of medium
changes without UVB irradiation.
Cell viability assay
Cell viability was measured using the 3-(4,5)-dimethy-
lthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay.
Cells that had been grown in 96-well plate at 70–80%
confluence were exposed to different doses of UVB irradiation
and incubated in complete medium for 24 h. In each well, 20 ml
of MTT solution (5mg/ml) was added and cultured for another
4 h. The reaction was stopped by adding 150 ml of 100%
dimethyl sulphoxide. Then, the absorbance of each well was
measured at 570 nm with an enzyme-linked immunosorbent
assay reader (BioTek ELX 800, Winooski, VT, USA).
Hoechst staining
Fibroblasts were cultured on cover slips to 70–80% confluence.
The controls were irradiated by UVB with different caspase
inhibitors and the experimental groups were irradiated without
any caspase inhibitors. At each indicated time points, the cells
were washed three times with ice-cold PBS, and then a Hoechst
33258 staining kit (Beyotime Institute of Biotechnology, Jiangsu,
China) was used to stain apoptotic cells. The cells were analyzed
under a fluorescence microscope to determine the percentage of
apoptotic cells, as described elsewhere previously (12).
Caspase activity assay
Caspase activity assays were performed using caspase-8 and -3
detection kits (Calbiochem, Darmstadt, Germany) according to
the manufacturers’ recommendations. UVB-irradiated cells were
collected at different time points and incubated with their own
substrate for 1 h at 37 1C in the dark. Measurements were then
performed using a fluorescencereader system (BioTek ELX 800)
at excitation and emission wavelengths of 485 and 528 nm. To
further investigate caspase activation, Z-VAD-FMK (general
caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor) and Z-
DEVD-FMK (caspase-3 inhibitor) (MBL Products, Nagoya,
Japan) were used. Cells were pre-incubated with 20 mM of Z-
VAD-FMK, Z-IETD-FMK or Z-DEVD-FMK for 1 h before UVB
irradiation. The data were expressed as the ratio of the
absorbance of treated samples to the absorbance of the samples
at 0 h after UVB irradiation.
Immunofluorescence staining of vimentin
Fibroblasts were cultured on cover slips to 70–80% confluence
and treated with UVB irradiation. After two washes with cold PBS
(pH 7.4), the cells were fixed with 4% paraformaldehyde for
30min at 4 1C and permeabilized with 0.5% Triton X-100
(Sigma, St Louis, MO, USA) in PBS for 3min at 4 1C. The cells
were then incubated with primary mouse monoclonal anti-
vimentin V9 (1 : 100; Santa Cruz Biotechnology, Santa Cruz, CA,
USA) for 90min at 4 1C, washed three times with cold PBS (pH
7.4) and further incubated with fluoresceine isothiocyanate-
labeled sheep anti-mouse immunoglobulin G (IgG) secondary
antibody (1 : 100; Vector, Burlingame, CA, USA) for 1 h at room
temperature. The stained cells were then washed with PBS and
visualized using an inverted microscope (Nikon TE 300; Nikon,
Tokyo, Japan).
Western blot
Fibroblasts were cultured and treated as described above. Proteins
were extracted from cells using a cell lysis buffer (50mmol/l
Tris(hydroxymethyl)aminomethane hydrochloride pH 7.4,
1mmol/l ethylene diamine tetraacetic acid, 150mmol/l
sodium chloride, 0.1% sodium dodecyl sulfate, 1% Triton-100,
1% sodium deoxycholate, 1mmol/l phenylmethanesulfonyl
fluoride). Using the bicinchoninic acid assay method, the
protein concentrations of the lysates were quantified. Twenty
microliters of cell lysate was subjected to sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and transferred onto
nitrocellulose membranes (Millipore, Billerica, MA, USA). The
membranes were blocked in 3% bovine serum albumin,
incubated with primary antibodies (vimentin mAb V9 1 : 200,
glyceraldehyde-3-phosphate dehydrogenase 1 : 200, Santa Cruz
Biotechnology) overnight at 4 1C and incubated with a secondary
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UVB-induced apoptosis of human skin fibroblasts
antibody for 1 h at room temperature. The secondary antibody
was horseradish peroxidase-conjugated anti-mouse IgG
(1 : 4000; Abgent, San Diego, CA, USA). Immunoreactive
proteins were detected by enhanced chemiluminescence
(BeyoECL Plus, Jiangsu, China). The signals were detected using
the FluorChem SP (Santa Clara, CA, USA) imaging system.
Statistical analysis
The results were presented as means� standard error of the
means. The independent samples t-test was used for statistical
analysis with SPSS 17.0 software. Probability values o 0.05 were
considered significant.
Results
UVB reduced viability of fibroblasts
To determine levels of UVB necessary for a cytotoxic effect,
cultured dermal fibroblasts were irradiated by UVB dosages of
50, 100, 150, 200, 250 and 300mJ/cm2. After 24 h of
irradiation, cell viability was reduced, showing a trend of
decreasing cell viability with increasing doses of UVB (Fig. 1a).
When cells were treated with 150mJ/cm2 UVB, cell viability
decreased as the length of time increased post-exposure (Fig. 1b).
Hence, 150mJ/cm2 UVB exposure was chosen in our following
experiments.
Caspases were involved in UVB-induced apoptosis of
fibroblasts
To examine the apoptotic effect of UVB on human fibro-
blasts, we treated the cells with 150mJ/cm2 UVB irradia-
tion. Apoptotic cells were apparent with typical nuclear
condensation and extensive brilliant blue nuclear staining, and
the number of apoptotic cells increased with time post-exposure
(Fig. 2a).
To test whether caspases were involved in the UVB-induced
apoptosis of fibroblasts, we used a general caspase inhibitor,
Z-VAD-FMK. When cells were pretreated with Z-VAD-FMK and
then irradiated by UVB, cell apoptosis was significantly inhibited
in the group pretreated with Z-VAD-FMK compared with the
control (Fig. 2b and c), confirming that caspases are involved in
UVB-triggered apoptosis of skin fibroblasts.
Caspase-8 and -3 were activated in UVB-induced apoptosis
of fibroblasts
To study whether caspase-8 and -3 were activated by 150mJ/
cm2 UVB, the activities of caspase-8 and -3 were detected. In cells
irradiated with 150mJ/cm2 UVB, caspase-8 and -3 activities
changed in a time-dependent manner after exposure. At 12 h
after irradiation, their activities were obviously higher than that
at other time points (Fig. 3a and b).
Caspase-8 and -3 interacted with each other in UVB-
induced apoptosis of fibroblasts
To further explore the roles of caspase-8 and -3 in UVB-triggered
apoptosis of fibroblasts, we pretreated the experimental groups
with their respective specific inhibitors before exposing them to
150mJ/cm2 UVB irradiation.
When caspase-8 activity was inhibited, the activity of caspase-
3 was reduced. Similarly, when caspase-3 was inhibited, the
activity of caspase-8 also decreased. Their activities increased
gradually with increasing time after UVB irradiation (Fig. 4a
and b).
Then, we compared different proportions of apoptotic cells.
The data reveal that either the caspse-3 or -8 inhibitor
significantly reduced the proportion of apoptotic cells compared
with the control after UVB irradiation. Compared with caspase-3
specific inhibition, at 36 and 48 h after UVB irradiation
*
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(a)
(b)
Fig. 1. Cell viability of fibroblasts irradiated by ultraviolet B (UVB). (a)
Cells were exposed to different irradiation intensities of UVB including
50, 100, 150, 200, 250 and 300mJ/cm2. After 24 h, cell viability
was detected by the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-
diphenytetrazoliumromide assay. (b) Cells were treated with 150mJ/cm2
UVB. After 12, 24, 36 and 48 h, cell viability was detected. Mock-
irradiated cells were used as controls. �Po 0.05 and ��Po 0.01 as
compared with controls.
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Xu et al.
exposure, the proportion of apoptotic fibroblasts exhibited
significantly more reduction with caspase-8 inhibition (Fig. 4c).
Vimentin expression increased in UVB-induced apoptosis
of fibroblasts
Vimentin could be disrupted by different caspases, and so the
changes of vimentin in apoptotic fibroblasts were detected. Using
cytoimmunofluorescence and Western blot, we found that
vimentin was not cleaved in fibroblasts at different time points
following UVB irradiation. In contrast, the amount of vimentin
increased in UVB-irradiated cells (Fig. 5a–c).
Discussion
UVB irradiation exerts cytotoxic effects by inducing the
production of reactive oxygen species and DNA photoproducts
including (6-4) photoproducts and cyclobutane pyrimidine
dimers. This study demonstrated that UVB-induced dermal
fibroblast apoptosis involved activation and interaction of
caspase-8 and -3 with an increased expression of vimentin.
Different frequencies of light irradiation may influence
caspase-8 and -3 activities in different ways. In normal primary
human gingival fibroblasts, the expression and activity of
caspase-8 and -3 increased in the UVC-irradiated group, but
their expression and activity were significantly recovered to
normal levels if the cells were pretreated with 635 nm light
irradiation (14). Similarly, Frank et al. (15) also reported that
infrared pre-irradiationinhibited the UVB-related activation of
caspase-3. In skin fibroblasts from individuals with xeroderma
pigmentosum, UVC irradiation induced the activation of
caspases-8 within 30min, which was followed by the activation
of caspase-3 (16).
(c)
(a) (b)
Fig. 2. Apoptotic fibroblasts treated with ultraviolet B (UVB) or UVB plus Z-VAD-FMK at different time points. Cells were irradiated with 150mJ/cm2
UVB (a), or pretreated with 20 mM Z-VAD-FMK (general caspase inhibitor) for 1 h and irradiated with 150mJ/cm2 UVB (b). Apoptotic cells showed
typical changes of morphology and condensed blue nuclear staining. (c) Fibroblasts were fixed with 10% buffered formalin and stained with Hoechst
dye. The percentage of apoptotic cells was determined by counting the total number of cells and the number of Hoechst-stained apoptotic cells in five
randomly selected fields. � Po 0.05 as compared with only UVB-irradiated groups.
201r 2010 John Wiley & Sons A/S � Photodermatology, Photoimmunology & Photomedicine 26, 198–204
UVB-induced apoptosis of human skin fibroblasts
Our results showed that the activities of caspase-8 and -3 were
significantly up-regulated at 12 h after UVB irradiation. However,
the proportion of apoptotic cells at 48 h was greater than that at
12 h after UVB treatment. This might be explained by the fact
that fibroblasts were not susceptible to UVB-induced apoptosis
(17) and that caspase-8 or -3 functioned at the early stage of
apoptosis. When caspase-8 activity was attenuated, UVB-induced
activation of caspase-3 was delayed. Activation of caspase-3
might also influence caspase-8 activity. In our study, when
caspase-3 activity was inhibited, the number of apoptotic cells
decreased and caspase-8 activity increased slowly. All these data
(a)
(b)
Fig. 3. Activity of caspase-8 and -3 in ultraviolet B (UVB)-irradiated
fibroblasts. Cells were irradiated with 150mJ/cm2 UVB. And then,
caspase-8 activity (a) and caspase-3 activity (b) were measured at
indicated time points. Results were expressed with respect to the level at
0 h after UVB irradiation. All data were presented as means� standard
error of the means of three independent experiments.
(a)
(b)
(c)
Fig. 4. Caspase-8 and -3 interacted with each other in ultraviolet B
(UVB)-irradiated apoptosis. Fibroblasts were pretreated with 20mM
caspase-8 inhibitor (Z-IETD-FMK) (a) or caspase-3 inhibitor (Z-DEVD-
FMK) (b) for 1 h, and then irradiated with 150mJ/cm2 UVB. Caspase-8
and -3 activities were measured at indicated time points. In (a), columns
marked caspase-8 activity were positive controls and columns marked
caspase-3 activity were test samples. In (b), columns marked caspase-3
activity were positive controls and columns marked caspase-8 activity
were test samples. Results were expressed with respect to the level at 0 h
after UVB irradiation. All data were presented as means� standard error
of the means of three independent experiments. (c) Fibroblasts seeded on
a cover slip in a six-well plate were treated with 20mM caspase-8
inhibitor (Z-IETD-FMK) or caspase-3 inhibitor (Z-DEVD-FMK) for 1 h,
and then irradiated with 150mJ/cm2 UVB. At different time points, cells
were fixed and stained with Hoechst dye. The percentage of apoptotic
cells was determined by counting the total number of cells and the
number of Hoechst-stained cells in five randomly selected fields of three
independent experiments. ��Po 0.01 as compared with only UVB-
irradiated controls.
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Xu et al.
indicated that the activation of caspase-8 might be increased by
caspase-3.
Vimentin is a vital cytoskeleton protein in fibroblasts that
protects cells from physical injury and DNA damage (18, 19).
Two major cleavage sites in vimentin can be recognized by
caspase-8 and -3 (11). Cleaved vimentin generates an amino-
terminal product (truncated vimentin) that is sufficient to induce
apoptosis (13). Byun and colleagues demonstrated that caspase
proteolysis of vimentin plays an active role in the execution of
apoptosis. Apoptotic cleavage of vimentin is likely to result in the
disruption of the cytoskeletal network, which may facilitate
nuclear condensation and subsequent fragmentation (11). Ge´lis
et al. (20) reported that the amount of vimentin increased in
hairless rat skin after 60 J/cm2 UVA exposure. We found that
12.5 J/cm2 UVA had no effect on vimentin expression in
fibroblasts (unpublished data). In this study, there was a UVA
component in our UV resource, but the accumulated UVA dose
was far o 12.5 J/cm2. Therefore, the increased protein level of
(a)
(b)
(c)
Fig. 5. UVB-induced expression of
vimentin in skin fibroblasts. (a)
Increased expression of vimentin was
detected by cell immunofluorescence in
fibroblasts at 48 h after 150mJ/cm2
ultraviolet (UVB) irradiation. (b) The
enhanced expression of vimentin in
UVB-treated cells was determined at
indicated time points by Western blot.
Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) protein was
used as an internal control. Mock-
irradiated cells were used as controls.
(c) Relative vimentin protein expression
levels. Data were calculated as vimentin
density at different time points after
UVB irradiation/vimentin density of
mock-irradiated controls. All data were
presented as means� standard error of
the means of four independent
experiments. The control meant that
cells were mock-irradiated. � Po 0.05
as compared with controls.
Fig. 6. Pathway in ultraviolet B (UVB)-induced apoptosis of skin
fibroblasts.
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UVB-induced apoptosis of human skin fibroblasts
vimentin might result from 150mJ/cm2 UVB irradiation. Based
on the knowledge that uncleaved vimentin contributes to
maintaining the integrity and dynamics of intracellular cell
structures (12, 13), we speculate that the accumulation of
vimentin may contribute to dermal fibroblasts’ resistance to
UVB irradiation.
In summary, caspase-8 and -3 were activated and interacted
with each other in UVB-induced apoptosis of fibroblasts, and
their substrate vimentin, which was an important cytoskeleton,
showed an increased expression (Fig. 6). However, the
mechanism and the role of over-expressed vimentin in UVB-
damaged skin fibroblasts remain unclear and need further
studies.
Acknowledgements
We are indebted to Dr Xuming Mao from the University of
Pennsylvania and Helena Chang from the University of
Wisconsin School of Medicine and Public Health for the critical
reading of the manuscript.
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