1
2
The photoaging is the effect of long-term UV exposure and sun damage superimposed 3
on intrinsically aged skin (Rabe et al., 2006). This process results in alteration of the skin 4
such as wrinkles, mottled pigmentation, telangiectasia, and malignancies of the skin. The 5
most notable change of the photoaging is wrinkles due to reduced dermal collagen and 6
dyspigmentation caused by increased melanogenesis.
7
Photodynamic therapy (PDT) is a therapeutic modality for cutaneous malignant and 8
premalignant condition. Topical PDT using 5-aminolevulinic acid (ALA) is based on the 9
photosensitization of the diseased tissue by ALA-induced porphyrins and subsequent 10
irradiation with light of specific wavelength. The excitation of the photosensitizer results in 11
the generation of reactive oxygen species (ROS) and it mediate cellular effects resulting in 12
cytotoxic reaction to the targeted cells (Brackett and Gollnick, 2011).
13
Recently, several clinical studies reported that the PDT has photorejuvenation effects on 14
the aged skin (Dover et al., 2005; Gold et al., 2006; Kohl et al., 2010). In clinical practice, 15
physicians also can notice photorejuvenated skin of elderly patients who had PDT to treat of 16
malignant or premalignant disease of skin. Peripheral skin of target lesion of PDT showed 17
improvement of wrinkles and mottled pigmentation. However, despite of several reports 18
about photorejuvenation effects of PDT, it remains controversial for the treatment of 19
photoaging skin because of limited data about its basic molecular mechanisms.
20
Recently, several reports were introduced about molecular mechanisms of increased 21
collagen synthesis after PDT in human skin and human dermal fibroblast (FB) (Park et al., 22
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2010; Jang et al., 2013). In human skin, the authors reported increased expressions of type I 1
and III procollagen, increased total collagen volume in the dermis and reduced expressions 2
of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-12 after PDT by 3
immunohistochemical stain (Park et al., 2010). In recent study, the condition of “low-level 4
PDT” which induced activation of extracellular signal–regulated kinase (ERK) in human 5
dermal FB without cytotoxic effects was introduced; ALA concentration of 0.1mM, 6
incubation time of 30 minutes, and 3 J/cm2 of irradiation. After this “low-level PDT”, FB 7
proliferation and increased secretion of the MMP-3 was noted mediated by prolonged 8
activation of ERK. Increasing MMP-3 expression may trigger destruction of old extracellular 9
matrix and FB proliferation may also lead to production of new collagen synthesis (Jang et 10
al., 2013).
11
Contrast to the studies about effect of PDT to FB, there are few data about molecular 12
mechanism of PDT to melanocyte (MC) which is induces inhibition of melanin synthesis. In 13
one study, light-emitting diodes (LED) irradiation at wavelengths of 830, 850, and 940 nm 14
effectively reduces melanogenesis, without any cytotoxic effects (Kim et al., 2012).
15
In photoaging response, there are cross-talks between cells, for example, keratinocyte 16
(KC), FB, and MC. UV induce various cytokines in KC such as tumor necrosis factor (TNF)-17
α, interleukin (IL)-1, IL-6 and transforming growth factor (TGF)-β. These cytokines regulate 18
MMPs and result in UV-mediated down-regulation of collagen synthesis in the dermal FB 19
(Karrer et al., 2004; Rabe et al., 2006). UV irradiation is also known to stimulate KC and FB 20
to induce numerous cytokines that is responsible for the proliferations and activation of MC;
21
stem cell factor (SCF), prostaglandin (PG), endothelin (ET) and granulocyte-macrophage 22
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colony stimulating factors (GM-CSF) in the KC; SCF, hepatocyte growth factor (HGF) and 1
basic fibroblast growth factor (bFGF) in the FB, respectively (Imokawa, 2004; Tanaka et al., 2
2010).
3
The PDT can affect FB and MC directly and indirectly. Because KC exists on the outer 4
most surface of the skin, PDT will affect KC firstly and then cytokines from the KC may 5
influence other cells around KC, for example, FB and MC. Therefore, interaction among 6
these three major cells of the skin, which are KC, FB and MC, is important to understand the 7
mechanisms of photorejuvenation induced by PDT.
8
In this study, I investigated the molecular mechanism of photorejuvenation after PDT, 9
focused on interaction between KC, FB, and MC.
10
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II. MATERIALS AND METHODS
1
Primary normal human KC was cultured as previously described (Pincelli et al., 1994).
5
Skin specimens obtained from repeat Caesarean section deliveries and circumcisions were 6
used for the cultures. The cells were suspended in Epilife medium (Cascade Biologics) with 7
human keratinocytes growth supplement (Cascade Biologics) at 37oC in an incubator 8
containing 5% CO2. Cells at passage 3-10th were used for experiments.
9
The HaCaT cells (human keratinocyte cell line) were cultured in DMEM (Gibco) 10
supplemented with 10% fetal bovine serum (Gibco) and 100 U/ml of penicillin/streptomycin 11
(Gibco) at 37oC in an incubator containing 5% CO2. 12
Primary normal human dermal FB was cultured as previously described (Palmetshofer 13
et al., 1995). Skin specimens obtained from circumcisions were used for the cultures. FBs 14
were cultured in DMEM supplemented with 10% fetal bovine serum, 2mM glutamine and 15
100 U/ml of penicillin/streptomycin (Gibco) at 37oC in an incubator containing 5% CO2. FB 16
were subcultured by trypsinization and used between the 5th and 20th passages.
17
To culture the MC, I purchased human neonatal epidermal MC (Cascade Biologics, 18
Portland, OR, USA) and maintained according to the manufacturer’s instructions. Briefly, 19
the cells were suspended in Medium 254 (Cascade Biologics) supplemented with bovine 20
pituitary extract, fetal bovine serum (FBS), bovine insulin, hydrocortisone, bFGF, bovine 21
transferrin, heparin and phorbol myristate acetate (all of supplements from Cascade 22
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Biologics) at 37oC in an incubator containing 5% CO2. Cells at passage 3-6th were used for 1
Anti-phospho-c-Jun N-terminal kinase (JNK), anti-phospho-ERK, anti-phospho-p38, 5
anti-ERK, and anti-p38 antibodies were purchased from Cell Signaling (Beverly, MA, USA).
6
Anti-JNK antibody was purchased from Invitrogen (Carlsbad, CA, USA). Anti-MMP-1 and 7
3 antibodies were purchased from Epitomics (Burlingame, CA, USA). Antitubulin and anti-8
collagen type I antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-TGF-9
PDT procedure was similar to the previous study (Jang et al., 2013). Briefly, cells were 15
plated in 60 mm dish. After the growth medium was removed, serum-free medium 16
containing 0.1mM of ALA was added, and cells were allowed to take up ALA for 30 minutes.
17
The medium containing ALA was removed; the cells were rinsed and then submerged with 18
phosphate-buffered saline. Irradiation of the cells within monolayer culture was performed 19
using an incoherent light source with 3 or 6 J/cm2 (Omnilux revive, 633nm, 20
Phototherapeutics, Montgomeryville, PA).
21 22
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2. Stimulation of fibroblasts with keratinocyte-conditioned medium 1
The FBs were stimulated with the KC-conditioned medium taken 48 h after PDT. After 2
3. Stimulation of melanocytes with keratinocyte-conditioned medium or fibroblast-6
conditioned medium 7
The MCs were stimulated with the KC-conditioned medium or FB-conditioned medium 8
taken 24 h after PDT. The cell pellets were taken at 48h after stimulation to determine 9
melanin content and dopa oxidase activity.
10 11
4. Assessment of cell viability 12
Cell viability was determined using the tetrazolium dye colorimetric test (MTT assay).
13
The MTT absorbance was then read at 570 nm. Using a phase-contrast microscope, the 14
representative images were taken.
15 16
5. Western blot analysis 17
Upon PDT, cells were lysed in M2 buffer (20mM Tris at pH 7, 0.5% NP-40, 250mM 18
NaCl, 3mM EDTA, 3mM EGTA, 2mM DTT, 0.5mM PMSF, 20mM b-glycerol phosphate, 19
1mM sodium vanadate, and 1 mg/ml leupeptin). Equal amounts of cell extracts were 20
resolved by 12% SDS-PAGE and analyzed by western blotting and visualized by enhanced 21
chemiluminescence (Amersham, Buckinghamshire, UK) 22
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6. Melanin content assay 2
Melanin content was measured according to the method of Tsuboi et al. (Tsuboi et al., 3
1998) with a slight modification. MC was cultured in a 60-mm culture dish at a density of 4
5.0×105 cells. Twenty-four hours after PDT or stimulation with KC-conditioned medium/FB-5
conditioned medium, cells were solubilized in 1 M NaOH and the relative melanin content 6
was measured at 490 nm using an enzyme-linked immunosorbent assay reader. The 7
absorbance was compared with a standard curve of synthetic melanin (Sigma).
8 9
7. Dopa oxidase activity assay 10
Dopa oxidase activity was determined by the method described by Tomita et al. (Tomita 11
et al., 1992) with slight modification. The enzyme induction was assayed 12
spectrophotometrically using L-DOPA as the substrate. MCs were solubilized with 1% SDS 13
+ 1% Tween20 in PBS PH 6.8 and 1 mM L-DOPA (Sigma) and incubated at 37oC for 90 14
minutes. The absorbance at 490 nm was measured to calculate the enzyme activity.
15 16
8. Enzyme-linked immunosorbent assay (ELISA) 17
KC and FB were cultured at a density of 3.0×105 cells/well in 6 well plates. The 18
secretion levels of cytokines including IL-1α, IL-6, TNF-α, TGF-β, SCF, ET-1, GM-CSF, 19
HGF, and bFGF in the conditioned medium were measured by ELISA kits (R&D Systems, 20
Minneapolis, MN, USA) at 24 and 48 h after PDT. The secretion levels are expressed as 21
pg/ml.
22
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9. Real-time polymerase chain reaction (PCR) 2
real-time PCR using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, 6
Foster, CA, USA) according to the manufacturer’s protocol and the 2–∆∆Ct method.
7
Normalization was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 8
for mRNA normalization by calculating ∆Ct = (∆Ctsample − ∆CtGAPDH). The calibrator sample 9
(∆Ct calibration) was assigned from the control group. Relative mRNA levels were 10
calculated by the expression 2–∆∆Ct, where ∆∆Ct= (∆Ctsample − ∆Ctcalibration). Primers and 11
internal probes for MMP-1, MMP-3, TGF-β and collagen type Iα were purchased as assays 12
on demand primer-probe sets (Applied Biosystems, Foster, CA, USA).
13
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melanocytes which induces activation of ERK 4
(data not shown). Therefore, I increased the dose of red light to obtain proper condition of 8
low-level PDT in KC and MC which induces activation of ERK similar to FB. PDT with 9
0.1mM of ALA and 6 J/cm2 of red light resulted in prolonged activation of phospho-ERK up 10
to 8 h in KC and up to 4h in HaCaT cell compare to control group and ALA alone group.
11
Cytotoxicity to KC and HaCaT cell was not observed (Fig. 1A and 1B). In this condition, 12
there was no significant change in the proliferation of KC and HaCaT cell in the PDT group 13
compared to the control group after 24 and 48 h (Fig. 1C). PDT with same condition (0.1mM 14
of ALA and 6 J/cm2 of red light) also induced activation of phospho-ERK up to 4 h in MC 15
compare to control group and ALA alone group. However, the protein of phospho-p38 and 16
phospho-JNK was not detected in all three groups. Cytotoxicity to MC was not observed 17
(Fig. 2A). In this condition, there was a no significant change in the cellular viability of MC 18
in the PDT group compared to control group after 24 and 48h (Fig. 2B). Because KC and 19
MC exists in the epidermis, KC and MC will receive more lights than FB in the dermis 20
during clinical application of PDT. Therefore, KC and MC may need higher dose of red light 21
than FB due to this structural position of these cells in the skin. Throughout this study, this 22
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“low level PDT” condition was used; 0.1mM of ALA and 6 J/cm2 of red light to KC and MC;
1
0.1mM of ALA, 3 J/cm2 of red light to FB.
2 3
- 11 - 1
2
Fig. 1. The determination of the optimal “low level-PDT” condition of keratinocytes 3
and HaCaT cell which induced activation of ERK. PDT with 0.1mM of ALA and 6 J/cm2 4
of red light induced prolonged activation of phospho-ERK up to 8 h in KC and up to 4h in 5
HaCaT cell. Cytotoxicity to KC and HaCaT cell was not observed (A and B). In this 6
condition, there was no significant change in the proliferation of KC and HaCaT cell in the 7
PDT group compared to the control group after 24 and 48 h (C).
8 9
- 12 - 1
2
Fig. 2. The determination of the optimal “low level-PDT” condition of melanocytes 3
which induced activation of ERK. PDT with 0.1mM of ALA and 6 J/cm2 of red light 4
induced prolonged activation of phospho-ERK up to 4 h in melanocytes without cytotoxicity 5
(A). In this condition, there was no significant difference in the cellular viability of 6
melanocytes in the PDT group compared to the control group after 24 and 48 h (B).
7 8 9
- 13 -
B. Indirect effect of photodynamic therapy on the fibroblasts through keratinocytes-1
fibroblasts interaction 2
In photoaging process, increased secretion of inflammatory cytokines such as TGF-β 3
induces expression of MMP-1 and MMP-3 which results in degradation of collagen I (Shin 4
et al., 2005). In previous report about photorejuvenation mechanism of PDT, the authors 5
examined direct effect of PDT on FB. In that report, PDT induced increase of MMP-3 and 6
collagen type Iα through prolonged activation of ERK. Increased MMP-3 may induce 7
breakage of old collagen and allow new collagen synthesis (Jang et al., 2013). In the present 8
study, the indirect effect of PDT on FB through KC-FB interaction was examined by using 9
KC-conditioned medium. The changes in MMP-1, MMP-3, TGF-β and collagen type Iα was 10
evaluated by real-time PCR after stimulation with KC-conditionied medium. FB were 11
stimulated with KC-conditioned medium (taken 48 h after PDT) for up to 24 h. FB were also 12
stimulated with supernatants of untreated KC to evaluate as controls. MMP-1 mRNA 13
significantly decreased in PDT group compared with control at 24 h (Fig. 3A). The mRNA 14
of MMP-3 has a tendency to decrease in PDT group compare to control up to 24 h. However, 15
it was not statistically significant (Fig. 3B). Although it was not significant, TGF-β mRNA 16
level has a tendency to increase after PDT at 6 and 12h (Fig. 3C). The mRNA of collagen 17
type Iα is significantly increased in PDT group compare to control at 12 h (Fig. 3D).
18 19
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Fig. 3. Time dependent changes of MMP-1, 3, TGF-β, and Collagen type Iα mRNA 2
levels in fibroblasts after stimulation with keratinocyte-conditioned medium taken after 3
photodynamic therapy. MMP-1 mRNA significantly decreased in PDT group compared 4
with control at 24 h (A). MMP- 3 mRNA has a tendency to decrease in PDT group compared 5
with control at 6, 12 and 24h (B). TGF-β mRNA level has a tendency to increase after PDT 6
at 6 and 12h (C). Collagen Iα mRNA level is significantly increased after PDT at 12 h (D).
7
Data represent the fold increase of mRNA over the untreated control (control is set=1) and 8
determined by four independent experiments for each case. KCM= fibroblasts stimulated 9
with keratinocyte-conditioned medium without PDT, KCM-PDT= fibroblasts stimulated 10
with keratinocyte-conditioned medium taken after PDT. *p < 0.05.
11 12
- 15 -
C. Changes of the amount of fibroblast stimulating cytokines secreted by keratinocytes 1
after photodynamic therapy 2
Cytokine of KC after PDT (0.1mM of ALA, 6 J/cm2 of red light) was determined by 3
ELISA in KC supernatants. The level of cytokines including IL-1α, IL-6, TNF-α and TGF-β 4
were evaluated 24 and 48 h after PDT. A significant elevation of IL-1α, IL-6, and TNF-α 5
level in KC was noted after PDT compared with the untreated controls (Fig. 4). TGF-β was 6
not significantly altered after PDT compared to the untreated control group.
7
It is possible that an elevated IL-1α, IL-6, and TNF-α may increase expression of 8
MMPs which induce degradation of old damaged collagen fibers. This phenomenon may 9
lead the FB to initiate formation of new collagen fibers to replace them.
10 11
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Fig. 4. Production of fibroblast stimulating cytokines in keratinocytes after 2
photodynamic therapy. A significant elevation of IL-1α, IL-6, and TNF-α level in 3
keratinocytes was noted after PDT compared with the untreated controls (A-C). TGF-β was 4
not significantly altered after PDT compared to the untreated control group (D). Data 5
represent the fold increase of cytokine over the untreated control (control is set=1) and 6
determined by five independent experiments for each case.*p < 0.05. Con=control group.
7 8 9
- 17 -
D. Direct effect of photodynamic therapy on the melanocytes 1
The melanin content and dopa oxidase activity in MC after PDT was examined to 2
determine the direct effect of PDT on the MC. After PDT, significantly reduced melanin 3
content was noted in PDT group compared to control group after 24 and 48h. Reduced 4
melanin content was also visualized in cell pellets (Fig. 5A). The cell number was not 5
changed by PDT. In addition, dopa oxidase activity was significantly decreased in PDT 6
group compared to the untreated control after 24 and 48h (Fig. 5B).
7 8
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Fig. 5. Reduced melanin content and dopa oxidase activity in melanocytes after 2
photodynamic therapy. After PDT, significantly reduced melanin content was noted in PDT 3
group compared to the control group after 24 and 48h (A). Dopa oxidase activity was also 4
significantly reduced in PDT group compared to the untreated control after 24 and 48h (B).
5
*p < 0.05, **p < 0.01.
6 7 8
- 19 -
E. Indirect effect of photodynamic therapy on the melanocytes through keratinocyte-1
melanocyte interaction 2
The indirect effect of PDT on MC through KC-MC interaction was examined using 3
KC-conditioned medium. The changes of melanin content and dopa oxidase activity were 4
evaluated in MC after stimulation with KC-conditioned medium (taken 24 h after PDT) for 5
48 h. As controls, MC was also stimulated with vehicle (keratinocyte medium itself; Epilife 6
medium) or untreated conditioned medium. MC stimulated with PDT treated KC-7
conditioned medium showed a significant reduction of melanin content and dopa oxidase 8
activity compared to the control groups (Fig. 6).
9 10
- 20 - 1
2
Fig. 6. Reduced melanin content and dopa oxidase activity in melanocytes stimulated 3
with keratinocyte-conditioned medium after photodynamic therapy. Significantly 4
reduced melanin content was noted in the melanocytes stimulated with KCM taken after 5
PDT compared to controls after 48h (A). Dopa oxidase activity was also significantly 6
reduced in the melanocytes stimulated with KCM taken after PDT compared to the control 7
groups after 48h (B). Data were determined by five independent experiments for each case.
8
Vehicle=keratinocyte medium itself (Epilife medium), KCM=keratinocyte-conditioned 9
medium, Unt=untreated, PDT=photodynamic therapy. *p < 0.05, ** p < 0.01.
10 11 12 13 14
- 21 -
F. Changes in the amount of melanocytes stimulating cytokines secreted by 1
keratinocytes after photodynamic therapy 2
The MC stimulating cytokines secreted by KC after PDT was determined by ELISA in 3
KC supernatants. The level of cytokines including SCF, ET-1 and GM-CSF were evaluated 4
at 24 and 48 h after PDT. The level of SCF in KC-conditioned medium was significantly 5
decreased at 24 and 48 h after PDT compared with the untreated controls (Fig. 7A). ET-1 and 6
GM-CSF were not significantly altered after PDT compared to the untreated control group 7
(Fig. 7B and 7C). Therefore, decreased secretion of SCF in KC may play crucial role in 8
inhibiting melanin synthesis in MC after PDT.
9 10
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2
Fig. 7. Production of melanocytes stimulating cytokines in keratinocytes after 3
photodynamic therapy. Significantly reduced level of SCF in keratinocytes was noted at 24 4
and 48 h after PDT compared with the untreated controls (A). ET-1 and GM-CSF were not 5
significantly changed after PDT compared to the untreated controls (B and C). Data 6
represent the fold increase of cytokine over the untreated control (control is set=1) and 7
determined by four to seven independent experiments for each case. *p < 0.05.
8 9 10 11 12
- 23 -
G. Indirect effect of photodynamic therapy on the melanocytes through fibroblast-1
melanocyte interaction 2
The indirect effect of PDT on MC through FB-MC interaction was evaluated by using 3
FB-conditioned medium. The level of melanin content and dopa oxidase activity were 4
examined in MC after stimulation with FB-conditioned medium (taken 24 h after PDT) for 5
48 h. As controls, MC was also stimulated with vehicle (1:1 mixture of fibroblast medium 6
and melanocyte medium) or untreated FB-conditioned medium. Contrast to result E, 1:1 7
mixture of FB medium and MC medium was used instead of FB medium alone, because MC 8
was not growing well in FB medium. MC stimulated with PDT treated FB-conditioned 9
medium showed a significant reduction of melanin content and dopa oxidase activity 10
compared with the control groups (Fig. 8).
11 12
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2
Fig. 8. Reduced melanin content and dopa oxidase activity in melanocytes stimulated 3
with fibroblast-conditioned medium after photodynamic therapy. Significantly reduced 4
melanin content was noted in the melanocytes stimulated with FCM taken after PDT 5
compared to the controls after 48h (A). Dopa oxidase activity was also significantly reduced 6
in the melanocytes stimulated with FCM taken after PDT compared with the controls after 7
48h (B). Data were determined by five independent experiments for each case. Vehicle=1:1 8
mixture of fibroblast medium and melanocyte medium, FCM=fibroblast-conditioned 9
medium, Unt=untreated, PDT=photodynamic therapy. *p < 0.05, ** p < 0.01.
10 11
- 25 -
H. Changes in the amount of melanocytes stimulating cytokines secreted by fibroblasts 1
after photodynamic therapy 2
The MC stimulating cytokines secreted by FB after PDT was determined by ELISA in 3
FB supernatants. The level of cytokines including SCF, HGF and bFGF were evaluated at 24 4
and 48 h after PDT. The HGF in FB-conditioned medium was significant decreased at 48 h 5
after PDT compared to the untreated control (Fig. 9A). The SCF was not significantly altered 6
after PDT compared to the untreated control (Fig. 9B). The bFGF, detected at very low level, 7
also showed no significant change after PDT compared with the untreated control (Fig. 9C).
8
Therefore, decreased secretion of HGF in FB may play a valuable part in inhibiting melanin 9
synthesis in MC after PDT.
synthesis in MC after PDT.