Various Wavelengths of Light-Emitting Diode Light Regulate the Proliferation of Human Dermal Papilla Cells and Hair Follicles via Wnt/β-Catenin and the Extracellular Signal-Regulated Kinase Pathways

LED light irradiation

A LED device was obtained from Korea Electronics Technology Institute (KETI, Seongnam, Korea). Four types of LED light sources were manufactured: 415 nm (4.23 mW/cm²); 525 nm (3.85 mW/cm²); 660 nm (2.42 mW/cm²); and 830 nm (24.72 mW/cm²) with 1, 5, or 10 J (Table 1).

Table 1
Characteristics of light sources

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Cell culture and irradiation

hDPCs (Promocell; GmbH, Heidlberg, Germany) were cultured as previously described6. The DPCs (2×10⁴ cells per well) were seeded into 24-well culture plates and then irradiated with LED in 10% fetal bovine serum (Gibco BRL; Life Technology, Karlsruhe, Germany), 1% penicillin/streptomycin (Gibco BRL) containing Dulbecco's modified Eagle medium (DMEM) for 48 hours. DPCs were used passage from 3 to 4 for the experiments. For LED irradiation, DPCs were seeded in on culture dish plates with serum free-DMEM and then the plates were exposed to LED light at distances of 2 cm. All dishes used in the experiments (including controls) were maintained at room temperature and atmosphere during light irradiation.

Cell proliferation assay

Cell proliferation was determined using a MTT assay that was performed by a slight modification of the method described by Twentyman and Luscombe7. Briefly, DPCs were seeded at a density of 2×10⁴ cells/well into 24-well plates and irradiated with light. After 48 hours of incubation, the cells were treated with an MTT reagent for 4 hours. The samples were assessed by measuring absorbance at 540 nm with an ELISA plate reader.

Real time-polymerase chain reaction

Total RNA from the DPCs using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA synthesis with QuantiTect Rev. Transcription kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The cDNA used for real time-polymerase chain reaction (RT-PCR), which was carried out with SYBR Green (Takara, Shiga, Japan). The primers sequences and PCR conditions are listed in Table 2. The PCR product was quantified with the use of analysis software (Quantity one 1-D analysis; Bio-Rad, Hercules, CA, USA).

Table 2
Primers used for polymerase chain reaction amplications

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Western blot analysis

Cells were harvested and lysed with RIPA lysis buffer (Pierce, Rockford, IL, USA). Protein concentration in the cells was measured using Bradford reagent (Bio-Rad) with bovine serum albumin (BSA) as a standard. Cell lysates containing equal amounts of total protein were separated by electrophoresis on SDS-polyacrylamide gel and then transferred to a PVDF membrane. The membranes were blocked with 5% BSA in TBS-T and subsequently incubated with primary antibodies against phospho-extracellular signal-regulated kinase (ERK), total-ERK, phospho-c-Jun N-terminal kinase (JNK), total-JNK, phosphorylated MEK, Cyclin D1 β-catenin and β-actin were obtained from Cell signaling Technology, Inc. (Cell Signal, Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Specific reactive bands were detected with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and the immunoreactive bands were visualized by the enhanced chemiluminescence detection system (Millipore Corp., Bedford, MA, USA).

HFs organ culture and assessment of hair elongation

Anagen HFs from human scalp skin specimens was obtained. The medical ethical committee of the Catholic University of Korea approved specimen collection. Three HFs per well in 24-well plates were cultured in Williams E medium at 37℃ in a humidified atmosphere with 5% CO₂ in 500 µl of basal medium supplemented with insulin 10 µg/ml, hydrocortisone 10 ng/ml, streptomycin 10 µg/ml, and penicillin 100 U/ml according to the Philpott's method as described8. Each experimental group contained at least 60 anagen HFs derived from one donor.

Statistical analysis

All data were expressed as mean±standard deviation (SD). Student's t-test was used to compare data between two groups, and one-way analysis of variance (ANOVA) with post hoc Duncan's multiple comparison test was used to compare data among groups. We considered a two-sided p<0.05 statistically significant. Because these were exploratory analyses, no p-value adjustment was made for multiple comparisons. All statistical analyses were performed using SigmaPlot 12.3 software.

LED light stimulated the proliferation of hDPCs

We irradiated various wavelengths of 415 nm, 525 nm, 660 nm, and 830 nm light at doses of 1, 5 and 10 J/cm² on hDPCs and performed MTT assay at 48 hours. All four wavelengths (415 nm of 1, 5 and 10 J/cm², 525 nm of 1, 5 and 10 J/cm², 660 nm of 5 and 10 J/cm² and 830 nm of 1 and 5 J/cm²) significantly increased hDPC proliferation (Fig. 1). 660 nm at 10 J/cm² was the most potent in promoting hDPC proliferation, as dose increased from 5 to 10 J/cm² with a maximal effect at 10 J/cm² (Fig. 1C).

Fig. 1
Effects of various wavelengths and doses of light-emitting diode light (A: 415 nm, B: 525 nm, C: 660 nm, D: 830 nm) on human dermal papilla cell (hDPC) proliferation. The hDPCs (2×10⁴ cells/well) were cultured in serum-free Dulbecco's modified Eagle medium for 24 hours. Then the cultured cells were irradiated immediately with indicated doses and wavelengths, and then incubated for 48 hours. MTT assay was assessed at 48 hours and the relative level is shown as mean±standard deviation from triplicate samples. Statistically significant differences were determined by one-way ANOVA (^*p<0.05) compared to control (CTL).

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LED light regulated Wnt/β-catenin signaling in hDPCs

Next, we examined changes in the mRNA expression of β-catenin, Axin2, Wnt3a, Wnt5a and Wnt10b. Both 660 nm and 830 nm significantly enhanced β-catenin mRNA expression compared to control (Fig. 2A). All four wavelengths significantly increased Axin2 expression (Fig. 2B). Wnt3a expression was increased by 660 nm and 830 nm of 10 J/cm² (Fig. 2C) and Wnt5a expression was increased by 415nm of 1 J/cm² and 660nm of 5 J/cm² compared to control (Fig. 2D). Wnt10b mRNA expression was markedly enhanced as much as 10- to 15-fold by 525 nm at 10 J/cm², 660 nm at 5 and 10 J/cm² and 830 nm at all doses (Fig. 2E).

Fig. 2
Effects of light-emitting diode (LED) light on the mRNA expression of β-catenin (A), Axin2 (B), Wnt3a (C), Wnt5a (D) and Wnt10b (E) in human dermal papilla cells (hDPCs). Effects of LED irradiation on the protein expression of β-catenin (F) and cyclin D1 (G) in hDPCs. The hDPCs (2×10⁵ cells/well) were cultured in serum-free Dulbecco's modified Eagle medium for 24 hours and then irradiated with indicated doses and wavelengths, and incubated for 24 hours. The mRNA expression was examined by using real time-polymerase chain reaction and normalized against GAPDH (A~E). The protein level of β-catenin and cyclin D1 was examined by Western blot and normalized against β-actin expression (F, G). The relative level is shown as mean±standard deviation from triplicate samples. Statistically significant differences were determined by one-way ANOVA (^*p<0.05) compared to control (CTL).

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We also measured the protein expression of β-catenin and cyclin D1 in hDPCs. 660 nm at 5 J/cm² induced the maximum peak expression of β- and 525 nm at 5 J/cm² also increased β-catenin protein expression (Fig. 2F). 415 nm at 5 J/cm², 525 nm at 10 J/cm², 660 nm and 830 nm at all doses increased cyclin D1 protein expression (Fig. 2G).

LED light promoted the proliferation of hDPCs by activating ERK signaling pathway

LED light induced significant increase of mitogen-activated protein kinase kinase (MEK) and ERK phosphorylation (Fig. 3A, B), in contrast to JNK (Fig. 3C) and p38 MAPK (data not shown). MEK phosphorylation was profoundly increased as much as 5- to 6-fold by 660 nm and 830 nm at 5 J/cm² (Fig. 3A). ERK phosphorylation was highly increased as much as 5- to 8-fold by 415 nm at 10/cm², 525 nm at 10/cm², 660 nm at 5 and 10 J/cm², and 830 nm at 5 and 10 J/cm² (Fig. 3B).

Fig. 3
Effects of light-emitting diode (LED) light on the phosphorylation of MEK (A), extracellular signal-regulated kinase (ERK) (B), and c-Jun N-terminal kinase (JNK) (C) in human dermal papilla cells (hDPCs). Effects of treatment with PD98059 on the proliferation of hDPCs and the counteract effect of LED irradiation on the inhibitory effect of PD98059 on hDPC proliferation (D~H). The hDPCs (2×10⁵ cells/well) were cultured in serum-free Dulbecco's modified Eagle medium for 24 hours and then irradiated with indicated doses and wavelengths, and incubated for 24 hours (A~C). The protein level of phosphorylated form of MEK (MEK1/2) (A), ERK (ERK1/2) (B) and JNK (C) was examined by Western blot and the relative level is shown as mean±standard deviation (SD) from triplicate samples. Next, the hDPCs were pretreated with 20 µM of PD98059 for 1 hour, and then irradiated with indicated doses and wavelengths, and incubated for 72 hours (D~H). Positive control group in which LED irradiation without PD98059 was shown in (I~L). MTT assay was assessed at 72 hours and the relative level is shown as mean±SD from triplicate samples. Statistically significant differences were determined by one-way ANOVA (^*p<0.05) compared to CTL (A~C, I~K). Statistically significant differences were determined by t-test compared to CTL (^†p<0.05); one-way ANOVA (^#p<0.05) compared to PD98059 treatment group (D~H). CTL: control, pMEK: phosphorylated MEK, pERK: phosphorylated ERK, pJNK: phosphorylated JNK.

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Furthermore, to determine that ERK signaling was involved in hDPC proliferation by LED light, hDPCs were pretreated with 20 µM PD98059, an ERK inhibitor, prior to light irradiation. LED light without PD98059 promoted hDPC proliferation in all wavelengths and doses (Fig. 3I~L). In the presence of the PD98059, however, the proliferative effect of LED light on hDPCs was inhibited (Fig. 3D; Statistically significant differences were determined by t-test compared to CTL (^†p<0.05). 415 nm, 525 nm and 660 nm of all doses, and 830 nm of 1 and 5 J/cm² significantly counteracted the inhibitory effect of PD98059 on hDPC proliferation (Fig. 3E~H; Statistically significant differences were determined by one-way ANOVA (^#p<0.05) compared to PD98059 treatment group). Therefore, LED light promoted the hDPC proliferation by activating the ERK signaling pathway.

LED light promoted hair growth ex vivo in a culture model

We performed ex vivo culture of whole human scalp HFs. Thirty nine HFs from one individual were cultured with light of various wavelengths and doses for 3 days. Elongation of each hair shaft was measured under a microscope on 3 day. The relative length of each hair shaft is shown as mean±SD from three HFs in percent change compared to control (Fig. 4). As shown in Fig. 4, HFs irradiated with 415 nm of 1 J/cm² grew 9 % longer than control (Fig. 4A) and those with 660 nm of 1 J/cm² and 10 J/cm² grew 20% longer than control (Fig. 4C). HFs irradiated with 830 nm of 5 J/cm² grew 17% longer than control (Fig. 4D). 525 nm irradiation produced no significant effect on hair shaft elongation compared to control (Fig. 4B). Statistically significant differences were determined by t-test (^*p<0.05) compared to 0 day (0 day; non-treatment).

Fig. 4
Effects of various wavelengths and doses of light-emitting diode light (A: 415 nm, B: 525 nm, C: 660 nm, D: 830 nm) on the elongation of hair follicles (HFs) in ex vivo culture of whole human scalp HFs. Thirty nine HFs from one individual were cultured with light of various wavelengths and doses for 3 days. Elongation of each hair shaft was measured under a microscope on 3 day. The relative length of each hair shaft is shown as mean±standard deviation from three HFs in percent change compared to control. Statistically significant differences were determined by t-test (^*p<0.05) compared to 0 day (0 day; non-treatment). CTL: control.

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