Effects of Extremely Low Frequency Electromagnetic Fields on Melanogenesis through p-ERK and p-SAPK/JNK Pathways in Human Melanocytes

Abstract

This study evaluated frequency-dependent effects of extremely low frequency electromagnetic fields (ELF-EMFs) on melanogenesis by melanocytes in vitro. Melanocytes were exposed to 2 mT EMFs at 30-75 Hz for 3 days before melanogenesis was examined. Exposure to ELF-EMFs at 50 and 60 Hz induced melanogenic maturation without cell damage, without changing cell proliferation and mitochondrial activity. Melanin content and tyrosinase activity of cells exposed to 50 Hz were higher than in controls, and mRNA expression of tyrosinase-related protein-2 was elevated relative to controls at 50 Hz. Phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) levels were higher than controls in cells exposed to ELF-EMFs at 50-75 Hz. Immunohistochemical staining showed that melanocyte-specific markers (HMB45, Melan-A) were strongly expressed in cells exposed to EMFs at 50 and 60 Hz compared to controls. Thus, exposure to ELF-EMFs at 50 Hz could stimulate melanogenesis in melanocytes, through activation of p-CREB and p-p38 and inhibition of phosphorylated extracellular signal-regulated protein kinase and phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase. The results may form the basis of an appropriate anti-gray hair treatment or be applied in a therapeutic device for inducing repigmentation in the skin of vitiligo patients.

Keywords: MITF; extremely low frequency electromagnetic fields (ELF-EMFs); melanogenesis; p-CREB; tyrosinase.

Figure 1

Photograph of the electromagnetic field (EMF) device. Pulsed EMF was generated using a pair of Helmholtz coils of 40 cm diameter and 15 cm separation.

Figure 2

Melanocyte morphology after stimulation by an extremely low frequency electromagnetic field (ELF-EMF) for 72 h. All groups were cultured in M254 media. Before ELF-EMF exposure, cells were cultured in modified M254 media containing forskolin, melanocyte-stimulating hormone (α-MSH), and 12-O-tetradecanoyl-phorbol-13-acetate for 72 h. After culturing in differentiation media, cells were treated with continuous ELF-EMFs. The α-MSH group was cultured in M254 media added to 5 nM α-MSH. (A) control; (B) α-MSH; (C) 30 Hz; (D) 50 Hz; (E) 60 Hz; and (F) 75 Hz. Original magnification was ×100; bar = 100 μm.

Figure 3

Cell proliferation, mitochondrial activity, and cytotoxicity of melanocytes after 72 h exposure to extremely low frequency electromagnetic fields. (A) Cell count (to measure cell proliferation); (B) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (to determine cell mitochondrial activity); (C) Lactate dehydrogenase (LDH) assay (to examine cellular damage). Each bar represents mean ± standard error from independent experiments performed in triplicate (n = 5). * p < 0.05, compared to the control.

Figure 4

Effect of extremely low frequency electromagnetic fields (ELF-EMFs) on melanogenesis of melanocytes after exposed to ELF-EMFs for 72 h. After ELF-EMFs exposure, melanin content and tyrosinase activity were determined. (A) Melanin content was detected by the melanin content assay; (B) Tyrosinase activity was measured by the tyrosinase assay. Each bar represents the mean ± standard error of independent experiments performed in triplicate (n = 3). * p < 0.05, ** p < 0.01, compared to the control.

Figure 5

Gene expression detected by reverse transcription polymerase chain reaction (RT-PCR) on melanocytes after exposure to extremely low frequency electromagnetic fields for 72 h. (A) Electrophoretic RT-PCR for melanogenesis-related genes; (B) mRNA expression of melanogenesis-related genes, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference protein. Each bar represents the mean ± standard error of independent experiments performed in triplicate (n = 5). * p < 0.05, compared to the control (Cont).

Figure 6

Western blot of cyclic adenosine monophosphate response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) expression in melanocytes after exposure to extremely low frequency electromagnetic fields for 72 h. (A) CREB and phosphorylated-CREB (p-CREB) banding; (B) p-CREB expression; (C) ERK and phosphorylated ERK (p-ERK) expression; (D) p-ERK expression. Each bar represents the mean ± standard error of independent experiments performed in triplicate (n = 5). * p < 0.05, compared to the control (Cont).

Figure 7

Western blot of stress-activated protein kinase/c-Jun N-terminal (SAPK/JNK) and p38 expression in melanocytes after exposure to extremely low frequency electromagnetic fields for 72 h. (A) Phosphorylated-SAPK (p-SAPK)/JNK and phosphorylated-38 (p-p38) banding; (B) p-SAPK/JNK expression; (C) p-p38 expression. Each bar represents the mean ± standard error of independent experiments performed in triplicate (n = 3). ** p < 0.01, compared to the control (Cont).

Figure 8

Western blot of protein expression levels detected in melanocytes after exposure to extremely low frequency electromagnetic fields for 72 h. (A) Melanogenesis-related protein banding, using β-actin as an internal control; (B) Melanogenesis-related protein expression. Each bar represents the mean ± standard error of independent experiments performed in triplicate (n = 5). * p < 0.05, compared to the control (Cont).

Figure 9

Light microscope photograph of human skin melanocytes attached to dish surface, stained with HMB45 and Melan-A after exposure to extremely low frequency electromagnetic field (ELF-EMF) for 72 h. (A–F) with HMB45 staining: (A) Control (Cont); (B) alpha-melanocyte-stimulating hormone (α-MSH); (C) 30 Hz; (D) 50 Hz; (E) 60 Hz; and (F) 75 Hz. (G–L) with Melan-A staining: (G) Control; (H) α-MSH; (I) 30 Hz; (J) 50 Hz; (K) 60 Hz; and (L) 75 Hz. Original magnification was ×400; bar = 50 μm. Quantitative analysis of expression levels of HMB45 (M) and Melan-A (N) after ELF-EMFs exposure. Each bar represents the mean ± standard error of independent experiments performed in triplicate (n = 4). ** p < 0.01, compared to the control.

Figure 10

The proposed mechanism by which an extremely low frequency electromagnetic field (ELF-EMF) induces melanin biosynthesis. Schematic shows that ELF-EMFs increases expression of microphthalmia-associated transcription factor (MITF) in human melanocytes. (p-CREB: phosphorylated cAMP response element-binding protein, p38: protein 38kDa, p-JNK: phosphorylated c-Jun N-terminal kinase, p-ERK: phosphorylated extracellular signal-regulated protein kinase, MITF microphthalmia-associated transcription factor, Tyr: tyrosinase, TRP: tyrosinase-related protein)