Exposure to Radiofrequency Electromagnetic Fields Enhances Melanin Synthesis by Activating the P53 Signaling Pathway in Mel-Ab Melanocytes

Abstract

The skin is the largest body organ that can be physiologically affected by exposure to radiofrequency electromagnetic fields (RF-EMFs). We investigated the effect of RF-EMFs on melanogenesis; Mel-Ab melanocytes were exposed to 1760 MHz radiation with a specific absorption rate of 4.0 W/kg for 4 h/day over 4 days. Exposure to the RF-EMF led to skin pigmentation, with a significant increase in melanin production in Mel-Ab melanocytes. The phosphorylation level of cAMP response element binding protein (CREB) and the expression of microphthalmia-associated transcription factor (MITF), which regulate the expression of tyrosinase, were significantly increased in Mel-Ab after RF-EMF exposure. Interestingly, the expression of tyrosinase was significantly increased, but tyrosinase activity was unchanged in the RF-EMF-exposed Mel-Ab cells. Additionally, the expression of p53 and melanocortin 1 receptor (MC1R), which regulate MITF expression, was significantly increased. These results suggest that the RF-EMF induces melanogenesis by increasing phospho-CREB and MITF activity. Importantly, when Mel-Ab cells were incubated at 38 °C, the melanin production and the levels of tyrosinase significantly decreased, indicating that the increase in melanin synthesis by RF-EMF exposure is not due to a thermal effect. In conclusion, RF-EMF exposure induces melanogenesis in Mel-Ab cells through the increased expression of tyrosinase via the activation of MITF or the phosphorylation of CREB, which are initiated by the activation of p53 and MC1R.

Keywords: MC1R; MITF; melanogenesis; p53; phospho-CREB; radiofrequency electromagnetic fields; tyrosinase.

Figure 1

Cellular morphology and pigmentation of Mel-Ab melanocytes after RF-EMF exposure. The Mel-Ab cells were cultured for 4 d with or without 1760 MHz radiofrequency electromagnetic field (RF-EMF) exposure (at 4.0 W/kg for 4 h/d) and heat treatment (38 °C). (A) Cellular morphology was observed using a microscope (Olympus, CKX53, Tokyo, Japan). Red arrows indicate darkened Mel-Ab cells. The 200× magnified photos with 100 µm scale bar are shown. (B) The cell pellets of control, RF-EMF-exposed, and 38 °C-incubated Mel-Ab melanocytes (lower panel) and dark intensity of each pellet was quantified using the ImageJ software bundled with 64-bit Java 8 (upper panel). (C) Melanin content was measured in Mel-Ab cells under each condition. (D) Cell viability of Mel-Ab cells under each condition. Data are expressed as mean ± standard error of the mean. * p and # p < 0.05, ** p and ## p < 0.01 compared to control (n = 6). RF-EMF, radiofrequency electromagnetic field.

Figure 2

Expression levels of heat shock proteins in Mel-Ab melanocytes after RF-EMF exposure. Expression of Hsp27 (A), Hsp70 (B), and Hsp90 (C) mRNA transcripts determined by quantitative real-time PCR. The relative mRNA levels of heat shock proteins were calculated by normalizing to the expression of Gapdh, using the 2^−ΔΔCt method. Data are expressed as mean ± standard error of the mean. ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to control (n = 4). RF-EMF, radiofrequency electromagnetic field.

Figure 3

Expression levels of tyrosinase and tyrosine hydroxylase were increased by RF-EMF exposure. (a). Expressional quantification of tyrosinase (A) and tyrosine hydroxylase (B) using Western blotting. (b). The graphs show the quantification of protein levels of tyrosinase (A), and tyrosine hydroxylase (B) normalized to that of β-actin. (C). Tyrosinase activity was measured in each condition. The data indicate the mean ± standard error of the mean. Levels of statistical significance were evaluated using one-way ANOVA. *** p < 0.001, * p and # p < 0.05 compared to the control (n = 4). RF-EMF, radiofrequency electromagnetic field; TH, tyrosine hydroxylase.

Figure 4

Expression levels of p53 and Mc1r were significantly increased by RF-EMF exposure in Mel-Ab melanocytes. (A). Expressional quantification of p53 by Western blotting (a). The graphs show the quantification of protein levels of p53 normalized to those of β-actin (b). (B). The mRNA levels of Mc1r were analyzed by quantitative real-time PCR. The relative mRNA levels of Mc1r were calculated by normalizing to the expression of Gapdh by the 2^-ddCt method. Data are expressed as the mean ± standard error of the mean. * p < 0.05, *** p < 0.001 compared to control (n = 3). RF-EMF, radiofrequency electromagnetic field.

Figure 5

The expression level of p53 was significantly increased by RF-EMF exposure in HaCaT keratinocytes. HaCaT cells were exposed to RF-EMF or treated with 38 °C heat. (A). Expressional quantification of p53 using Western blotting. (B). The graphs show the quantification of protein levels of p53 normalized to those of β-actin. Data are expressed as the mean ± standard error of the mean. * p < 0.05 compared to control (n = 3). RF-EMF, radiofrequency electromagnetic field.

Figure 6

Expression levels of MITF and phospho-CREB were increased by RF-EMF exposure. Quantification of MITF (A), p-CREB (Ser133), and CREB (B) expression by Western blotting. Data are presented as mean ± standard error of the mean. Statistical significance was evaluated using one-way ANOVA; * p < 0.05, ** p < 0.01 compared to control (n = 6). RF-EMF, radiofrequency electromagnetic field.