Radiofrequency Irradiation Attenuated UVB-Induced Skin Pigmentation by Modulating ATP Release and CD39 Expression

Introduction, Page 2, Line 43 in the revised manuscript

The skin has protective roles as a barrier organ. The skin also has a neuroendocrine function that stimulates the central nervous, endocrine, and immune systems to coordinate body homeostasis [1–3]. External stimuli, such as ultraviolet (UV) radiation lead to the upregulation of various cytokines, uro-cortins, corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), and enkephalins, which further stimulate various skin responses, including melanogenesis [1–4].

Introduction, Page 15, Line 567 in the revised manuscript

2.          Slominski, A.; Tobin, D.J.; Shibahara, S.; Wortsman, J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 2004, 84, 1155–1228.

3.          Slominski, R.M.; Sarna, T.; Płonka, P.M.; Raman, C.; Brożyna, A.A.; Slominski, A.T. Melanoma, Melanin, and Melanogenesis: The Yin and Yang Relationship. Front Oncol 2022, 12, 842496.

Result, Page 4, Line 127 in revised manuscript

2.1. RF irradiation decreased ATP release from UV-radiated keratinocytes

First, we evaluated whether RF irradiation decreased ATP release from UVB-exposed keratinocytes. After human keratinocytes were subjected to UVB radiation, RF irradiation was performed. Forty-eight hours after RF irradiation, ATP levels in the supernatant of keratinocyte were measured (Figure S1A). The ATP level was significantly increased by UVB radiation and significantly de-creased by RF treatment (Figure 1A).

Result, Page 4, Line 148 in revised manuscript

2.2. RF decreased the expression of CD39, CD73 and the A_2A/A_2BARs in melanocytes and UV-irradiated animal skin

Next, we evaluated whether RF irradiation decreased the expressions of CD39, CD73, and A_2A/A_2BARs in melanocytes by decreasing ATP release from keratinocytes. The conditioned media (CM) obtained from CON-, UVB-, or UVB and RF-irradiated keratinocytes were used to treat human melanocytes (Figure S1B).

The expression of CD39 and CD73 in melanocytes was significantly increased with treatment of CM from UVB-irradiated keratinocytes (CM-UVB) and significantly decreased with treatment of CM from UVB- and RF-irradiated keratinocytes (CM-UVB/RF) (Figures 1B–1D).

The expression of the A_2A/A_2BARs in melanocytes significantly increased under treatment with CM-UVB and significantly decreased under treatment with CM-UVB/RF (Figures 1B, 1E, and 1F).

The expression of CD39 and CD73 was also increased by UVB radiation in the skin of animals subjected to UVB radiation compared with that of control animals that did not receive UVB radiation. The expressions of CD39 and CD73 were significantly decreased by RF irradiation (Figures 2A–2C).

Moreover, the expression of A_2A/A_2BARs was increased in the skin of animals subjected to UVB radiation and decreased by RF irradiation (Figure 2A, 2D, and 2E).

Figure 1, Page 4, Line 134 in revised manuscript

Figure 1. CD39/CD73/A_2AAR/A_2BAR reduction in melanocytes due to ATP release from UVB/RF-exposed keratinocytes. HEKn were not exposed or exposed to UVB for 5 min (CON or UVB group), and then RF was irradiated after exposure to UVB (UVB/RF group). (A) ATP release in the CON group, UVB group and UVB/RF group was verified using an ATP release assay. (B–F) HEMn were treated with a supernatant of HEKn (CM-CON), UVB-exposed HEKn (CM-UVB), or UVB and RF-treated HEKn (CM-UVB/RF) for 48 h. (B) The protein expression of CD39/CD73/A_2AAR/A_2BAR in CM-treated HEMn was measured by western blot. (C–F) Quantitative graph for western blot of (B). Data are presented as the mean ± SD of three independent experiments. **, p < 0.01; ***, p < 0.001, second bar vs. first bar; $$, p < 0.01, third bar vs. second bar (Mann–Whitney U test). A_2AAR, A_2A adenosine receptor; A_2BAR, A_2B adenosine receptor; ATP, adenosine triphosphate; CD39, ectonucleoside triphosphate diphosphohydrolase-1; CD73, 5′-nucleotidase; CM, conditioned media; CON, control; HEKn, human epidermal primary keratinocytes; h, hours; HEMn, human epidermal primary melanocytes; MW, molecular weight; RF, radiofrequency; UVB, ultraviolet B.

Figure 2, Page 5, Line 169 in revised manuscript

Figure 2. RF irradiation reduced CD39/CD73/A_2AAR/A_2BAR expression in UVB-exposed mouse skin. Mice were either not exposed or exposed to UVB 9 times for 5 min each for 14 d (CON or UVB group), and then RF irradiation was applied (UVB/RF group). (A) The protein expression of CD39/CD73/A_2AAR/A_2BAR in UVB/RF-exposed mouse skin was validated using western blot. (B–E) Quantitative graph for western blot of (A). Data are presented as the mean ± SD of three independent experiments. ***, p < 0.001, second bar vs. first bar; $$, p < 0.01; $$$, p < 0.001, third bar vs. second bar (Mann–Whitney U test). A_2AAR, A_2A adenosine receptor; A_2BAR, A_2B adenosine receptor; CD39, ectonucleoside triphosphate diphosphohydrolase-1; CD73, 5′-nucleotidase; CM, conditioned media; CON, control; d, days; min, minutes; MW, molecular weight; RF, radiofrequency; UVB, ultraviolet B.

Result, Page 8, Line 245 in revised manuscript

Next, we evaluated whether RF irradiation decreased melanin synthesis. Tyrosinase activity in melanocytes was increased by the administration of CM-UVB. Upon silencing CD39, tyrosinase activity was not increased by the administration of CM-UVB compared with the CON. The increased tyrosinase activity caused by the administration of CM-UVB was significantly decreased by CM-UVB/RF treatment. However, among silencing CD39 melanocyte, tyrosinase activity was not different when either CM-UVB or CM-UVB/RF were treated (Figure 5A).

The melanin content in melanocytes was increased by the administration of CM-UVB. Upon silencing CD39, melanin content was not increased by the administration of CM-UVB compared with that of the CON. The increased melanin content by administration of CM-UVB was significantly decreased by CM-UVB/RF administration. However, among silencing CD39 melanocyte, melanin content was not different following treatment by either CM-UVB or CM-UVB/RF (Figure 5B).

Figure 5, Page 9, Line 266 in revised manuscript

Figure 5. RF irradiation reduced tyrosinase activity and melanin accumulation through regulation of CD39. (A­ and B) HEMn were treated with the supernatant of HEKn cells (CM-CON), UVB-exposed HEKn (CM-CON), or UVB- and RF-treated HEKn (CM-UVB/RF) for 48 h. To confirm the regulation by CD39, after silencing CD39, HEMn were treated with CM-UVB (siCD39/CM-UVB) or CM-UVB/RF (CM-siCD39/UVB/RF). (A) The tyrosinase activity in CM-treated HEMn was measured using a tyrosinase activity assay. (B) Melanin accumulation was confirmed by a melanin assay in CM-treated HEMn. (C–E) Mice were either not exposed or exposed to UVB 9 times for 5 min each for 14 d (CON or UVB group), and then RF was applied (UVB/RF group). (C) The tyrosinase activity in mouse skin was measured by tyrosinase activity assay. (D) Quantitative graph for Fontana Masson staining (E). (E) Melanin accumulation was determined by Fontana Masson staining in mouse skin. (F) Summary. Data are presented as the mean ± SD of three independent experiments. ***, p < 0.001, second bar vs. first bar; $$, p < 0.01; $$$, p < 0.001, vs. second bar, #, p < 0.05, vs. third bar (Mann–Whitney U test). cAMP, cyclic adenosine monophosphate; CD39, ectonucleoside triphosphate diphosphohydrolase-1; CD73, 5′-nucleotidase; CM, conditioned media; CON, control; CREB, cyclic adenosine monophosphate response element-binding protein; MFN2, mitofusin 2; MITF, microphthalmia‑associated transcription factor; MW, molecular weight; OPA1, optic atrophy type 1; pDRP1, phosphorylation of dynamin-related protein 1; RF, radiofrequency; siCD39, silencing CD39; UVB, ultraviolet B.

Discussion, Page 10, Line 299 in revised manuscript

The purpose of our study was to evaluate whether RF could decrease UV-B induced melanogenesis via decreasing ATP release from keratinocytes. Thus, we designed an in vitro model in which CM from UVB-irradiated keratinocytes was administered to melanocytes. First, we sought to evaluate whether UV radiation increased ATP release from keratinocytes. ATP levels in the supernatant from UVB-irradiated keratinocytes increased compared with those of control keratinocytes (Figure 1A). Next, we evaluated how elevated ATP secretion from keratinocytes affects melanocytes to increase melanogenesis. Upon treating melanocytes with CM from UVB-irradiated keratinocytes, the expressions of CD39, CD73, and A_2A/A_2BARs increased (Figures 1B–1F). Moreover, AC activity and cAMP levels were increased by CM from UVB-irradiated keratinocytes. It seemed that UVB radiation increased ATP release from keratinocytes, and released extracellular ATP was converted into adenosine by CD39 and CD73, which stimulated A_2A/A_2BARs. The upregulation of A_2A/A_2BARs led to increased AC activity and increased cAMP levels in melanocytes (Figures 3A and 3B). Moreover, increased cAMP was accompanied by an increased expression of PKA in melanocytes treated with CM from UVB-irradiated keratinocytes (Figure 3C).

Discussion, Page 10, Line 316 in revised manuscript

Our results also show that increased tyrosinase activity and melanin contents in melanocytes via treatment with CM from UVB radiated keratinocytes. When CD39 was silenced by siCD39, tyrosinase activity and melanin content were not increased by treatment with CM from UVB radiated keratinocytes (Figures 5A–5B). This result suggests that melanogenesis triggered in melanocytes by ATP secretion from keratinocytes could be mediated by CD39. To the best of our knowledge, this is the first study that shows the role of CD39 and ARs in UV radiation-induced melanogenesis. UV radiation also led to increased expressions of CD39, CD73, and A_2A/A_2BARs, as well as AC activity, cAMP levels, and PKA expression in animal skin (Figures 2 and 3D–G). Moreover, melanogenesis-related factors such as CREB and MITF were increased in UV-irradiated animal skin (Figures 4A, 4H, and 4I).

Discussion, Page 11, Line 355 in revised manuscript

Since the phosphorylation of DRP1 at Ser637 via cAMP-mediated PKA activation is known to downregulate DRP1 and eventually lead to mitochondrial elongation [27], we evaluated whether UV-induced DRP1 phosphorylation at Ser637 modulates mitochondrial dynamics via cAMP-mediated PKA activation. UV-irradiated animal skin had an increased expression of phosphorylated DRP at Ser637, and RF irradiation decreased this expression (Figures 4B and 4C). We did not directly evaluate mitochondrial morphology using any imaging technique. However, mitochondrial fusion-related proteins such as OPA1 and MFN2 were increased by UV radiation in animal skin and decreased by RF irradiation (Figures 4B, 4D, and 4E). Although we did not observe mitochondrial elongation with imaging, we could infer that increased phosphorylation of DRP at Ser637 affects mitochondrial dynamics.

Since mitochondrial fission activates ERK and degrades MITF [31], we also evaluated ERK changes after RF irradiation. UVB radiation decreased the pERK1/2/ERK1/2 ratio, which was increased by RF irradiation (Figure 4F–4G).

Material and Method, Page 11, Line 361 in revised manuscript

HEKn (ATCC, Manassas, VA, USA) were cultivated in a Dermal Cell Basal Medium (ATCC) containing a keratinocyte growth kit (ATCC) in an incubator at 37 °C with 5% CO₂. When HEKn reached 70% confluence, RF (2 MHz, 10 W/100 ms) was applied after exposure to UVB (200 mJ/cm²) for 5 min. UVB used an instrument with a peak wavelength of 306 nm (G15T8E; SANKYO DENKI, Yokohama, Japan). RF-treated HEKn were cultured in an incubator at 37 °C with 5% CO₂ for 48 h, and the cell lysate and supernatant (conditioned media, CM) were obtained (Figure S1A).

Introduction, Page 3, Line 104 in revised manuscript

Radiofrequency (RF) is a form of electromagnetic energy that generates molecular agitation in tissues [35]. Moreover, a conducted electric current caused by RF is transformed into heat [35]. Since heat generated by RF stimulates collagen synthesis by increasing the expression of various heat shock proteins (HSPs), such as HSP47 and HSP70, RF has been used for skin rejuvenation [36,37].

Materials and Method, Page 10, Line 348 in revised manuscript

4.1. Radiofrequency irradiation system

A radiofrequency device (POTENZA, Jeisys, Seoul, Korea) was used in this study. It has an impedance checking and feedback system which was used to determine the compensation value by automatically measuring impedance. The most common configurations of electrode systems are monopolar, bipolar, and multipolar, including fractional systems where this effect is achieved by the superposition of RF current paths between paired electrodes. Bipolar radiofrequency energy was delivered to the skin surface and epidermis via a noninvasive electrode tip, SFA tip. The tip consisted of 192 electrodes with a diameter of 0.2 mm arranged at a spacing of 1 mm, and it is designed to deliver RF energy at a shallow depth. The RF conditions was treated 10W, 100 ms, and 2 pulse. Additionally, the frequency was 2 MHz, and the wavelength is inversely proportional to the frequency [53].

Introduction, Page 2, Line 50 in revised manuscript

Melanogenesis is a complex process that produces melanin in the melanocytes. The initial steps of melanogenesis involve the hydroxylation of phenylalanine to L-tyrosine [3–5]. L-tyrosine is then hydroxylated to L-dihydroxyphenylalanine (L-DOPA) by tyrosinase. Tyrosinase further oxidizes L-DOPA to L-DOPAquinone [3–5]. Depending on the presence of cysteine, L-DOPAquinone changes into either yellow-to-reddish pheomelanin or brown-to-blackish eumelanin [6–8]. Without cysteine, L-DOPAquinone is changed into DOPAchrome by the tyrosinase‑related protein (TRP)‑1 and TRP‑2, which is then further synthesized to eumelanin [6–8].

UV upregulates tumor suppressor protein p53, which further stimulates POMC. POMC are cleaved into adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (MSH), β-MSH, and γ-MSH [9–14].

Secreted α-MSH binds to a melanocortin receptor (MC1R) in melanocytes, which promotes the dissociation of the α subunit and eventually upregulates the activity of adenylate cyclase (AC) [15]. ACs generate the second messenger cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) [15]. cAMP leads to the increased activity of the cAMP-dependent protein kinase (PKA) and the transcription factor cAMP response element-binding protein (CREB), which sequentially increases the expression of microphthalmia-associated transcription factor (MITF) [16].

MITF is the main regulator of melanogenesis, modulating the survival, proliferation, and growth of melanocytes [17]. MITF also stimulates tyrosinase, TRP‑1, and TRP‑2 and eventually increases melanogenesis [17]. Aside from the cAMP-dependent pathway, Wnt/β-catenin, ERK/MAPK, and nitric oxide/cyclic guanosine monophosphate (cGMP) pathway lead to upregulation of MITF in melanogenesis [18].

Introduction, Page 2, Line 43 in revised manuscript

The skin has protective roles as a barrier organ. The skin also has a neuro-endocrine function that stimulates the central nervous, endocrine, and immune systems to coordinate body homeostasis [1]. External stimuli, such as ultraviolet (UV) radiation lead to the upregulation of various cytokines, urocortins, corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), and enkephalins, which further stimulate various skin responses, including melanogenesis [1,2].

Result, Page 7, Line 225 in revised manuscript

Additionally, pMITF and MITF expression levels increased in the skin of animals subjected to UVB radiation and decreased by RF irradiation (Figures 4H–4J).

Figure 4, Page 7, Line 228 in revised manuscript

Figure 4. RF application decreased CREB/pDRP1/OPA1/MFN2/MITF in UVB-exposed mouse skin. Mice were either not exposed or exposed to UVB 9 times for 5 min each for 14 d (CON or UVB group), and then RF was irradiated (UVB/RF group). (A) The mRNA levels of CREB in mouse skin were measured by qRT‒PCR. (B) The protein expression of pDRP1/OPA/MFN2 in mouse skin was measured by western blot. (C–E) Quantitative graph for western blot of (B). (F) The protein expression of pERK1/2/ERK1/2 in mouse skin was determined by western blot. (G) Quantitative graph of the western blot of (F). (H) The protein expression of pMITF and MITF in mouse skin were measured by western blot. (I–J) Quantitative graph for western blot of (H). Data are presented as the mean ± SD of three independent experiments. **, p < 0.01; ***, p < 0.001, second bar vs. first bar; $$, p < 0.01, third bar vs. second bar (Mann–Whitney U test). CON, control; CREB, cyclic adenosine monophosphate response element-binding protein; d, days; MFN2, mitofusin 2; min, minutes; MITF, microphthalmia‑associated transcription factor; MW, molecular weight; OPA1, optic atrophy type 1; pDRP1, phosphorylation of dynamin-related protein 1; pMITF, phosphorylation of microphthalmia‑associated transcription factor; qRT-PCR, quantitative real-time polymerase chain reaction; RF; RF, radiofrequency; UVB, ultraviolet B.

Introduction, Page 3, Line 104 in revised manuscript

Radiofrequency (RF) is a form of electromagnetic energy that generates molecular agitation in tissues [35]. Moreover, a conducted electric current caused by RF is transformed into heat [35]. Since heat generated by RF stimulates collagen synthesis by increasing the expression of various heat shock proteins (HSPs), such as HSP47 and HSP70, RF has been used for skin rejuvenation [36,37].

Materials and Method, Page 10, Line 348 in revised manuscript

4.1. Radiofrequency irradiation system

A radiofrequency device (POTENZA, Jeisys, Seoul, Korea) was used in this study. It has an impedance checking and feedback system which was used to determine the compensation value by automatically measuring impedance. The most common configurations of electrode systems are monopolar, bipolar, and multipolar, including fractional systems where this effect is achieved by the superposition of RF current paths between paired electrodes. Bipolar radiofrequency energy was delivered to the skin surface and epidermis via a noninvasive electrode tip, SFA tip. The tip consisted of 192 electrodes with a diameter of 0.2 mm arranged at a spacing of 1 mm, and it is designed to deliver RF energy at a shallow depth. The RF conditions was treated 10W, 100 ms, and 2 pulse. Additionally, the frequency was 2 MHz, and the wavelength is inversely proportional to the frequency [53].

Figure 5, Page 10, Line 265 in revised manuscript

Figure 5. RF irradiation reduced tyrosinase activity and melanin accumulation through regulation of CD39. (A­ and B) HEMn were treated with the supernatant of HEKn cells (CM-CON), UVB-exposed HEKn (CM-CON), or UVB- and RF-treated HEKn (CM-UVB/RF) for 48 h. To confirm the regulation by CD39, after silencing CD39, HEMn were treated with CM-UVB (siCD39/CM-UVB) or CM-UVB/RF (CM-siCD39/UVB/RF). (A) The tyrosinase activity in CM-treated HEMn was measured using a tyrosinase activity assay. (B) Melanin accumulation was confirmed by a melanin assay in CM-treated HEMn. (C–E) Mice were either not exposed or exposed to UVB 9 times for 5 min each for 14 d (CON or UVB group), and then RF was applied (UVB/RF group). (C) The tyrosinase activity in mouse skin was measured by tyrosinase activity assay. (D) Quantitative graph for Fontana Masson staining (E). (E) Melanin accumulation was determined by Fontana Masson staining in mouse skin (scale bar = 50 μm). (F) Summary. Data are presented as the mean ± SD of three independent experiments. ***, p < 0.001, second bar vs. first bar; $$, p < 0.01; $$$, p < 0.001, vs. second bar, #, p < 0.05, vs. third bar (Mann–Whitney U test). cAMP, cyclic adenosine monophosphate; CD39, ectonucleoside triphosphate diphosphohydrolase-1; CD73, 5′-nucleotidase; CM, conditioned media; CON, control; CREB, cyclic adenosine monophosphate response element-binding protein; MFN2, mitofusin 2; MITF, microphthalmia‑associated transcription factor; MW, molecular weight; OPA1, optic atrophy type 1; pDRP1, phosphorylation of dynamin-related protein 1; RF, radiofrequency; siCD39, silencing CD39; UVB, ultraviolet B.

Introduction, Page 2, Line 50 in revised manuscript

Melanogenesis is a complex process that produces melanin in the melanocytes. The initial steps of melanogenesis involve the hydroxylation of phenylalanine to L-tyrosine [3–5]. L-tyrosine is then hydroxylated to L-dihydroxyphenylalanine (L-DOPA) by tyrosinase. Tyrosinase further oxidizes L-DOPA to L-DOPAquinone [3–5]. Depending on the presence of cysteine, L-DOPAquinone changes into either yellow-to-reddish pheomelanin or brown-to-blackish eumelanin [6–8]. Without cysteine, L-DOPAquinone is changed into DOPAchrome by the tyrosinase‑related protein (TRP)‑1 and TRP‑2, which is then further synthesized to eumelanin [6–8].

UV upregulates tumor suppressor protein p53, which further stimulates POMC. POMC are cleaved into adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (MSH), β-MSH, and γ-MSH [9–14].

Secreted α-MSH binds to a melanocortin receptor (MC1R) in melanocytes, which promotes the dissociation of the α subunit and eventually upregulates the activity of adenylate cyclase (AC) [15]. ACs generate the second messenger cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) [15]. cAMP leads to the increased activity of the cAMP-dependent protein kinase (PKA) and the transcription factor cAMP response element-binding protein (CREB), which sequentially increases the expression of microphthalmia-associated transcription factor (MITF) [16].

MITF is the main regulator of melanogenesis, modulating the survival, proliferation, and growth of melanocytes [17]. MITF also stimulates tyrosinase, TRP‑1, and TRP‑2 and eventually increases melanogenesis [17]. Aside from the cAMP-dependent pathway, Wnt/β-catenin, ERK/MAPK, and nitric oxide/cyclic guanosine monophosphate (cGMP) pathway lead to upregulation of MITF in melanogenesis [18].

Discussion, Page 11, Line 286 in revised manuscript

Melanin in the skin, as a photoprotective pigment, absorbs UV and acts as scavenger against reactive oxygen/nitrogen species (ROS/RNS) [40]. Proper melanogenesis after UV exposure is essential for skin protection; however, the overproduction of melanin causes various cosmetic problems, such as senile lentigines, post-inflammatory hyperpigmentation, freckles, and dots [41].

Introduction, Page 2, Line 43 in revised manuscript

The skin has protective roles as a barrier organ. The skin also has a neuro-endocrine function that stimulates the central nervous, endocrine, and immune systems to coordinate body homeostasis [1]. External stimuli, such as ultraviolet (UV) radiation lead to the upregulation of various cytokines, urocortins, corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), and enkephalins, which further stimulate various skin responses, including melanogenesis [1,2].