Low-Temperature Argon Plasma Regulates Skin Moisturizing and Melanogenesis-Regulating Markers through Yes-Associated Protein

Extensive water loss and melanin hyperproduction can cause various skin disorders. Low-temperature argon plasma (LTAP) has shown the possibility of being used for the treatment of various skin diseases, such as atopic dermatitis and skin cancer. However, the role of LTAP in regulating skin moisturizing and melanogenesis has not been investigated. In this study, we aimed to determine the effect of LTAP on yes-associated protein (YAP), a major transcriptional coactivator in the Hippo signaling pathway that is involved in skin moisturizing and melanogenesis-regulating markers. In normal human epidermal keratinocytes (NHEKs), the human epidermal keratinocyte line HaCaT, and human dermal fibroblasts (HDFs), we found that LTAP exhibited increased expression levels of YAP protein. In addition, the expression levels of filaggrin (FLG), which is involved in natural moisturizing factors (NMFs), and hyaluronic acid synthase (HAS), transglutaminase (TGM), and involucrin (IVL), which regulate skin barrier and moisturizing, were also increased after exposure to LTAP. Furthermore, collagen type I alpha 1 and type III alpha 1 (COL1A1, COL3A1) were increased after LTAP exposure, but the expression level of matrix metalloproteinase-3 (MMP-3) was reduced. Moreover, LTAP was found to suppress alpha-melanocyte stimulating hormone (α-MSH)-induced melanogenesis in murine melanoma B16F10 cells and normal human melanocytes (NHEMs). LTAP regulates melanogenesis of the melanocytes through decreased YAP pathway activation in a melanocortin 1 receptor (MC1R)-dependent manner. Taken together, our data show that LTAP regulates skin moisturizing and melanogenesis through modulation of the YAP pathway, and the effect of LTAP on the expression level of YAP varies from cell to cell. Thus, LTAP might be developed as a treatment method to improve the skin barrier, moisture content, and wrinkle formation, and to reduce melanin generation.


Introduction
The skin protects the body from trauma, regulates body temperature, and balances moisture and electrolytes. Moisture in the skin circulates from the blood to the dermal and epidermal layers, reduces transepidermal water loss, and increases the thickness and density of the skin to prevent aging factors such as pigmentation and wrinkles [1]. Repeated exposure to ultraviolet (UV) rays creates elasticity and wrinkles in the skin and causes dehydration of cells [2]. Dehydration of skin cells decreases skin barrier functions [3]. Natural moisturizing factor (NMF) synthesis-related proteins such as transglutaminase (TGM), filaggrin (FLG), and involucrin (IVL) play a pivotal role in skin moisturizing and barrier function [4][5][6]. Similarly, hyaluronic acid (HA) is known to increase moisture content in the skin by regulating hyaluronic acid synthase (HAS)-encoding genes [7]. TGM catalyzes the formation of isopeptide bonds between proteins to prevent water loss, and

LTAP Induces Skin Barrier and Moisturizing Factors
We first investigated the effect of LTAP exposure for 1, 3, or 5 min on the skin barrier and NMF synthesis-related gene expression in HaCaT cells and NHEKs. On LTAP exposure, the expression levels of skin barrier and moisturizing-related genes, including TGM, FLG, IVL, and HAS-1, 2 were found to increase at the protein and transcript levels in both HaCaT cells and NHEKs ( Figure 1A HaCaT cells and NHEKs were exposed to LTAP for 1, 3, or 5 min, and further incubated for 24 h. (A) Expression of filaggrin (FLG) and involucrin (IVL) was determined by Western blotting. Numerical values on the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments. (B) Expression of skin barrier and moisturizing-related genes (tansglutaminases (TGM), FLG, IVL and hyaluronic acid synthase-1, 2 (HAS-1, 2)) was measured by quantitative real-time PCR (qRT-PCR) in HaCaT cells and NHEKs. Results are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (control vs. LTAP-exposed cells).

Effects of LTAP on the Skin Proliferation and Aging-Related Gene
Next, we aimed to determine the effect of LTAP exposure on proliferation and aging factors in HDFs. Transforming growth factor-β (TGF-β) is a cytokine that promotes collagen synthesis and regulates the vascular endothelial growth factor (VEGF)-encoding gene, which is involved in cell proliferation and skin tissue regeneration [17,18]. Here, we found that LTAP exposure increases the expression levels of TGF-β and Collagen type I/III, alpha 1 (COL1A1, COL3A1), and decreases the matrix metallopeptidase-3 (MMP-3) expression at protein level ( Figure 2A). Moreover, LTAP exposure also increases the expression levels of TGF-β, VEGF and COL1A1, COL3A1, and decreases the expression level of MMP-3 at transcript level ( Figure 2B). HaCaT cells and NHEKs were exposed to LTAP for 1, 3, or 5 min, and further incubated for 24 h. (A) Expression of filaggrin (FLG) and involucrin (IVL) was determined by Western blotting. Numerical values on the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments. (B) Expression of skin barrier and moisturizing-related genes (tansglutaminases (TGM), FLG, IVL and hyaluronic acid synthase-1, 2 (HAS-1, 2)) was measured by quantitative real-time PCR (qRT-PCR) in HaCaT cells and NHEKs. Results are expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (control vs. LTAP-exposed cells).

Effects of LTAP on the Skin Proliferation and Aging-Related Gene
Next, we aimed to determine the effect of LTAP exposure on proliferation and aging factors in HDFs. Transforming growth factor-β (TGF-β) is a cytokine that promotes collagen synthesis and regulates the vascular endothelial growth factor (VEGF)-encoding gene, which is involved in cell proliferation and skin tissue regeneration [17,18]. Here, we found that LTAP exposure increases the expression levels of TGF-β and Collagen type I/III, alpha 1 (COL1A1, COL3A1), and decreases the matrix metallopeptidase-3 (MMP-3) expression at protein level ( Figure 2A). Moreover, LTAP exposure also increases the expression levels of TGF-β, VEGF and COL1A1, COL3A1, and decreases the expression level of MMP-3 at transcript level ( Figure 2B). . HDFs were exposed to LTAP for 1, 3, or 5 min, and further incubated for 24 h. (A) Expression levels of transforming growth factor-β (TGF-β), matrix metallopeptidase-3 (MMP-3), and collagen type I alpha 1 (COL1A1) were analyzed by Western blotting. β-actin was used as a loading control. Numerical values on the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments. (B) mRNA expression analysis for TGF-β, vascular endothelial growth factor (VEGF), MMP-3, COL1A1, and COL3A1 was performed by qRT-PCR. Data represent three independent experiments and are the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (control vs. LTAP-exposed cells).

The Skin Moisturizing and Aging Factors are Mediated by YAP
To understand the mechanism by which LTAP affects skin moisturizing and aging factors, HaCaT cells, NHEKs, and HDFs were exposed to LTAP for 1, 3, or 5 min, and then further incubated for 24 h. As shown in Figure 3A, LTAP increased the activation of YAP protein and significantly higher phosphorylation levels were observed in the group exposed for 5 min compared with exposure for 1 or 3 min. Verteporfin (VP), an inhibitor of YAP, is a sensitizer approved by the U.S. Food and Drug Administration for use in photodynamic therapy for age-related macular degeneration [19]. HaCaT cells and HDFs were treated with VP at concentrations of 5 and 10 μM and incubated for 24 h. The expression levels of both the phosphorylated and total form of YAP were found to be inhibited with VP treatment ( Figure 3B). In addition, the mRNA expression level of the skin barrier and moisturizing-related genes and factors related to aging were found to be suppressed on VP treatment in HaCaT cells and HDFs, respectively ( Figure 3C,D). LTAP regulates proliferating and aging factors in human dermal fibroblasts (HDFs). HDFs were exposed to LTAP for 1, 3, or 5 min, and further incubated for 24 h. (A) Expression levels of transforming growth factor-β (TGF-β), matrix metallopeptidase-3 (MMP-3), and collagen type I alpha 1 (COL1A1) were analyzed by Western blotting. β-actin was used as a loading control. Numerical values on the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments. (B) mRNA expression analysis for TGF-β, vascular endothelial growth factor (VEGF), MMP-3, COL1A1, and COL3A1 was performed by qRT-PCR. Data represent three independent experiments and are the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (control vs. LTAP-exposed cells).

The Skin Moisturizing and Aging Factors Are Mediated by YAP
To understand the mechanism by which LTAP affects skin moisturizing and aging factors, HaCaT cells, NHEKs, and HDFs were exposed to LTAP for 1, 3, or 5 min, and then further incubated for 24 h. As shown in Figure 3A, LTAP increased the activation of YAP protein and significantly higher phosphorylation levels were observed in the group exposed for 5 min compared with exposure for 1 or 3 min. Verteporfin (VP), an inhibitor of YAP, is a sensitizer approved by the U.S. Food and Drug Administration for use in photodynamic therapy for age-related macular degeneration [19]. HaCaT cells and HDFs were treated with VP at concentrations of 5 and 10 µM and incubated for 24 h. The expression levels of both the phosphorylated and total form of YAP were found to be inhibited with VP treatment ( Figure 3B). In addition, the mRNA expression level of the skin barrier and moisturizing-related genes and factors related to aging were found to be suppressed on VP treatment in HaCaT cells and HDFs, respectively ( Figure   HDFs were treated with either 5 or 10 μM VP, and further incubated for 24 h. Cells were harvested for mRNA isolation, and expression of YAP, TGF-β, VEGF, COL1A1 and COL3A1 was determined by qRT-PCR. Data represent the mean ± SD. ** p < 0.01, *** p < 0.001. All data represent three independent experiments.

YAP Overexpression or Knockdown in HaCaT Cells Regulates Skin Barrier and Moisturizing Factors
To further evaluate the YAP-mediated regulation of the skin barrier and moisture regulation, we transfected HaCaT cells with YAP plasmid and small interfering RNA (siRNA) and determined the expression levels of the skin barrier and moisturizing-related genes. As shown in Figure 4A  NHEKs, and HDFs were exposed to LTAP for 1, 3, or 5 min, and further incubated for 24 h. Cells were harvested and the levels of phosphorylated YAP (pYAP), and YAP were evaluated by Western blotting. β-actin was used as a loading control. (B) HaCaT cells were treated with either 5 or 10 µM verteporin (VP), and further incubated for 24 h. The levels of pYAP and YAP were evaluated by Western blotting. Numerical values on the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments. (C) The expression of YAP, TGM, FLG, IVL and HAS-1, 2 in HaCaT cells was analyzed by qRT-PCR. (D) HDFs were treated with either 5 or 10 µM VP, and further incubated for 24 h. Cells were harvested for mRNA isolation, and expression of YAP, TGF-β, VEGF, COL1A1 and COL3A1 was determined by qRT-PCR. Data represent the mean ± SD. ** p < 0.01, *** p < 0.001. All data represent three independent experiments.

YAP Overexpression or Knockdown in HaCaT Cells Regulates Skin Barrier and Moisturizing Factors
To further evaluate the YAP-mediated regulation of the skin barrier and moisture regulation, we transfected HaCaT cells with YAP plasmid and small interfering RNA (siRNA) and determined the expression levels of the skin barrier and moisturizing-related genes. As shown in Figure 4A  Data are the means ± SD and represent three independent experiments. ** p < 0.01, *** p < 0.001.

LTAP Inhibits Melanin Production in B16F10 Cells and NHEMs
To understand the effect of LTAP on melanogenesis, we exposed B16F10 cells and NHEMs to LTAP for 1, 3, or 5 min. Our results showed that the melanin content in B16F10 cells was significantly decreased on LTAP exposure ( Figure 5A,B). The main factors that produce melanin are microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related proteins-1, 2 (TYRP-1, TYRP-2) [20]. Here, we found that LTAP exposure significantly decreased the expression levels of MITF, TYR and TYRP-1, but not TYRP-2, in both B16F10 cells and NHEMs ( Figure 5C). Furthermore, the melaninproducing factors other than TYRP-2 were significantly reduced at the protein level in cells exposed for 5 min compared with cells exposed for 1 or 3 min in both B16F10 cells and NHEMs ( Figure 5D).

LTAP Inhibits Melanin Production in B16F10 Cells and NHEMs
To understand the effect of LTAP on melanogenesis, we exposed B16F10 cells and NHEMs to LTAP for 1, 3, or 5 min. Our results showed that the melanin content in B16F10 cells was significantly decreased on LTAP exposure ( Figure 5A,B). The main factors that produce melanin are microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and tyrosinase-related proteins-1, 2 (TYRP-1, TYRP-2) [20]. Here, we found that LTAP exposure significantly decreased the expression levels of MITF, TYR and TYRP-1, but not TYRP-2, in both B16F10 cells and NHEMs ( Figure 5C). Furthermore, the melaninproducing factors other than TYRP-2 were significantly reduced at the protein level in cells exposed for 5 min compared with cells exposed for 1 or 3 min in both B16F10 cells and NHEMs ( Figure 5D). (A) B16F10 cells were exposed to LTAP for 1, 3, or 5 min, and incubated further for 72 h. Cells were harvested and images were taken. (B) The melanin content in B16F10 cells and NHEMs was measured at 450 nm. * p < 0.05, ** p < 0.01, *** p < 0.001 (control vs. LTAP-exposed cells). (C) B16F10 cells and NHEMs were exposed to LTAP for 1, 3, or 5 min, and further incubated for 24 h. The expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein-1, 2 (TYRP-1, 2) were measured by qRT-PCR analysis. Data are the means ± SD and represent three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (control vs. LTAP-exposed cells). (D) Cells were exposed to LTAP for 1, 3, or 5 min, and incubated for 72 h. The expression of MITF, TYR and TYRP-1, 2 was evaluated by Western blotting. β-actin was used as a loading control. Numerical values on the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments. (A) B16F10 cells were exposed to LTAP for 1, 3, or 5 min, and incubated further for 72 h. Cells were harvested and images were taken. (B) The melanin content in B16F10 cells and NHEMs was measured at 450 nm. * p < 0.05, ** p < 0.01, *** p < 0.001 (control vs. LTAP-exposed cells). (C) B16F10 cells and NHEMs were exposed to LTAP for 1, 3, or 5 min, and further incubated for 24 h. The expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein-1, 2 (TYRP-1, 2) were measured by qRT-PCR analysis. Data are the means ± SD and represent three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (control vs. LTAP-exposed cells). (D) Cells were exposed to LTAP for 1, 3, or 5 min, and incubated for 72 h. The expression of MITF, TYR and TYRP-1, 2 was evaluated by Western blotting. β-actin was used as a loading control. Numerical values on the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments.

LTAP Inhibits Receptors and Genes Involved in Melanin Production and Melanoma Proliferation
The proliferation and differentiation of melanoblasts is regulated by regulatory factors, such as endothelin (EDN) and Wingless-type protein (WNT) [21], and melanocortin 1 receptor (MC1R) is reported to be a major regulator of human pigmentation and a melanoma-related gene [22]. To elucidate the mechanism of the anti-melanogenesis effect of LTAP, we exposed B16F10 cells to LTAP and determined the expression levels of MC1R, EDN1, and WNT by Western blot analysis. As shown in Figure 6A, exposure to LTAP inhibited the expression of MC1R, EDN1, and WNT. Alpha-melanocyte stimulating hormone (α-MSH) is a ligand of MC1R that is known to activate the downstream protein kinase A (PKA), cyclic adenosine monophosphate (cAMP), and mitogen-activated protein kinase (MAPK) signaling pathways [23,24]. Here, we found that LTAP exposure suppressed the α-MSH-mediated activation of downstream PKA, cAMP response element-binding protein (CREB), and MAPK signaling, including of p38, extracellular signal-regulated kinase (ERK), and c-Jun-N-terminal kinase (JNK) (Figure 6B,C). In addition, to elucidate the anti-proliferative mechanism of LTAP, we determined the expression levels of cell cycle-related proteins. We found that LTAP exposure down-regulated the expression of Cyclin A, Cyclin B1, Cyclin D1, Cyclin-dependent kinase 1 (CDK1), and CDK2 ( Figure 6D). 1 receptor (MC1R) is reported to be a major regulator of human pigmentation and a melanoma-related gene [22]. To elucidate the mechanism of the anti-melanogenesis effect of LTAP, we exposed B16F10 cells to LTAP and determined the expression levels of MC1R, EDN1, and WNT by Western blot analysis. As shown in Figure 6A, exposure to LTAP inhibited the expression of MC1R, EDN1, and WNT. Alpha-melanocyte stimulating hormone (α-MSH) is a ligand of MC1R that is known to activate the downstream protein kinase A (PKA), cyclic adenosine monophosphate (cAMP), and mitogen-activated protein kinase (MAPK) signaling pathways [23,24]. Here, we found that LTAP exposure suppressed the α-MSH-mediated activation of downstream PKA, cAMP response element-binding protein (CREB), and MAPK signaling, including of p38, extracellular signal-regulated kinase (ERK), and c-Jun-N-terminal kinase (JNK) (Figure 6B,C). In addition, to elucidate the antiproliferative mechanism of LTAP, we determined the expression levels of cell cycle-related proteins. We found that LTAP exposure down-regulated the expression of Cyclin A, Cyclin B1, Cyclin D1, Cyclin-dependent kinase 1 (CDK1), and CDK2 ( Figure 6D).

Melanogenesis Related Genes Are Regulated by Verteporine on YAP Inhibitors
Next, we conducted an experiment to find out the association between LTAP and YAP in B16F10 and NHEM cells. Interestingly, we found that exposure of LTAP inhibited the expression levels of YAP in both B16F10 and NHEM cells ( Figure 7A). In addition, VP, an inhibitor of YAP, was found to downregulate the expression of YAP and melanogenesis regulating factors such as MC1R and MITF in B16F10 cells ( Figure 7B). To further elucidate the inhibitory mechanism, we investigated the effect of VP on the SRY (sex determining region Y)-box 10 (SOX10) and paired box gene 3 (PAX3) transcription factors that regulate melanoma markers such as protein melan-A (MART-1) and MITF expressed in melanoma cells [25][26][27]. Our result showed that the gene expression levels of MC1R, MITF, MART-1, SOX10 and PAX3 were all significantly suppressed by VP in a concentration-dependent manner ( Figure 7C). These results showed that the anti-melanogenesis effect of LTAP is mediated by YAP.

Overexpression and Knockdown of YAP Regulates Melanogenesis-Related Molecule Expression in B16F10 Cells
To confirm YAP-mediated regulation of melanogenesis-related molecules, we either over-expressed YAP in B16F10 cells from a plasmid or knocked down its expression using siRNA. As Figure 8A,B show, the overexpression of YAP in B16F10 cells significantly increased the expression of MITF, TYR and TYRP-1 at both proteins and mRNA levels. However, LTAP did not affect the expression level of TYRP-2. Knockdown of the YAP gene suppressed the mRNA and protein levels of MITF, TYR, and TYRP-1 but not TYRP-2 ( Figure 8C,D). These data indicate that the amount of YAP expression in B16F10 cells and NHEMs regulates the growth of melanoma cells and melanogenic factors in melanocytes. Overall, the data suggests that LTAP may be an effective treatment for melanoma and hyperpigmentation through regulation of YAP.
cidate the inhibitory mechanism, we investigated the effect of VP on the SRY (sex determining region Y)-box 10 (SOX10) and paired box gene 3 (PAX3) transcription factors that regulate melanoma markers such as protein melan-A (MART-1) and MITF expressed in melanoma cells [25][26][27]. Our result showed that the gene expression levels of MC1R, MITF, MART-1, SOX10 and PAX3 were all significantly suppressed by VP in a concentration-dependent manner ( Figure 7C). These results showed that the anti-melanogenesis effect of LTAP is mediated by YAP. increased the expression of MITF, TYR and TYRP-1 at both proteins and mRNA levels. However, LTAP did not affect the expression level of TYRP-2. Knockdown of the YAP gene suppressed the mRNA and protein levels of MITF, TYR, and TYRP-1 but not TYRP-2 ( Figure 8C,D). These data indicate that the amount of YAP expression in B16F10 cells and NHEMs regulates the growth of melanoma cells and melanogenic factors in melanocytes. Overall, the data suggests that LTAP may be an effective treatment for melanoma and hyperpigmentation through regulation of YAP.

Discussion
This study investigated the effects of LTAP exposure on the skin barrier, moisturizing function, and the mechanisms that potentially mediate this effect. As shown in Figure 1, exposure of HaCaT cells and NHEKs to LTAP increased the expression of skin barrier and moisturizing-related genes (TGM, FLG, IVL, and HAS-1, 2) that enhance moisturizing power. In addition, exposure of HDFs to LATP increased the expression of TGF-β, which is known to promote collagen synthesis, and VEGF, which is involved in cell proliferation and regeneration [18]. The expression level of MMP-3 was suppressed, and the expression levels of COL1A1 and COL3A1 were increased (Figure 2). These results suggest that LTAP up-regulates the expression of TGM, FLG, IVL, and HAS-1, 2 in keratinocytes, supplying moisture to the epidermal layer, which would help to maintain a stronger skin barrier and prevent skin aging and wrinkle formation.
It was reported that LTAP exposure improves the transdermal transduction of epidermal growth factors by regulating E-cadherin-mediated cell junctions [28]. In that study LTAP exposure inhibited the expression of E-cadherin, an adhesive molecule, in skin cells, inducing a temporary decrease in skin barrier function, thereby improving transdermal delivery of drugs and cosmetics. Inhibition of E-cadherin expression and reduction in skin barrier function were reported to be completely recovered within 3 h of LTAP exposure.
FLG is another protein involved in the skin barrier and has a function similar to that of E-cadherin, but the mechanism of interaction between the two molecules has not been accurately established. Indeed, much research has been done on E-cadherin and FLG, and there are conflicting reports about their interactions. In one study, a decrease in the expression of E-cadherins and occludin was reported to be due to FLG and loricrin deficiency; in another, inhibition of E-cadherin function induced FLG and loricrin expression [29,30]. For clear interpretation of our results, further experiments on the interaction of FLG and E-cadherin should be conducted.
The Hippo pathway plays an important role in controlling cell proliferation and size. YAP, an effector of the Hippo pathway, is expressed in the epidermis and is known to be an important gene for skin cell development and proliferation [31][32][33]. Studies show that YAP expression is significantly up-regulated during cutaneous wound healing and is localized to the keratinocyte nucleus around the wound [34], and that deletion of YAP reduced skin expansion [35,36]. It has been found that adhesion to the adhesion junction and the basement membrane plays an important role in the regulation of epidermal YAP activity [32,34]. VP, an inhibitor of YAP, has been reported to inhibit proliferation and induce G0/G1 arrest in pancreatic ductal adenocarcinoma cells, hepatocellular carcinoma cells, melanoma cells and leukemia cells [37][38][39][40]. However, few studies have focused on skin barrier and moisturizing-related and aging-related factors. Our data show that LTAP can enhance the activation of YAP protein, and the use of VP downregulated skin moisturizing and aging factors in skin cells (Figure 3). Similarly, overexpression of YAP stimulated the expression of skin barrier and moisturizing-related factors, whereas knockdown of YAP resulted in the downregulation of such genes (Figure 4). Taken together, our results indicate that skin barrier and moisturizing-related and aging-related genes are mediated by YAP, and that YAP plays an important role in maintaining skin homeostasis. Thus, we believe that LTAP can be considered as a therapy for cutaneous wound healing and maintenance of moisture content in skin through the regulation of YAP.
As mentioned above, YAP maintains skin homeostasis and regulates various types of cancer cells; the antitumor activity of VP has been reported in numerous studies [37][38][39][40]. Among them, malignant melanoma is the most fatal skin cancer, with a high risk of metastasis. There is evidence that YAP contributes to maintaining the transforming phenotype of melanoma cells [41,42], and VP contributes to the inhibition of melanoma cell proliferation [19,43]. However, little is known about the mechanism by which YAP regulates melanoma function. Here, we investigated the effects of LTAP exposure on YAP expression and anti-melanogenesis in B16F10 cells and NHEMs. LTAP exposure significantly inhibited mRNA and protein levels of the factors involved in anti-melanogenesis (MITF, TYR, and TYRP-1). However, it did not affect the expression level of TYRP-2 ( Figure 5).
Melanogenesis in the skin is mediated through several melanogenesis signaling pathways, including MAPK signaling, and cAMP and protein kinase C-mediated pathways. Upregulation of intracellular cAMP activates PKA, which phosphorylates CREB, resulting in the increased expression of MITF [44]. In this study, we demonstrated that LTAP inhibited melanogenesis, its effect was mediated via the PKA, MAPK and CREB signaling pathways, and this effect inhibited cell proliferation ( Figure 6). These results are in good agreement with the previously reported effects of microplasma on melanoma cell death, although the types of cells and plasma are different [16,45,46]. MITF is known as a central regulator of melanoma cell survival, proliferation, and differentiation [47,48], and the factors that regulate it include MC1R, MART-1, SOX10 and PAX3 [25][26][27]. Our data confirmed that LTAP exposure downregulated MITF and YAP at the protein level in both B16F10 cells and NHEMs. In addition, the expression levels of MC1R, MART-1, SOX10 and PAX3, which are transcription factors that regulate MITF, were significantly suppressed by VP treatment in a concentration-dependent manner (Figure 7). In fact, it has been reported that YAP depletion reduces the expression of MITF [48], and we have specifically demonstrated a mechanism for the antitumor activity of VP. In addition, YAP overexpression and knockdown were investigated to determine the role of YAP in the factors related to melanogenesis in B16F10 cells. YAP overexpression resulted in a significant increase in the expression level of melanogenesis-related factors, whereas YAP knockdown resulted in significant down-regulation of melanogenesis-related factors at both the protein and mRNA levels ( Figure 8). Taken together, our results demonstrate that LTAP mediates YAP to inhibit the proliferation of melanoma cells and regulate anti-melanogenesis activity. However, this was an in vitro system, and further research will be needed to see if these observations extend in vivo.
Our study investigated the role of an LTAP device in regulating skin moisture content, aging factors, melanoma cell proliferation and anti-melanogenesis in different types of skin cells. In summary, our results show that, as summarized in Figure 9, LTAP significantly increased the barrier, moisture content-related, and aging factors of skin cells, and suppressed melanogenesis in melanoma cell lines through YAP regulation. These results suggest that LTAP has the potential to be developed as an effective device to control skin moisturization, and for anti-melanogenesis.

LTAP Device
A microwave plasma generator was used for plasma exposure of cells and was kindly provided by Professor Gyu-Chun Kim of Pusan National University, Republic of Korea. For plasma generation, a 900 MHz coaxial transmission line resonator and ~2.5 standard L/min of argon gas were used together. The microwave plasma jet had a radius of 2.5 mm and a length of 7 mm, and the bulk power density of the plasma jet was estimated to be 14.55 W.mm −3 . More information on this device can be found in ref 28.

LTAP Device
A microwave plasma generator was used for plasma exposure of cells and was kindly provided by Professor Gyu-Chun Kim of Pusan National University, Republic of Korea. For plasma generation, a 900 MHz coaxial transmission line resonator and~2.5 standard L/min of argon gas were used together. The microwave plasma jet had a radius of 2.5 mm and a length of 7 mm, and the bulk power density of the plasma jet was estimated to be 14.55 W.mm −3 . More information on this device can be found in ref [28]. CCT TGC TGC TCT ACC TCC A, 3 -GAT GGC AGT AGC TGC GCT GAT A; human  MMP-3, 5 -CAC TCA CAG ACC TGA CTC GGT T, 3 -AAG CAG GAT CAC AGT TGG  CTG G; human COLA1A, 5 -GAT TCC CTG GAC CTA AAG GTG C, 3 -AGC CTC TCC  ATC TTT GCC AGC A; human COLA3A1, 5 -TGG TCT GCA AGG AAT GCC TGG A,  3 -TCT TTC CCT GGG ACA CCA TCA G; human MITF, 5 -ACC TTC TCT TTG CCA GTC  CA, 3 -TTG GGC TTG CTG TAT GTG GT; human TYR, 5 -CTT GTG AGC TTG CTG TGT  CG, 3 -GTG AGG TCA GGC TTT TTG GC; human TYRP-1, 5 -ATG TCG CTC AGT GCT  TGG AA, 3 -GAC TTC GAA CAG CAG GGT CA; human TYRP-2, 5 -AAC TCC CTT CCC  TGC ATG TG, 3 -TTG TGA CCA TAG GGG CCA G;

Western Blotting
Western blotting was performed as previously described [20]. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Elpis-biotech, Daejeon, Republic of Korea), and proteins were detected by enhanced chemiluminescence (Advansta, San Jose, CA, USA) using the LAS 3000 detection system

Statistical Analysis
The results of this study were repeated three or more times. Statistical significance was evaluated by one way analysis of variance, and the data represent the mean ± SD. Statistically significant differences were considered as p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.