Korean J Physiol Pharmacol.  2022 Mar;26(2):113-123. 10.4196/kjpp.2022.26.2.113.

Diarylpropionitrile inhibits melanogenesis via protein kinase A/cAMP-response element-binding protein/microphthalmiaassociated transcription factor signaling pathway in α-MSHstimulated B16F10 melanoma cells

• Lee HJ
• An S
• Bae S
• Lee JH

• KMID: 2526730
• DOI: http://doi.org/10.4196/kjpp.2022.26.2.113

Abstract

Diarylpropionitrile (DPN), a selective agonist for estrogen receptor β(ERβ), has been reported to regulate various hormonal responses through activation of ERβ in tissues including the mammary gland and brain. However, the effect of DPN on melanogenesis independent of ERβ has not been studied. The aim of this study is to examine the possibility of anti-melanogenic effect of DPN and its underlying mechanism. Melanin contents and cellular tyrosinase activity assay indicated that DPN inhibited melanin biosynthesis in alpha-melanocyte stimulating hormonestimulated B16F10 melanoma cell line. However, DPN had no direct influence on in vitro tyrosinase catalytic activity. On the other hand, 17β-estradiol had no effect on inhibition of melanogenesis, suggesting that the DPN-mediated suppression of melanin production was not related with estrogen signaling pathway. Immunoblotting analysis showed that DPN down-regulated the expression of microphthalmiaassociated transcription factor (MITF), a central transcription factor of melanogenesis and its down-stream genes including tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. Also, DPN attenuated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). Additionally, DPN suppressed the melanin synthesis in UVB-irradiated HaCaT conditioned media culture system suggesting that DPN has potential as an anti-melanogenic activity in physiological conditions. Collectively, our data show that DPN inhibits melanogenesis via downregulation of PKA/CREB/MITF signaling pathway.

Keyword

CREB; Diarylpropionitrile; Melanogenesis; MITF; PKA

Figure

• 

  Fig. 1 Cell viability of B16F10 melanoma cells after treatment with DPN. (A) Chemical structure of DPN. (B) B16F10 cells were treated with DPN (1–100 μM). Cells were further cultured for 24 h and 48 h, then the cell viability was determined by WST-1 assay. Data are shown as mean ± SD from three independent experiments. DPN, diarylpropionitrile; WST-1, water-soluble tetrazolum salt. *p < 0.05 were considered as statistically significant.

• 

  Fig. 2 Effect of DPN on melanogenesis in α-MSH-stimulated B16F10 cells. B16F10 cells were co-treated with α-MSH (100 nM) and DPN (20 and 50 μM). (A) After 48 h, the melanin contents were examined by measuring absorbance at 450 nm. (B) Cellular tyrosinase activities were examined by analyzing conversion of L-DOPA into dopachrome by measuring absorbance at 490 nm. (C) The effects of DPN (1–100 μM) on enzymatic activity of tyrosinase were examined using mushroom tyrosinase in cell free system. Tyrosinase activities were determined by analyzing conversion of L-DOPA into dopachrome. (D) B16F10 cells were co-treated with α-MSH (100 nM) and 17β-estradiol (1 and 10 μM) or α-MSH (100 nM) and DPN (50 μM). Cells were further cultured for 48 h and the melanin contents were determined by measuring absorbance at 450 nm. Arbutin (500 μM) was used as positive control. Data are shown as mean ± SD from three independent experiments. DPN, diarylpropionitrile; α-MSH, alpha-melanocyte stimulating hormone; DOPA, dihydroxyphenylalanine; ns, not significant. *p < 0.05 were considered as statistically significant from vehicle treated group. #p < 0.05 were considered as statistically significant from α-MSH-treated group.

• 

  Fig. 3 Effect of DPN on the expression of tyrosinase and its related genes in α-MSH-stimulated B16F10 cells. (A) B16F10 cells were co-treated with α-MSH (100 nM) and DPN (20 and 50 μM) for 24 h. The expression levels of tyrosinase, TRP-1 and TRP-2 were examined by immunoblotting with their specific antibodies. Actin was used as a loading control. Indicated immunoblot intensities were analyzed by Image-J. (B–D) The transcriptional expression levels of tyrosinase, TRP-1 and TRP-2 were examined by qRT-PCR. GAPDH was used for normalizing mRNA expression level. Data are shown as mean ± SD from three independent experiments. DPN, diarylpropionitrile; α-MSH, alpha-melanocyte stimulating hormone; TRP, tyrosinase-related protein. *p < 0.05 were considered as statistically significant from vehicle treated group. #p < 0.05 were considered as statistically significant from α-MSH-treated group.

• 

  Fig. 4 Effect of DPN on the cAMP/PKA/CREB signaling pathway in α-MSH-stimulated B16F10 cells. (A) B16F10 cells were co-treated with α-MSH (100 nM) and DPN (20 and 50 μM). After 24 h, the expression level of MITF was examined by immunoblotting. (B) Cells were co-treated with α-MSH (100 nM) and DPN (20 and 50 μM). The cells were further cultured for 24 h and mRNA expression level of MITF was examined by qRT-PCR. Data are shown as mean ± SD from three independent experiments. (C) The expressions of PKA, pPKA, CREB, pCREB, ERK, pERK, p38, and pp38 were examined by immunoblotting with their specific antibodies. Indicated immunoblot intensities were analyzed by Image-J. DPN, diarylpropionitrile; PKA, protein kinase A; CREB, cAMP-response element-binding protein; MITF, microphthalmia-associated transcription factor; α-MSH, alpha-melanocyte stimulating hormone; ERK, extracellular signal-regulated kinase. *p < 0.05 were considered as statistically significant from vehicle treated group. #p < 0.05 were considered as statistically significant from α-MSH-treated group.

• 

  Fig. 5 Effect of DPN on melanogenesis using UVB-irradiated HaCaT conditioned media system. HaCaT keratinocytes were irradiated with 30 mJ/cm2 of UVB and further cultured for 24 h, then cultured media which referred to as conditioned media (UV-CM) were harvested. B16F10 cells were treated with DPN (20 and 50 μM) and UV-CM and further cultured for 48 h. The melanin contents were examined by measuring the absorbance at 450 nm. HaCaT cultured media not irradiated with UVB were used as a negative control (NUV-CM). Arbutin was used as a positive control. *p < 0.05 were considered as statistically significant from vehicle treated group. Data are shown as mean ± SD from three independent experiments. DPN, diarylpropionitrile. #p < 0.05 were considered as statistically significant from the conditioned media-treated group.

• 

  Fig. 6 Schematic illustration of inhibitory effect on melanogenesis via PKA/CREB/MITF axis by DPN. DPN inhibits melanin synthesis by down-regulating the activation of PKA and CREB in α-MSH-stimulated B16F10. DPN, diarylpropionitrile; PKA, protein kinase A; CREB, cAMP-response element-binding protein; MITF, microphthalmia-associated transcription factor; α-MSH, alpha-melanocyte stimulating hormone; TRP, tyrosinase-related protein; MC1R, melanocortin 1 receptor.

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