Rottlerin Reduces cAMP/CREB-Mediated Melanogenesis via Regulation of Autophagy

Abstract

Melanogenesis is the sequential process of melanin production by melanocytes in order to protect the skin from harmful stimuli. Melanogenesis is disrupted by radiation exposure, which results in the differentiation of melanocytes into melanoma. Recently, some methods have been developed to maintain the instability of melanogenesis in melanoma by activating cellular autophagy. However, there is still a lack of knowledge about how autophagy is involved in the regulation of melanogenesis in melanoma cells. Here, we used rottlerin as an autophagy inducer to investigate the role of the cyclic adenosine monophosphate (cAMP)/cAMP response element binding (CREB) signaling pathway in melanogenesis. We found that rottlerin can inhibit melanin production by targeting cAMP, which is initially activated by alpha-melanocyte stimulating hormone (α-MSH). Our findings suggest that rottlerin has a pivotal role as an autophagy inducer in the regulation of melanogenesis by targeting the cAMP/CREB signaling pathway.

Keywords: autophagy; cAMP/CREB signaling pathway; melanogenesis; rottlerin.

Figure 1

Chemical structure of rottlerin.

Figure 2

Effect of rottlerin on alpha-melanocyte stimulating hormone (α-MSH)-induced melanogenesis in melanoma cells. (a) and (b) Viability of B16-F10 cells after rottlerin treatment (5 and 10 µM) and arbutin (1 mM at 48 h) was assessed with MTT solution. (c) Extracellular and (d) intracellular melanin contents in B16-F10 cells (10⁵ cells/mL) induced with α-MSH (100 nM), treated with rottlerin (5 and 10 µM) or arbutin (1 mM) as a positive control for 48 h were determined by spectrophotometry. All data (a–c) are expressed as the mean ± the standard deviation (SD) of three replicates. * p < 0.05 compared to control or normal groups.

Figure 3

Effect of rottlerin on genes involved in melanogenesis and CREB (cAMP response element binding) transcription factor. (a) Semi-quantitative PCR was carried out to measure mRNA expression of MITF, TYR (tyrosinase), TYRP1 (tyrosinase related protein 1), and TYRP2 in B16-F10 cells (10⁵ cells/mL) stimulated by α-MSH in the presence or absence of 5 and 10 µM of rottlerin. (b) and (c) B16-F10 cells (10⁵ cells/mL) were transfected with CREB-luciferase (CREB-Luc) and beta-galactosidase (β-gal, 0.8 µg) for 48 h, activated with α-MSH 24 h after CREB-Luc transfection, and (c) treated with forskolin (200 nM) with or without 5 and 10 µM of rottlerin. All data (b,c) are expressed as the mean ± SD of three replicates. ** p < 0.01 compared to control groups.

Figure 4

Rottlerin inhibits melanogenesis via the cAMP/CREB signaling pathway. (a) B16-F10 cells (10⁵ cells/mL) were activated by α-MSH in the presence or absence of rottlerin (5 and 10 µM) or arbutin (1 mM) for 48 h. Phosphorylated and total CREB were evaluated by immunoblotting. (b) Intracellular cAMP levels were analyzed using a cAMP immunoassay kit. All data (b) are expressed as the mean ± SD of three replicates. WCL: Whole cell lysates. * p < 0.05 and ** p < 0.01 compared to control groups.

Figure 5

Rottlerin downregulates CREB-mediated melanogenesis by the activity of autophagy. (a) Confirmation of rottlerin as an autophagy inducer was achieved by assessing levels of autophagy-related proteins, such as LC3 and ATG5, by immunoblotting analysis under the same conditions for 24 h. (b) B16-F10 cells (10⁵ cells/mL) were transfected with CREB-Luc and β-gal (0.8 µg) for 24 h, activated with α-MSH 24 h after CREB-Luc transfection, and (c) treated with α-MSH with or without 5 and 10 µM of rottlerin and 3-MA (10 mM). Luciferase activity was measured with a luminometer. (c) B16-F10 cells (10⁵ cells/mL) were induced by α-MSH in the presence or absence of rottlerin (5 and 10 µM) or arbutin (1 mM) as well as 3-MA (10 mM) for 48 h, and level of intracellular melanin content was measured by spectrophotometry. All data (b,c) are expressed as the mean ± SD of three replicates. * p< 0.05 and ** p < 0.01 compared to control groups. # p < 0.01 compared to selected groups.

Figure 6

Potential pathway by which rottlerin regulates melanogenesis in melanoma cells.