Sargahydroquinoic Acid Suppresses Hyperpigmentation by cAMP and ERK1/2-Mediated Downregulation of MITF in α-MSH-Stimulated B16F10 Cells

Abstract

Hyperpigmentation diseases of the skin require topical treatment with depigmenting agents. We investigated the hypopigmented mechanisms of sargahydroquinoic acid (SHQA) in alpha-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 cells. SHQA reduced cellular tyrosinase (TYR) activity and melanin content in a concentration-dependent manner and attenuated the expression of TYR and tyrosinase-related protein 1 (TRP1), along with their transcriptional regulator, microphthalmia-associated transcription factor (MITF). SHQA also suppressed α-MSH-induced cellular production of cyclic adenosine monophosphate (cAMP), which inhibited protein kinase A (PKA)-dependent cAMP-responsive element-binding protein (CREB) activation. Docking simulation data showed a potential binding affinity of SHQA to the regulatory subunit RIIβ of PKA, which may also adversely affect PKA and CREB activation. Moreover, SHQA activated ERK1/2 signaling in B16F10 cells, stimulating the proteasomal degradation of MITF. These data suggest that SHQA ameliorated hyperpigmentation in α-MSH-stimulated B16F10 cells by downregulating MITF via PKA inactivation and ERK1/2 phosphorylation, indicating that SHQA is a potent therapeutic agent against skin hyperpigmentation disorders.

Keywords: ERK; MITF; cAMP; hyperpigmentation; melanin; sargahydroquinoic acid.

Figure 1

SHQA suppresses TYR activity and melanin production in B16F10 cells. (A) SHQA structure is isolated from S. serratifolium. (B) Cytotoxicity was measured by an MTS assay after SHQA treatment for 24 h. (C) After B16F10 cells were pre-treated with SHQA for 1 h, the cells were exposed to α-MSH (1.0 μM) for 72 h with and without SHQA. The cellular tyrosinase activity and melanin content was tested by measuring absorbance. (D) Measurement of melanin release in the culture medium after 72 h of SHQA treatment. Data are expressed as the mean ± standard deviation (SD) (n = 3). Different superscripts represent statistical significance at p < 0.05.

Figure 2

SHQA suppresses the protein levels of enzymes and transcription factors related to melanogenesis in B16F10 cells. (A) After pre-treatment of SHQA for 1 h, cells were treated with α-MSH (1.0 μM) for 48 h with or without SHQA. Western blotting was performed for TYR, TRP1, and TRP2. After pre-treatment of SHQA for 1 h, cells were treated with α-MSH for 6 h with or without SHQA. (B) Western blotting was performed for nuclear MITF and CREB. Data represent the mean ± standard deviation SD (n = 3). Data are expressed as the mean ± standard deviation (SD) (n = 3). Different superscripts represent statistical significance at p < 0.05.

Figure 3

SHQA prevents cAMP production and PKA activation in B16F10 cells. (A) After pre-treatment of SHQA for 1 h, cells were treated with α-MSH (1.0 μM) for 30 min with or without SHQA. Intracellular cAMP concentration was measured by a cAMP ELISA kit. (B) Cell lysates were immunoprecipitated with anti-PKA-RIIβ antibody and PKA-Cα co-precipitation was tested by western blotting. The band images were measured by densitometry and shown as bar graphs. Data are expressed as the mean ± standard deviation (SD) (n = 3). Different superscripts represent statistical significance at p < 0.05.

Figure 4

Protein-ligand docking simulation of SHQA to the cAMP binding domain of PKA. (A) Docking simulation of SHQA to the regulatory subunit IIβ of PKA. cAMP and SHQA are shown in the gray and black colors, respectively. (B) Binding mode analysis between SHQA and PKA (RIIβ). (C) Docking simulation of BISA and the regulatory subunit IIβ of PKA. (D) Binding mode analysis between BISA and PKA (RIIβ).

Figure 5

Effect of SHQA on melanogenic upstream signaling pathways in B16F10 cells. After pre-treatment of SHQA for 1 h, cells were treated with α-MSH (1.0 μM) for 1 h with or without SHQA. Western blotting was performed for (A) MAPKs and (B) AKT. (C) After pre-treatment of SHQA for 1 h with or without PD98059 (15.0 μM), cells were treated with α-MSH for 48 h. Western blotting was performed to detect tyrosinase. (D) After pre-treatment of SHQA for 1 h with or without MG-132 (70 nM), cells were treated with α-MSH for 48 h. Western blotting was performed for MITF and tyrosinase. The band images were measured by densitometry and shown as bar graphs. Data are expressed as the mean ± standard deviation (SD) (n = 3). Different superscripts represent statistical significance at p < 0.05.

Figure 6

Hypothetical mechanism showing the anti-melanogenic effect of SHQA in α-MSH-stimulated B16F10 cells. The illustration represents the MITF downregulation by SHQA via the inhibition of cAMP- and PKA-dependent CREB activation and the proteasomal degradation of MITF via the activation of ERK1/2 signaling. Downregulation of MITF leads to the suppressed production of TYR and TRP1 enzymes, leading to the suppression of melanin production.