Betaine reduces cellular melanin content via suppression of microphthalmia-associated transcription factor in B16-F1 murine melanocytes

Abstract

Long-term topical skin care by traditional anti-melanogenic agents can raise several safety concerns. An understanding of the molecular mechanisms of active compounds on melanogenesis is, therefore, necessary to address pigmentation issues. Here we revealed that stimulation with 1 mM betaine, an abundant component in rice bran, significantly reduced 21% of intracellular melanin content by suppressing tyrosinase activity and microphthalmia-associated transcription factor (MITF) expression in B16-F1 murine melanocytes. The expression of MITF was suppressed at both mRNA and protein levels by 43 and 44%, respectively. Subsequently, the betaine-stimulated melanocytes showed inhibition of PKA-CREB signaling axis but activation of extracellular-signal-regulated kinase and AKT-GSK3β signaling pathways. This inhibition and activation led to downregulation of MITF expression at both the transcriptional and post-translational levels to suppress melanin synthesis. These findings collectively suggested that betaine is a potential anti-melanogenic compound for functional foods and cosmetics.

Keywords: Betaine; MITF; Melanocyte; Melanogenesis; Tyrosinase.

Fig. 1

Betaine reduces intracellular melanin content in B16-F1 murine melanocytes. (A) Cell viability of B16-F1 murine melanocytes stimulated with 1 mM arbutin and 0.5 mM or 1 mM betaine. (B) Tyrosinase activity. Cell-free mushroom tyrosinase and cellular tyrosinase activities were measured. (C) Cellular melanin content in B16-F1 murine melanocytes stimulated with 1 mM arbutin and 0.5 mM or 1 mM betaine. (D) Fontana-Masson staining of melanin contents in B16-F1 murine melanocytes stimulated with 1 mM arbutin or 1 mM betaine. Melanin (dark brown) was visualized after 72 h of the stimulation. P values compared with controls are denoted as *P < 0.05, **P < 0.01. (Color figure online)

Fig. 2

Betaine suppresses MITF activity and melanogenic enzymes. Gene expression level (A) and protein (B) expression levels of MITF and its transcriptional targets, tyrosinase and dopachrome tautomerase

Fig. 3

Betaine regulates cellular signaling molecules suppressing MITF activity. (A) and (B) Cellular cAMP concentration and PKA kinase activity were quantified after 30 min and 1 h of the betaine stimulation, respectively. (C) Protein expression levels of CREB, ERK, AKT, GSK-3β and their phosphorylated forms

Fig. 4

Betaine suppresses MITF expression by regulation of several cellular signaling pathways. (A) MITF protein expression in the melamocytes stimulated by either betaine or/and LY294002. (B) Proposed mechanism of anti-melanogenesis effect of betaine in B16-F1 melanocytes