Melasma is one of the most common patterns of hyperpigmentation, particularly during the reproductive lifespan of women. In terms of pigmentation, melasma does not differ from other conditions with hyperpigmentation, however, the outcome is different from the hyperpigmentation caused by inflammation or by UV exposure, suggesting different regulatory mechanisms being involved in these conditions. Recently, we performed microarray analysis to compare hyperpigmented with normally pigmented skin specimens from patients with melasma to identify specific factors associated with the hyperpigmentation of melasma. Down-regulation of H19 RNA was one of the identified factors associated with melasma. There was also a 2-fold or greater down-regulation in the expression of the Wnt inhibitory factor-1 (WIF-1) gene in the hyperpigmented skin of melasma patients. The real time-PCR and immunohistochemistry using anti-WIF-1 antibody confirmed the significant reduction of WIF-1 gene expression in the hyperpigmented skin from melasma patients, but not in healthy controls, regardless of UV irradiation.
Wnts, a family of secreted glycoproteins, regulate a vast array of biological processes, including skin pigmentation through either the canonical or the non-canonical pathways. In the canonical pathway, β-catenin plays a critical role, which is also called the Wnt/β-catenin pathway; the binding of Wnts to their receptor generates a signaling pool of β-catenin and translocate β-catenin to the nucleus to stimulate transcription of target genes, whereas β-catenin without the binding is degraded by the destruction complex, which is composed of four proteins including GSK3 (glycogen synthase kinase3). Non-canonical Wnt pathway is β-catenin-independent and involves a diverse signal transduction, such as JNK or calcium flux, the latter is called the Wnt/calcium pathway which activates PKC (protein kinase C) and NFAT (nuclear factor of activated T cells). The extracellular antagonists of the Wnt signaling pathway can be divided into two classes, the secreted Frizzled-related protein class (sFRP) and the Dickkopf class. WIF-1 belongs to the sFRP class family, and inhibits both canonical and non-canonical Wnt pathways.
WIF-1 is expressed in cultured skin keratinocytes and fibroblasts, but not in melanocytes. Melanocyte growth and differentiation is affected by neighboring fibroblasts as well as by keratinocytes. Therefore, we examined whether WIF-1 knockdown in neighboring keratinocytes and fibroblasts plays a role in melasma. For the examination, keratinocyte-melanocyte co-cultures were used to examine the effect of WIF-1 down-regulated keratinocytes or fibroblasts on normal melanocytes. Additionally, the effect of WIF-1 over-expression on the amelioration of hyperpigmentation was examined by the treatment of cultured melanocytes with rhWnt-1. WIF-1 knockdown, either in fibroblasts or in keratinocytes significantly stimulated tyrosinase expression and melanosome transfer, whereas melanocytes with WIF-1 over-expression, significantly reduced those parameters. The WIF-1 knockdown decreased GSK-3β, β-catenin and NFATc2 phosphorylation and increased MITF expression as in melanocytes with Wnt-1 over-expression, whereas the WIF-1 over-expression reversed the results. Expression of Wnts, both canonical and non-canonical, was increased in the hyperpigmented skin of melasma patients. Collectively, WIF-1 down-regulation, which may occur in epidermal keratinocytes and in dermal fibroblasts, is involved in melasma development due to the stimulation of melanogenesis and melanosome transfer through up-regulation of the canonical and the non-canionical Wnt signaling pathway.