Withania somnifera extract attenuates stem cell factor-stimulated pigmentation in human epidermal equivalents through interruption of ERK phosphorylation within melanocytes
We previously demonstrated that mitogen-activated protein kinase (MAPK) signaling, including microphthalmia-associated transcription factor (MITF) and cAMP response element-binding protein (CREB) phosphorylation, is a major pathway involved in up-regulating melanogenesis within human melanocytes in several hyperpigmentary disorders such as UVB melanosis and lentigo senilis. Recently, a redox imbalance was shown to be closely linked to a variety of altered cellular responses in which the precise balance between levels of oxidizing and reducing equivalents that reflect the intracellular redox condition profoundly affects intracellular signaling pathways, especially the MAPK pathway. To elucidate the effects of redox balance regulation on epidermal pigmentation, we used an antioxidant-rich extract of the herb Withania somnifera to assess its effect on stem cell factor (SCF)-stimulated pigmentation in human epidermal equivalents and analyzed its biological mechanism of action. Addition of the W. somnifera extract (WSE) caused a marked reduction in SCF-stimulated pigmentation in a dose-dependent manner after 14 days of treatment, which was accompanied by a significant decrease in eumelanin content. In WSE-treated human epidermal equivalents, melanocyte-specific proteins (including tyrosinase) were significantly suppressed at the gene and protein levels by WSE. Signaling analysis with immunoblots revealed that in human melanocytes or human melanoma cells treated with WSE, there was a marked deficiency in SCF-stimulated phosphorylation of ERK, MITF and CREB, but not of Raf-1 and MEK. Since WSE had no direct inhibitory effect on tyrosinase activity and no melano-cytotoxic effect on melanocytes present in the human epidermal equivalents or on cultured human melanocytes, the sum of these findings indicates that WSE attenuates SCF-stimulated pigmentation by preferentially interrupting ERK phosphorylation within melanocytes and can serve as a therapeutic tool for SCF-associated hyperpigmentary disorders.
KeywordsWithania somnifera Melanocytes Stem cell factor MAPK Pigmentation
Withania somnifera extract
Stem cell factor
Endothelin B receptor
Mitogen-activated protein kinase
Microphthalmia-associated transcription factor
Normal human melanocytes
Protein kinase C
Reactive oxygen species
We thank Dr. Hiroshi Murata of the Department of Dermatology, School of Medicine, Shinshu University for providing us with acral lentigo malignant melanoma (SM2-1) cells.
Conflict of interest
The authors have declared no conflict of interest.
- 7.Imokawa G, Yada Y, Kimura M (1996) Signaling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes. Biochem J J314:305–312Google Scholar
- 9.Imokawa G, Kobayashi T, Miyagishi M (2000) Intracellular signaling mechanisms leading to synergistic effects of endothelin-1 and stem cell factor on proliferation of cultured human melanocytes: cross-talk via trans-activation of the tyrosine kinase c-kit receptor. J Biol Chem 275:33321–33328PubMedCrossRefGoogle Scholar
- 26.Ichikawa H, Takada Y, Shishodia S, Jayaprakasam B, Nair MG, Aggarwal BB (2006) Withanolides potentiate apoptosis, inhibit invasion, and abolish osteoclastogenesis through suppression of nuclear factor-nB (NF-nB) activation and NF-nB-regulated gene expression. Mol Cancer Ther 5:1434–1445PubMedCrossRefGoogle Scholar
- 28.Aalinkeel R, Hu Z, Nair BB, Sykes DE, Reynolds JL, Mahajan SD, Schwartz SA (2010) Genomic analysis highlights the role of the JAK-STAT signaling in the anti-proliferative effects of dietary flavonoid ‘Ashwagandha’ in prostate cancer cells. Evid Based Complement Altern Med 7:177–187PubMedCrossRefGoogle Scholar
- 35.Amae S, Fuse N, Yasumoto K, Sato S, Yajima I, Yamamoto H, Udono T, Durlu YK, Tamai M, Takahashi K, Shibahara S (1998) Identification of a novel isoform of microphthalmia-associated transcription factor that is enriched in retinal pigment epithelium. Biochem Biophys Res Commun 247:710–715PubMedCrossRefGoogle Scholar
- 38.Lennartsson J, Blume-Jensen P, Hermanson M, Ponten E, Carlberg M, Ronnstrand L (1999) Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction. Oncogene 18:5546–5553PubMedCrossRefGoogle Scholar
- 44.Hiroaki N, Yuki W, Kazumasa W, Genji I.(2011) An extract of Withania somnifera attenuates endothelin-1-stimulated pigmentation in human epidermal equivalents through the interruption of PKC activity within melanocytes. Phytother Res. doi: 10.1002/ptr.3552