Withaferin A abolishes the stem cell factor-stimulated pigmentation of human epidermal equivalents by interrupting the auto-phosphorylation of c-KIT in human melanocytes.
We characterized the mechanism(s) underlying the abrogating effect of withaferin A (WFA) on the stem cell factor (SCF)-stimulated pigmentation of human epidermal equivalents (HEEs). Increased gene and protein expression levels of tyrosinase, tyrosinase-related protein1, dopachrome tautomerase, PMEL17, c-KIT and their targeted transcription factor, microphthalmia-associated transcription factor (MITF) were significantly reversed at days 7 and 10, respectively, by treatment with WFA. In WFA-treated normal human melanocytes (NHMs), there was a marked deficiency in the SCF-stimulated series of phosphorylations of c-KIT, Shc, Raf-1, MEK, ERK, MITF and CREB. Treatment with dithiothreitol (DTT) distinctly abolished the suppressive effect of WFA on the SCF-stimulated phosphorylation of c-KIT in NHMs. On the other hand, even after incubation at 4 °C for 2 h with 5 nM SCF, followed by the removal of unbound SCF by washing and then raising the temperature to 37 °C to start the signaling reaction, c-KIT was distinctly phosphorylated to a similar extent by incubation for 15 min with SCF only or with SCF + WFA. These findings indicate that WFA attenuates the SCF-induced activation of c-KIT in NHMs by interrupting the auto-phosphorylation of c-KIT through DTT-suppressible Michael addition thioalkylation reactions without interrupting the binding of SCF to the c-KIT receptor.
Research Institute for Biological Functions, Chubu University, 1200 Matsumoto, Kasugai, Aichi, 487-8501, Japan.
Shuko
S
Terazawa
Hiroaki
H
Nakajima
Katsunori
K
Fukasawa
Genji
G
Imokawa
D002478:Cells, Cultured; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects* / Q000201:enzymology; D005786:Gene Expression Regulation; D006801:Humans; D008543:Melanins / Q000096:biosynthesis; D008544:Melanocytes / Q000187:drug effects* / Q000201:enzymology; D014442:Monophenol Monooxygenase / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D062327:Skin Lightening Preparations / Q000494:pharmacology*; D012880:Skin Pigmentation / Q000187:drug effects*; D019089:Stem Cell Factor / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors; D054358:Withanolides / Q000494:pharmacology*
D008543:Melanins; D062327:Skin Lightening Preparations; D019089:Stem Cell Factor; D054358:Withanolides; D014442:Monophenol Monooxygenase; D019009:Proto-Oncogene Proteins c-kit; C009684:withaferin A