We established a model for the stem cell factor (SCF)-associated stimulation of human epidermal equivalent (HEE) pigmentation. The addition of SCF (at 5 nM) gradually stimulated the visible pigmentation of HEEs over 14 days of treatment. A time course study using real-time RT-PCR and western blotting analysis demonstrated that the expression of all melanocyte-specific genes and proteins examined was gradually up-regulated over 7-10 days of treatment with SCF. The addition of astaxanthin (Ax) at concentrations of 1, 4, or 8 μM markedly abolished the SCF- but not the endothelin (EDN)1-elicited increase in visible pigmentation over 14 days in a dose-dependent manner, with almost complete inhibition at 8 μM. While no degeneration of the epidermal tissue was visible at day 14 by HE staining, melanin deposition throughout the epidermis was markedly reduced in the Ax-treated HEEs at day 14 compared to untreated controls. Ax significantly reduced the eumelanin content of HEEs to the non-SCF-stimulated level at concentrations of 4 or 8 μM compared with untreated controls. Real-time RT-PCR and western blotting of Ax-treated HEEs revealed that the SCF-stimulated expression of tyrosinase (TYR), TYR-related protein-1 (TYRP1), and Pmel17, as well as microphthalmia-associated transcription factor (MITF), is significantly suppressed by Ax at the transcriptional and translational levels. Studies using cultured normal human melanocytes revealed that pre-treatment with Ax interrupts the SCF- but not the EDN1-induced stimulation of TYR activity, and there was no direct inhibitory effect of Ax on TYR activity in vitro. These findings indicate that Ax attenuates SCF-stimulated pigmentation by directly interrupting SCF-associated intracellular signaling linkages through increased expression of MITF, which leads to the stimulated expression of melanogenic genes and proteins in a reactive oxygen species depletion-independent mechanism.
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.
The orexigenic agouti-related protein (AgRP) and the anorexigenic pro-opiomelanocortin (POMC) are crucial players in the control of feed intake in vertebrates, yet their role in teleosts has not been fully established. Triplicate groups of Atlantic salmon (Salmo salar) post smolts were subjected to (1) fasting for 3 days (fast) and (2) normal feeding (fed), resulting in a significant (p < 0.05) upregulation of hypothalamic agrp1 transcripts levels in the fast group. Moreover, the mRNA abundance of agrp1 was significantly (p < 0.05) correlated with the stomach dry weight content. Corresponding inverse patterns were observed for pomca2, albeit not statistically significant. No significant differences were found for the other paralogues, agrp2 and pomca1 and b, between fed and fast groups. The significant correlation between stomach fullness and agrp1 mRNA expression suggests a possible link between the stomach filling/distension and satiety signals. Our study indicates that hypothalamic agrp1 acts as an orexigenic signal in Atlantic salmon.
To elucidate the effects of redox balance regulation on epidermal pigmentation, we used an antioxidant-rich extract of the herb Melia toosendan (dried mature fruits) to assess its effect on endothelin-1 (EDN1)-stimulated pigmentation in human epidermal equivalents and analyzed its biological mechanism of action. Addition of the Melia toosendan extract elicited a marked depigmenting effect on EDN1-stimulated pigmentation after 14 days of treatment, which was accompanied by a significant decrease in eumelanin content. Real-time RT-PCR and Western blotting revealed that the EDN1-stimulated expression of melanocyte-specific proteins (including tyrosinase) was significantly suppressed at the gene and protein levels by the extract. Signaling analysis with specific inhibitors and immunoblots revealed that in melanoma cells treated with the extract, there was a marked deficiency in the EDN1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB. Since all those proteins are downstream phosphorylation targets of PKC activity, these findings indicate that the Melia toosendan extract attenuates the EDN1-stimulated pigmentation by preferentially inhibiting PKC activity within melanocytes.
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