Melanocyte Activation Mechanisms and Rational Therapeutic Treatments of Solar Lentigos.

To characterize the pathobiology of solar lentigos (SLs), analyses by semiquantitative RT-PCR, Western blotting, and immunohistochemistry revealed the upregulated expression of endothelin (EDN)-1/endothelin B receptors (EDNBRs), stem cell factor (SCF)/c-KIT, and tumor necrosis factor (TNF)α in the lesional epidermis, which contrasted with the downregulated expression of interleukin (IL) 1α. These findings strongly support the hypothesis that previous repeated UVB exposure triggers keratinocytes to continuously produce TNFα. TNFα then stimulates the secretion of EDNs and the production of SCF in an autocrine fashion, leading to the continuous melanogenic activation of neighboring melanocytes, which causes SLs. A clinical study of 36 patients with SLs for six months treated with an M. Chamomilla extract with a potent ability to abrogate the EDN1-induced increase in DNA synthesis and melanization of human melanocytes in culture revealed a significant improvement in pigment scores and color differences expressed as L values. Another clinical study using a tyrosinase inhibitor L-ascorbate-2-phosphate 3 Na (ASP) demonstrated that L values of test lotion (6% APS)-treated skin significantly increased in SLs and in non-lesional skin with a significantly higher ΔL value in SLs when compared with non-lesional skin. The sum of these findings strongly suggests that combined topical treatment with EDN signaling blockers and tyrosinase inhibitors is a desirable therapeutic choice for SLs.

Strawberry seed extract and its major component, tiliroside, promote ceramide synthesis in the stratum corneum of human epidermal equivalents.

The activation of peroxisomeproliferator-activated receptor (PPAR) α can stimulate the expression of ceramide-related enzymes, and a major component of strawberry seed extract (SSE) tiliroside enhances the expression of PPARα. We determined whether SSE and tiliroside may stimulate ceramide synthesis in the stratum corneum (SC) of the human epidermal equivalents (HEEs) culture model. Treatment with SSE at 1.0 and 3.0 µg/mL elicited a significant increase in the total ceramide content in the SC, which was accompanied by a significant increase in almost all ceramide species except for ceramide [EOS] and [AP]. Treatment with tiliroside at 0.3 µg/mL slightly accentuated the total ceramide content in the SC together with a significant increase in the ceramide [NS, NDS] content. Messenger RNA analysis demonstrated that SSE at 1 or 3 µg/mL significantly stimulated the gene expression of serine palmitoyltransferase (SPT) 2, ceramide synthase (CerS) 3, glucosylceramide synthase (GCS), and ß-glucocerebrosidase (GBA) but not of SPT1, sphingomyelin synthase (SMS) 1/2 and acid sphingomyelinase (ASM). In contrast, tiliroside elicited significant increases in the gene expression levels of GCS and GBA only at 0.3 and/or 0.1 µg/mL. Western blotting analysis revealed that both SSE and tiliroside enhanced the protein expression levels of GCS and GBA but not of SPT2 at 1 or 3 and 0.1 or 0.3 µg/mL, respectively. These findings suggested that both SSE and tiliroside have a distinct potential to stimulate the level of ceramide [NS, NDS] in the SC by enhancing the expression of GCS and GBA. The higher stimulatory effect with SSE than tiliroside on SC ceramide synthesis correlates with the significant increase observed with SSE but not tiliroside in the gene expression levels of SPT2 and CerS3. Therefore, it is anticipated that SSE is effective in improving skin barrier function and moisture retention in several ceramide-deficit skin conditions, including surfactant-induced roughened skin, xerosis, and atopic dermatitis.

Amelioration of lactic acid sensations in sensitive skin by stimulating the barrier function and improving the ceramide profile.

We determined whether compensating ceramides in the stratum corneum (SC) may ameliorate the impaired barrier function and subsequently attenuate the enhanced skin sensitivity. Treatment for 4 weeks with the ceramide complex cream or the placebo cream significantly ameliorated the intensity of lactic acid sensations in 39 female subjects with sensitive skin, the degree of which was attenuated to a greater extent at 1 week by the ceramide complex cream compared with the placebo cream. The amelioration of skin sensations was accompanied by a significant increase in total ceramide content in the SC elicited by the ceramide complex cream that was significantly more effective than the placebo cream at 4 weeks. Consistently, TEWL and conductance values were significantly decreased or increased at 1 and 4 weeks, respectively, to a greater extent by the ceramide complex cream than by the placebo cream. TEWL levels were significantly correlated with the increased levels of SC total ceramide in the ceramide complex cream-treated skin but not in the placebo cream-treated skin. Thus, the amelioration of lactic acid sensations by topical application of a ceramide complex cream, provides a deep insight into the pathophysiology of sensitive skin as a reduced barrier function-dependent sub-clinical sensory response.

Epithelial-mesenchymal interaction mechanisms leading to the over-expression of neprilysin are involved in the UVB-induced formation of wrinkles in the skin.

In clinical studies, the formation of facial wrinkles has been closely linked to the loss of elastic properties of the skin. Repetitive UVB irradiation of animal skin at suberythemal doses significantly reduces its elastic properties, resulting in the formation of wrinkles. That also elicits a marked alteration in the three-dimensional structure of elastic fibres, which is closely associated with a subsequent reduction in the elastic properties of the skin. While UVB irradiation stimulates the activity of skin fibroblast-derived elastase in the dermis, a synthetic inhibitor specific for skin fibroblast-derived elastase as well as an extract of Zingiber officinale (L.) Rose capable of inhibiting skin fibroblast-derived elastase, but not neutrophil elastase, prevented wrinkle formation in our studies of animal and human facial skin, respectively. The close interrelationship among wrinkle formation, elastic properties and elastic fibre linearity is revealed by the effects of different concentrations of the elastase inhibitor, which indicates that enhanced elastase activity by dermal fibroblasts plays a pivotal role in the UVB wrinkling mechanism. Fortunately, we were able to identify human skin fibroblast-derived elastase as the previously known enzyme neprilysin/neutral endopeptidase. Using both a UVB-conditioned medium assay and a co-culture system, we characterized the epithelial-mesenchymal interaction between keratinocytes and fibroblasts which leads to increased expression of neprilysin at the transcriptional, translational and enzymatic levels. Our results demonstrate that interleukin-1α and granulocyte-macrophage colony-stimulating factor are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by skin fibroblasts.

UVB Stimulates the Expression of Endothelin B Receptor in Human Melanocytes via a Sequential Activation of the p38/MSK1/CREB/MITF Pathway Which Can Be Interrupted by a French Maritime Pine Bark Extract through a Direct Inactivation of MSK1.

Melanogenesis is the physiological process by which melanin is synthesized to protect the skin from UV damage. While paracrine interactions between keratinocytes and melanocytes are crucial for regulating epidermal pigmentation, the endothelin (EDN)-endothelin B-receptor (EDNRB) interaction is one of the key linkages. In this study, we found that a single exposure of normal human melanocytes (NHMs) with UVB stimulates the expression of EDNRB and its upstream transcription factor microphthalmia-associated transcription factor (MITF) at the transcriptional and translational levels. That stimulation can be abrogated by post-irradiation treatment with a French maritime pine bark extract (PBE). UVB stimulated the phosphorylation of p38 and c-jun N-terminal kinase (JNK), but not ERK, followed by the increased phosphorylation of MSK1 and CREB. The post-irradiation treatment with PBE did not affect the increased phosphorylation of p38 and JNK, but distinctly abrogated the phosphorylation of MSK1 and CREB. Post-irradiation treatment with the MSK1 inhibitor H89 significantly down-regulated the increased gene expression of MITF and EDNRB in UVB-exposed NHMs. Our findings indicate for the first time that the increased expression of MITF that leads to the up-regulation of melanocyte-specific proteins in UVB-exposed NHMs is mediated via activation of the p38/MSK1/CREB pathway but not the ERK/RSK/CREB pathway. The mode of action by PBE demonstrates that interrupting MSK1 activation is a new target for antioxidants including PBE which can serve as anti-pigmenting agents in a reactive oxygen species-depletion-independent manner.

Inhibitors of intracellular signaling pathways that lead to stimulated epidermal pigmentation: perspective of anti-pigmenting agents.

Few anti-pigmenting agents have been designed and developed according to their known hyperpigmentation mechanisms and corresponding intracellular signaling cascades. Most anti-pigmenting agents developed so far are mechanistically involved in the interruption of constitutional melanogenic mechanisms by which skin color is maintained at a normal and unstimulated level. Thus, owing to the difficulty of confining topical application to a specific hyperpigmented skin area, potent anti-pigmenting agents capable of attenuating the natural unstimulated pigmentation process have the risk of leading to hypopigmentation. Since intracellular signaling pathways within melanocytes do not function substantially in maintaining normal skin color and are activated only by environmental stimuli such as UV radiation, specifically down-regulating the activation of melanogenesis to the constitutive level would be an appropriate strategy to develop new potent anti-pigmenting agents with a low risk of hypopigmentation. In this article, we review the hyperpigmentation mechanisms and intracellular signaling pathways that lead to the stimulation of melanogenesis. We also discuss a screening and evaluation system to select candidates for new anti-melanogenic substances by focusing on inhibitors of endothelin-1 or stem cell factor-triggered intracellular signaling cascades. From this viewpoint, we show that extracts of the herbs Withania somnifera and Melia toosendan and the natural chemicals Withaferin A and Astaxanthin are new candidates for potent anti-pigmenting substances that avoid the risk of hypopigmentation.

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.

An extract of Withania somnifera attenuates endothelin-1-stimulated pigmentation in human epidermal equivalents through the interruption of PKC activity within melanocytes.

Redox imbalances have been shown to be closely linked to a variety of altered cellular responses and profoundly affect intracellular signaling pathways, especially the PKC/MAPK pathway which is a major pathway involved in regulating melanogenesis within human melanocytes. To elucidate the effects of redox balance regulation on epidermal hyperpigmentary disorders, an antioxidant-rich herb extract of Withania somnifera was used to assess its effect on endothelin-1 (EDN1)-stimulated pigmentation in human epidermis equivalents and its biological mechanisms analysed. Addition of the Withania somnifera extract (10 µg/mL) elicited a marked depigmenting effect on EDN1 (10 nm)-stimulated pigmentation which was accompanied by a significant decrease in eumelanin content. Real-time RT-PCR and western blotting revealed that the stimulated expression of melanocyte-specific mRNAs and proteins, including microphthalmia associated transcription factor (MITF), was significantly suppressed at days 7-10 of culture by the Withania somnifera extract (10 µg/mL), suggesting an impairment in intracellular signaling upstream of gene expression. Signaling analysis revealed that in Withania somnifera extract (10 µg/mL)-treated human melanoma cells in culture, there was a marked deficiency in EDN1 (10 nm)-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and Cyclic AMP responsive element binding protein (CREB) at 15 min after EDN1 treatment. Consistently, treatment with withaferin A, a major component of the Withania somnifera extract, at concentrations of 10-50 µm also significantly down-regulated the EDN1 stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB at 15 min after EDN1 treatment. Since Raf-1 is phosphorylated by protein kinase C (PKC) activity, these findings indicate that the Withania somnifera extract attenuates EDN1-stimulated pigmentation by preferentially inhibiting EDN1-triggered PKC activity.

An extract of Melia toosendan attenuates endothelin-1-stimulated pigmentation in human epidermal equivalents through the interruption of PKC activity within melanocytes.

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.

Recent advances in characterizing biological mechanisms underlying UV-induced wrinkles: a pivotal role of fibrobrast-derived elastase.

In clinical studies, the formation of facial wrinkles has been closely linked to the loss of elastic properties of the skin. Cumulative irradiation with ultraviolet (UV) B at suberythemal doses significantly reduces the elastic properties of the skin, resulting in the formation of wrinkles. In in vitro studies, we identified a paracrine pathway between keratinocytes and fibroblasts, which leads to wrinkle formation via the up-regulation of fibroblast elastases that degrade elastic fibers. UVB irradiation stimulates the activity of fibroblast elastases in animal skin. Scanning electron microscopy revealed that cumulative UVB irradiation elicits a marked alteration in the three-dimensional structure of elastic fibers, which is closely associated with the subsequent reduction in the elastic properties of the skin, resulting in wrinkle formation. Studies using anti-wrinkle treatments suggest a close relationship between the recovery of wrinkles and an improvement in the linearity of elastic fibers. Those studies also suggest a close correlation between the recovery in the linearity of elastic fibers and the improvement in skin elasticity. In a study using ovariectomized animals, we characterized the important role of elastase in their high vulnerability to UV-induced wrinkle formation. A synthetic inhibitor specific for fibroblast elastases significantly prevents wrinkle formation without reducing the elastic properties of the skin, accompanied by minor damage in elastic fibers. Finally, we identified an effective extract of Zingiber officinale (L.) Rose from a screen of many herb extracts, which has a safe and potent inhibitory activity against fibroblast elastases. Animal studies using the L. Rose extract revealed that it has significant preventive effects against UVB-induced wrinkle formation, which occur in concert with beneficial effects on skin elasticity. A 1-year clinical study on human facial skin to determine the efficacy of the L. Rose extract demonstrated that it inhibits the UV-induced decrease in skin elasticity and prevents or improves wrinkle formation in skin around the corner of the eye without changing the water content of the stratum corneum. Our long-term studies support our hypothesis for a mechanism of wrinkle formation in which cytokine expression is activated by UV irradiation and triggers dermal fibroblasts to increase the expression of elastase. That increase in elastase results in the deterioration of the three-dimensional architecture of elastic fibers, reducing skin elasticity and finally leading to the formation of wrinkles.

Inhibition of ultraviolet-B-induced wrinkle formation by an elastase-inhibiting herbal extract: implication for the mechanism underlying elastase-associated wrinkles.

BACKGROUND: Previously, we have demonstrated that fibroblast-derived elastase plays an essential role in the increased three-dimensional tortuosity of elastic fibers, contributing to the loss of skin elasticity in UV-B-exposed skin. This decrease in skin elasticity is closely associated with the formation of wrinkles induced by UV exposure. OBJECTIVE: To further clarify the role of elastase in the formation of wrinkles induced by UV exposure, we assessed the effects of an extract of Zingiber officinale (L.) Rose (which inhibits fibroblast-derived elastase) on the wrinkle formation induced by chronic UV-B irradiation. RESULTS: Topical application of an extract of Zingiber officinale (L.) Rose to rat or hairless mouse skin significantly inhibited the wrinkle formation induced by chronic UV-B irradiation at a suberythemal dose, which was accompanied by a significant prevention of the decrease in skin elasticity in both types of animal skin. In the rat hind limb skin, consistent with the inhibition of reduced skin elasticity, wrinkle prevention occurred concomitantly with a significant decrease in the curling and three-dimensional tortuosity of dermal elastic fibers. CONCLUSION: Our results indicate that herbal extracts with an ability to inhibit fibroblast-derived elastase may prove to be effective as anti-wrinkling agents, confirming the important role of elastase in UV-B-induced wrinkle formation.