Hiroaki Nakajima

Astaxanthin attenuates the UVA-induced up-regulation of matrix-metalloproteinase-1 and skin fibroblast elastase in human dermal fibroblasts

BACKGROUND Repetitive exposure of the skin to UVA radiation elicits sagging more frequently than wrinkling, which is mainly attributed to its biochemical mechanism to up-regulate the expression of matrix-metalloproteinase (MMP)-1 and skin fibroblast elastase (SFE)/neutral endopeptidase (NEP), respectively. OBJECTIVE In this study, we examined the effects of a potent antioxidant, astaxanthin (AX), on the induction of MMP-1 and SFE by UVA treatment of cultured human dermal fibroblasts. METHODS Those effects were assessed by real-time RT-PCR, Western blotting and enzymic activity assays. RESULTS UVA radiation elicited a significant increase in the gene expression of MMP-1 as well as SFE/NEP (to a lesser extent) which was followed by distinct increases in their protein and enzymatic activity levels. The addition of AX at concentrations of 4-8 microM immediately after UVA exposure significantly attenuated the induction of MMP-1 and SFE/NEP expression elicited by UVA at the gene, protein and activity levels although both the UVA stimulation and the subsequent AX inhibition were greater for MMP-1 than for SFE/NEP. Analysis of the UVA-induced release of cytokines revealed that UVA significantly stimulated only the secretion of IL-6 among the cytokines tested and that AX significantly diminished only the IL-6 secretion. CONCLUSION These findings indicate that, based on different effective concentrations of AX, a major mode of action leading to the inhibition elicited by AX depends on inhibition of UVA effects of the reactive oxygen species-directed signaling cascade, but not on interruption of the IL-6-mediated signaling cascade. We hypothesize that AX would have a significant benefit on protecting against UVA-induced skin photo-aging such as sagging and wrinkles.

Astaxanthin attenuates the UVB‐induced secretion of prostaglandin E2 and interleukin‐8 in human keratinocytes by interrupting MSK1 phosphorylation in a ROS depletion–independent manner

Abstract:  To elucidate the effects of redox balance regulation on cutaneous inflammation, we used the potent antioxidant astaxanthin (AX) to assess its effect on the UVB‐induced secretion of PGE2 and IL‐8 in human keratinocytes and analysed its biological mechanism of action. The addition of AX (at 8 μm) to human keratinocytes even after UVB irradiation significantly down‐regulated the increased secretion of PGE2 or IL‐8. Those suppressive effects were accompanied by significantly decreased expression of genes encoding COX‐2 or IL‐8 as well as COX‐2 protein. Analysis using a specific NF‐κB tanslocation inhibitor demonstrated that the UVB‐stimulated secretion of PGE2 and IL‐8 was significantly abolished by its treatment prior to UVB irradiation. Western blotting of phosphorylated signalling molecules revealed that UVB irradiation (80 mJ/cm2) significantly stimulated the phosphorylation of p38, ERK and JNK, which was not suppressed by treatment with AX after irradiation. In contrast, AX significantly inhibited the UVB‐increased phosphorylation of mitogen‐ and stress‐activated protein kinase (MSK)‐1, NF‐kBp65 or CREB even when treated postirradiation. Further, the MSK1 inhibitor H89 significantly down‐regulated the increased secretion of PGE2 and IL‐8 in UVB‐exposed human keratinocytes, following post‐irradiation treatment. These findings suggests that AX attenuates the auto‐phosphorylation of MSK1 required for its activation, which results in the decreased phosphorylation of NF‐kBp65, which in turn probably leads to a deficiency of NF‐kB DNA binding activity. This may be associated with the significant suppression of PGE2/IL‐8 secretion via the down‐regulated expression of COX‐2 and IL‐8 at the gene and/or protein levels.

Biological Mechanisms Underlying the Ultraviolet Radiation-Induced Formation of Skin Wrinkling and Sagging II: Over-Expression of Neprilysin Plays an Essential Role

Our previous studies strongly indicated that the up-regulated activity of skin fibroblast-derived elastase plays a pivotal role in wrinkling and/or sagging of the skin via the impairment of elastic fiber configuration and the subsequent loss of skin elasticity. Fortunately, we succeeded in identifying human skin fibroblast-derived elastase as a previously known enzyme, neprilysin or neutral endopeptidase (NEP). We have also characterized epithelial-mesenchymal paracrine cytokine interactions between UVB-exposed-keratinocytes and dermal fibroblasts and found that interleukin-1α and granulocyte macrophage colony stimulatory factor (GM-CSF) are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by fibroblasts. On the other hand, direct UVA exposure of human fibroblasts significantly stimulates the secretion of IL-6 and also elicits a significant increase in the gene expression of matrix metallo-protease(MMP)-1 as well as neprilysin (to a lesser extent), which is followed by distinct increases in their protein and enzymatic activity levels. Direct UVA exposure of human keratinocytes also stimulates the secretion of IL-6, IL-8 and GM-CSF but not of IL-1 and endothelin-1. These findings suggest that GM-CSF secreted by UVA-exposed keratinocytes as well as IL-6 secreted by UVA-exposed dermal fibroblasts play important and additional roles in UVA-induced sagging and wrinkling by up-regulation of neprilysin and MMP-1, respectively, in dermal fibroblasts.

The UVB-Stimulated Expression of Transglutaminase 1 Is Mediated Predominantly via the NFκB Signaling Pathway: New Evidence of Its Significant Attenuation through the Specific Interruption of the p38/MSK1/NFκBp65 Ser276 Axis.

The influence of ultraviolet B (UVB) radiation on transglutaminase 1 (TGase 1), a major factor that regulates skin keratinization, has not been sufficiently characterized especially at the gene or protein level. Thus, we determined whether UVB affects the expression of TGase 1 in human keratinocytes and clarified the intracellular stress signaling mechanism(s) involved. Exposure of human keratinocytes to UVB significantly up-regulated the expression of TGase 1 at the gene and protein levels. Treatment with inhibitors of p38, MEK, JNK or NFκB significantly abolished the UVB-stimulated protein expression of TGase 1. Treatment with astaxanthin immediately after UVB irradiation did not attenuate the increased phosphorylation of Ser536/Ser468NFκBp65, c-Jun, ATK-2 and CK2, and did not abrogate the increased or diminished protein levels of c-Jun/c-Fos or I-κBα, respectively. However, the same treatment with astaxanthin significantly abolished the UVB-stimulated expression of TGase 1 protein, which was accompanied by the attenuated phosphorylation of Thr565/Ser376/Ser360MSK1, Ser276NFκBp65 and Ser133CREB. The MSK1 inhibitor H89 significantly down-regulated the increased protein expression of TGase 1 in UVB-exposed human keratinocytes, which was accompanied by an abrogating effect on the increased phosphorylation of Ser276NFκBp65 and Ser133CREB but not Thr565/Ser376/Ser360MSK1. Transfection of human keratinocytes with MSK1 siRNA suppressed the UVB-stimulated protein expression of TGase 1. These findings suggest that the UVB-stimulated expression of TGase 1 is mediated predominantly via the NFκB pathway and can be attenuated through a specific interruption of the p38/MSK1/NFκBp65Ser276 axis.

Astaxanthin and withaferin A block paracrine cytokine interactions between UVB-exposed human keratinocytes and human melanocytes via the attenuation of endothelin-1 secretion and its downstream intracellular signaling.

BACKGROUND Paracrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect. OBJECTIVE To resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action. METHODS AND RESULTS RT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity of tyrosinase in melanocytes. Western blot analysis of intracellular signaling factors revealed that withaferin A but not astaxanthin significantly abolished the endothelin-1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB in human melanocytes. CONCLUSIONS These results demonstrate that this co-culture system is an appropriate model to characterize melanogenic paracrine interactions and that astaxanthin and withaferin A serve as potent inhibitors of those interactions. Their effects are caused not only by down-regulating the increased secretion of an intrinsic melanogenic cytokine, endothelin-1, by UVB-exposed human keratinocytes, but also by interrupting the endothelin-1-triggered downstream intracellular signaling between protein kinase C and Raf-1 in human melanocytes (only for withaferin A).

Abrogating effect of N-linked carbohydrate modifiers on the stem cell factor and endothelin-1-stimulated epidermal pigmentation in human epidermal equivalents.

BACKGROUND We previously demonstrated that the hyperpigmentation that occurs in UVB-melanosis as well as in solar lentigos is associated with the increased production of melanogenic cytokines, such as endothelin (EDN)-1 and stem cell factor (SCF), by keratinocytes in those areas of the skin. OBJECTIVE We developed a model for these hyperpigmentary disorders in EDN1+SCF stimulated human epidermal equivalents (HEEs) and characterized the effects of the N-linked carbohydrate core synthesis inhibitor glucosamine or N-linked carbohydrate processing inhibitors deoxynojirimycin or monensin on the stimulated HEE pigmentation. METHODS Those effects were assessed by melanin analysis, real-time RT-PCR and Western blotting. RESULTS The addition of these N-linked carbohydrate modifiers (NCMs) markedly abolished the EDN1+SCF-elicited increase in HEE pigmentation over 14 days. Real-time RT-PCR and Western blotting of these NCM-treated HEEs unexpectedly revealed that the EDN1+SCF-stimulated steady-state levels of tyrosinase (TYR), TYR-related protein-1, dopachrome tautomerase and PMEL17 as well as microphthalmia-associated transcription factor (MITF) were significantly attenuated at the transcriptional and translational levels without any cytotoxic effects on keratinocytes and melanocytes in the HEEs. Pre-treatment of cultured normal human melanocytes with the NCMs interrupted the EDN1+SCF-induced stimulation of steady-state levels of MITF at the transcriptional and translational levels and TYR activity without any direct inhibitory effect on the catalytic activity of TYR in vitro. CONCLUSION This study provides evidence that NCMs have a potential to attenuate the EDN1+SCF-stimulated pigmentation of HEEs by abrogating the increased steady-state levels of MITF mRNA, which results in the attenuation of the increased steady-state levels of these melanocyte-specific proteins.

An Extract of Withania somnifera Attenuates Endothelin-1-stimulated Pigmentation in Human Epidermal Equivalents through the Interruption of PKC Activity Within Melanocytes: Withania Somnifera Attenuates pigmentation by interrupting PKC

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. Copyright

Reduced glutathione disrupts the intracellular trafficking of tyrosinase and tyrosinase-related protein-1 but not dopachrome tautomerase and Pmel17 to melanosomes, which results in the attenuation of melanization

We previously reported that treatment of B16 melanotic melanoma cells with reduced glutathione (GSH) converts them to amelanotic cells without any significant down-regulation of tyrosinase activity. To characterize the cellular mechanism(s) involved, we determined the intracellular distribution of melanocyte-specific proteins, especially in melanin synthesis-specific organelles, termed melanosomes by subcellular fractionation followed by Western blotting and confocal laser microscopy (CFLM). In the melanosome-rich large granule fraction and in highly purified melanosome fractions, while GSH-induced amelanotic B16 cells have significantly diminished levels of protein/activity of tyrosinase and tyrosinase-related protein-1 compared with control melanized B16 cells, there was substantially no difference in the distribution and levels of dopachrome tautomerase and the processed isoform of Pmel17 (HMB45) between control melanized and GSH-induced amelanotic B16 cells. Analysis of merged images obtained by CFLM revealed that whereas tyrosinase, Pmel17 and dopachrome tautomerase colocalize with each other in the control melanized B16 cells, tyrosinase does not colocalize with Pmel17 or its processed isoform and with dopachrome tautomerase in GSH-induced amelanotic B16 cells. The sum of these findings suggests that reduced glutathione selectively disrupts the intracellular trafficking of tyrosinase and tyrosinase-related protein-1 but not dopachrome tautomerase and Pmel17 to melanosomes, which results in the attenuation of melanization, probably serving as a putative model for oculocutaneous albinism type 4.

Paracrine cytokine mechanisms underlying the hyperpigmentation of seborrheic keratosis in covered skin areas

We previously reported that increased expression of the endothelin (EDN)1/EDNB receptor (EDNBR) as well as the stem cell factor (SCF)/SCF receptor (c‐KIT) linkages is mainly responsible for the activation of melanocytes in the epidermal hyperpigmentation of ultraviolet (UV)‐B melanosis and lentigo senilis (LS). In this study, we characterized seborrheic keratosis (SK) to examine the paracrine cytokine mechanism(s) involved in its epidermal hyperpigmentation by reverse transcription polymerase chain reaction, immunohistochemistry and western blotting analyses. In contrast to our previous study which showed the upregulated expression of EDN1 and EDNBR at the transcriptional and translational levels in the epidermis of SK, we observed unexpectedly that the cytokine SCF and its receptor c‐KIT are not upregulated, but are downregulated at both the gene and protein levels. We established SK cell lines to examine whether SK basaloid cells are less sensitive to SCF‐inducible stimulation than are normal human keratinocytes (NHK). Comparison of the stimulatory effects of interleukin (IL)‐1α or tumor necrosis factor (TNF)‐α on SCF production between SK cells and NHK demonstrated that SK cells do not respond to IL‐1α or TNF‐α to stimulate production of SCF, whereas a significant stimulation of SCF is elicited by those same cytokines in NHK. These finding underscore a role of phenotypic changes in melanogenic cytokine production in the epidermis between SK and LS/UV‐B melanosis.

RETRACTED: Paracrine cytokine interaction between UVB-exposed epidermal keratinocytes and dermal fibroblasts in stimulating expression of skin fibroblast-derived elastase

BACKGROUND We recently reported that over-expression of skin fibroblast-derived elastase (SFE) plays a pivotal role in the mechanism of UVB-induced skin wrinkling. Since UVB penetrates only modestly to the dermis, we hypothesized that factors secreted by UVB-exposed keratinocytes in the epidermis trigger fibroblasts in the dermis to increase their expression of SFE which then degrades the elastic fibers. OBJECTIVE In this study, we characterized the paracrine interaction between human keratinocytes (HK) and human fibroblasts (HF) which leads to increased expression of SFE. METHODS AND RESULTS Medium conditioned by UVB-exposed HK contained increased levels of IL-1α, GM-CSF, IL-6, TNFα and IL-8. While HF cultured with those conditioned medium slightly down-regulated the gene expression of collagen and elastin, they significantly increased their expression of SFE at the transcriptional, translational and enzymatic levels. Neutralizing antibodies to IL-1α or GM-CSF significantly abolished the increased expression of SFE at the translational and/or enzymatic levels in HF cultured with those conditioned medium, while neutralizing antibodies to IL-6, IL-8 or TNFα had no such effect. The addition of IL-1α or GM-CSF, but not TNFα, IL-6 or IL-8, at concentrations ranging from 1 to 10nm, significantly stimulated the enzymatic levels of SFE in HF. CONCLUSIONS The sum of these findings suggests that IL-1α and GM-CSF are intrinsic cytokines secreted by UVB-exposed HK that stimulate expression of SFE by HF, leading to UVB-induced wrinkle formation.