Intracellular Mg2+ protects mitochondria from oxidative stress in human keratinocytes.

Reactive oxygen species (ROS) are harmful for the human body, and exposure to ultraviolet irradiation triggers ROS generation. Previous studies have demonstrated that ROS decrease mitochondrial membrane potential (MMP) and that Mg2+ protects mitochondria from oxidative stress. Therefore, we visualized the spatio-temporal dynamics of Mg2+ in keratinocytes (a skin component) in response to H2O2 (a type of ROS) and found that it increased cytosolic Mg2+ levels. H2O2-induced responses in both Mg2+ and ATP were larger in keratinocytes derived from adults than in keratinocytes derived from newborns, and inhibition of mitochondrial ATP synthesis enhanced the H2O2-induced Mg2+ response, indicating that a major source of Mg2+ was dissociation from ATP. Simultaneous imaging of Mg2+ and MMP revealed that larger Mg2+ responses corresponded to lower decreases in MMP in response to H2O2. Moreover, Mg2+ supplementation attenuated H2O2-induced cell death. These suggest the potential of Mg2+ as an active ingredient to protect skin from oxidative stress.

Magnesium Supplementation Attenuates Ultraviolet-B-Induced Damage Mediated through Elevation of Polyamine Production in Human HaCaT Keratinocytes.

Magnesium ions (Mg2+) have favorable effects such as the improvement of barrier function and the reduction of inflammation reaction in inflammatory skin diseases. However, its mechanisms have not been fully understood. Microarray analysis has shown that the gene expressions of polyamine synthases are upregulated by MgCl2 supplementation in human HaCaT keratinocytes. Here, we investigated the mechanism and function of polyamine production. The mRNA and protein levels of polyamine synthases were dose-dependently increased by MgCl2 supplementation, which were inhibited by U0126, a MEK inhibitor; CHIR-99021, a glycogen synthase kinase-3 (GSK3) inhibitor; and Naphthol AS-E, a cyclic AMP-response-element-binding protein (CREB) inhibitor. Similarly, reporter activities of polyamine synthases were suppressed by these inhibitors, suggesting that MEK, GSK3, and CREB are involved in the transcriptional regulation of polyamine synthases. Cell viability was reduced by ultraviolet B (UVB) exposure, which was rescued by MgCl2 supplementation. The UVB-induced elevation of reactive oxygen species was attenuated by MgCl2 supplementation, which was inhibited by cysteamine, a polyamine synthase inhibitor. Our data indicate that the expression levels of polyamine synthases are upregulated by MgCl2 supplementation mediated through the activation of the MEK/GSK3/CREB pathway. MgCl2 supplementation may be useful in reducing the UVB-induced oxidative stress in the skin.

Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells.

Skin barrier damage is present in the patients with hereditary disorders of the magnesium channel, but the molecular mechanism has not been fully understood. We found that the expressions of hyaluronan synthase (HAS), HAS2 and HAS3 are influenced by MgCl2 concentration in human keratinocyte-derived HaCaT cells. The exposure of cells to a high concentration (5.8 mM) of MgCl2 induced the elevation of HAS2/3 expression, which was inhibited by mRNA knockdown of nonimprinted in Prader-Willi/Angelman syndrome-like domain containing 4 (NIPAL4). Similarly, the content of hyaluronic acid (HA) was changed according to MgCl2 concentration and the expression of NIPAL4. The MgCl2 supplementation increased the reporter activities of HAS2/3, which were inhibited by NIPAL4 knockdown, indicating that the expressions of HAS2/3 are up-regulated at the transcriptional level. The reporter activities and mRNA levels of HAS2/3, and the production of HA were inhibited by CHIR-99021, a glycogen synthase kinase-3 (GSK3) inhibitor, and naphthol AS-E, a cyclic AMP-response element binding protein (CREB) inhibitor. Furthermore, the mutation in putative CREB-binding sites of promoter region in HAS2/3 genes inhibited the MgCl2 supplementation-induced elevation of promoter activity. Our results indicate that the expressions of HAS2/3 are up-regulated by MgCl2 supplementation in HaCaT cells mediated through the activation of GSK3 and CREB. Magnesium may play a pivotal role in maintaining the skin barrier function and magnesium supplementation may be useful to enhance moisturization and wound repair in the skin.

Japanese Cedar (Cryptomeria japonica) pollen allergen induces elevation of intracellular calcium in human keratinocytes and impairs epidermal barrier function of human skin ex vivo.

Cry j1 is the major peptide allergen of Japanese cedar (Sugi), Cryptomeria japonica. Since some allergens disrupt epidermal permeability barrier homeostasis, we hypothesized that Cry j1 might have a similar effect. Intracellular calcium level in cultured human keratinocytes was measured with a ratiometric fluorescent probe, Fura-2 AM. Application of Cry j1 significantly increased the intracellular calcium level of keratinocytes, and this increase was inhibited by trypsin inhibitor or a protease-activated receptor 2 (PAR-2) antagonist. We found that Cry j1 itself did not show protease activity, but application of Cry j1 to cultured keratinocytes induced a rapid (within 30 s) and transient increase of protease activity in the medium. This transient increase was blocked by trypsin inhibitor or PAR-2 antagonist. The effect of Cry j1 on transepidermal water loss (TEWL) of cultured human skin was measured in the presence and absence of a trypsin inhibitor and PAR-2 antagonist. Cry j1 significantly impaired the barrier function of human skin ex vivo, and this action was blocked by co-application of trypsin inhibitor or PAR-2 antagonist. Our results suggested that interaction of Cry j1 with epidermal keratinocytes leads to the activation of PAR-2, which induces elevation of intracellular calcium and disruption of barrier function. Blocking the interaction of Cry j1 with epidermal keratinocytes might ameliorate allergic reaction and prevent disruption of epidermal permeability barrier homeostasis.

Coculture system of keratinocytes and dorsal-root-ganglion-derived cells for screening neurotrophic factors involved in guidance of neuronal axon growth in the skin.

The density of peripheral nerve fibres is increased in atopic dermatitis. Moreover, reduction in the fibres in a mouse model of atopic dermatitis reduces scratching behaviour. Thus, regulation of nerve fibre extension could be an effective strategy to reduce itching in pruritus dermatosis. In this study, we established a new coculture system of keratinocytes and dorsal-root-ganglion-derived cells using an apparatus, AXIS(™) , which consists of two different channels connected via a set of microgrooves, through which signalling molecules and axons, but not living cells, can pass. When we seeded keratinocytes in one chamber, extension of nerve fibres was observed from dorsal root ganglion cells seeded in the other chamber. Addition of anti-BDNF antibody in the keratinocyte-seeded chamber significantly reduced the extension. Application of Semaphorin 3A also reduced the extension by approximately 50%. We suggest that this coculture system may be useful for screening of anti-itching drugs.

Phosphodiesterase inhibitors block the acceleration of skin permeability barrier repair by red light.

We previously demonstrated that exposure to red light (550-670 nm) accelerates epidermal permeability barrier recovery after barrier disruption. Furthermore, we showed that photosensitive proteins, originally found in retina, are also expressed in epidermis. In retina, transducin and phosphodiesterase 6 play key roles in signal transmission. In this study, we evaluate the role of phosphodiesterese 6 in the acceleration by red light of epidermal permeability barrier recovery. Immunohistochemical study and reverse transcription-PCR assays confirmed the expression of both transducin and phosphodiesterase 6 in epidermal keratinocytes. Topical application of 3-isobutyl-1-methylxanthine, a non-specific phosphodiesterase inhibitor, blocked the acceleration of the barrier recovery by red light. Topical application of zaprinast, a specific inhibitor of phosphodiesterases 5 and 6, also blocked the acceleration, whereas T0156, a specific inhibitor of phosphodiesterase 5, had no effect. Red light exposure reduced the epidermal hyperplasia induced by barrier disruption under low humidity, and the effect was blocked by pretreatment with zaprinast. Our results indicate phosphodiesterase 6 is involved in the recovery-accelerating effect of red light on the disrupted epidermal permeability barrier.