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.

Cuf2 boosts the transcription of APC/C activator Fzr1 to terminate the meiotic division cycle.

The number of nuclear divisions in meiosis is strictly limited to two. Although the precise mechanism remains unknown, this seems to be achieved by adjusting the anaphase-promoting complex/cyclosome (APC/C) activity to degrade cyclin. Here, we describe a fission yeast cuf2 mutant that enters into a third nuclear division cycle, represented by ectopic spindle assembly and abnormal chromosome segregation. Cuf2 is a meiotic transcription factor, and its critical target is fzr1(+)/mfr1(+), which encodes a meiotic APC/C activator. fzr1Δ also enters a third nuclear division. Thus, Cuf2 ensures termination of the M-phase cycle by boosting Fzr1 expression to generate functional gametes.

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.

Kaempferol enhanced the intracellular thioredoxin system in normal cultured human keratinocytes.

The effects of kaempferol on redox-associated enzymes in normal human keratinocytes were studied. It enhanced the gene expression of thioredoxin reductase 1 and thioredoxin. Flavonols had higher ability to induce the expression of these genes than the other anti-oxidants and the other flavonoids tested. Furthermore, kaempferol and quercetin increased the activity of thioredoxin reductase in normal human keratinocytes.

Fibulin-5 accelerates elastic fibre assembly in human skin fibroblasts.

Fibulin-5 null mice display abnormalities in the elastic fibres in the dermis. We postulated, therefore, that fibulin-5 might be a regulator of elastic fibre assembly and stability. To clarify the role of fibulin-5 in elastic fibre formation, we employed in vitro systems that allowed increasing expression of elastic fibre components by gene transduction using retroviral vector constructs. First, the human tropoelastin gene (ELN) was transduced into human dermal fibroblasts, which resulted in elevated gene expression. These cells were then cultured in monolayer, but the overexpression of ELN in this system did not alter the assembly of elastic fibres. However, incubation of fibroblasts with TGF-beta1 resulted in elastic fibre accumulation, and the expression of fibulin-5 was enhanced by TGF-beta1. Thus, we overexpressed human fibulin-5 cDNA in dermal fibroblasts using a retroviral vector containing a cytomegalovirus (CMV) promoter. These cells deposited elastic fibres. These results suggest that fibulin-5 is a critical component in the control of elastic fibre assembly by dermal fibroblasts.

Unsaturated fatty acids induce calcium influx into keratinocytes and cause abnormal differentiation of epidermis.

Abnormal follicular keratinization is involved in comedogenesis in acne vulgaris. We recently demonstrated that calcium influx into epidermal keratinocytes is associated with impaired skin barrier function and epidermal proliferation. Based on these results, we hypothesized that sebum components affect calcium dynamics in the keratinocyte and consequently induce abnormal keratinization. To test this idea, we first observed the effects of topical application of sebum components, triglycerides (triolein), saturated fatty acids (palmitic acid and stearic acid), and unsaturated fatty acids (oleic acid and palmitoleic acid) on hairless mouse skin. Neither triglyceride nor saturated fatty acids affected the skin surface morphology or epidermal proliferation. On the other hand, application of unsaturated fatty acids, oleic acid, and palmitoleic acid induced scaly skin, abnormal keratinization, and epidermal hyperplasia. Application of triglycerides and saturated fatty acids on cultured human keratinocytes did not affect the intracellular calcium concentration ([Ca(2+)](i)), whereas unsaturated fatty acids increased the [Ca(2+)](i) of the keratinocytes. Moreover, application of oleic acid on hairless mouse skin induced an abnormal calcium distribution in the epidermis. These results suggest that unsaturated fatty acids in sebum alter the calcium dynamics in epidermal keratinocytes and induce abnormal follicular keratinization.

Urokinase-type plasminogen activator is activated in stratum corneum after barrier disruption.

The plasminogen/plasmin system in epidermis is thought to be the major protease involved in the delay of barrier recovery. However, little is known about the mechanism through which this system is activated. In order to clarify this mechanism, we first determined the distribution of proteolytic activity by using in situ zymography. As a result, plasminogen-activator activity was found to be present in the stratum corneum (SC) after barrier disruption. Next, SC subjected to repeated barrier disruption was collected to identify the protease. The protease was identified as urokinase-type plasminogen activator, because flybrinolytic activity of the collected SC was abolished by addition of anti-urokinase antibody. Urokinase activation in SC was confirmed by means of an in vitro assay, in which the precursor of urokinase (pro-uPA) became active after incubation with the insoluble component of SC homogenate. These findings indicated that urokinase-type plasminogen activator is activated in SC after barrier disruption and this activation might trigger the plasminogen/plasmin system in the epidermis.