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.

Protective Effects of Ethanol Extract of Brazilian Green Propolis and Apigenin against Weak Ultraviolet Ray-B-Induced Barrier Dysfunction via Suppressing Nitric Oxide Production and Mislocalization of Claudin-1 in HaCaT Cells.

Once weak ultraviolet ray-B (UVB) irradiates the skin cells, the generation of reactive nitrogen species (RNS), but not reactive oxygen species (ROS), is stimulated for the mislocalization of claudin-1 (CLDN1), an essential protein for forming tight junctions (TJs). Since our skin is constantly exposed to sunlight throughout our lives, an effective protection strategy is needed to maintain the skin barrier against weak UVB. In the present study, we investigated whether an ethanol extract of Brazilian green propolis (EBGP) and flavonoids had a protective effect against weak UVB irradiation-induced barrier dysfunction in human keratinocyte-derived HaCaT cells. A pretreatment with EBGP suppressed TJ permeability, RNS production, and the nitration level of CLDN1 in the weak UVB-exposed cells. Among the propolis components, apigenin and apigenin-like flavonoids have potent protective effects against NO production and the mislocalization of CLDN1 induced by UVB. The analyses between structures and biological function revealed that the chemically and structurally characteristic flavonoids with a hydroxyl group at the 4' position on the B-ring might contribute to its protective effect on barrier dysfunction caused by weak UVB irradiation. In conclusion, EBGP and its component apigenin protect HaCaT cells from weak UVB irradiation-induced TJ barrier dysfunction mediated by suppressing NO production.

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.

Flavonol glycosides of Rosa multiflora regulates intestinal barrier function through inhibiting claudin expression in differentiated Caco-2 cells.

Eijitsu, the fruits of Rosa multiflora Thunberg, is a traditional Japanese natural medicine and used as purgatives. The active constituents were identified as flavonol glycosides, multiflorin A (MF), and multinoside A (MSA), but mechanism of the purgative action is still unknown. We hypothesized that the flavonol glycosides 1 and 2 may exhibit the purgative actions through modulating intestinal epithelial barrier function. Then, this study aimed to investigate their effects on intestinal epithelial barrier function and possible molecular mechanisms in human intestinal Caco-2 cells. MF and MSA decreased transepithelial electrical resistance and increased paracellular permeability of Caco-2 cell monolayers. Expression of claudins (CLDNs) involved in paracellular permeability of ions and low-molecular substances was significantly decreased by the treatment with MF or MSA. The compounds increased the ratio of N-cadherin/E-cadherin, expression of transforming growth factor-ß and Slug, and phosphorylation level of Smad3, suggesting epithelial-mesenchymal transition activation, and epithelial-mesenchymal transition inhibition by transforming growth factor-ß receptor kinase inhibitors completely recovered the decreased CLDNs expression caused by MF and MSA. Moreover, the increased paracellular permeability and the decreased CLDNs expression by the treatment with MF or MSA for 24 hours recovered to the same extent as the untreated group with the compounds by continuous culture in the growth medium alone for 48 hours. These results suggest that Eijitsu may be effective in preventing or relieving constipation symptoms, unless used chronically.

Autophagy inhibition enhances anticancer efficacy of artepillin C, a cinnamic acid derivative in Brazilian green propolis.

Propolis, a resinous substance produced by honeybees, possesses various biological actions including anticancer activity towards tumor cells. Recently, the ethanol extract of Brazilian green propolis has been shown to induce autophagy, which is known to be induced in treatment of cancer cells with anticancer drugs, leading to cancer cell survival and decreased sensitivity to anticancer agents. In this study, we aimed to identify autophagy-inducing components of the propolis and elucidated the reciprocal relationship between anticancer cytotoxicity and protective autophagy in prostate cancer CWR22Rv1 cells. Among eight cinnamic acid derivatives [chlorogenic acid, p-coumaric acid, caffeic acid, 3,4-caffeoylquinic acid, artepillin C (ArtC), baccharin, drupanin and caffeic acid phenethyl ester] in propolis, only ArtC showed high autophagy-inducing activity accompanying LC3-II upregulation. ArtC was also induced apoptosis as revealed by DNA fragmentation and increases in cleaved caspase-3 and poly ADP-ribose polymerase. The apoptosis induced by ArtC was exacerbated by cotreatment with autophagy inhibitors (chloroquine, wortmannin and U0126). The cotreatment further induced necroptosis accompanying increased expression of receptor-interacting serine/threonine protein kinases 1 and 3. These data indicate that cytotoxicity of ArtC to the prostate cancer cells is dampened by induced autophagy, but is markedly augmented by inhibition of autophagy. Therefore, the combination of ArtC and autophagy inhibitors may be a novel complementary-alternative treatment for prostate cancer.

Structure-activity relationship of flavonoids as potent inhibitors of carbonyl reductase 1 (CBR1).

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily, reduces a variety of carbonyl compounds including therapeutic drugs. CBR1 is involved in the reduction of the anthracycline anticancer drugs to their less anticancer C-13 hydroxy metabolites, which are cardiotoxic. CBR1 inhibitors are thought to be promising agents for adjuvant therapy with twofold beneficial effect in prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity, a side effect of the drugs. In this study, we evaluated 27 flavonoids for their inhibitory activities of CBR1 in order to explore the structure-activity relationship (SAR). Among them, luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one) showed the most potent inhibition (IC5095nM), which is also more potent compared to all known classes of CBR1 inhibitors. The inhibition of luteolin was noncompetitive with respect to the substrate in the NADPH-dependent reduction direction, but CBR1 exhibited moderate NADP(+)-dependent dehydrogenase activity for some alicyclic alcohols, in which the luteolin inhibition was competitive with respect to the alcohol substrate (Ki59nM). The SAR of the flavonoids indicated that the 7-hydroxy group of luteolin was responsible for the potent inhibition of CBR1. The molecular docking of luteolin in CBR1-NADPH complex showed that theflavonoid binds to the substrate-binding cleft, in which its 7-hydroxy group formed a H-bond with main-chain oxygen of Met234, in addition to H-bond interactions (of its 5-hydroxy and 4-carbonyl groups with catalytically important residues Tyr193 and/or Ser139) and a π-stacking interaction (between its phenyl ring and Trp229).

Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway.

Claudin-16 (CLDN-16) is involved in the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle. The tight junctional localization and Mg(2+) transport of CLDN-16 are regulated by cAMP/PKA-dependent phosphorylation. Here, we examined whether PKA phosphorylates CLDN-16 in a direct or indirect manner. CLDN-16 was stably expressed in Madin-Darby canine kidney (MDCK) cells using a Tet-OFF system. The phosphorylation of CLDN-16 is upregulated by fetal calf serum (FCS). This phosphorylation was completely inhibited by a PKA inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Without FCS, dibutyryl cAMP (DBcAMP) increased the phosphoserine level of CLDN-16 in a concentration-dependent manner. The phosphorylated CLDN-16 elicited increases of transepithelial electrical resistance (TER) and transepithelial transport of Mg(2+). Vasodilator-stimulated phosphoprotein (VASP) was also phosphorylated in the presence of FCS or DBcAMP. In the glutathione-S-transferase (GST) pull down assay, a cytosolic carboxyl domain of CLDN-16 was associated with PKA, but not with VASP. Furthermore, PKA was immunoprecipitated with CLDN-16 in MDCK cells, but VASP was not. In cells expressing a dephosphorylated mutant (Ser160Ala) of VASP, CLDN-16 was phosphorylated by DBcAMP and was associated with ZO-1, a tight junctional-scaffolding protein, without integral cell-cell junctions. We suggest that PKA directly phosphorylates CLDN-16, resulting in the localization to tight junctions (TJs) and the maintenance of Mg(2+) reabsorption.