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Ultraviolet (UV) irradiation causes cellular oxidative stress. Under redox imbalance, Keap1-dependent Nrf2 degradation is minimal. In this study, we examined the role of Ca2+ in Nrf2 homeostasis after UVB irradiation using human dermal fibroblasts. UVB irradiation stimulates 12-lipoxygenase and the product 12-hydroxyeicosatetraenoic acid then activates TRPV1 increasing the cell's cytosolic Ca2+ concentration. UVB irradiation induced reactive oxygen species generation and apoptosis are inhibited in the absence of Ca2+ or in the presence of either a 12-lipoxygenase inhibitor or a TRPV1 inhibitor during and after UVB irradiation. Thus, the Ca2+ increase via TRPV1 is a critical factor in UVB irradiation induced oxidative stress. UVB irradiation induces a Ca2+ dependent Nrf2 degradation and thus activation of TRPV1 with 12-hydroxyeicosatetraenoic acid also decreasing Nrf2 levels. UVB irradiation induced Nrf2 degradation is inhibited by co-treatment of cells with W-7, cyclosporin A, SB-216763 or MG-132, which are inhibitors of calmodulin, calcineurin, GSK3β and the proteasome, respectively. Furthermore, UVB irradiation in parallel induces GSK3β dephosphorylation in a Ca2+ dependent manner. Co-immunoprecipitation showed that UVB irradiation induces an increase in Nrf2 phosphorylation, an increase in the binding of β-TrCP and Nrf2, and an increase in Nrf2 ubiquitination; these effects are all Ca2+ dependent. These findings suggest that UVB irradiation induced GSK3β activation in a Ca2+ dependent manner, which then stimulates the phosphorylation and ubiquitination of Nrf2 via β-TrCP. Indeed, silencing of β-TrCP was found to inhibit UVB irradiation-induced oxidative stress, Nrf2 degradation and apoptosis, while it had no effect on the Ca2+ increase. Taken together, our results suggest that a Ca2+ influx via TRPV1 is responsible for UVB irradiation-induced Nrf2 degradation and that modulation of the Ca2+-calmodulin-calcineurin-GSK3β-Nrf2-β-TrCP-Cullin-1 pathway may explain Ca2+ dependent Nrf2 degradation. Image 1 • UVB irradiation causes Ca2+ increase via TRPV1 after 12-lipoxygenase activation. • UVB irradiation induced Nrf2 degradation, ROS generation and apoptosis requires Ca2+. • Calcineurin dephosphorylates and activates GSK3β, which then phosphorylates Nrf2. • The P-Nrf2-β-TrCP complex ubiquitinates Nrf2 and targets it for proteasomal degradation. [ABSTRACT FROM AUTHOR]

Increased matrix metalloproteinase 1 (MMP‐1) expression is a feature of photo‐aged skin. We investigated the effects of baicalein and sulphoraphane on ultraviolet B (UVB) irradiation–induced MMP‐1 expression and apoptosis using human dermal fibroblasts. UVB irradiation not only increased MMP‐1 expression, but also caused apoptosis. Both baicalein and sulphoraphane protected cells from UVB irradiation–induced apoptosis, but only baicalein inhibited MMP‐1 expression. UVB irradiation activated 12‐lipoxygenase, and its product, 12‐hydroxyeicosatetraenoic acid, activated TRPV1 channels. The resulting UVB irradiation–induced Ca2+ increase was blocked by the 12‐lipoxygenase inhibitor baicalein and the TRPV1 blocker capsazepine, but not by the Nrf2 inducer sulphoraphane. UVB irradiation also increased ROS generation and decreased Nrf2 protein levels. UVB irradiation–induced MMP‐1 expression was blocked by the Ca2+ chelator BAPTA, by capsazepine and by TRPV1 silencing. However, induction was unaffected by the antioxidant N‐acetylcysteine. ERK phosphorylation and JNK phosphorylation were induced by UVB irradiation, but only ERK phosphorylation was Ca2+ sensitive. Increased MMP‐1 expression was blocked by PD98059, but not by SP600125. Thus, increased MMP‐1 expression is mediated by increased cytosolic Ca2+ and ERK phosphorylation. UVB irradiation–induced ROS generation is also Ca2+ sensitive, and UVB irradiation–induced apoptosis is caused by increased ROS. Thus, baicalein, by blocking the UVB irradiation–induced cytosolic Ca2+ increase, protects cells from UVB irradiation–induced MMP‐1 expression and apoptosis. In contrast, sulphoraphane, by decreasing cellular ROS, protects cells from only UVB‐induced apoptosis. Thus, targeting 12‐lipoxygenase may provide a therapeutic approach to improving the health of photo‐aged human skin. [ABSTRACT FROM AUTHOR]

Copper is more likely than iron to generate reactive oxygen species (ROS) in a redox reaction due to its higher electrochemical reactivity. This study examined the effect of a newly synthesized Cu2+ binding compound, (E)‐2‐(4‐(dimethylamino)phenylimino)methyl)quinolin‐8‐ol (DPMQ), on ultraviolet B (UVB) irradiation–induced cytotoxicity in human dermal fibroblasts. DPMQ induced Cu2+ influx as effectively as disulfiram, a Cu2+ ionophore anticancer drug. However, disulfiram induced ROS generation, mitochondrial dysfunction, and apoptosis in fibroblasts in a Cu2+‐dependent manner, whereas DPMQ was not only nontoxic, but protected cells against UVB irradiation–induced apoptosis in a Cu2+‐independent manner. UVB irradiation induced a Ca2+‐dependent increase in ROS generation, a decrease in Nrf2 levels, and activation of the mitochondrial apoptotic pathway, and these effects were prevented by DPMQ, which also increased Nrf2 nuclear translocation in a Cu2+‐independent manner. UVB irradiation activated 12‐lipoxygenase and 12‐hydroxyeicosatetraenoic acid (12‐HETE), a product of 12‐lipoxygenase, activated the TRPV1 channel. DMPQ did not act as a Ca2+ chelator, but inhibited the cytosolic Ca2+ increase induced by 12‐HETE or capsaicin, but not that induced by bradykinin or ATP. Blockade of Ca2+ influx by pharmacological inhibition or silencing of the TRPV1 channel or chelation of cytosolic Ca2+ inhibited the UVB irradiation–induced Nrf2 reduction, ROS generation, mitochondrial dysfunction, and apoptosis. Taken together, our results suggest that Ca2+ influx via the TRPV1 channel is responsible for UVB irradiation–induced cytotoxicity and that DPMQ protects cells against UVB irradiation by inhibiting the TRPV1 channel and stabilizing Nrf2, and could thus be a potentially useful compound for the treatment of free radical‐induced diseases. Treatment of cells with ultraviolet B (UVB) irradiation results in increased reactive oxygen species (ROS) generation and decreased Nrf2 levels by a mechanism involving lipoxygenase stimulation and TRPV1 channel activation. Increased ROS results in mitochondrial dysfunction and apoptosis. DPMQ inhibits the TRPV1 channel and scavenges ROS, and hence protects cells against UVB irradiation–induced cytotoxicity, whereas disulfiram induces copper influx and increases ROS generation, and therefore causes apoptosis. [ABSTRACT FROM AUTHOR]

In this study, we characterized the effect of baicalein on the regulation of keratinocyte differentiation and proliferation, which are abnormal in atopic dermatitis or psoriasis. Treatment of HaCaT keratinocytes with 10 μ m baicalein slightly inhibited cell growth, caused morphological differentiation and increased expression of keratins 1 and 10 (K1/K10) without affecting ROS generation, cytochrome c release or apoptosis. Baicalein treatment caused growth arrest in G0/G1 phase and also induced Ca2+ influx via TRPV4 receptor activation. Phosphorylation of ERK, Akt and p38 MAPK, but not JNK, was increased by baicalein, and inhibition of phosphorylation of ERK, but not that of Akt or p38 MAPK, blocked the baicalein-induced increase in K1/K10 expression, suggesting that ERK activation is involved in this increase. Removal of extracellular Ca2+ or blockade of Ca2+ influx by pharmacological inhibition or silencing of the TRPV4 receptor did not affect growth arrest, ROS generation or apoptosis, but inhibited baicalein-induced ERK phosphorylation and K1/K10 expression. Thus, baicalein treatment increases differentiation, and decreases proliferation, of keratinocytes. The mechanism of differentiation of keratinocytes is distinct from that of proliferation, the former being Ca2+ dependent and the latter Ca2+ independent. [ABSTRACT FROM AUTHOR]