Control of the signaling role of PtdIns(4)P at the plasma membrane through H2O2-dependent inactivation of synaptojanin 2 during endocytosis.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is implicated in various processes, including hormone-induced signal transduction, endocytosis, and exocytosis in the plasma membrane. However, how H2O2 accumulation regulates the levels of PtdIns(4,5)P2 in the plasma membrane in cells stimulated with epidermal growth factors (EGFs) is not known. We show that a plasma membrane PtdIns(4,5)P2-degrading enzyme, synaptojanin (Synj) phosphatase, is inactivated through oxidation by H2O2. Intriguingly, H2O2 inhibits the 4-phosphatase activity of Synj but not the 5-phosphatase activity. In EGF-activated cells, the oxidation of Synj dual phosphatase is required for the transient increase in the plasma membrane levels of phosphatidylinositol 4-phosphate [PtdIns(4)P], which can control EGF receptor-mediated endocytosis. These results indicate that intracellular H2O2 molecules act as signaling mediators to fine-tune endocytosis by controlling the stability of plasma membrane PtdIns(4)P, an intermediate product of Synj phosphoinositide dual phosphatase.

Diclofenac: A Nonsteroidal Anti-Inflammatory Drug Inducing Cancer Cell Death by Inhibiting Microtubule Polymerization and Autophagy Flux.

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) used to treat inflammatory diseases induces cellular toxicity by increasing the production of reactive oxygen species (ROS) and impairing autophagic flux. In this study, we investigated whether diclofenac induces cancer cell death and the mechanism by which diclofenac causes cell death. We observed that diclofenac induces mitotic arrest with a half-maximal effective concentration of 170 µM and cell death with a half-maximal lethal dose of 200 µM during 18-h incubation in HeLa cells. Cellular microtubule imaging and in vitro tubulin polymerization assays demonstrated that treatment with diclofenac elicits microtubule destabilization. Autophagy relies on microtubule-mediated transport and the fusion of autophagic vesicles. We observed that diclofenac inhibits both phagophore movement, an early step of autophagy, and the fusion of autophagosomes and lysosomes, a late step of autophagy. Diclofenac also induces the fragmentation of mitochondria and the Golgi during cell death. We found that diclofenac induces cell death further in combination with 5-fuorouracil, a DNA replication inhibitor than in single treatment in cancer cells. Pancreatic cancer cells, which have high basal autophagy, are particularly sensitive to cell death by diclofenac. Our study suggests that microtubule destabilization by diclofenac induces cancer cell death via compromised spindle assembly checkpoints and increased ROS through impaired autophagy flux. Diclofenac may be a candidate therapeutic drug in certain type of cancers by inhibiting microtubule-mediated cellular events in combination with clinically utilized nucleoside metabolic inhibitors, including 5-fluorouracil, to block cancer cell proliferation.

Endosomal mTORC2 Is Required for Phosphoinositide-Dependent AKT Activation in Platelet-Derived Growth Factor-Stimulated Glioma Cells.

The serine/threonine kinase AKT is a major effector during phosphatidylinositol 3-kinase (PI3K)-driven cell signal transduction in response to extracellular stimuli. AKT activation mechanisms have been extensively studied; however, the mechanism underlying target of rapamycin complex 2 (mTORC2) phosphorylation of AKT at Ser473 in the cellular endomembrane system remains to be elucidated. Here, we demonstrate that endocytosis is required for AKT activation through phosphorylation at Ser473 via mTORC2 using platelet-derived growth factor-stimulated U87MG glioma cells. mTORC2 components are localized to early endosomes during growth factor activation, and the association of mTORC2 with early endosomes is responsible for the local activation of AKT, which is critical for specific signal transduction through glycogen synthase kinase-3 beta and forkhead box O1/O3 phosphorylation. Furthermore, endosomal phosphoinositide, represented by PtdIns(3,4)P2, provides a binding platform for mTORC2 to phosphorylate AKT Ser473 in endosomes through mammalian Sty1/Spc1-interacting protein (mSIN), a pleckstrin homology domain-containing protein, and is dispensable for AKT phosphorylation at Thr308. This PtdIns(3,4)P2-mediated endosomal AKT activation provides a means to integrate PI3K activated by diverse stimuli to mTORC2 assembly. These early endosomal events induced by endocytosis, together with the previously identified AKT activation by PtdIns(3,4,5)P3, contribute to the strengthening of the transduction of AKT signaling through phosphoinositide.

A Basic Domain-Derived Tripeptide Inhibits MITF Activity by Reducing its Binding to the Promoter of Target Genes.

The keratinocytes in UV-irradiated skin produce and secrete α-melanocyte-stimulating hormone. α-Melanocyte-stimulating hormone upregulates the expression of MITF in melanocytes through the cAMP‒protein kinase A‒CREB signaling pathway. Thereafter, MITF induces the expression of melanogenic genes, including the tyrosinase gene TYR and TYRP-1 and TYRP-2 genes, which leads to the synthesis and accumulation of melanin. In this study, we examined whether MITF basic region-derived tripeptides can bind to the DNA-binding domain of MITF and inhibit MITF-induced melanogenesis through the inhibition of MITF‒DNA binding. MITF-KGR, a representative MITF-derived tripeptide, suppressed the transcriptional activity of MITF by disrupting its binding to the promoter region of the target genes, which resulted in the inhibition of skin epidermis thickness and melanin synthesis in vivo and in vitro. Our results indicate that MITF-KGR exerts an inhibitory effect on melanogenesis by targeting MITF.

The Role of Hydrogen Peroxide and Peroxiredoxins throughout the Cell Cycle.

Hydrogen peroxide (H2O2) is an oxidizing agent that induces cellular damage at inappropriate concentrations and gives rise to an arrest during cell cycle progression, causing cell death. Recent evidence indicates that H2O2 also acts as a promoter for cell cycle progression by oxidizing specific thiol proteins. The intracellular concentration of H2O2 is regulated tightly, enabling its use as a cellular signaling molecule while minimizing its potential to cause cellular damage. Peroxiredoxins (Prxs) have peroxidase activity toward H2O2, organic hydroperoxides, and peroxynitrite for protecting cells from oxidative stress. They are suggested to work as signaling mediators, allowing the local accumulation of H2O2 by inactivating their peroxidase activity uniquely compared with other antioxidant proteins such as catalase and glutathione peroxidase. Given that Prxs are highly sensitive to oxidation by H2O2, they act as sensors and transducers of H2O2 signaling via transferring their oxidation state to effector proteins. The concentrations of intracellular H2O2 increase as the cell cycle progresses from G1 to mitosis. Here, we summarize the roles of Prxs with regard to the regulation of cell cycle-dependent kinase activity and anaphase-promoting complex/cyclosome in terms of changes in H2O2 levels. Protection of the cell from unwanted progression of the cell cycle is suggested to be a role of Prx. We discuss the possible roles of Prxs to control H2O2 levels.

Quantitative and qualitative analysis of autophagy flux using imaging.

As an intracellular degradation system, autophagy is an essential and defensive cellular program required for cell survival and cellular metabolic homeostasis in response to various stresses, such as nutrient deprivation and the accumulation of damaged organelles. In general, autophagy flux consists of four steps: (1) initiation (formation of phagophore), (2) maturation and completion of autophagosome, (3) fusion of autophagosomes with lysosomes (formation of autolysosome), and (4) degradation of intravesicular components within autolysosomes. The number of genes and reagents that modulate autophagy is increasing. Investigation of their effect on autophagy flux is critical to understanding the roles of autophagy in many physiological and pathological processes. In this review, we summarize and discuss ways to analyze autophagy flux quantitatively and qualitatively with the use of imaging tools. The suggested imaging method can help estimate whether each modulator is an inhibitor or a promoter of autophagy and elucidate the mode of action of specific genes and reagents on autophagy processes. [BMB Reports 2020; 53(5): 241-247].

Inactivation of the PtdIns(4)P phosphatase Sac1 at the Golgi by H2O2 produced via Ca2+-dependent Duox in EGF-stimulated cells.

Binding of epidermal growth factor (EGF) to its cell surface receptor induces production of H2O2, which serves as an intracellular messenger. We have shown that exogenous H2O2 reversibly inactivates the phosphatidylinositol 4-phosphate [PtdIns(4)P] phosphatase Sac1 (suppressor of actin 1) at the Golgi complex of mammalian cells by oxidizing its catalytic cysteine residue and thereby increases both the amount of Golgi PtdIns(4)P and the rate of protein secretion. Here we investigated the effects of EGF on Sac1 oxidation and PtdIns(4)P abundance at the Golgi in A431 cells. EGF induced a transient increase in Golgi PtdIns(4)P as well as a transient oxidation of Sac1 in a manner dependent on elevation of the intracellular Ca2+ concentration and on H2O2. Oxidation of Sac1 occurred at the Golgi, as revealed with the use of the Golgi-confined Sac1-K2A mutant. Knockdown of Duox enzymes implicated these Ca2+-dependent members of the NADPH oxidase family as the major source of H2O2 for Sac1 oxidation. Expression of a Golgi-targeted H2O2 probe revealed transient EGF-induced H2O2 production at this organelle. Our findings have thus uncovered a previously unrecognized EGF signaling pathway that links intracellular Ca2+ mobilization to events at the Golgi including Duox activation, H2O2 production, Sac1 oxidation, and PtdIns(4)P accumulation.