Enhanced glycogen synthase kinase-3β activity mediates podocyte apoptosis under diabetic conditions

Glycogen synthase kinase-3β (GSK-3β) is involved in the pathogenesis of various kidney diseases. This study was undertaken to examine the changes in GSK-3β activity in podocytes under diabetic conditions and to elucidate the functional role of GSK-3β in podocyte apoptosis. In vivo, 32 rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with 6-bromoindirubin-3′-oxime (BIO) for 3 months. In vitro, immortalized mouse podocytes were exposed to 5.6 mM glucose or 30 mM glucose (HG) with or without 10 μM BIO. Western blot analysis and TUNEL or Hoechst 33342 staining were performed to identify apoptosis. Urinary albumin excretion was significantly higher in DM rats, and this increase was significantly abrogated in DM rats by BIO treatment. The protein expression of Tyr216-phospho-GSK-3β was significantly increased in DM glomeruli and in cultured podocytes exposed to HG. Western blot analysis revealed that the protein expression of Bax and active fragments of caspase-3 were significantly increased, whereas phospho-Akt, β-catenin, and Bcl-2 protein expression were significantly decreased in DM glomeruli and HG-stimulated podocytes. Apoptosis, determined by TUNEL assay and Hoechst 33342 staining, was also significantly increased in podocytes under diabetic conditions. The changes in the expression of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG-stimulated podocytes were significantly ameliorated by BIO. These findings suggest that enhanced GSK-3β activity within podocytes under diabetic conditions is associated with podocyte loss in diabetic nephropathy.     

A Splicing Variant of NME1 Negatively Regulates NF-κB Signaling and Inhibits Cancer Metastasis by Interacting with IKKβ

IKKβ functions as a principal upstream activator of the canonical NF-κB pathway by phosphorylating IκB, leading to its proteasomal degradation. Because IKKβ is considered a therapeutic target, understanding its regulation may facilitate the design of efficient regulators of this molecule. Here, we report a novel IKKβ-interacting molecule, NME1L, a splicing variant of the NME1 protein. NME1 has attracted attention in cancer research because of its antimetastatic activity and reduced expression in multiple aggressive types of cancer. However, the effect was just moderate but not dramatic in anti-cancer activities. We found that only NME1L interacts with IKKβ. Exogenous expression of NME1L resulted in a potent decrease in TNFα-stimulated NF-κB activation, whereas knockdown of NME1/NME1L with shRNA enhanced activity of NF-κB. NME1L down-regulates IKKβ signaling by blocking IKKβ-mediated IκB degradation. When NME1L was introduced into highly metastatic HT1080 cells, the mobility was efficiently inhibited. Furthermore, in a metastasis assay, NME1L-expressing cells did not colonize the lung. Based on these results, NME1L is a potent antimetastatic protein and may be a useful weapon in the fight against cancers.     

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.     

Retinal pigment epithelial cells undergoing mitotic catastrophe are vulnerable to autophagy inhibition

The increased mitochondrial DNA damage leads to altered functional capacities of retinal pigment epithelial (RPE) cells. A previous study showed the increased autophagy in RPE cells caused by low concentrations of rotenone, a selective inhibitor of mitochondrial complex I. However, the mechanism by which autophagy regulates RPE cell death is still unclear. In the present study, we examined the mechanism underlying the regulation of RPE cell death through the inhibition of mitochondrial complex I. We report herein that rotenone induced mitotic catastrophe (MC) in RPE cells. We further observed an increased level of autophagy in the RPE cells undergoing MC (RPE-MC cells). Importantly, autophagy inhibition induced nonapoptotic cell death in RPE-MC cells. These findings indicate that autophagy has a pivotal role in the survival of RPE-MC cells. We next observed PINK1 accumulation in the mitochondrial membrane and parkin translocation into the mitochondria from the cytosol in the rotenone-treated RPE-MC cells, which indicates that increased mitophagy accompanies MC in ARPE-19 cells. Noticeably, the mitophagy also contributed to the cytoprotection of RPE-MC cells. Although there might be a significant gap in the roles of autophagy and mitophagy in the RPE cells in vivo, our in vitro study suggests that autophagy and mitophagy presumably prevent the RPE-MC cells from plunging into cell death, resulting in the prevention of RPE cell loss.Cell death is a process that is both complementary and antagonistic to cell division in order to maintain tissue homeostasis, and cell death has a pivotal role in several physiological processes and diseases.¹ The most extensively studied category, apoptosis, is characterized by the massive activation of caspases, chromatin condensation, and a reduction in cell volume. Necrosis is characterized by an increase in cell volume, the swelling of organelles, and the rupture of the plasma membrane and is largely considered an accidental, uncontrolled type of cell death.² Necroptosis is a regulated necrotic cell death that is triggered by broad caspase inhibition in the presence of death receptor ligands and is characterized by necrotic cell death morphology. Autophagy is a degradative lysosomal pathway that is characterized by the accumulation of cytoplasmic material in the vacuoles for bulk degradation by lysosomal enzymes. Although autophagy has a pivotal role in cell survival, increased autophagic activity is often associated with cell death.² Mitotic catastrophe (MC) is a type of cell death that results from a failure to undergo mitosis after DNA damage, leading to tetraploidy or endopolyploidy. Cells undergoing MC usually form large cells with multiple micronuclei.³Retinal pigment epithelial (RPE) cells form a single layer of cells adjacent to the photoreceptor outer segment (POS) of the retina, and these cells have pivotal roles in the maintenance of the POS cells. RPE cell death is a significant factor in several ocular pathological conditions, such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). AMD is a progressive degeneration of the macula and is broadly classified as either dry or wet. The dry form of AMD is more common and is characterized by the presence of drusen in the macula. Mitochondrial DNA variants of respiratory complex I are associated with an increased risk of AMD.⁴ Because damage to and the death of RPEs are crucial and perhaps even triggering events in AMD,⁵ protection against RPE cell death could delay the onset of AMD. Conversely, RPE cells significantly contribute to the formation of the epiretinal membrane in PVR. Thus, the induction of RPE cell death in the epiretinal membranes could be a new approach to inhibit cellular proliferation in PVR.⁶ Most studies concerning RPE cell death in the context of these ocular pathological conditions have focused on two types of cell death, apoptosis and necrosis.Although advances have been made in the understanding of RPE cell death, there is little information concerning the role of autophagy in the RPE cell death associated with these ocular pathological conditions. Each day, RPE cells phagocytose and digest the distal parts of the POS, which are ultimately degraded in the lysosomes.^{7, 8, 9} The interplay of phagocytosis and autophagy within the RPE is required for both POS degradation and the maintenance of retinoid levels to support vision.⁹ In the RPE cells of old eyes, this physiological lysosomal load may be further increased to remove damaged material, and insufficient digestion of the damaged macromolecules and organelles by old RPE cells will lead to progressive accumulation of biological ‘garbage'', such as lipofuscin.¹⁰ Thus, abnormalities in the lysosome-dependent degradation of shed POS debris can contribute to the degeneration of RPE cells. A previous study suggested that age-related changes in autophagy may underlie the genetic susceptibility found in AMD patients and may be associated with the pathogenesis of AMD.¹⁰ However, the mechanism by which autophagy regulates RPE cell demise in AMD is still unclear. The role of autophagy in the proliferation of the RPE cells in PVR and its regulation as a therapeutic strategy for PVR have not been documented yet.Rotenone, a natural isoflavonoid produced by plants, is a selective and stoichiometric inhibitor of mitochondrial complex I.¹¹ More specifically, rotenone blocks NADH oxidation by the NADH-ubiquinone oxide reductase enzymatic complex, which results in the inhibition of mitochondrial respiration and a reduction in ATP synthesis.^{12, 13, 14} Rotenone treatment also results in the production of reactive oxygen species (ROS), eventually leading to cell death.^{15, 16} Several studies have shown that rotenone causes an accumulation of autophagic vacuoles, perhaps in response to the inhibition of mitochondrial function and the generation of oxidative stress.^{17, 18, 19} Irrespective of that activity of rotenone has been lively studied in various cells, the effect of rotenone on RPE cells has rarely been studied. A previous study using an in vitro system revealed that low concentrations of rotenone resulted in mtDNA damage in RPE cells and suggested that the increased autophagy caused by rotenone treatment in aged RPE cells could affect the formation of drusen and AMD.¹⁰ However, the mechanism by which rotenone regulates RPE cell demise remains unclear.We undertook this study to elucidate the mechanism regulating the demise of RPE cells that are damaged by mitochondrial complex I inhibition. We report herein that rotenone induces MC in RPE cells. Additionally, we show that RPE cells undergoing mitotic catastrophe (RPE-MC cells) induced by mitochondrial complex I inhibition are vulnerable to autophagy inhibition.     

Overexpression of an alkaline lipase gene from Proteus vulgaris K80 in Escherichia coli BL21/pKLE

Over-expression of Proteus vulgaris K80 lipase gene in Escherichia coli BL21 (DE3)/pKLE was achieved, with the enzyme being produced in an as active soluble form, using the T7 RNA polymerase system in a modified M9 salt or M9ZB medium. d-Lactose (55 mM) was used to induce gene expression and gave twice the lipase activity achieved with 0.4 mM IPTG. The expression of the lipase gene depended on the feeding rate of glucose being optimal at 12 g l^–1 h^–1.     

Direct colorimetric assay of microcystin using protein phosphatase

A new direct colorimetric assay of microcystin in water and algal samples is proposed consisting of two procedures as follows: 1) the elimination of phosphorus in the sample and concentration of microcystin using a C₁₈ cartridge, 2) the detection of the released phosphorus by the ascorbic acid method and determination of protein phosphatase (PP) inhibition by microcystin. The optimum amounts of phosphorylase α and PP-1 in 50 μL concentrated sample were 50 μg/50 μL buffer and 1.0 unit/50 μL buffer, respectively, for the best assay. The pH for the maximum activity of PP-1 was 8. The minimum detectable concentration for this method was about 0.02 μg/L, which is sufficient to meet the proposed guideline level of 1 μg microcystin/L in drinking water. Consequently, it would seem that the proposed direct colorimetric assay using PP is a rapid, easy, and convenient method for the detection of microcystin in water and algal samples.     

Ligand binding inhibitors of A1 adenosine receptor from Rana rugosa are phospholipase A2s.

Inhibitors of the A1 adenosine receptor were isolated from the skin extract of Korean frog, Rana rugosa. The frog-skin extract was prepared by an electrical shock and fractionated with C4 followed by C18 reverse-phase HPLC. Two A1 receptor inhibitors were isolated using a filter binding assay and the molecular masses of the proteins were estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be 15 347 and 15 404 Da, respectively. The inhibitory activity was also measured against other membrane receptors, such as the A2 adenosine receptor, muscarinic acetylcholine receptor and capsaicin receptor. Ligand binding to the A2 and muscarinic receptors was also severely inhibited by these proteins. However, they did not inhibit the functional activation of the capsaicin receptor by its ligand, capsaicin, suggesting that inhibition of ligand-receptor binding occurs specifically. Their N-terminal sequences were determined by Edman degradation. Surprisingly, they showed sequence similarity to the secretory protein, phospholipase A2 from various organisms. The phospholipase A2 activity of both proteins was tested using Dole's assay technique. Both proteins showed phospholipase A2 activity, and therefore, they were designated as PLA2-R1 and PLA2-R2, respectively. In addition, their ligand-binding inhibitory activity depended on their phospholipase A2 activity. This is the first finding that the frog secretes a phospholipase A2 similar to that of snake venoms, which posess inhibitory activity against the adenosine A1, adenosine A2 and muscarinic receptors.