Inhibition of sodium glucose cotransporter 2 (SGLT2) has been reported as a new therapeutic strategy for treating diabetes. However, the effect of SGLT2 inhibitors on the kidney is unknown. In addition, whether SGLT2 inhibitors have an anti-inflammatory or antioxidative stress effect is still unclear. In this study, to resolve these issues, we evaluated the effects of the SGLT2 inhibitor, dapagliflozin, using a mouse model of type 2 diabetes and cultured proximal tubular epithelial (mProx24) cells. Male db/db mice were administered 0.1 or 1.0 mg/kg of dapagliflozin for 12 weeks. Body weight, blood pressure, blood glucose, hemoglobin A1c, albuminuria and creatinine clearance were measured. Mesangial matrix accumulation and interstitial fibrosis in the kidney and pancreatic ?-cell mass were evaluated by histological analysis. Furthermore, gene expression of inflammatory mediators, such as osteopontin, monocyte chemoattractant protein-1 and transforming growth factor-?, was evaluated by quantitative reverse transcriptase-PCR. In addition, oxidative stress was evaluated by dihydroethidium and NADPH oxidase 4 staining. Administration of 0.1 or 1.0 mg/kg of dapagliflozin ameliorated hyperglycemia, ?-cell damage and albuminuria in db/db mice. Serum creatinine, creatinine clearance and blood pressure were not affected by administration of dapagliflozin, but glomerular mesangial expansion and interstitial fibrosis were suppressed in a dose-dependent manner. Dapagliflozin treatment markedly decreased macrophage infiltration and the gene expression of inflammation and oxidative stress in the kidney of db/db mice. Moreover, dapagliflozin suppressed the high-glucose-induced gene expression of inflammatory cytokines and oxidative stress in cultured mProx24 cells. These data suggest that dapagliflozin ameliorates diabetic nephropathy by improving hyperglycemia along with inhibiting inflammation and oxidative stress.
Dynasore, a specific dynamin GTPase inhibitor, suppresses lamellipodia formation and cancer cell invasion by destabilizing actin filaments. In search for novel dynamin inhibitors that suppress actin dynamics more efficiently, dynasore analogues were screened. N-[4-(dipropylamino)benzylidene]-2-hydroxybenzohydrazide (DBHA) markedly reduced in vitro actin polymerization, and dose-dependently inhibited phosphatidylserine-stimulated dynamin GTPase activity. DBHA significantly suppressed both the recruitment of dynamin 2 to the leading edge in U2OS cells and ruffle formation in H1299 cells. Furthermore, DBHA suppressed both the migration and invasion of H1299 cells by approximately 70%. Furthermore, intratumoral DBHA delivery significantly repressed tumor growth. DBHA was much less cytotoxic than dynasore. These results strongly suggest that DBHA inhibits dynamin-dependent actin polymerization by altering the interactions between dynamin and lipid membranes. DBHA and its derivative may be potential candidates for potent anti-cancer drugs.
Oxidative stress and inflammation play important roles in diabetic complications, including diabetic nephropathy. Metallothionein (MT) is induced in proximal tubular epithelial cells as an antioxidant in the diabetic kidney; however, the role of MT in renal function remains unclear. We therefore investigated whether MT deficiency accelerates diabetic nephropathy through oxidative stress and inflammation. Diabetes was induced by streptozotocin injection in MT-deficient (MT(-/-)) and MT(+/+) mice. Urinary albumin excretion, histological changes, markers for reactive oxygen species (ROS), and kidney inflammation were measured. Murine proximal tubular epithelial (mProx24) cells were used to further elucidate the role of MT under high-glucose conditions. Parameters of diabetic nephropathy and markers of ROS and inflammation were accelerated in diabetic MT(-/-) mice compared with diabetic MT(+/+) mice, despite equivalent levels of hyperglycemia. MT deficiency accelerated interstitial fibrosis and macrophage infiltration into the interstitium in the diabetic kidney. Electron microscopy revealed abnormal mitochondrial morphology in proximal tubular epithelial cells in diabetic MT(-/-) mice. In vitro studies demonstrated that knockdown of MT by small interfering RNA enhanced mitochondrial ROS generation and inflammation-related gene expression in mProx24 cells cultured under high-glucose conditions. The results of this study suggest that MT may play a key role in protecting the kidney against high glucose-induced ROS and subsequent inflammation in diabetic nephropathy.
Dynamin GTPase, a key molecule in endocytosis, mechanically severs the invaginated membrane upon GTP hydrolysis. Dynamin functions also in regulating actin cytoskeleton, but the mechanisms are yet to be defined. Here we show that dynamin 1, a neuronal isoform of dynamin, and cortactin form ring complexes, which twine around F-actin bundles and stabilize them. By negative-staining EM, dynamin 1-cortactin complexes appeared as "open" or "closed" rings depending on guanine nucleotide conditions. By pyrene actin assembly assay, dynamin 1 stimulated actin assembly in mouse brain cytosol. In vitro incubation of F-actin with both dynamin 1 and cortactin led to the formation of long and thick actin bundles, on which dynamin 1 and cortactin were periodically colocalized in puncta. A depolymerization assay revealed that dynamin 1 and cortactin increased the stability of actin bundles, most prominently in the presence of GTP. In rat cortical neurons and human neuroblastoma cell line, SH-SY5Y, both dynamin 1 and cortactin localized on actin filaments and the bundles at growth cone filopodia as revealed by immunoelectron microscopy. In SH-SY5Y cell, acute inhibition of dynamin 1 by application of dynamin inhibitor led to growth cone collapse. Cortactin knockdown also reduced growth cone filopodia. Together, our results strongly suggest that dynamin 1 and cortactin ring complex mechanically stabilizes F-actin bundles in growth cone filopodia. Thus, the GTPase-dependent mechanochemical enzyme property of dynamin is commonly used both in endocytosis and regulation of F-actin bundles by a dynamin 1-cortactin complex.
The sorting machinery in early endosomes is crucial for intracellular homeostasis and signal transduction and its disruption leads to the development of various diseases. In spite of its significance, the molecular mechanism underlying this machinery remains largely unknown. Actin filaments are implicated in intracellular trafficking, including membrane fission at endocytosis, membrane stretching at the Golgi complex, and maturation of endosomes. We have recently found that actin is required for receptor sorting in early endosomes and identified cortactin as a candidate for actin regulation in early endosomes. Inhibition of actin dynamics leads to enlargement of early endosomes and impairment of the sorting; the latter is also observed in cortactin-depleted cells. The endosomal localization of cortactin was enhanced by dynasore, a dynamin inhibitor that effectively inhibits endosomal sorting, indicating that cortactin is involved in the sorting machinery in early endosomes. Here we discuss the role of actin filaments in early endosomes and other molecules implicated in endosomal trafficking.
A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.
The early endosome acts as a sorting station for internalized molecules destined for recycling or degradation. While recycled molecules are sorted and delivered to tubular endosomes, residual compartments containing molecules to be degraded undergo "maturation" before final degradation in the lysosome. This maturation involves acidification, microtubule-dependent motility, and perinuclear localization. It is currently unknown how sorting and the processes of maturation cooperate with each other. Here, we show that fission of a tubular endosome triggers the maturation of the residual endosome, leading to degradation. Use of the dynamin inhibitor dynasore to block tubular endosome fission inhibited acidification, endosomal motility along microtubules, perinuclear localization, and degradation. However, tubular endosome fission was not affected by inhibiting endosomal acidification or by depolymerizing the microtubules. These results demonstrate that the fission of recycling tubules is the first important step in endosomal maturation and degradation in the lysosome. We believe this to be the first evidence of a cascade from sorting to degradation.
Early endosomes (EEs) are known to be a sorting station for internalized molecules destined for degradation, recycling, or other intracellular organelles. Segregation is an essential step in such sorting, but the molecular mechanism of this process remains to be elucidated. Here, we show that actin is required for efficient recycling and endosomal maturation by producing a motile force. Perturbation of actin dynamics by drugs induced a few enlarged EEs containing several degradative vacuoles and also interfered with their transporting ability. Actin repolymerization induced by washout of the drug caused the vacuoles to dissociate and individually translocate toward the perinuclear region. We further elucidated that cortactin, an actin-nucleating factor, was required for transporting contents from within EEs. Actin filaments regulated by cortactin may provide a motile force for efficient sorting within early endosomes. These data suggest that actin filaments coordinate with microtubules to mediate segregation in EEs.
Dynamin, a ~100 kDa large GTPase, is known as a key player for membrane traffic. Recent evidence shows that dynamin also regulates the dynamic instability of microtubules by a mechanism independent of membrane traffic. As microtubules are highly dynamic during mitosis, we investigated whether the regulation of microtubules by dynamin is essential for cell cycle progression. Dynamin 2 intensely localized at the mitotic spindle, and the localization depended on its proline-rich domain (PRD), which is required for microtubule association. The deletion of PRD resulted in the impairment of cytokinesis, whereby the mutant had less effect on endocytosis. Interestingly, dominant-negative dynamin (K44A), which blocks membrane traffic but has no effect on microtubules, also blocked cytokinesis. On the other hand, the deletion of the middle domain, which binds to ?-tubulin, impaired the entry into mitosis. As both deletion mutants had no significant effect on endocytosis, dynamin 2 may participate in cell cycle progression by regulating the microtubules. These data suggest that dynamin may play a key role for cell cycle progression by two distinct pathways, membrane traffic and cytoskeleton.
The tumor suppressor REIC/Dkk-3 is a secretory protein which was originally identified to be downregulated in human immortalized cells. In the present study, we investigated the expression pattern of REIC/Dkk-3 in various cell types to characterize its physiological functions. We first examined the expression level of REIC/Dkk-3 in a broad range of cancer cell types and confirmed that it was significantly downregulated in all of the cell types. We also examined the tissue distribution pattern in a variety of normal mouse organs. Ubiquitous REIC/Dkk-3 protein expression was observed in the organs. The expression was abundant in the liver, heart and brain tissue, but was absent in the spleen and peripheral blood mononuclear cells. The immunohistochemical analyses revealed that the subcellular localization of REIC/Dkk-3 had a punctate pattern around the nucleus, indicating its association with secretory vesicles. In cancer cells stably transfected with REIC/Dkk-3, the protein was predominantly localized to the endoplasmic reticulum (ER) under observation with confocal microscopy. Because REIC/Dkk-3 was found to be abundantly expressed in the acinar epithelial cells of the mouse prostate, we analyzed the effects of recombinant REIC/Dkk-3 protein on the acinar morphogenesis of RWPE-1 cells, which are derived from human normal prostate epithelium. Statistically significant acinar growth was observed in the culture condition with 10 µg/ml REIC/Dkk-3 protein, implicating the soluble form in prostatic acinar development. Current results suggest that REIC/Dkk-3 may play a role in regulating the morphological process of normal tissue architecture through an autocrine and/or paracrine manner.
Dynamic remodeling of actin filaments are bases for a variety of cellular events including cell motility and cancer invasion, and the regulation of actin dynamics implies dynamin, well characterized endocytotic protein. Here we report that dynasore, a inhibitor of dynamin GTPase, potently destabilizes F-actin in vitro, and it severely inhibits the formation of pseudopodia and cancer cell invasion, both of which are supported by active F-actin formation. Dynasore rapidly disrupted F-actin formed in brain cytosol in vitro, and the dynasores effect on F-actin was indirect. Dynasore significantly suppressed serum-induced lamellipodia formation in U2OS cell. Dynasore also destabilized F-actin in resting cells, which caused the retraction of the plasma membrane. A certain amount of dynamin 2 in U2OS cells localized along F-actin, and co-localized with cortactin, a physiological binding partner of dynamin and F-actin. However, these associations of dynamin were partially disrupted by dynasore treatment. Furthermore, invasion activity of H1080 cell, a lung cancer cell line, was suppressed by approximately 40% with dynasore treatment. These results strongly suggest that dynasore potently destabilizes F-actin, and the effect implies dynamin. Dynasore or its derivative would be suitable candidates as potent anti-cancer drugs.
Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidic liposome-triggered, N-WASP-dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.
Dynamin is a fission protein that participates in endocytic vesicle formation. Although dynamin was originally identified as a microtubule-binding protein, the physiological relevance of this function was unclear. Recently, mutations in the ubiquitously expressed dynamin 2 (dyn2) protein were found in patients with Charcot-Marie-Tooth (CMT) disease, which is an inherited peripheral neuropathy. In this study, we show that one of these mutations, 551Delta3, induces prominent decoration of microtubules with the mutant dyn2. Dyn2 was required for proper dynamic instability of microtubules, and this was impaired in cells expressing the 551Delta3 mutant, which showed a remarkable increase in microtubule acetylation, a marker of stable microtubules. Depletion of endogenous dyn2 with a small interfering RNA also resulted in the accumulation of stable microtubules. Furthermore, the formation of mature Golgi complexes, which depends on microtubule-dependent membrane transport, was impaired in both dyn2 knockdown cells and cells expressing the 551Delta3 mutant. Collectively, our results suggest that dyn2 regulates dynamic instability of microtubules, which is essential for organelle motility, and that this function may be impaired in CMT disease.
Dynamin 2 has been reported to be implicated in phagocytosis. However, the mode of action of dynamin is poorly understood. In this study, we examined whether dynamin 2 participates in actin assembly during phagocytosis in Sertoli cells. In the presence of dynasore, a dynamin inhibitor, phagocytosis was reduced by 60-70% in Sertoli cells and macrophages. Scanning electron microscopy revealed that Sertoli cells treated with dynasore were unable to form phagocytic cups. In addition, dysfunction of dynamin 2 reduced both actin polymerization and recruitment of actin and dynamin 2 to phosphatidylinositol (4,5) bisphosphate [PI(4,5)P(2)]-containing liposomes. The formation of dynamin 2-positive ruffles of Sertoli cells was decreased by 60-70% by sequestering PI(4,5)P(2) either by expression of PH domain of PLCdelta or treatment with neomycin. These results strongly suggest that dynamin 2 is involved in actin dynamics and the formation of dynamin 2-positive ruffles during phagocytosis.
Charcot-Marie-Tooth disease (CMT) is an inherited neuronal disorder, and is induced by mutations of various genes associated with intracellular membrane traffic and cytoskeleton. A large GTPase, dynamin, which is known as a fission protein for endocytic vesicles, was identified as a gene responsible for dominant-intermediate CMT type 2B (DI-CMT2B). Of these mutants, the PH domain, which is required for interaction with phosphoinositides, was mutated in several families. Interestingly, the expression of a deletion mutant, 551?3, did not impair endocytosis, but induced abnormal accumulation of microtubules. Recent evidence has shown that dynamin 2 regulates the dynamic instability of microtubules, and 551?3 lacks this function. We propose a model for the regulation of the dynamic instability of microtubules by dynamin 2 and discuss the relationship between dynamin 2 and CMT.
Bladder cancer is one of the most common urogenital malignancies. The intravesical instillation of anticancer agents is an attractive strategy to treat a superficial lesion or floating/disseminated cancer cells after transurethral operation. An adenovirus carrying REIC/Dkk-3, a tumor suppressor gene (Ad-REIC), exhibits cancer-specific apoptotic effects in various types of cancer cells. The aim of the present study was to examine the potential of Ad-REIC as a therapeutic agent for bladder cancer. KK47 and RT4 human bladder cancer cells were sensitive to the Ad-REIC treatment for apoptosis induction, but some human bladder cancer cell lines (T24, J82 and TccSup) were resistant. Significant cell growth inhibition was observed when these resistant cancer cell lines were treated with Ad-REIC in a condition of floating cells, which is clinically observed after transurethral operation and becomes a cause of intravesical cancer dissemination. The therapeutic potential of Ad-REIC for the treatment of multidrug-resistant bladder cancer was investigated. The adriamycin-resistant KK47 bladder cancer cells (KK47/ADM), which also present multidrug resistance, showed induction of significant apoptosis following Ad-REIC treatment. The Ad-REIC treatment induced downregulation of P-glycoprotein in KK47/ADM cells and restored the sensitivity to doxorubicin (adriamycin). Ad-REIC suppressed P-glycoprotein expression in a c-Jun-NH2-kinase (JNK)-dependent manner. Therefore, the current study indicated two therapeutic aspects of the Ad-REIC agent against human bladder cancer cells, as an apoptosis inducer/cell growth inhibitor and as a sensitizer of chemotherapeutic agents in multidrug-resistant cancer cells. The intravesical instillation of Ad-REIC could be an attractive therapeutic method in human bladder cancer where the treatment of superficial lesions and floating/disseminated or multidrug-resistant cancer cells is necessary.
A novel transcriptional system was developed that can robustly enhance cancer-specific gene expression. In the system, hTERT promoter-driven gene expression was enhanced by an advanced two-step transcriptional amplification (TSTA). This construct was used to develop a novel system for detection of bladder cancer cells. The current study evaluated the advanced TSTA system by examining the cancer-specific gene transcription in various bladder cancer cell lines. The system significantly enhanced cancer-specific luciferase gene expression in the bladder cancer cell lines in comparison to the previous expression system of one-step or conventional TSTA. The fold gain of the enhancement was significantly correlated to the telomerase activity of the cell lines. A green fluorescent protein (GFP) gene encoding plasmid vector was constructed where hTERT promoter-driving transcription is enhanced by the advanced TSTA to utilize the system for the imaging and detection of viable bladder cancer cells. The advanced TSTA-hTERT-GFP plasmid successfully induced cancer-specific gene expression, showing robust GFP expression in human bladder cancer cell lines, but no visible GFP expression in normal bladder urothelial cells. The control GFP plasmid with a CMV promoter yielded GFP expression in both normal bladder cells and cancer cells. The advanced TSTA-hTERT-GFP plasmid allowed selective visualization of viable human bladder cancer cells in mixed cell culture containing 10- and 100-fold more normal bladder urothelial cells. These findings indicate that the advanced TSTA-hTERT expressional system is a valuable tool for detecting viable bladder cancer cells. The current system can be applied for in vitro detection of bladder cancer cells in urine and other types of cancer cells disseminated in vivo.
Phosphatidylinositol binding clathrin assembly protein (PICALM), also known as clathrin assembly lymphoid myeloid leukemia protein (CALM), was originally isolated as part of the fusion gene CALM/AF10, which results from the chromosomal translocation t(10;11)(p13;q14). CALM is sufficient to drive clathrin assembly in vitro on lipid monolayers and regulates clathrin-coated budding and the size and shape of the vesicles at the plasma membrane. However, the physiological role of CALM has yet to be elucidated. Here, the role of CALM in vivo was investigated using CALM-deficient mice. CALM-deficient mice exhibited retarded growth in utero and were dwarfed throughout their shortened life-spans. Moreover, CALM-deficient mice suffered from severe anemia, and the maturation and iron content in erythroid precursors were severely impaired. CALM-deficient erythroid cells and embryonic fibroblasts exhibited impaired clathrin-mediated endocytosis of transferrin. These results indicate that CALM is required for erythroid maturation and transferrin internalization in mice.
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