Eri Takahashi

CD44 Variant Regulates Redox Status in Cancer Cells by Stabilizing the xCT Subunit of System xc− and Thereby Promotes Tumor Growth

CD44 is an adhesion molecule expressed in cancer stem-like cells. Here, we show that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH). Human gastrointestinal cancer cells with a high level of CD44 expression showed an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS). Ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. It also induced activation of p38(MAPK), a downstream target of ROS, and expression of the gene for the cell cycle inhibitor p21(CIP1/WAF1). These findings establish a function for CD44v in regulation of ROS defense and tumor growth.

Tumor Necrosis Factor-α Regulates Transforming Growth Factor-β-dependent Epithelial-Mesenchymal Transition by Promoting Hyaluronan-CD44-Moesin Interaction

Aberrant epithelial-mesenchymal transition (EMT) is involved in development of fibrotic disorders and cancer invasion. Alterations of cell-extracellular matrix interaction also contribute to those pathological conditions. However, the functional interplay between EMT and cell-extracellular matrix interactions remains poorly understood. We now show that the inflammatory mediator tumor necrosis factor-α (TNF-α) induces the formation of fibrotic foci by cultured retinal pigment epithelial cells through activation of transforming growth factor-β (TGF-β) signaling in a manner dependent on hyaluronan-CD44-moesin interaction. TNF-α promoted CD44 expression and moesin phosphorylation by protein kinase C, leading to the pericellular interaction of hyaluronan and CD44. Formation of the hyaluronan-CD44-moesin complex resulted in both cell-cell dissociation and increased cellular motility through actin remodeling. Furthermore, this complex was found to be associated with TGF-β receptor II and clathrin at actin microdomains, leading to activation of TGF-β signaling. We established an in vivo model of TNF-α-induced fibrosis in the mouse eye, and such ocular fibrosis was attenuated in CD44-null mice. The production of hyaluronan and its interaction with CD44, thus, play an essential role in TNF-α-induced EMT and are potential therapeutic targets in fibrotic disorders.

Molecular mechanisms regulating dissociation of cell–cell junction of epithelial cells by oxidative stress

Oxidative stress is regarded as a causative factor in aging and various degenerative diseases. Here, we show the mechanism by which oxidative stress induces disruption of cell–cell junctions using retinal pigment epithelial cells. We demonstrated that reactive oxygen species (ROS)‐mediated activation of Src kinase increases the tyrosine phosphorylation state of p120‐catenin and rapidly triggers translocation of p120‐catenin and internalization of N‐cadherin from the cell–cell adhesion sites to an early endosomal compartment. Endosomal accumulation of p120‐catenin resulted in stress fiber formation and cell–cell dissociation through the activation of Rho/Rho kinase pathway. However, these cytoskeletal remodeling and cell–cell dissociation induced by oxidative stress were transient, due to the activation of nuclear factor‐κB (NF‐κB) and the expression of manganese superoxide dismutase (Mn‐SOD). Using the NF‐κB specific inhibitor DHMEQ, we found that NF‐κB is part of a negative feedback loop to control intracellular ROS levels. Finally, we demonstrated that H2O2 treatment alone does not induce the epithelial mesenchymal transition (EMT) in retinal pigment epithelial cells, which can be induced by TNF‐α treatment. These findings suggest that oxidative stress is a crucial factor to induce the cell–cell dissociation, an initial step of EMT, but does not provide sufficient signals to establish and to maintain the EMT.

Elevated erythropoietin in vitreous with ischemic retinal diseases

The aim of the current study was to measure the concentrations of erythropoietin in the vitreous fluid and analyze its association with vascular endothelial growth factor (VEGF) in ischemic vitreoretinal diseases. Vitreous fluid samples were collected from patients with proliferative diabetic retinopathy, branch retinal vein occlusion and idiopathic macular hole. Concentrations of erythropoietin and VEGF in vitreous fluid were significantly elevated in patients with proliferative diabetic retinopathy and branch retinal vein occlusion as compared to patients with macular hole. There were no differences in serum concentrations of erythropoietin and VEGF among patient groups. There was significant correlation between erythropoietin and VEGF concentrations in vitreous fluid. Erythropoietin was up-regulated in ischemic disorders and may act as an endogenous neuroprotective factor against ischemic retinal disorders.

Myristoylated alanine-rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C-dependent pathway

(Cancer Sci 2010; 101: 658–665)

Cloning and functional expression of ASIC-β2, a splice variant of ASIC-β

We have isolated a cDNA encoding a splice variant of ASIC (acid-sensing ion channel)-β from the rat trigeminal ganglion. This clone, designated ASIC-β2, showed a 342 base deletion just after the first transmembrane domain in ASIC-β. RT–PCR experiments revealed that ASIC-β2 was expressed exclusively in the trigeminal ganglion and dorsal root ganglion. In situ hybridization showed that ASIC-β2 mRNA was concentrated in both small diameter and large diameter neurons and co-localized with ASIC-β mRNA within single sensory neurons in the trigeminal ganglion. When expressed in Xenopus oocytes, ASIC-β2 was inactive by itself. However, it associated with ASIC-β to form heteromers, which display lower affinity for protons than ASIC-β alone.

Epithelial mesenchymal transition-like phenomenon in trabecular meshwork cells

The trabecular meshwork (TM) is a major component of the conventional outflow pathway and the excess extracellular matrix (ECM), and fibrosis in the TM causes increased outflow resistance. In this study, we first investigated the effects of several ECM components in the induction of an epithelial mesenchymal transition (EMT)-like phenomenon in TM cells. TM cells were isolated from cynomolgus monkeys (Macaca fascicularis). The cells were cultured in ECM-coated dishes and then subjected to both western blot analysis and immunocytochemistry to measure the levels of EMT-associated markers. Cell motility was assessed using wound healing and chemotaxis assays. We found that type I collagen, fibronectin and laminin induced the dissociation of cell-cell contact and elongation of actin stress fibers in the cultured monkey TM cells. In addition, following the same stimulation of the ECM, the expression of mesenchymal markers, such as fibronectin and α-smooth muscle actin, and the phosphorylation of Smad2 increased in the TM cells. Our results showed the significant acceleration of TM cellular motility following stimulation with type I collagen, fibronectin and laminin. These phenomena were inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. In addition, siRNA against paxillin was transfected to evaluate the association between paxillin and the EMT-like phenomenon. The knockdown of paxillin expression by transfection with siRNA blocked the EMT-like alteration of the cellular characteristics and chemotaxis toward transforming growth factor-β2 in the cultured TM cells. Our results showed that the ECM-JNK-paxillin pathway induced an EMT-like phenomenon in TM cells, resulting in the abundant expression of fibronectin and activation of motility in TM cells. This EMT-like phenomenon could result in aberrant conditions in the aqueous outflow pathway in glaucomatous eyes.

Involvement of hyaluronan and its receptor CD44 with choroidal neovascularization

PURPOSE CD44 is a cell-surface adhesion molecule and receptor for hyaluronan (HA), one of the major extracellular matrix components. The purpose of the present study was to clarify a role of HA and CD44 in the development of choroidal neovascularization (CNV). METHODS Laser photocoagulation was used to induce CNV in C57BL/6 mice or CD44-deficient mice. The mRNA expression of CD44 and HA synthase (HAS)-2 in the retinal pigment epithelium (RPE)-choroid complex was evaluated by DNA microarray and real-time RT-PCR analyses 3 days after laser treatment. HA synthesis and CD44 expression were examined by immunohistochemistry 1 week after photocoagulation. Mice with laser-induced CNV were systemically administered the HA synthesis inhibitor 4-methylumbelliferone (MU) or an anti-CD44-neutralizing antibody. The response of CNV was analyzed by volumetric measurements 1 week after photocoagulation. Macrophage infiltration into CNV lesions was evaluated by real-time RT-PCR for F4/80 3 days after laser-induced injury. RESULTS The induction of CNV led to a significant increase in expression of CD44 and HAS2 mRNA. HA and CD44 were immunopositive in the CNV lesions. Compared with vehicle treatment, the systemic application of MU significantly attenuated CNV volume in a dose-dependent fashion, together with macrophage infiltration into the lesions. Consistently, antibody-based blockade of CD44 resulted in a significant reduction of CNV volume, compared with the isotype control. In contrast, genetic ablation of CD44 significantly augmented CNV formation together with HA accumulation and macrophage infiltration, compared with wild-type mice. CONCLUSIONS These results indicate a significant role of HA and its receptor CD44 in the development of CNV.

Factors Influencing Aqueous Proinflammatory Cytokines and Growth Factors in Uveitic Glaucoma.

Purpose To analyze the effects of factors on aqueous humor proinflammatory cytokine and growth factor levels in patients with uveitic glaucoma (UG). Methods In this cross-sectional study, we enrolled 143 participants: 1) UG patients (n = 39); 2) primary open-angle glaucoma (POAG) patients (n = 36); and 3) cataract surgery patients, as a comparative group (n = 68). Aqueous humor samples were obtained at the start of surgery. Aqueous cytokine levels were determined using a multiplex immunoassay (xMAP and the Human Cytokine/Chemokine Panel I). Results In UG cases, mean interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, and VEGF levels were 171.1, 214.5, 2791.7, 3.5, 23.9, 5.4, and 168.9 pg/mL, respectively, and were higher than those in cataract (non-glaucomatous) cases except PDGF. Levels of IL-6, MCP-1, and VEGF were higher in UG cases than in POAG cases. UG cases with a history of phacoemulsification displayed significantly higher levels of IL-6 (P = 0.0164), IL-8 (P = 0.0003), MCP-1 (P = 0.0465), and PDGF-AB/BB (P = 0.0062) compared to the phakic cases. The presence of cells in the anterior chamber was related to higher levels of IL-8 (P = 0.0002), TNF-α (P = 0.0037), and PDGF-AB/BB (P = 0.0009). The level of PDGF-AB/BB was higher in infectious uveitis than in non-infectious uveitis (P = 0.0211). The level of transforming growth factor (TGF)-β2 was negatively correlated with the levels of MCP-1 (adjusted R2 = 0.28, t = -2.45, P = 0.031) and TNF-α (adjusted R2 = 0.27, t = -2.43, P = 0.032). Conclusion A history of phacoemulsification, the presence of cells in the anterior chamber, and infectious uveitis were related to aqueous proinflammatory cytokine levels in patients with UG. TGF-β2 might be an anti-inflammatory factor in aqueous humor of UG patients.

Suppression of choroidal neovascularization in lectin-like oxidized low-density lipoprotein receptor type 1-deficient mice

PURPOSE To elucidate the role of the scavenger receptor, lectin-like oxidized low-density lipoprotein receptor type 1 (LOX-1), in the formation of choroidal neovascularization (CNV). METHODS CNV was induced by laser photocoagulation of the ocular fundus in mice. The expression of LOX-1 mRNA and protein after laser injury was determined by real-time RT-PCR and Western blot analysis. Gelatin zymography was used to measure the activity of matrix metalloproteinase (MMP)-2 and pro-MMP-9, and ELISA was used to determine monocyte chemoattractant protein (MCP)-1 and vascular endothelial growth factor (VEGF) levels. At 14 days after laser injury, the extent of CNV was evaluated by fluorescein angiography and lectin staining using confocal microscopy. RESULTS In wild-type mice, the relative expression level of LOX-1 mRNA compared with the control increased significantly 6 hours after laser injury and peaked 12 hours after laser injury (P = 0.011 and P = 0.0006, respectively), and the expression of LOX-1 protein was also detected 1 and 3 days after laser injury. Increases in MMP-2, pro-MMP2, and pro-MMP-9 after laser injury were reduced in LOX-1-deficient mice compared with wild-type mice. At 3 days after laser injury, increases in MCP-1 and VEGF significantly decreased in LOX-1-deficient mice compared with wild-type mice (P = 0.014 and P = 0.001, respectively). Morphometric analyses revealed that the induction of CNV formation was significantly inhibited in LOX-1-deficient mice. CONCLUSIONS These results suggest that LOX-1 plays an important role in the formation of CNV. This scavenging system might thus be a novel therapeutic target for CNV.