Endothelial Cell-Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature.

Objective- Blood-CNS (central nervous system) barrier defects are implicated in retinopathies, neurodegenerative diseases, stroke, and epilepsy, yet, the pathological mechanisms downstream of barrier defects remain incompletely understood. Blood-retina barrier (BRB) formation and retinal angiogenesis require ß-catenin signaling induced by the ligand norrin (NDP [Norrie disease protein]), the receptor FZD4 (frizzled 4), coreceptor LRP5 (low-density lipoprotein receptor-like protein 5), and the tetraspanin TSPAN12 (tetraspanin 12). Impaired NDP/FZD4 signaling causes familial exudative vitreoretinopathy, which may lead to blindness. This study seeked to define cell type-specific functions of TSPAN12 in the retina. Approach and Results- A loxP-flanked Tspan12 allele was generated and recombined in endothelial cells using a tamoxifen-inducible Cdh5-CreERT2 driver. Resulting phenotypes were documented using confocal microscopy. RNA-Seq, histopathologic analysis, and electroretinogram were performed on retinas of aged mice. We show that TSPAN12 functions in endothelial cells to promote vascular morphogenesis and BRB formation in developing mice and BRB maintenance in adult mice. Early loss of TSPAN12 in endothelial cells causes lack of intraretinal capillaries and increased VE-cadherin (CDH5 [cadherin5 aka VE-cadherin]) expression, consistent with premature vascular quiescence. Late loss of TSPAN12 strongly impairs BRB maintenance without affecting vascular morphogenesis, pericyte coverage, or perfusion. Long-term BRB defects are associated with immunoglobulin extravasation, complement deposition, cystoid edema, and impaired b-wave in electroretinograms. RNA-sequencing reveals transcriptional responses to the perturbation of the BRB, including genes involved in vascular basement membrane alterations in diabetic retinopathy. Conclusions- This study establishes mice with late endothelial cell-specific loss of Tspan12 as a model to study pathological consequences of BRB impairment in an otherwise intact vasculature.

Norrin-induced Frizzled4 endocytosis and endo-lysosomal trafficking control retinal angiogenesis and barrier function.

Angiogenesis and blood-brain barrier formation are required for normal central nervous system (CNS) function. Both processes are controlled by Wnt or Norrin (NDP) ligands, Frizzled (FZD) receptors, and ß-catenin-dependent signalling in vascular endothelial cells. In the retina, FZD4 and the ligand NDP are critical mediators of signalling and are mutated in familial exudative vitreoretinopathy. Here, we report that NDP is a potent trigger of FZD4 ubiquitination and induces internalization of the NDP receptor complex into the endo-lysosomal compartment. Inhibition of ubiquitinated cargo transport through the multivesicular body (MVB) pathway using a dominant negative ESCRT (endosomal sorting complexes required for transport) component VPS4 EQ strongly impairs NDP/FZD4 signalling in vitro and recapitulates CNS angiogenesis and blood-CNS-barrier defects caused by impaired vascular ß-catenin signalling in mice. These findings provide evidence for an important role of FZD4 endocytosis in NDP/FZD4 signalling and in CNS vascular biology and disease.

TSPAN12 Is a Norrin Co-receptor that Amplifies Frizzled4 Ligand Selectivity and Signaling.

Accessory proteins in Frizzled (FZD) receptor complexes are thought to determine ligand selectivity and signaling amplitude. Genetic evidence indicates that specific combinations of accessory proteins and ligands mediate vascular ß-catenin signaling in different CNS structures. In the retina, the tetraspanin TSPAN12 and the ligand norrin (NDP) mediate angiogenesis, and both genes are linked to familial exudative vitreoretinopathy (FEVR), yet the molecular function of TSPAN12 remains poorly understood. Here, we report that TSPAN12 is an essential component of the NDP receptor complex and interacts with FZD4 and NDP via its extracellular loops, consistent with an action as co-receptor that enhances FZD4 ligand selectivity for NDP. FEVR-linked mutations in TSPAN12 prevent the incorporation of TSPAN12 into the NDP receptor complex. In vitro and in Xenopus embryos, TSPAN12 alleviates defects of FZD4 M105V, a mutation that destabilizes the NDP/FZD4 interaction. This study sheds light on the poorly understood function of accessory proteins in FZD signaling.

Glycolytic enzyme inhibitors affect pancreatic cancer survival by modulating its signaling and energetics.

BACKGROUND AND AIM: The importance of glycolysis in cancer cells is well documented. The effects of inhibiting glycolysis using metabolic inhibitors iodoacetate (IAA), an inhibitor of GAPDHase, and 3-bromopyruvate (3BP), an inhibitor of hexokinase-II, on survival and signaling of pancreatic cancer cells (Panc-1) were investigated. MATERIALS AND METHODS: Cellular survival was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Lactate dehydrogenase (LDH) assay was used to analyze the induced necrosis and protein levels were evaluated using Western blot analysis. RESULTS: The results show that the inhibitors lowered cellular survival and increased cellular necrosis. Mitogenic signaling pathways were affected by 3BP but not by IAA. CONCLUSION: We conclude that there may be a cross-talk between signaling pathways and glycolysis in regulating pancreatic cancer cell survival and signaling. Thus, a combination of agents that inhibit both energy production and cell signaling may provide a novel and effective approach to target pancreatic cancer effectively.

Inhibition of Cell Proliferation and MAP Kinase and Akt Pathways in Oral Squamous cell Carcinoma by Genistein and Biochanin A.

High morbidity and mortality associated with oral squamous cell carcinoma (OSCC) are largely attributable to late stage diagnosis. Despite significant advances in therapeutic strategies, the five-year survival rate for oral cancer remains at about 50%. A chemopreventive approach may be an effective alternative or adjunct to current therapies. Previous studies have shown anti-tumor effects of isoflavones in several cancers, including oral cancer. However, their mechanisms of action are still unclear. We hypothesized that isoflavones inhibit multiple signaling pathways implicated in oral carcinogenesis. To address our hypothesis, we investigated the effects of three isoflavone derivatives, genistein, biochanin A and daidzein, on SCC15 and SCC25 squamous cell carcinoma cell lines. In cell proliferation experiments, we found that genistein and biochanin A inhibited SCC15 and SCC25 cell growth with an IC50 of 50 µM. We also investigated the effect of isoflavones on ERK and Akt pathways. Our results, from western blot analysis, suggest that both genistein and biochanin A induced decreases in phosphorylation of ERK and Akt at treatment concentrations of 20, 50 and 100 µM. Taken together, our results clearly demonstrate a differential regulation of signaling pathways by various isoflavones in OSCC cell lines. Thus, tumor progression models can be utilized to study the preventive and therapeutic roles of isoflavones in oral cancer cell lines.

Exposure to titanium dioxide and other metallic oxide nanoparticles induces cytotoxicity on human neural cells and fibroblasts.

The use of titanium dioxide (TiO(2)) in various industrial applications (eg, production of paper, plastics, cosmetics, and paints) has been expanding thereby increasing the occupational and other environmental exposure of these nanoparticles to humans and other species. However, the health effects of exposure to TiO(2) nanoparticles have not been systematically assessed even though recent studies suggest that such exposure induces inflammatory responses in lung tissue and cells. Because the effects of such nanoparticles on human neural cells are unknown, we have determined the putative cytotoxic effects of these nanoparticles on human astrocytes-like astrocytoma U87 cells and compared their effects on normal human fibroblasts. We found that TiO(2) micro- and nanoparticles induced cell death on both human cell types in a concentration-related manner. We further noted that zinc oxide (ZnO) nanoparticles were the most effective, TiO(2) nanoparticles the second most effective, and magnesium oxide (MgO) nanoparticles the least effective in inducing cell death in U87 cells. The cell death mechanisms underlying the effects of TiO(2) micro- and nanoparticles on U87 cells include apoptosis, necrosis, and possibly apoptosis-like and necrosis-like cell death types. Thus, our findings may have toxicological and other pathophysiological implications on exposure of humans and other mammalian species to metallic oxide nanoparticles.