Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

The Roles of ZnT1 and ZnT4 in Glucose-Stimulated Zinc Secretion in Prostate Epithelial Cells.

PURPOSE: We have previously demonstrated by MRI that high glucose stimulates efflux of zinc ions from the prostate. To our knowledge, this phenomena had not been reported previously and the mechanism remains unknown. Here, we report some initial observations that provide new insights into zinc processing during glucose-stimulated zinc secretion (GSZS) in the immortalized human prostate epithelial cell line, PNT1A. Additionally, we identified the subtypes of zinc-containing cells in human benign prostatic hyperplasia (BPH) tissue to further identify which cell types are likely responsible for zinc release in vivo. PROCEDURE: An intracellular fluorescence marker, FluoZin-1-AM, was used to assess the different roles of ZnT1 and ZnT4 in zinc homeostasis in wild type (WT) and mRNA knockdown PNT1A cell lines. Additionally, Bafilomycin A1 (Baf) was used to disrupt lysosomes and assess the role of lysosomal storage during GSZS. ZIMIR, an extracellular zinc-responsive fluorescent marker, was used to assess dynamic zinc efflux of WT and ZnT1 mRNA knockdown cells exposed to high glucose. Electron microscopy was used to assess intracellular zinc storage in response to high glucose and evaluate how Bafilomycin A1 affects zinc trafficking. BPH cells were harvested from transurtheral prostatectomy tissue and stained with fluorescent zinc granule indicator (ZIGIR), an intracellular zinc-responsive fluorescent marker, before being sorted for cell types using flow cytometry. RESULTS: Fluorescent studies demonstrate that ZnT1 is the major zinc efflux transporter in prostate epithelial cells and that loss of ZnT1 via mRNA knockdown combined with lysosomal storage disruption results in a nearly 4-fold increase in cytosolic zinc. Knockdown of ZnT1 dramatically reduces zinc efflux during GSZS. Electron microscopy (EM) reveals that glucose stimulation significantly increases lysosomal storage of zinc; disruption of lysosomes via Baf or ZnT4 mRNA knockdown increases multi-vesicular body (MVB) formation and cytosolic zinc levels. In human BPH tissue, only the luminal epithelial cells contained significant amounts of zinc storage granules. CONCLUSIONS: Exposure of prostate epithelial cells to high glucose alters zinc homeostasis by inducing efflux of zinc ions via ZnT1 channels and increasing lysosomal storage via ZnT4. Given that prostate cancer cells undergo profound metabolic changes that result in reduced levels of total zinc, understanding the complex interplay between glucose exposure and zinc homeostasis in the prostate may provide new insights into the development of prostate carcinogenesis.

SR-B1 drives endothelial cell LDL transcytosis via DOCK4 to promote atherosclerosis.

Atherosclerosis, which underlies life-threatening cardiovascular disorders such as myocardial infarction and stroke1, is initiated by passage of low-density lipoprotein (LDL) cholesterol into the artery wall and its engulfment by macrophages, which leads to foam cell formation and lesion development2,3. It is unclear how circulating LDL enters the artery wall to instigate atherosclerosis. Here we show in mice that scavenger receptor class B type 1 (SR-B1) in endothelial cells mediates the delivery of LDL into arteries and its accumulation by artery wall macrophages, thereby promoting atherosclerosis. LDL particles are colocalized with SR-B1 in endothelial cell intracellular vesicles in vivo, and transcytosis of LDL across endothelial monolayers requires its direct binding to SR-B1 and an eight-amino-acid cytoplasmic domain of the receptor that recruits the guanine nucleotide exchange factor dedicator of cytokinesis 4 (DOCK4)4. DOCK4 promotes internalization of SR-B1 and transport of LDL by coupling the binding of LDL to SR-B1 with activation of RAC1. The expression of SR-B1 and DOCK4 is increased in atherosclerosis-prone regions of the mouse aorta before lesion formation, and in human atherosclerotic arteries when compared with normal arteries. These findings challenge the long-held concept that atherogenesis involves passive movement of LDL across a compromised endothelial barrier. Interventions that inhibit the endothelial delivery of LDL into artery walls may represent a new therapeutic category in the battle against cardiovascular disease.

NDRG1 regulates neutral lipid metabolism in breast cancer cells.

BACKGROUND: Altered lipid metabolism is an emerging hallmark of aggressive breast cancers. The N-myc downstream regulated gene (NDRG1) gene plays a critical role in peripheral nervous system myelination, as inactivating mutations cause severe demyelinating neuropathy. In breast cancer, elevated NDRG1 expression has been linked to clinical outcomes, but its functional role in breast cancer physiology has remained unclear. METHODS: A meta-analysis of NDRG1 expression in multiple large publicly available genomic databases was conducted. Genome-wide expression correlation and Cox proportional hazards and Kaplan-Meier modeling of clinical outcomes associated with elevated expression were assessed. To study NDRG1 function, gene silencing and overexpression phenotypic studies were carried out in a panel of cell lines representing all major breast cancer molecular subtypes. Changes in cell proliferation, morphology, and neutral lipid accumulation due to altered NDRG1 expression were assessed by high throughput, quantitative microscopy. Comprehensive lipidomics mass spectrometry was applied to characterize global changes in lipid species due to NDRG1 silencing. Labeled fatty acids were used to monitor cellular fatty acid uptake and subcellular distribution under nutrient replete and starvation culture conditions. RESULTS: NDRG1 overexpression correlated with glycolytic and hypoxia-associated gene expression, and was associated with elevated rates of metastasis and patient mortality. Silencing NDRG1 reduced cell proliferation rates, causing lipid metabolism dysfunction including increased fatty acid incorporation into neutral lipids and lipid droplets. Conversely, NDRG1 expression minimized lipid droplet formation under nutrient replete and starvation conditions. CONCLUSIONS: Here we report that NDRG1 contributes to breast cancer aggressiveness by regulating the fate of lipids in cells that exhibit an altered lipid metabolic phenotype. In line with its role in promoting myelination and its association with altered metabolism in cancer, our findings show that NDRG1 is a critical regulator of lipid fate in breast cancer cells. The association between NDRG1 and poor prognosis in breast cancer suggests it should play a more prominent role in patient risk assessment. The function of NDRG1 in breast cancer lipid metabolism may represent a promising therapeutic approach in the future.

Mpl traffics to the cell surface through conventional and unconventional routes.

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA-mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER-tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER-Golgi and autolysosome secretory pathways, as well as recycling.

The architectural relationship of components controlling mast cell endocytosis.

Eukaryotic cells use multiple routes for receptor internalization. Here, we examine the topographical relationships of clathrin-dependent and clathrin-independent endocytic structures on the plasma membranes of leukemia-derived mast cells. The high affinity IgE receptor (FcεRI) utilizes both pathways, whereas transferrin receptor serves as a marker for the classical clathrin-mediated endocytosis pathway. Both receptors were tracked by live-cell imaging in the presence or absence of inhibitors that established their differential dependence on specific endocytic adaptor proteins. The topology of antigen-bound FcεRI, clathrin, dynamin, Arf6 and Eps15-positive structures were analyzed by 2D and 3D immunoelectron microscopy techniques, revealing their remarkable spatial relationships and unique geometry. We conclude that the mast cell plasma membrane has multiple specialized domains for endocytosis. Their close proximity might reflect shared components, such as lipids and adaptor proteins, that facilitate inward membrane curvature. Intersections between these specialized domains might represent sorting stations that direct cargo to specific endocytic pathways.

Low temperature acclimation of photosynthetic capacity and leaf morphology in the context of phloem loading type.

Carbon export from leaf mesophyll to sugar-transporting phloem occurs via either an apoplastic (across the cell membrane) or symplastic (through plasmodesmatal cell wall openings) pathway. Herbaceous apoplastic loaders generally exhibit an up-regulation of photosynthetic capacity in response to growth at lower temperature. However, acclimation of photosynthesis to temperature by symplastically loading species, whose geographic distribution is particularly strong in tropical and subtropical areas, has not been characterized. Photosynthetic and leaf anatomical acclimation to lower temperature was explored in two symplastic (Verbascum phoeniceum, Cucurbita pepo) and two apoplastic (Helianthus annuus, Spinacia oleracea) loaders, representing summer- and winter-active life histories for each loading type. Regardless of phloem loading type, the two summer-active species, C. pepo and H. annuus, exhibited neither foliar anatomical nor photosynthetic acclimation when grown under low temperature compared to moderate temperature. In contrast, and again irrespective of phloem loading type, the two winter-active mesophytes, V. phoeniceum and S. oleracea, exhibited both a greater number of palisade cell layers (and thus thicker leaves) and significantly higher maximal capacities of photosynthetic electron transport, as well as, in the case of V. phoeniceum, a greater foliar vein density in response to cool temperatures compared to growth at moderate temperature. It is therefore noteworthy that symplastic phloem loading per se does not prevent acclimation of intrinsic photosynthetic capacity to cooler growth temperatures. Given the vagaries of weather and climate, understanding the basis of plant acclimation to, and tolerance of, low temperature is critical to maintaining and increasing plant productivity for food, fuel, and fiber to meet the growing demands of a burgeoning human population.

H2O2 localization in the green alga Micrasterias after salt and osmotic stress by TEM-coupled electron energy loss spectroscopy.

Reactive oxygen species (ROS), including hydrogen peroxide (H(2)O(2)), are constantly generated as by-products of normal metabolic cellular pathways and can be overproduced in response to stress. In this study, we investigated ROS production and localization of H(2)O(2) after salt (200 mM KCl) and osmotic (iso-osmotic sorbitol concentration) stress in the unicellular green alga Micrasterias. By means of the dye H(2)DCFDA and confocal laser scanning microscopy, most ROS production could be detected in KCl-treated cells when compared to sorbitol-exposed cells and controls. For ultrastructural detection of H(2)O(2), CeCl(3), which reacts with H(2)O(2) and produces cerium perhydroxide deposits, has been used. Cerium was identified by transmission electron microscopy (TEM)-coupled electron energy loss spectroscopy (EELS) in organelles of KCl- and sorbitol-treated cells and in controls. Statistical measurements of the presence of the cerium M(4,5) edge were performed in mitochondria, chloroplasts, cell walls, and cytoplasmic sites of five individual cells after each treatment. The most pronounced increase in H(2)O(2) production was found in chloroplasts of KCl- and sorbitol-treated cells. This shows that the chloroplast reveals the strongest response in H(2)O(2) production after stress induction in Micrasterias. Significant elevation of H(2)O(2) production also occurred in mitochondria and cytoplasm, whereas H(2)O(2) levels remained unchanged or even slightly decreased in cell walls of treated cells. Additionally, TEM micrographs and EELS analyses provided indirect evidence for an increased H(2)O(2) production at the plasma membrane of KCl-treated cells, indicating an involvement of the plasma membrane NADPH oxidase in H(2)O(2) generation.

PCD and autophagy in the unicellular green alga Micrasterias denticulata.

Programmed cell death (PCD) plays a central role in normal plant development and is also induced by various biotic and abiotic stress factors. In the unicellular freshwater green alga Micrasterias denticulata morphological and biochemical hallmarks such as the appearance of autophagosomes, increased production of ROS and degradation of genomic DNA into small fragments ("DNA laddering") indicate PCD. Our data not only demonstrate that Micrasterias is capable of performing PCD under salt stress, but also that it is triggered by the ionic and not osmotic component of salinity. Additionally, results from the present and previous studies suggest that different inducers may lead to different cell death pathways in one and the same organism.

Salt stress-induced cell death in the unicellular green alga Micrasterias denticulata.

Programmed cell death (PCD) is a key element in normal plant growth and development which may also be induced by various abiotic and biotic stress factors including salt stress. In the present study, morphological, biochemical, and physiological responses of the theoretically immortal unicellular freshwater green alga Micrasterias denticulata were examined after salt (200 mM NaCl or 200 mM KCl) and osmotic stress induced by iso-osmotic sorbitol. KCl caused morphological changes such as cytoplasmic vacuolization, extreme deformation of mitochondria, and ultrastructural changes of Golgi and ER. However, prolonged salt stress (24 h) led to the degradation of organelles by autophagy, a special form of PCD, both in NaCl- and KCl-treated cells. This was indicated by the enclosure of organelles by ER-derived double membranes. DNA of NaCl- and KCl-stressed cells but not of sorbitol-treated cells showed a ladder-like pattern on agarose gel, which means that the ionic rather than the osmotic component of salt stress leads to the activation of the responsible endonuclease. DNA laddering during salt stress could be abrogated by addition of Zn(2+). Neither cytochrome c release from mitochondria nor increase in caspase-3-like activity occurred after salt stress. Reactive oxygen species could be detected within 5 min after the onset of salt and osmotic stress. Respiration, photosynthetic activity, and pigment composition indicated an active metabolism which supports programmed rather than necrotic cell death in Micrasterias after salt stress.