Keishi Narita

Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells

We recently showed that human skin fibroblasts internalize fluorescent analogues of the glycosphingolipids lactosylceramide and globoside almost exclusively by a clathrin-independent mechanism involving caveolae. In contrast, a sphingomyelin analogue is internalized approximately equally via clathrin-dependent and caveolar routes. Here, we further characterized the caveolar pathway for glycosphingolipids, showing that Golgi targeting of sphingolipids internalized via caveolae required microtubules and phosphoinositol 3-kinases and was inhibited in cells expressing dominant-negative Rab7 and Rab9 constructs. In addition, overexpression of wild-type Rab7 or Rab9 (but not Rab11) in Niemann-Pick type C (NP-C) lipid storage disease fibroblasts resulted in correction of lipid trafficking defects, including restoration of Golgi targeting of fluorescent lactosylceramide and endogenous GM(1) ganglioside, and a dramatic reduction in intracellular cholesterol stores. Our results demonstrate a role for Rab7 and Rab9 in the Golgi targeting of glycosphingolipids and suggest a new therapeutic approach for restoring normal lipid trafficking in NP-C cells.

A Role of the Amino Acid Residue Located on the Fifth Position before the First Aspartate-rich Motif of Farnesyl Diphosphate Synthase on Determination of the Final Product

Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the ω-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate.

HSulf-1 inhibits angiogenesis and tumorigenesis in vivo.

We previously identified HSulf-1 as a down-regulated gene in several tumor types including ovarian, breast, and hepatocellular carcinomas. Loss of HSulf-1, which selectively removes 6-O-sulfate from heparan sulfate, up-regulates heparin-binding growth factor signaling and confers resistance to chemotherapy-induced apoptosis. Here we report that HSulf-1 expression in MDA-MB-468 breast carcinoma clonal lines leads to reduced proliferation in vitro and reduced tumor burden in athymic nude mice in vivo. Additionally, xenografts derived from HSulf-1-expressing stable clones of carcinoma cells showed reduced vessel density, marked necrosis, and apoptosis, indicative of inhibition of angiogenesis. Consistent with this observation, HSulf-1-expressing clonal lines showed reduced staining with the endothelial marker CD31 in Matrigel plug assay, indicating that HSulf-1 expression inhibits angiogenesis. More importantly, HSulf-1 expression in the xenografts was associated with a reduced ability of vascular endothelial cell heparan sulfate to participate in a complex with fibroblast growth factor 2 (FGF-2) and its receptor tyrosine kinase FGF receptor 1c. In vitro, short hairpin RNA-mediated down-regulation of HSulf-1 in human umbilical vein endothelial cells (HUVEC) resulted in an increased proliferation mediated by heparan sulfate-dependent FGF-2, hepatocyte growth factor, and vascular endothelial growth factor 165 (VEGF165) but not by heparan sulfate-independent VEGF121. HSulf-1 down-regulation also enhanced downstream signaling through the extracellular signal-regulated kinase pathway compared with untreated cells. Consistent with the role of heparan sulfate glycosaminoglycan sulfation in VEGF-mediated signaling, treatment of HUVEC cells with chlorate, which inhibits heparan sulfate glycosaminoglycan sulfation and therefore mimics HSulf-1 overexpression, led to an attenuated VEGF-mediated signaling. Collectively, these observations provide the first evidence of a novel mechanism by which HSulf-1 modulates the function of heparan sulfate binding VEGF165 in proliferation and angiogenesis.

Protein transduction of Rab9 in Niemann-Pick C cells reduces cholesterol storage

Niemann‐Pick disease type C (NPC) is a genetic disorder in which patient cells exhibit lysosomal accumulation of cholesterol and sphingolipids (SLs) caused by defects in either NPC1 or NPC2 proteins. We previously demonstrated that NPC1 human skin fibroblasts overexpressing endosomal Rab proteins (Rab7 or Rab9) showed a correction in the storage disease phenotype. In the current study, we used protein transduction to further investigate Rab9‐mediated reduction of stored lipids in NPC cells. Recombinant human Rab9 fused with the herpes simplex virus VP22 protein fragment was overexpressed, purified, and added to culture medium to induce protein transduction. When VP22‐Rab9 was transduced into NPC1 fibroblasts, nearly all cells showed significant reduction in cellular free cholesterol levels, with no cytotoxicity up to 5 µM. A fraction of the VP22‐Rab9 that was transduced into the cells was shown to bind to rab GDP dissociation inhibitor, suggesting that this pool of VP22‐Rab9 had become prenylated. The reduction in cellular free cholesterol was associated with correction of abnormal intracellular trafficking of BODIPY‐lactosylceramide and an increase of sterols in the culture media. The clearance of lysosomal free cholesterol was also associated with a decrease in LDL‐receptor levels. In addition, we demonstrated reduction of intracellular cholesterol by VP22‐Rab9 transduction in NPC2 fibroblasts and in cultured mouse NPC1 neurons. These observations provide important new information about the correction of membrane traffic in NPC cells by Rab9 overexpression and may lead to new therapeutic approaches for treatment of this disease.

Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance

To investigate the mechanism by which HSulf-1 expression is downregulated in ovarian cancer, DNA methylation and histone acetylation of HSulf-1 was analysed in ovarian cancer cell lines and primary tumors. Treatment of OV207 and SKOV3 by 5-aza-2′-deoxycytidine resulted in increased transcription of HSulf-1. Sequence analysis of bisulfite-modified genomic DNA from ovarian cell lines and primary tumors without HSulf-1 expression revealed an increase in the frequency of methylation of 12 CpG sites in exon 1A. Chromatin immunoprecipitation assays showed an increase in histone H3 methylation in cell lines without HSulf-1 expression. To assess the significance of HSulf-1 downregulation in ovarian cancer, OV167 and OV202 cells were transfected with HSulf-1 siRNA. Downregulation of HSulf-1 expression in OV167 and OV202 cells lead to an attenuation of cisplatin-induced cytotoxicity. Moreover, patients with ovarian tumors expressing higher levels of HSulf-1 showed a 90% response rate (27/30) to chemotherapy compared to a response rate of 63% (19/30) in those with weak or moderate levels (P=0.0146, χ2 test). Collectively, these data indicate that HSulf-1 is epigenetically silenced in ovarian cancer and that epigenetic therapy targeting HSulf-1 might sensitize ovarian tumors to conventional first-line therapies.

Loss of HSulf-1 expression enhances autocrine signaling mediated by amphiregulin in breast cancer.

Heparan sulfate (HS) glycosaminoglycans are the oligosaccharide chains of heparan sulfate proteoglycans. The sulfation of HS glycosaminoglycan residues is required for its interaction with various heparin-binding growth factors to promote their biological activities to activate their high affinity receptor tyrosine kinases. We have identified HS glycosaminoglycan-6-O-endosulfatase HSulf-1 as a down-regulated gene in ovarian, breast, and several other cancer cell lines. Here we have shown that HSulf-1 inhibits autocrine activation of the EGFR-ERK (epidermal growth factor receptor-extracellular signal-regulated kinase) pathway induced by serum withdrawal in MDA-MB-468 breast cancer cells. Short hairpin RNA-mediated down-regulation of HSulf-1 in HSulf-1 clonal lines of MDA-MB-468 led to a significant increase in autocrine activation of ERK compared with vector only control. The autocrine signaling was also inhibited with neutralization antibodies against amphiregulin and HB-EGF, the heparin-binding growth factor family of the EGF superfamily. Furthermore, HSulf-1-mediated inhibition of autocrine signaling was associated with reduced cyclin D1 levels, leading to decreased S phase fraction and increased G2–M fraction, as well as increased cell death. Finally, evaluation of HSulf-1 expression levels in primary invasive breast tumors by RNA in situ hybridization indicated that HSulf-1 is down-regulated in the majority (60%) of tumors, with a predominant association with lobular histology. These data suggest a potential role of HSulf-1 down-regulation in mammary carcinogenesis.

Genetically encoded calcium indicator illuminates calcium dynamics in primary cilia

Visualization of signal transduction in live primary cilia constitutes a technical challenge owing to the organelles submicrometer dimensions and close proximity to the cell body. Using a genetically encoded calcium indicator targeted to primary cilia, we visualized calcium signaling in cilia of mouse fibroblasts and kidney cells upon chemical or mechanical stimulation with high specificity, high sensitivity and wide dynamic range.

Multiple Primary Cilia Modulate the Fluid Transcytosis in Choroid Plexus Epithelium

Functional defects in cilia are associated with various human diseases including congenital hydrocephalus. Previous studies suggested that defects in cilia not only disrupt the flow of cerebrospinal fluid (CSF) generated by motile cilia in ependyma lining the brain ventricles, but also cause increased CSF production at the choroid plexus. However, the molecular mechanisms of CSF overproduction by ciliary dysfunction remain elusive. To dissect the molecular mechanisms, choroid plexus epithelial cells (CPECs) were isolated from porcine brain. These cells expressed clusters of primary cilia on the apical surface. Deciliation of CPECs elevated the intracellular cyclic AMP (cAMP) levels and stimulated basolateral‐to‐apical fluid transcytosis, without detrimental effects on other morphological and physiological features. The primary cilia possessed neuropeptide FF (NPFF) receptor 2. In deciliated cells, the responsiveness to NPFF was reduced at nanomolar concentrations. Furthermore, CPECs expressed NPFF precursor along with NPFFR2. An NPFFR antagonist, BIBP3226, increased the fluid transcytosis, suggesting the presence of autocrine NPFF signaling in CPECs for a tonic inhibition of fluid transcytosis. These results suggest that the clusters of primary cilia in CPECs act as a sensitive chemosensor to regulate CSF production.

Structure and function of vertebrate cilia, towards a new taxonomy

In this review, we propose a new classification of vertebrate cilia/flagella and discuss the evolution and prototype of cilia. Cilia/flagella are evolutionarily well-conserved membranous organelles in eukaryotes and serve a variety of functions, including motility and sensation. Vertebrate cilia have been traditionally classified into conventional motile cilia and sensory primary cilia. However, an avalanche of emerging evidence on the variations of cilia has made it almost impossible to classify them in a simple dichotomic manner. For example, conventional motile cilia are also involved in the sensation of bitter taste to facilitate the beating of cilia as a defense system of the respiratory system. On the other hand, the primary cilium, often regarded as a non-motile sensory organelle, has been revealed to be motile in vertebrate embryonic nodes, where they play a crucial role in the determination of left-right asymmetry of the body. Moreover, choroid plexus epithelial cells in the cerebral ventricular system exhibit multiple primary cilia on a single cell. Considering these lines of evidence on the diversity of cilia, we believe the classification of cilia should be based on their structure and function, and include more detailed criteria. Another intriguing issue is how in the evolution of cilia, their function and morphology are combined. For example, has motility been acquired from originally sensory cilia, or vice versa? Alternatively, were they originally hybrid in nature? These questions are inseparable from the classification of cilia per se. We would like to address these conundrums in this review article, principally from the standpoint of differentiation of the animal cell.

Differential Neuroprotective Activity of Two Different Grape Seed Extracts

Glutamate excitotoxicity is one of the major events that takes place during various neurotoxic injuries such as brain ischemia. We prepared grape seed extracts, from two different varieties, containing high amounts of polyphenols but little resveratrol. Their neuroprotective effects were investigated using primary culture of neonatal mouse hippocampal neurons treated with an excitotoxic concentration of glutamate. Koshu, a white, local variety of V. vinifera, alleviated the acute inactivation of Erk1/2 and dendrite retraction in cultured hippocampal neurons exposed to a toxic concentration of glutamate (1.0 ng/ml). By contrast, Muscat Bailey A, a red, hybrid variety (Muscat Humburg × Bailey), failed to show any neuroprotective effect. Unlike brain-derived neurotrophic factor and other neuroprotective cytokines, Koshu extract did not induce Akt phosphorylation. Koshu extract also augmented neuron survival rate 24 hours after glutamate toxicity. The comparison of polyphenols between the two samples by liquid chromatography/time-of-flight mass spectrometry demonstrated that Koshu had higher amounts of low molecular weight polyphenols along with several Koshu-specific procyanidin oligomers. These data suggest the presence of high affinity molecular targets for polyphenols in hippocampal neurons, which induce neuroprotective effects in a manner different from BDNF, and the importance of low molecular weight polyphenols and/or procyanidin oligomers for neuroprotection.