Taiji Kato

Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors.

The blood-brain barrier (B-BB) protects the free passage of substances into the brain and maintains the homeostasis of the central nervous system. It is commonly accepted that astrocytes surrounding brain endothelial cells influence the B-BB formation and the exhibition of B-BB function of capillaries. To begin the in vitro study on the B-BB, it is essential to obtain a homogenous and sufficient supply of brain endothelial cells as well as astrocytes. We thus immortalized the bovine brain endothelial cell (BBEC) by transfection of the SV40 large T antigen and obtained a single clone, t-BBEC-117, which retained the brain endothelial cell phenotype. Astrocyte in co-culture was found to tighten the intercellular contacts of the immortal cells resulting in a reduced L-glucose permeability, and its conditioned medium (CM) augmented a B-BB phenotype, alkaline phosphatase (ALP) activity. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes as measured by L-glucose permeability and ALP activity. Moreover, anti-bFGF antibody canceled 90% of ALP activation by astrocyte CM. Basic FGF, however, failed to induce other B-BB phenotypes such as the expressions of multidrug resistance (mdr) and glucose transporter (GLUT-1) genes. These data suggest that bFGF is one of the most plausible astrocytic factors to induce the B-BB properties of immortal brain endothelial cells together with some unknown factors in the astrocyte CM.

Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [Cell 39 (1984) 49], that increase plasma membrane water permeability in secretory and absorptive cells. In the central nervous system (CNS), we detected the transcripts of AQP3, 5 and 8 in addition to the previously reported transcripts of AQP4 and 9 in astrocytes, of AQP3, 5 and 8 in neurons, of AQP8 in oligodendrocytes, and none of them in microglia using RNase protection assay and the reverse transcription-polymerase chain reaction (RT-PCR). Hypoxia evoked a marked decrease in the expression levels of AQP4, 5 and 9, but not of AQP3 and 8 mRNAs, and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP4 and 9 to their basal levels. Interestingly, AQP5 showed a transient up-regulation (about 3-fold) and subsequent down-regulation of its expression within 20 h of reoxygenation after hypoxia. The changes in the profiles of AQP expression during hypoxia and reoxygenation were also observed by Western blot analysis. These results suggest that AQP5 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury.

Experimental implication of celiac ganglionotropic invasion of pancreatic-cancer cells bearing c-ret proto-oncogene with reference to glial-cell-line-derived neurotrophic factor (GDNF)

Perineural invasion is a prominent clinical feature of pancreatic cancer which causes difficulty in curative resection. In the present study, the human pancreatic cancer cell lines, PaCa‐2, AsPC‐1, SW1990 and Capan‐2, were all found to express abundant c‐ret proto‐oncogene mRNA and RET protein, a member of the receptor‐tyrosine‐kinase superfamily, identified as being a receptor for glial‐cell‐line‐derived neurotrophic factor (GDNF). In an invasion assay, the migration of pancreatic cancer cells was markedly induced by co‐cultivation with human glioma cells, T98G or A172, capable of producing and secreting GDNF. Anti‐GDNF antibody in conditioned media of glioma cells suppressed much of the migratory activity. Checkerboard analysis of the migration showed both chemotactic and chemokinetic activity of GDNF. There was no detectable expression of another GDNF receptor component, a glycosyl‐phosphatidylinositol‐linked receptor (GFRα‐1), in pancreatic‐cancer cell lines, suggesting that the neural invasion of pancreatic‐cancer cells spreads along a concentration gradient of GDNF produced from peripheral ganglions through direct interaction of GDNF with its receptor, the c‐ret proto‐oncogene product. Immunochemical localization of GDNF in human celiac ganglionic tissue supported this contention. Int. J. Cancer 81:67–73, 1999.

Alpha-fetoprotein producing gastric cancer lacks transcription factor ATBF1.

Alpha-fetoprotein (AFP) producing gastric cancer (AFP–GC) is very malignant and highly metastatic compared with common gastric cancer. However, the causal relationship between AFP production and the high malignancy of AFP-GC is unclear. We investigated AFP gene regulation in AFP-GC by an active transcription factor, HNF1 (hapatocyte nuclear factor 1) and a repressive transcription factor, ATBF1 (AT motif binding factor 1). RNase protection assays revealed that the production of AFP in gastric cancer cells did not directly associate with HNF1 expression. An inverse relation between the expressions of ATBF1 and AFP was clearly observed in gastric cancer cells. CAT assays showed the direct inhibition of AFP gene expression by ATBF1. Methylation analysis of the AFP promoter region in gastric cancer cells suggested that methylation itself could not explain the silencing of the AFP gene. Immunohistochemistry of resected clinical samples revealed that AFP producing cells lacked ATBF1 immunoreactivity. Our data suggests that the absence of ATBF1 is responsible for AFP gene expression in gastric cancer, and the absence of ATBF1 is a distinct characteristic of AFP-GC and might be important for its highly malignant nature.

Heat-induced Apoptosis in Human Glioblastoma Cell Line A172

OBJECTIVEnHyperthermia has been clinically applied to some types of brain tumors. However, the detailed mechanisms of this growth inhibition are not clear. The effect of mild hyperthermia on cultured human glioblastoma cell line, A172, was studied.nnnMETHODSnA172 cells were heat treated (43-44.5 degrees C) for 1 hour in the growing phase. Cell viability was assessed by trypan blue dye exclusion assay. The presence of apoptosis was determined by the morphological changes observed using phase contrast microscopy and nuclear changes observed using HOECHST 33342 stain. For the evaluation of cellular deoxyribonucleic acid fragmentation, the TUNEL method was used. The expression of p53 and bax proteins was evaluated by Western blot, and the bax messenger ribonucleic acid was detected by Northern blot.nnnRESULTSnHeat treatment induced cell death in time- and temperature-dependent manners. The nuclear staining with HOECHST 33342 demonstrated morphological changes consistent with apoptosis. The TUNEL stain also demonstrated damages in the deoxyribonucleic acid. These morphological changes were accompanied by the accumulation of p53 protein, bax protein, and messenger ribonucleic acid.nnnCONCLUSIONnThese results indicate that mild hyperthermia induces apoptosis in A172 glioblastoma cells.

Interleukin-1β induces the expression of lipocortin 1 mRNA in cultured rat cortical astrocytes

Lipocortin 1 (LC1) has been shown to increase in neuronal damage and act as a neuroprotectant and a neurotrophic factor. IL-1beta acts as a mediator of inflammation and has been reported as a potent inducer of various neurotrophic factors including nerve growth factor and fibroblast growth factor. In this study, we investigated the relationship between LC1 and IL-1beta in cultured rat astrocytes. Time-and dose-dependent experiments of IL-1beta on rat cortical astrocytes in culture revealed that the expression of LC1 mRNA was significantly augmented by IL-1beta at 8 h, 10 ng/ml. In addition, IL-1beta evoked an extracellular secretion of LC1 without its cytotoxic effects. The effect of IL-1beta was completely abolished when we treated cells with inhibitor of mitogen-activated protein kinases (MAPKs) (PD98059) (25 microM), phospholipase A(2) inhibitor mepacrine (30 microM) and protein synthesis inhibitor cycloheximide (CHX) (10 microg/ml). This suggests that induction of LC1 by IL-1beta is through a MAPKs and phospholipaseA(2) pathway and requires protein synthesis. These results indicate that IL-1beta released in the central nervous system (CNS) injury can stimulate the transcription of the LC1 gene. Subsequent synthesis and release of LC1 may provide trophic support to neurons and modulate the action of IL-1beta in brain damage.

Regulation of aquaporin-4 expression in astrocytes

Aquaporin-4 (AQP4), a mercury-insensitive water channel protein, is abundant in the central nervous system and is localized in astrocytes and ependymal cells. AQP4 is speculated to maintain the homeostasis of intracellular and extracellular water in the brain, but little is known about the mechanism of induction of its expression. To investigate the expressional regulation of AQP4, we analyzed changes in its expression during chemically induced differentiation of embryonal carcinoma cells (P19) to neuronal and astrocytic cells, and during the cell cycle of glioma cells. After exposure to retinoic acid for 4 days AQP4 mRNA expression started at the initiation of astrocytic differentiation of P19 cells at 6 days, and increased markedly by 21 days. AQP4 expression was parallel to that of GFAP, a marker intermediate filament of astrocytes. In glioma cell lines, AQP4 mRNA was not detected in the growing phase, but was induced when the cell cycle was arrested at G0/G1 by transient expression of p21. Although quiescent astrocytes in the G0/G1-phase cultured under the serum-free condition exhibited a high expression of AQP4, serum supplement moved them to the S-phase and markedly decreased the AQP expression. These results suggest that AQP4 expression may be induced not only at the initiation of astrocytic differentiation of neural stem cells, but also at the G0/G1-phase during the cell cycle of astrocytes.

Membrane-Bound Form of ADP-Ribosyl Cyclase in Rat Cortical Astrocytes in Culture

Abstract: ADP‐ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [3H]cyclic ADP‐ribose production converted from [3H]NAD+ by ADP‐ribosyl cyclase (2.015 ± 0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained ∼50% of the total cyclase activity in astrocytes. The formation rate of [3H]ADP‐ribose from cyclic ADP‐ribose by cyclic ADP‐ribose hydrolase and/or from NAD+ by NAD glycohydrolase was low and enriched in the cytosolic fraction. Although NAD+ in the extracellular medium was metabolized to cyclic ADP‐ribose by incubating cultures of intact astrocytes, the presence of Triton X‐100 in the medium for permeabilizing cells increased cyclic ADP‐ribose production three times as much. Isoproterenol and GTP increased [3H]cyclic ADP‐ribose formation in crude membrane‐associated cyclase activity. This isoproterenol‐induced stimulation of membrane‐associated ADP‐ribosyl cyclase activity was confirmed by cyclic GDP‐ribose formation fluorometrically. This stimulatory action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin. These results suggest that ADP‐ribosyl cyclase in astrocytes has both extracellular and intracellular actions and that signals of β‐adrenergic stimulation are transduced to membrane‐bound ADP‐ribosyl cyclase via G proteins within cell surface membranes of astrocytes.

Molecular cloning of two bovine aquaporin-4 cDNA isoforms and their expression in brain endothelial cells.

Two cDNA isoforms of bovine aquaporin-4 (bAQP4-A and bAQP4-B) were newly isolated. Sequence analysis of both cDNAs revealed open reading frames of 972 (bAQP4-A) and 906 nucleotides (bAQP4-B) with deduced proteins of 323 (bAQP4-A) and 301 amino acid residues (bAQP4-B). Partial 5-genomic sequence analysis showed that the 5-noncoding sequences specific to bAQP4-A and -B transcripts were contained in distinct exons, exon 0 for bAQP4-A and new exon X for bAQP4-B. RNase protection assay demonstrated the definite expression of both isoforms in bovine brain. The deduced amino acid sequence of bAQP4-A was highly homologous to the human (97%), rat (95%), and mouse (93%) AQP4. Reverse transcription-PCR detected the expression of AQP4 mRNAs in bovine brain endothelial cells as well as in a variety of bovine organs such as brain, lung, spleen, and kidney. Northern blot analysis indicated that a 6.0 kb message is predominantly expressed in bovine brain and lung.

Expression of glia maturation factor during retinal development in the rat

Glia maturation factor plays important roles in the development and growth of glia and neurons. We investigated the expression and localization of Glia maturation factor-beta (GMFB) and Glia maturation factor-gamma (GMFG) in the rat retina. By northern blot analysis, both GMFB and GMFG mRNAs were detected in retina as early as embryonic day (E) 18 and persisted until adult. The expression of GMFB mRNA was always much greater than that of GMFG mRNA. In situ hybridization showed that the GMFB mRNA signal was positive in the retina from E14 till adult. Immunostaining revealed that GMFB protein was present in the inner layer of retina at E14 and P1, and in Müller cells in adult. GMFG immunoreactivity was observed only in the inner limiting membrane from E14 to P1 rat retina, and was not detected in the adult retina. These results show that GMFs are synthesized and localized mainly in Müller cells in the rat retina, and suggest that they may contribute to the development and growth of glia and neurons.