Cell Structure and Function

REGULAR ARTICLES

Establishment of Conditionally Immortalized Rat Retinal Pericyte Cell Lines (TR-rPCT) and Their Application in a Co-culture System Using Retinal Capillary Endothelial Cell Line (TR-iBRB2)

Tetsu Kondo, Ken-ichi Hosoya, Satoko Hori, Masatoshi Tomi, Sumio Ohtsu ...

Article type: scientific monograph
Subject area: Cell Structure and Function
2003 Volume 28 Issue 3 Pages 145-153
Published: 2003
Released on J-STAGE: August 29, 2003

DOIhttps://doi.org/10.1247/csf.28.145

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The purpose of this study was to establish and characterize a retinal pericyte cell line from retinal capillaries of transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), and to apply this to the co-culture with a retinal capillary endothelial cell line. The conditionally immortalized rat retinal pericyte cell lines (TR-rPCTs), which express a temperature-sensitive large T-antigen, were obtained from two tsA58 Tg rats. These cell lines had a multicellular nodule morphology and reacted positively with von Kossa staining, a marker of calcification. TR-rPCTs cells expressed mRNA of pericyte markers such as rat intercellular adhesion molecule-1, platelet-derived growth factor-receptor β, angiopoietin-1, and osteopontin. Western blot analysis indicated that α-smooth muscle actin (α-SMA) was expressed in TR-rPCT3 and 4 cells. In contrast, α-SMA was induced by transforming growth factor-β1 and its enhancement was reduced by basic fibroblast growth factor in TR-rPCT1 and 2 cells. When TR-rPCT1 cells were cultured with a rat retinal endothelial cell line (TR-iBRB2) in a contact co-culture system, the number of TR-iBRB2 cells were significantly reduced in comparison with that of a single culture of TR-iBRB2 cells, suggesting that physical contact between pericytes and retinal endothelial cells is important for the growth of retinal endothelial cells. In conclusion, conditionally immortalized retinal pericyte cell lines were established from tsA58 Tg rats. These cell lines exhibited the properties of retinal pericytes and can be applied in co-culture systems with a retinal capillary endothelial cell line.

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Clusters of VIP36-Positive Vesicles between Endoplasmic Reticulum and Golgi Apparatus in GH3 Cells

Osamu Shimada, Sayuri Hara-Kuge, Katsuko Yamashita, Hisami Tosaka-Shim ...

Article type: scientific monograph
Subject area: Cell Structure and Function
2003 Volume 28 Issue 3 Pages 155-163
Published: 2003
Released on J-STAGE: August 29, 2003

DOIhttps://doi.org/10.1247/csf.28.155

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The vesicular integral membrane protein VIP36 belongs to the family of animal lectins and may act as a cargo receptor trafficking certain glycoproteins in the secretory pathway. Immunoelectron microscopy of GH3 cells provided evidence that endogenous VIP36 is localized mainly in 70-100-nm-diameter uncoated transport vesicles between the exit site on the ER and the neighboring cis-Golgi cisterna. The thyrotrophin-releasing hormone (TRH) stimulation and treatment with actin filament-perturbing agents, cytochalasin D or B or latrunculin-B, caused marked aggregation of the VIP36-positive vesicles and the appearance of a VIP36-positive clustering structure located near the cis-Golgi cisterna. The size of this structure, which comprised conspicuous clusters of VIP36, depended on the TRH concentration. Confocal laser scanning microscopy confirmed the electron microscopically demonstrated distribution and redistribution of VIP36 in these cells. Furthermore, VIP36 colocalized with filamentous actin in the paranuclear Golgi area and its vicinity. This is the first study to show the ultrastructural distribution of VIP36 in the early secretory pathway in GH3 cells. It suggests that actin filaments are involved in glycoprotein transport between the ER and cis-Golgi cisterna by using the lectin VIP36.

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In vivo Analysis of the Cyclin D1 Promoter during Early Embryogenesis in Xenopus

Toshiaki Tanaka, Makoto Kubota, Kazufumi Shinohara, Kunio Yasuda, Jun- ...

Article type: scientific monograph
Subject area: Cell Structure and Function
2003 Volume 28 Issue 3 Pages 165-177
Published: 2003
Released on J-STAGE: August 29, 2003

DOIhttps://doi.org/10.1247/csf.28.165

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Cyclin D1 plays an important role in the regulation of G1 progression of the somatic cell cycle by functioning as a regulatory subunit of cdk4 and 6. Since both cyclin D1-mRNA and -protein turn over very rapidly, the expression of cyclin D1 is mostly regulated at the transcription level. Detection of the cyclin D1-specific mRNA during early embryogenesis in Xenopus laevis revealed that expression was induced soon after the midblastula stage but not during the cleavage stage, and was mainly found in the neural plate and eye vesicles. The recently-developed transgenic frog technique enabled us to analyze the frog cyclin D1 promoter activity in vivo by using green fluorescent protein (GFP) as a marker. During early embryogenesis, a GFP signal was detected at the neural plate in the neurula of the transgenic embryos. Transgenic analysis using sequential-deletion and point mutants of the cyclin D1 promoter revealed that a single, putative TCF/LEF binding site, located approximately 3.5 kb upstream of the transcriptional initiation site, was required for neural plate-specific expression. Our results suggest that the TCF/LEF signaling pathway participates in the regulation of cyclin D1 induction during the generation of the dorsal nervous system in early frog embryogenesis.

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Cholesteryl Glucoside-induced Protection against Gastric Ulcer

Shohko Kunimoto, Wataru Murofushi, Isao Yamatsu, Yukie Hasegawa, Narie ...

Article type: scientific monograph
Subject area: Cell Structure and Function
2003 Volume 28 Issue 3 Pages 179-186
Published: 2003
Released on J-STAGE: August 29, 2003

DOIhttps://doi.org/10.1247/csf.28.179

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The cytoprotective effect of heat shock proteins (HSPs) promises new therapeutic modalities for medical treatment. We examined the anti-ulcer effect of cholesteryl glucoside (1-O-cholesteryl-β-D-glucopyranoside, CG) on cold-restraint stress-induced gastric ulcer in rats, in terms of its correlative ability to activate heat shock factor (HSF) and to induce HSP70. Rapid induction of CG occurred in animal tissues, especially in stomach, after exposure to stress, indicating that this glycolipid might act as an anti-stress, lipid mediator involved in the very early stages of stress-induced signal transduction. Orally administered CG apparently showed anti-ulcer activity in rats via HSF activation and HSP70 induction. When compared with geranylgeranylacetone (GGA), the well known as an effective, synthetic anti-ulcer agent, CG proved to have the same level of strength on ulcer inhibition. GGA caused CG and HSP70 induction in gastric mucosa, indicating that GGA induced HSP70 via CG production. CG thus might be useful for medical treatment of stress-induced diseases, and as an anti-stress supplement for daily diet.

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Analysis of Yeast Prion Aggregates with Amyloid-staining Compound In Vivo

Yoko Kimura, Sumiko Koitabashi, Takashi Fujita

Article type: scientific monograph
Subject area: Cell Structure and Function
2003 Volume 28 Issue 3 Pages 187-193
Published: 2003
Released on J-STAGE: August 29, 2003

DOIhttps://doi.org/10.1247/csf.28.187

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Yeast prions are protein-based genetic elements whose non-Mendelian patterns of inheritance are explained by their inheritance of altered conformations. Here we showed that aggregates made by overexpression of two different prion domains of Sup35 and Rnq1, were stained in yeast by thioflavin-S, an amyloid binding compound. These results suggested that yeast prion domains take the form of amyloid in vivo, and supported the idea that the self-propagating property of amyloids is responsible for the heritable traits of yeast prions.

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c-myc Induces Autophagy in Rat 3Y1 Fibroblast Cells

Makoto Tsuneoka, Toshiyuki Umata, Hiroshi Kimura, Yoshiro Koda, Masayu ...

Article type: scientific monograph
Subject area: Cell Structure and Function
2003 Volume 28 Issue 3 Pages 195-204
Published: 2003
Released on J-STAGE: August 29, 2003

DOIhttps://doi.org/10.1247/csf.28.195

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The proto-oncogene c-myc is a multifunctional gene that regulates cell division, cell growth, and apoptosis. Here we report a new function of c-myc: induction of autophagy. Autophagy is a bulk degradation system for intracellular proteins. Autophagy proceeds with characteristic morphologies, which begins with the formation of a double-membrane structure called the autophagosome surrounding a portion of the cytoplasm, after which its outer membrane then fuses with the lysosomal membrane to become an autolysosome. Autophagosomes and autolysosomes are generally called autophagic vacuoles. When c-Myc protein was overexpressed in rat 3Y1 fibroblasts or when the chimeric protein c-MycER was activated by estrogen, the number of autophagic vacuoles in cells increased significantly. The formation of autophagic vacuoles induced by c-Myc was completely blocked by a specific inhibitor of autophagosome formation, 3-methyladenine. A c-Myc mutant lacking Myc Box II induced neither apoptosis nor oncogenic transformation, but still stimulated autophagy. An inhibitor of caspases suppressed apoptosis but not autophagy. These results suggest that the autophagy caused by c-myc is not due to the apoptosis or tumorigenesis induced by c-myc. Taken together, our results suggest that the induction of autophagy is a novel function of c-myc.

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