Cytochemical studies on the membrane biogenesis

膜生物发生的细胞化学研究

• 批准号: 02454112
• 负责人: HIRANO Hiroshi
• 金额: $ 4.35万
• 依托单位: Kyorin University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454112/
• 关键词: Membrane; Immunocytochemistry; In situ hybridization; Endogenous beta-galactoside binding lectin; Glucose transporters; differentiation; development; malignancy; in situハイブリダイゼイション; 生体膜; 生合成; 組織細胞化学; グルコ-ス輸送体; ビタミンA; 細胞内骨格

Followings were obtained mainly by means of immunohisto- & cytochemistry at the light and electron microscopic level.1)Chick embryos have two different kinds of beta-galactoside binding lectins, the 14- and 16-kDa lectins. In the present study, localization of endogenous beta-galactoside binding lectins and their gene expression patterns were investigated during differentiation of chick embryonic skin in vivo and in vitro. By light microscopy, immunostaining of the lectins was weak in the undifferentiated epidermis, while it became intense in the keratinized epidermis, particularly in the intermediate cells. The gene expression pattern as revealed by in situ hybridization technique was consistent with these immunohistochemical observations. In the Vitamin A-pretreated cultured skin, mucous metaplasia of the epidermis was induced and marked changes in localization of these two isolectins were observed. In the metaplastic epidermis, the 16-kDa lectin expression was increased especially i … More n the superficial cells, and the gene expression was detected in all layers of the epidermis, while distribution of 14-kDa lectin and its gene expression was markedly reduced. In the keratinized epidermis, detailed localization of two isolectins under the electron microscope was almost the same. Both of them were located primarily along the plasma membrane, in the intercellular space, and in the desmosomes. In the mucous metaplastic epidermis, however, the localization of the 16-kDa lectin was completely different from that of the 14-kDa lectin; e.g., the former was detected in the while the later was scarcely observed in the epidermis. To characterize the complementary sugar residue recognized by these endogenous lectins, conventional plant lectins such as PNA, RCA and ECA were applied and found that binding patterns of these exogenous lectins were coincided with the expression of the 16-kDa lectin, but not with that of 14-kDa lectin in the mucous metaplastic skin. The present results indicated that 1)the two isolectins are expressed during the epidermal differentiation, 2)much 16-kDa lectin is produced and secreted from the Vitamin A-pretreated culture epidermis, while 14-kDa lectin disappears, and 3)the gene expression of 14- and 16-kDa lectins is regulated independently.2)The production of extracellular matrix components such as laminin, Type IV collagen, fibronectin, and tenascin during the formation of basement membrane in cultured epidermis-dermis recombinant skin of 13-day-old chick embryo was analyzed immunohistochemically. The epidermis and dermis were separated from each other by treatment with EDTA and/or dispase. The basal lamina of the basement membrane was thus removed from both epidermis and dermis. The isolated epidermis was overlaid onto the isolated dermis, i.e., recombined, and then cultured for 1-7 days in a chemically defined medium (BGJb) on a Millipore filter. Immunofluorescence labeling was used for light microscopy and HRP or colloidal gold labeling for electron microscopy. In specimens from 2-day cultures, positive sites of anti-laminin and anti-fibronectin reaction were observed light microscopically as patches which, at the electron microscopic level, corresponded to fragments of the basal lamina located immediately beneath and in the vicinity of the attachment plaques of the hemidesmosomes. The staining pattern became continuous 7 days after recombination. Fluorescence labeling of laminin and fibronectin appeared somewhat earlier than that of Type IV collagen and tenascin. All of the four components were found localized primarily in the basal lamina. Furthermore, fibronectin and tenascin were also distributed in the extracellular matrix of the dermis. The expression of tenascin, which does not exist in the basement membrane of 13-day-old intact embryonic skin, was induced in vitro. These results suggest that hemidesmosomes may play an important role in the reconstruction of the basement membrane appeared at different times during the reconstruction.3)Recently, it has been revealed that changes in carbohydrate structures of glycolipids and glycoproteins constituting the plasma membrane occur in association with canceration of the cell. GAT (galactosyltransferase associated with tumor) is one of the glycosyltransferases which are regarded to be responsible for malignant changes. Differences were detected in ultrastructural location of GAT by light and electron microscopy in the normal and malignant endometrium.4)Glucose transporters are integral membrane proteins that transport glucose across the plasma membrane. At least five isoforms of facilitated diffusion glucose transporter (GLUT1-5) have been identified in mammalian cells. Using isoform-specific antibodies, we determined their localization using immunofluorescence and immunogold staining of frozen sections. GLUT1 was found at the plasma membrane in various cells, while GLUT4, an isoform expressed in insulin sensitive cells, was found intracellularly in skeletal muscle cells in the absence of insulin. Ultracytochemical examination revealed that GLUT4 was localized in the trans side of the Golgi apparatus and vesicles and tubules near the plasma membrane. Insulin treatment induced the translocation of GLUT4 to the plasma membrane. To see whether their different intracellular localization is dependent on the cell types that express these glucose transporter isoforms, or on the isoform molecules themselves, GLUT1 and GLUT4 were stably expressed in Chinese hamster ovary cells by the transfection of their cDNAs. GLUT1 was localized at the plasma membrane, while GLUT4 remained intracellularly in the trans-Golgiarea as well as vesicles and tubules, suggesting that each isoform has a signal to determine its cellular localization. To elucidate these differential intracellular targeting mechanisms, several chimeric glucose transporters in which portions of GLUT4 are replaced with corresponding portions of GLUT1, are expressed in Chinese hamster ovary cells. Two domains of GLUT4 seems to determine its targeting to the cytoplasmic vesicles. One is the second transmembrane domain, which contains the consensus sequence of leucine zipper structure. The other is the portion including the eighth transmembrane domain. These domains would be specifically recognized by the intracellular targeting mechanism, resulting the differential localization of the isoforms. Less

以下主要是通过免疫组织和细胞化学在光镜和电镜水平上获得的。1)鸡胚有两种不同的β -半乳糖苷结合凝集素，14- kda和16-kDa凝集素。本研究在体内和体外研究了鸡胚皮肤分化过程中内源性β -半乳糖苷结合凝集素的定位及其基因表达模式。光镜下，凝集素在未分化表皮的免疫染色较弱，而在角质化表皮，特别是中间细胞中，凝集素的免疫染色变得强烈。原位杂交技术显示的基因表达模式与免疫组化观察结果一致。在维生素a预处理的培养皮肤中，诱导表皮粘膜化生，观察到这两种隔离素的定位发生明显变化。在化生表皮中，16-kDa凝集素的表达量明显增加，特别是在表皮表层细胞中，且该基因在表皮各层均有表达，而14-kDa凝集素的分布及其基因表达量明显减少。在角质化的表皮中，电镜下两种隔离素的详细定位几乎相同。它们主要位于质膜、细胞间隙和桥粒内。而在粘液化表皮中，16-kDa凝集素与14-kDa凝集素的定位完全不同；例如，前者在表皮中检测到，而后者在表皮中几乎没有观察到。为了表征这些内源性凝集素识别的互补糖残基，我们应用了传统的植物凝集素如PNA、RCA和ECA，发现这些外源性凝集素的结合模式与16 kda凝集素的表达一致，而与14 kda凝集素在粘液化生皮肤中的表达不一致。结果表明：1)两种凝集素在表皮分化过程中表达；2)经维生素a预处理的培养表皮产生并分泌大量16-kDa凝集素，而14- kda凝集素消失；3)14-和16-kDa凝集素的基因表达是独立调控的。2)用免疫组织化学方法分析了13日龄鸡胚表皮-真皮层重组皮肤基底膜形成过程中层粘连蛋白、IV型胶原、纤维连接蛋白、腱素等细胞外基质成分的产生。通过EDTA和（或）疾病治疗，表皮和真皮层相互分离。因此，基底膜的基底层从表皮和真皮层中被去除。将分离的表皮覆盖在分离的真皮层上，即重组，然后在Millipore过滤器上的化学定义培养基（BGJb）中培养1-7天。光镜用免疫荧光标记，电镜用HRP或胶体金标记。在培养2天的标本中，光镜下观察到抗层粘连蛋白和抗纤维连接蛋白反应的阳性位点为斑块，在电子显微镜下，这些斑块对应于位于半底粒附着斑块下方和附近的基板碎片。重组后7天染色模式连续。层粘连蛋白和纤维连接蛋白的荧光标记出现时间稍早于IV型胶原和腱蛋白。这四种成分主要分布于基底膜。此外，纤维连接蛋白和腱素也分布在真皮细胞外基质中。在体外诱导13日龄完整胚胎皮肤基底膜中不存在腱黄蛋白的表达。这些结果表明，半粒酶可能在基底膜的重建中起重要作用，在重建过程中出现的时间不同。3)最近发现，构成质膜的糖脂和糖蛋白的碳水化合物结构的变化与细胞的癌变有关。与肿瘤相关的半乳糖转移酶（galactosyltransferase with tumor， GAT）是一种被认为与恶性变化有关的糖基转移酶。光镜和电镜观察正常和恶性子宫内膜GAT超微结构定位的差异。葡萄糖转运蛋白是一种完整的膜蛋白，它在质膜上运输葡萄糖。已在哺乳动物细胞中鉴定出至少五种促进扩散的葡萄糖转运蛋白（GLUT1-5）同工型。使用同种异构体特异性抗体，我们通过免疫荧光和免疫金染色对冷冻切片进行定位。GLUT1存在于多种细胞的质膜上，而GLUT4是在胰岛素敏感细胞中表达的异构体，在缺乏胰岛素的情况下存在于骨骼肌细胞的细胞内。超细胞化学检查显示，GLUT4定位于高尔基体的反侧和质膜附近的囊泡和小管。胰岛素治疗诱导GLUT4转运至质膜。为了了解它们在细胞内的不同定位是否取决于表达这些葡萄糖转运蛋白亚型的细胞类型，还是取决于亚型分子本身，我们通过转染GLUT1和GLUT4的cdna在中国仓鼠卵巢细胞中稳定表达。GLUT1定位于质膜，而GLUT4则停留在细胞内的跨高尔基区以及囊泡和小管中，这表明每种异构体都有确定其细胞定位的信号。为了阐明这些不同的细胞内靶向机制，几种嵌合葡萄糖转运体在中国仓鼠卵巢细胞中表达，其中GLUT4的部分被GLUT1的相应部分所取代。GLUT4的两个结构域似乎决定了它对细胞质囊泡的靶向性。一个是第二跨膜结构域，包含亮氨酸拉链结构的一致序列。另一部分是包含第八个跨膜结构域的部分。这些结构域将被细胞内靶向机制特异性识别，从而导致同种异构体的差异定位。少

项目成果

帯刀 章子,秋元 義弘,平野 寛,遠藤 浩良: "レチノイドの短時間作用による表皮粘液化生誘導ーーーアデニレ-トシクラ-ゼ・cAMP系およびプロスタグランジンE_2による促進." 第3回レチノイドシンポジウム,東京,平成3年12月6ー7日.

Akiko Tateto、Yoshihiro Akimoto、Hiroshi Hirano、Hiroyoshi Endo：“类视黄醇短期作用诱导表皮粘液化生 - 腺苷酸环化酶/cAMP 系统和前列腺素 E_2 的促进，第 3 届类视黄醇研讨会，东京，平成 12 月 6 日至 7 日”第三年。

福島 久喜、鈴木 昇、関原 正、松田 実、花岡 建夫、鍋谷 欣市、北島 政樹、立川 勲、川上 速人、平野 寛: "乳癌組織における免疫組織化学的検討ーーーとくに抗ラミニン抗体結合反応について." 乳癌の臨床.5. 111-116 (1990)

Hisaki Fukushima、Noboru Suzuki、Tadashi Sekihara、Minoru Matsuda、Takeo Hanaoka、Kinichi Nabetani、Masaki Kitajima、Isao Tachikawa、Hayato Kawakami、Hiroshi Hirano：“乳腺癌组织的免疫组织化学研究 - 特别是关于抗层粘连蛋白抗体的结合反应。” “临床乳腺癌.5。111-116（1990）

Susumu N, Aoki D, Nozawa S, Kawakami H & Hirano H: "Immuno-histochemical studies of endometrial adenocarcinomas using a monoclonal antibody against an endometrial cancer cell line." J Clin Electron Microsc. 23. 910-911 (1990)

Susumu N、青木 D、野泽 S、川上 H

Takata K,Kasahara T,Kasahara M,Ezaki O & Hirano H:"Ultracytochemical localization of the erythrocyte/HepG2ーtype glucose transporter (GLUT1) in cells of the bloodーretinal barrier in the rat." Invest Ophthalmol Vis Sci. 33. 377-383 (1992)

Takata K、Kasahara T、Kasahara M、Ezaki O 和 Hirano H：“大鼠血-视网膜屏障细胞中红细胞/HepG2 型葡萄糖转运蛋白 (GLUT1) 的超细胞化学定位。” Invest Olookingmol Vis Sci。 377-383 (1992)

Asano T,Takata K,Katagiri H,Tsukuda K,Lin JーL, Ishihara H,Inukai K,Hirano H,Yazaki Y & Oka Y:"Domains responsible for the differential targeting of glucose transporter isoforms." J Biol Chem. 267. 19636-19641 (1992)

Asano T、Takata K、Katagiri H、Tsukuda K、Lin J-L、Ishihara H、Inukai K、Hirano H、Yazaki Y 和 Oka Y：“负责葡萄糖转运蛋白亚型差异靶向的结构域。”J Biol Chem。 -19641 (1992)