Studies on insulin secretory granule formation capacity by the control of proprotein-processing endoprotease furin.

通过控制前蛋白加工内切蛋白酶弗林蛋白酶来研究胰岛素分泌颗粒形成能力。

• 批准号: 09470213
• 负责人: TAKEUCHI Toshiyuki
• 金额: $ 8.64万
• 依托单位: Gunma University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470213/
• 关键词: Pancreatic β cells; Insulin secretion; Furin; PTHrP; TGFβ; MIN6 cells; 膵β細胞; フューリン; MAPキナーゼ; フォスファターゼ; TGF-β; furin; インスリン

We previously reported that the islet cells with higher furin expression induces increased production of growth factors, which result in an increase in cell growth using an autocrine/paracrine mechanism. When searching for substrates for furin in pancreatic β cells, we noticed that parathyroid hormone-related protein (PTHrP) and transforming growth factor β (TGFb) possess a furin cleavage motif Arg-X-(Lys/Arg)-Arg. PTHrP is frequently produced in pancreatic endocrine tumors. PTHrP is synthesized as the precursor pro-PTHrP and the cleavage of the propeptide from its precursor is crucial for its biological activation. We demonstrated that furin is highly expressed in rat pancreatic islets during the perinatal stage growth. From this, we suspected that furin may be co-expressed with PTHrP in insulinomas. We examined 21 human endocrine tumors, and furin was positively stained in all 10 insulinomas. Likewise, PTHrP was detected in the same insulinomas. But other non-insulinomas endocrine tu … More mors did not display furin- and PTHrP- positivity. Thus, furin and pro-PTHrP are co-expressed specifically in insulinomas.Production of PTHrP is reportedly higher in more differentiated β cell culture lines, although insulinomas are less differentiated than normal islet β cells. PTHrP induces differentiation in some cell-types and de-differentiation or growth in others. We examined whether PTHrP production is greater in growing β cells or in well-differentiated β cells, and whether PTHrP induces differentiation or growth in β cells. We used four sublines of the well-differentiated mouse β cell line, MIN6, with 17, 25, 31, and 41 passages, and mouse pancreatic islets. With insreasing MIN6 passage number, β cell-specific differentiated features such as insulin content diminished together with TGFβ expression, whereas the expression of PTHrP, furin, and cell growth gradually increased. Both PTHrP(1-34) and PTHrP(1-86) increased insulin content and mRNA levels more in MIN6-17 cells than in MIN6-41 cells. A PTHrP-induced increase in insulin content was also noted in primary-cultured islets. In contrast, PTHrP increased DNA synthesis more extensively in MIN6-41 cells than in MIN6-17 cells. PTHrP increased TGFβ expression in MIN6-17 cells and decreased its expression in MIN6-41 cells. The expression of PTH/PTHrP receptor and TGFβ type II receptor were similar in all MIN6 sublines. Dibutyryl cAMP reproduced PTHrP's effect on insulin content and DNA synthesis in the MIN6 sublines. PTHrP appears to induce insulin expression by a cAMP pathway. We conclude that PTHrPb increases differentiated functions such as insulin and TGFβ expressions in well-differentiated β cells through the cAMP pathway, and stimulates growth in growing β cells. Less

我们以前报道过，高表达Furin的胰岛细胞诱导生长因子的产生增加，从而通过自分泌/旁分泌机制导致细胞生长增加。在胰腺β细胞中寻找呋喃底物时，我们注意到甲状旁腺激素相关蛋白(PTHrP)和转化生长因子β(TGFb)含有一个切割呋喃的基序Arg-X-(Lys/Arg)-Arg。PTHrP常见于胰腺内分泌肿瘤。PTHrP被合成为前体PTHrP，其前体的裂解是其生物活性的关键。我们证明了在围产期生长的大鼠胰岛中呋喃的表达水平很高。由此，我们怀疑在胰岛素瘤中可能与PTHrP共表达。我们检查了21个人类内分泌肿瘤，10个胰岛素瘤中均呈阳性染色。同样，PTHrP在相同的胰岛素瘤中也被检测到。但其他非胰岛素瘤的内分泌却是tu…更多MORS未显示FURIN和PTHrP阳性。因此，尽管胰岛素瘤的分化程度低于正常的胰岛β细胞，但在胰岛素瘤中，呋喃和甲状旁腺素原在胰岛素瘤中的表达是特异的。据报道，在分化程度较高的β细胞系中，甲状旁腺素受体的产量较高。甲状旁腺素能诱导某些类型的细胞分化，而另一些类型的细胞则去分化或生长。我们检测了生长中的β细胞或分化良好的β细胞中甲状旁腺素rp的产量是否较高，以及甲状旁腺素rp是否诱导β细胞分化或生长。我们使用了分化良好的小鼠β细胞系MIN6的四个亚系，分别传代17、25、31和41代，以及小鼠胰岛。随着MIN6传代次数的增加，β细胞特异性分化特征如胰岛素含量和转化生长因子β的表达逐渐减少，而PTHrP、Furin的表达和细胞生长逐渐增加。PTHrP(1-34)和PTHrP(1-86)对MIN6-17细胞的胰岛素含量和mRNA水平的增加均高于MIN6-41细胞。在原代培养的胰岛中，PTHrP诱导的胰岛素含量也被观察到增加。相反，甲状旁腺素rP对MIN6-41细胞的DNA合成的促进作用比对MIN6-17细胞的促进作用更大。甲状旁腺素能增加转化生长因子β在MIN6-17细胞中的表达，降低其在MIN6-41细胞中的表达。甲状旁腺激素/甲状旁腺激素受体和转化生长因子βII型受体在所有MIN6亚系中的表达相似。二丁酰cAMP复制了甲状旁腺素对MIN6亚系胰岛素含量和DNA合成的影响。PTHrP似乎通过cAMP途径诱导胰岛素表达。我们认为甲状旁腺素通过cAMP途径促进分化良好的β细胞胰岛素和转化生长因子的表达，并刺激生长中的β细胞生长。较少

项目成果

Wang J.,Takeuchi T.,Yokota H.et al.: "Novel rabphillin3-like protein associated with insulin-containing granules in pancreatic beta cells"J. Biol. Chem.. 274. 28542-28548 (1999)

Wang J.，Takeuchi T.，Yokota H.等人：“与胰腺β细胞中含胰岛素颗粒相关的新型rabphillin3样蛋白”J。

S. Yamada, H. Nishigori, H. Onda, T. Utsugi, T. Yanagawa, T. Maruyama, K. Onigata, K. Nagashima, R. Nagai, A. Morikawa, T. Takeuchi, and J. Takeda.: "Identification of mutants in the hepatocyte nudear factor-1α (HNF-1α) gene I Japanese subjects with IDDM

S. Yamada、H. Nishigori、H. Onda、T. Utsugi、T. Yanakawa、T. Maruyama、K. Onigata、K. Nagashima、R. Nagai、A. Morikawa、T. Takeuchi 和 J. Takeda： “肝细胞核因子 1α (HNF-1α) 基因 I 突变体的鉴定 日本 IDDM 受试者

H. Kamimura, Y. Konda, H. Yokota, Y. Nagamachi, H. Kuwano, and T. Takeuchi.: "Kex2-family endoprotease furin is expressed spesifically in the pit region-parietal cells of the gastric mucosa."Am. J. Physiol.. 277. G183-G190 (1999)

H. Kamimura、Y. Konda、H. Yokota、Y. Nagamachi、H. Kuwano 和 T. Takeuchi.：“Kex2 家族内切蛋白酶弗林蛋白酶在胃粘膜的凹坑区域 - 壁细胞中特异性表达。”

Wang,J.,Takeuchi,T.,Tanaka,S.,et al.: "A mutation of the insulin 2 gene at a cysteine residue,which forms an intramolecular disulfide bond,induces diabetes with severe pancreatic β-cell dysfunction in the Mody mouse." J.Clin.Invest.103. 27-37 (1999)

Wang, J.、Takeuchi, T.、Tanaka, S. 等人：“胰岛素 2 基因半胱氨酸残基发生突变，形成分子内二硫键，诱发糖尿病并伴有严重的胰腺 β 细胞功能障碍在么小鼠中。”J.Clin.Invest.103. 27-37 (1999)

T.Kayo, Y.Sawada, M.Suda, et al.: "Propeotein-processing endoprotease furin controls growth of pancreatic β cells." Diabetes. 46. 1296-1304 (1997)

T.Kayo、Y.Sawada、M.Suda 等人：“Propeotein 加工内切蛋白酶弗林蛋白酶控制胰腺 β 细胞的生长。”46. 1296-1304 (1997)