|Budget Amount *help
¥13,500,000 (Direct Cost : ¥13,500,000)
Fiscal Year 1999 : ¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1998 : ¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1997 : ¥8,700,000 (Direct Cost : ¥8,700,000)
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
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.
Differential display法によりM1N6-17と、PTHrPを作用させたM1N6-17でMAPキナーゼに特異的なフォスファターゼが高発現していることを見出した。このフォスファターゼをアデノウィルスベクターを用いてMlN6-17で発現させると、インスリン発現とインスリン含量が増加した。このインスリン含量は正常膵β細胞の含量に匹敵するものであり、M1N6-41を用いた場合にはこの現象は観察できないことから、MAPキナーゼフォスファターゼはβ細胞高次機能の完成に近い時期に作用しているものと考えられる。 Less