A novel strategy to increase the proliferative potential of adult human β-cells while maintaining their differentiated phenotype.
A novel strategy to increase the proliferative potential of adult human β-cells while maintaining their differentiated phenotype.
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DOI：
10.1371/journal.pone.0066131
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
McDaniel ML
中科院分区：
文献类型：
--
作者：
Aly H;Rohatgi N;Marshall CA;Grossenheider TC;Miyoshi H;Stappenbeck TS;Matkovich SJ;McDaniel ML
我们以前的研究表明，Wnt/GSK-3/β-catenin和mTOR信号传导是刺激成人β细胞增殖过程所必需的。GSK-3的直接抑制，其参与Wnt受体下游的Wnt信号传导，增加β-连环蛋白核转位和β-细胞增殖，但导致胰岛素含量降低。我们目前的目标是在受体水平上参与经典和非经典Wnt信号传导，以显著增加人β细胞增殖，同时保持完整胰岛中的β细胞表型。我们采用了一个利用来自表达Wnt 3a、R-spondin-3和Noggin的L细胞的条件培养基（L-WRN条件培养基）的系统。此外，我们在这些培养物中使用ROCK抑制剂（Y-27632）和SB-431542（其导致RhoA抑制）。用L-WRN条件培养基加抑制剂处理完整的人胰岛，以雷帕霉素敏感的方式显著增加DNA合成2.6倍。此外，这种治疗显著增加了人β细胞增殖，比单独使用葡萄糖高出20倍。只有L-WRN条件培养基与RhoA/ROCK抑制剂的组合导致显著的增殖。全转录组基因表达谱分析表明，L-WRN培养基在几个信号传导家族中引起了强烈的变化，包括增强的β-连环蛋白介导的和β-细胞特异性基因表达。这种处理也增加了Nr 4a 2和Irs 2的表达，并导致Akt的磷酸化。重要的是，葡萄糖刺激的胰岛素分泌和含量不下调L-WRN培养基处理。我们的数据表明，通过该方法在受体水平上参与Wnt信号传导导致多个信号传导途径之间的必要串扰，所述多个信号传导途径包括Akt、mTOR、Wnt/β-连环蛋白、PKA/CREB的激活和RhoA/ROCK的抑制，其显著增加人β细胞增殖，同时维持β细胞表型。
Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype.
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影响因子：
4.3
作者：
Kim, KA;Zhao, JS;Funk, WD
通讯作者：
Funk, WD
DOI：
10.1073/pnas.220413597
发表时间：
2000-10-24
影响因子：
11.1
作者：
Fang, XJ;Yu, SX;Mills, GB
通讯作者：
Mills, GB
影响因子：
4.6
作者：
Kazanskaya, Olga;Ohkawara, Bisei;Niehrs, Christof
通讯作者：
Niehrs, Christof
影响因子：
7.7
作者：
Cozar-Castellano, I;Takane, KK;Stewart, AF
通讯作者：
Stewart, AF
影响因子：
64.8
作者：
Dor, Y;Brown, J;Melton, DA
通讯作者：
Melton, DA