INS-1 cells undergoing caspase-dependent apoptosis enhance the regenerative capacity of neighboring cells.
INS-1 cells undergoing caspase-dependent apoptosis enhance the regenerative capacity of neighboring cells.
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作者：
Bonner C;Bacon S;Concannon CG;Rizvi SR;Baquié M;Farrelly AM;Kilbride SM;Dussmann H;Ward MW;Boulanger CM;Wollheim CB;Graf R;Byrne MM;Prehn JH
在糖尿病中，β细胞团不是静态的，而是处于细胞死亡和更新的持续过程中。转录因子1 (tcf-1)/肝细胞核因子1a （hnf1a）失活突变导致β-细胞质量下降和hnf1a成熟型糖尿病（hnf1a - mody）。在这里，我们研究了显性阴性HNF1A突变体（DN-HNF1A）诱导的细胞凋亡对INS-1细胞再生能力的影响。DN-HNF1A在INS-1细胞中通过逆四环素依赖的反激活剂系统表达。采用实时定量RT-PCR、Western blotting、免疫组织化学等方法研究参与β细胞再生的基因/蛋白。采用酶联免疫吸附法检测人血清胰石蛋白/再生蛋白（PSP/reg）水平。在dn - hnf1a诱导的细胞凋亡过程中，我们检测到PSP/reg在基因和蛋白水平上的显著诱导。在转基因HNF1A-MODY小鼠的胰岛和HNF1A-MODY患者的血清中也检测到PSP/reg水平升高。PSP/reg的诱导是葡萄糖依赖性的，在细胞凋亡过程中由caspase激活介导。有趣的是，上清来自表达dn - hnf1a的细胞，而不是表达dn - hnf1a的细胞。在naïve未处理的INS-1细胞中，fmk足以诱导PSP/reg基因表达并增加细胞增殖。进一步的实验表明，源自INS-1细胞凋亡的膜联蛋白v阳性微粒介导了PSP/reg的诱导。重组PSP/reg处理通过刺激细胞增殖和增加胰岛素基因表达逆转dn - hnf1a诱导的细胞表型。我们的研究结果表明，凋亡的INS-1细胞脱落的微粒可能刺激邻近细胞的PSP/reg诱导，这一机制可能促进HNF1A-MODY中β-细胞质量的恢复。
In diabetes, β-cell mass is not static but in a constant process of cell death and renewal. Inactivating mutations in transcription factor 1 (tcf-1)/hepatocyte nuclear factor1a (hnf1a) result in decreased β-cell mass and HNF1A–maturity onset diabetes of the young (HNF1A-MODY). Here, we investigated the effect of a dominant-negative HNF1A mutant (DN-HNF1A) induced apoptosis on the regenerative capacity of INS-1 cells. DN-HNF1A was expressed in INS-1 cells using a reverse tetracycline-dependent transactivator system. Gene(s)/protein(s) involved in β-cell regeneration were investigated by real-time quantitative RT-PCR, Western blotting, and immunohistochemistry. Pancreatic stone protein/regenerating protein (PSP/reg) serum levels in human subjects were detected by enzyme-linked immunosorbent assay. We detected a prominent induction of PSP/reg at the gene and protein level during DN-HNF1A–induced apoptosis. Elevated PSP/reg levels were also detected in islets of transgenic HNF1A-MODY mice and in the serum of HNF1A-MODY patients. The induction of PSP/reg was glucose dependent and mediated by caspase activation during apoptosis. Interestingly, the supernatant from DN-HNF1A–expressing cells, but not DN-HNF1A–expressing cells treated with zVAD.fmk, was sufficient to induce PSP/reg gene expression and increase cell proliferation in naïve, untreated INS-1 cells. Further experiments demonstrated that annexin-V–positive microparticles originating from apoptosing INS-1 cells mediated the induction of PSP/reg. Treatment with recombinant PSP/reg reversed the phenotype of DN-HNF1A–induced cells by stimulating cell proliferation and increasing insulin gene expression. Our results suggest that apoptosing INS-1 cells shed microparticles that may stimulate PSP/reg induction in neighboring cells, a mechanism that may facilitate the recovery of β-cell mass in HNF1A-MODY.
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7.3
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Hagenfeldt-Johansson, KA;Herrera, PL;Wollheim, CB
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