Rubinek Tami, Chesnokova Vera, Wolf Ido, Wawrowsky Kolja, Vlotides George, Melmed Shlomo
Academic Affairs, Rm. 2015, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA.
Am J Physiol Cell Physiol. 2007 Sep;293(3):C1082-92. doi: 10.1152/ajpcell.00145.2007. Epub 2007 Jul 11.
The mammalian securin, pituitary tumor-transforming gene (Pttg), regulates sister chromatid separation during mitosis. Mice deficient in Pttg expression exhibit organ-specific hypoplasia of the testis, spleen, pituitary, and postmaturity pancreatic beta-cells, pointing to a possible adult stem cell defect. Bone marrow stem cells (BMSCs) contribute to bone, cartilage, and fat tissue repair and regeneration, and multipotent adult progenitor cells (MAPCs) have broader differentiation ability. Bone marrow cells derived under MAPC conditions are involved in a spectrum of tissue repair. We therefore tested whether Pttg deletion affects stem cell proliferation and differentiation. BMSCs were isolated under MAPC conditions, although unlike MAPCs, wild-type (WT) and Pttg(-/-) BMSCs do not express octamer-binding transcription factor 4 and are stem cell antigen-I positive. WT and Pttg(-/-) cells did not differ in their ability to differentiate into adipogenic, osteogenic, or hepatocyte-like cells or in phenotypic markers. Cells underwent >100 population doublings, with no observed transforming events. Pttg-null BMSCs replicated 27% slower than WT BMSCs, and under hypoxic conditions, this difference widened. Although apoptosis was not enhanced in Pttg(-/-) cells, Pttg(-/-) BMSC senescence-associated beta-galactosidase activity was elevated, consistent with enhanced p21 protein levels. Using gene array assays, DNA repair genes were shown to be upregulated in Pttg(-/-) BMSCs, whereas genes involved in cell cycle progression, including cyclin D(1), were decreased. Separase, the protease regulated by Pttg, has been implicated in DNA damage repair and was downregulated in Pttg(-/-) BMSCs. Separase was constitutively phosphorylated in Pttg(-/-) cells, a modification likely serving as a compensatory mechanism for Pttg deletion. The results indicate that Pttg deletion reduces BMSC proliferation, renders cells more sensitive to hypoxia, and enhances senescent features, thus pointing to a role for Pttg in the maintenance and proliferation of BMSCs.
哺乳动物的分离酶抑制蛋白，垂体肿瘤转化基因（Pttg），在有丝分裂期间调节姐妹染色单体的分离。Pttg表达缺陷的小鼠表现出睾丸、脾脏、垂体和过成熟胰腺β细胞的器官特异性发育不全，这表明可能存在成体干细胞缺陷。骨髓干细胞（BMSC）有助于骨骼、软骨和脂肪组织的修复与再生，而多能成体祖细胞（MAPC）具有更广泛的分化能力。在MAPC条件下获得的骨髓细胞参与一系列组织修复。因此，我们测试了Pttg缺失是否会影响干细胞的增殖和分化。在MAPC条件下分离出BMSC，尽管与MAPC不同，野生型（WT）和Pttg（-/-）BMSC不表达八聚体结合转录因子4，且干细胞抗原-I呈阳性。WT和Pttg（-/-）细胞在分化为脂肪细胞、成骨细胞或肝细胞样细胞的能力或表型标记方面没有差异。细胞经历了超过100次群体倍增，未观察到转化事件。Pttg基因缺失的BMSC比WT BMSC的复制速度慢27%，在缺氧条件下，这种差异会扩大。虽然Pttg（-/-）细胞中的凋亡没有增强，但Pttg（-/-）BMSC衰老相关β-半乳糖苷酶活性升高，这与p21蛋白水平升高一致。使用基因阵列分析表明，DNA修复基因在Pttg（-/-）BMSC中上调，而参与细胞周期进程的基因，包括细胞周期蛋白D（1），则减少。由Pttg调节的蛋白酶分离酶与DNA损伤修复有关，在Pttg（-/-）BMSC中下调。分离酶在Pttg（-/-）细胞中持续磷酸化，这种修饰可能是Pttg缺失的一种补偿机制。结果表明，Pttg缺失会降低BMSC的增殖，使细胞对缺氧更敏感，并增强衰老特征，从而表明Pttg在BMSC的维持和增殖中起作用。
