Identification of apoptotic genes mediating TGF-β/Smad3-induced cell death in intestinal epithelial cells using a genomic approach
Identification of apoptotic genes mediating TGF-β/Smad3-induced cell death in intestinal epithelial cells using a genomic approach
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DOI：
10.1152/ajpgi.00437.2005
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发表时间：
2007-01-01
影响因子：
4.5
通讯作者：
Ko, Tien C.
中科院分区：
文献类型：
--
作者：
Cao, Yanna;Chen, Lu;Ko, Tien C.
转化生长因子（TGF）-β依赖性细胞凋亡对于消除体内正常组织中受损或异常细胞非常重要。此前，我们已经证明TGF-β抑制大鼠肠上皮（RIE）-1细胞的生长。然而，由于内源性 Smad3 与 Akt 的比率较低，RIE-1 细胞对 TGF-β 诱导的细胞凋亡具有相对抵抗力。 Smad3 的过表达通过改变 Smad3 与 Akt 的比例以促进细胞凋亡，从而使 RIE-1 细胞 (RIE-1/Smad3) 对 TGF-β 诱导的细胞凋亡敏感。在这项研究中，我们利用基因组方法来鉴定受 TGF-β/Smad3 调控的潜在下游靶基因。使用 Affymetrix 寡核苷酸微阵列分析总 RNA 样品。我们发现TGF-β调节与其靶基因相对应的518个探针组。有趣的是，在微阵列分析中包含的已知凋亡基因中，只有 caspase-3 被诱导，这通过实时 RT-PCR 得到了证实。此外，TGF-β 通过蛋白质裂解激活 caspase-3。在 caspase-3 上游，TGF-β 诱导线粒体去极化、细胞色素 c 释放和 caspase-9 裂解，这表明内在凋亡途径介导 TGF-β 诱导 RIE-1/Smad3 细胞凋亡。
Transforming growth factor (TGF)-beta-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Previously, we have shown that TGF-beta inhibits the growth of rat intestinal epithelial (RIE)-1 cells. However, RIE-1 cells are relatively resistant to TGF-beta-induced apoptosis due to a low endogenous Smad3-to-Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF-beta-induced apoptosis by altering the Smad3-to-Akt ratio in favor of apoptosis. In this study, we utilized a genomic approach to identify potential downstream target genes that are regulated by TGF-beta/Smad3. Total RNA samples were analyzed using Affymetrix oligo-nucleotide microarrays. We found that TGF-beta regulated 518 probe sets corresponding to its target genes. Interestingly, among the known apoptotic genes included in the microarray analyses, only caspase-3 was induced, which was confirmed by real-time RT-PCR. Furthermore, TGF-beta activated caspase-3 through protein cleavage. Upstream of caspase-3, TGF-beta induced mitochondrial depolarization, cytochrome c release, and cleavage of caspase-9, which suggests that the intrinsic apoptotic pathway mediates TGF-beta-induced apoptosis in RIE-1/Smad3 cells.