Cao Yanna, Chen Lu, Zhang Weili, Liu Yan, Papaconstantinou Harry T, Bush Craig R, Townsend Courtney M, Thompson E Aubrey, Ko Tien C
Department of Surgery, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0737, USA.
Am J Physiol Gastrointest Liver Physiol. 2007 Jan;292(1):G28-38. doi: 10.1152/ajpgi.00437.2005. Epub 2006 Aug 10.
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 oligonucleotide 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.
转化生长因子（TGF）-β依赖的细胞凋亡在体内正常组织中清除受损或异常细胞方面具有重要作用。此前，我们已表明TGF-β可抑制大鼠肠上皮（RIE）-1细胞的生长。然而，由于内源性Smad3与Akt的比例较低，RIE-1细胞对TGF-β诱导的细胞凋亡具有相对抗性。Smad3的过表达通过改变Smad3与Akt的比例，使其有利于细胞凋亡，从而使RIE-1细胞（RIE-1/Smad3）对TGF-β诱导的细胞凋亡敏感。在本研究中，我们采用基因组学方法来鉴定受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样本。我们发现TGF-β调控了518个与其靶基因相对应的探针组。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通过蛋白切割激活caspase-3。在caspase-3的上游，TGF-β诱导线粒体去极化、细胞色素c释放以及caspase-9的切割，这表明内源性凋亡途径介导了TGF-β诱导的RIE-1/Smad3细胞凋亡。
