Submitted on September 15, 2005
Accepted on July 31, 2006
IDENTIFICATION OF APOPTOTIC GENES MEDIATING TRANSFORMING GROWTH FACTOR (TGF)-
/Smad3-INDUCED CELL DEATH IN INTESTINAL EPITHELIAL CELLS USING A GENOMIC APPROACH
Yanna Cao1, Lu Chen2, Weili Zhang1, Yan Liu2, Harry T. Papaconstantinou3, Craig R. Bush2, Courtney M. Townsend, Jr.1, E. Aubrey Thompson2, and Tien C. Ko4*
1 Surgery, University of Texas Medical Branch, Galveston, Texas, United States
2 Mayo Clinic, Jacksonville, Florida, United States
3 Surgery, Texas A&M Health Science Center, United States
4 Surgery, University of Texas Medical Branch, Galveston, Texas, United States; Human Biological Chemistry & Genetics, Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, Texas, United States
* To whom correspondence should be addressed. E-mail: tko{at}utmb.edu.
TGF-
-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Previously, we have shown that TGF- inhibits the growth of rat intestinal epithelial cells (RIE-1). However, RIE-1 cells are relatively resistant to TGF--induced apoptosis due to a low endogenous Smad3/Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF--induced apoptosis by altering the Smad3/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-/Smad3. Total RNA samples were analyzed using Affymetrix Oligonucleotide Microarrays. We found that TGF- 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- activated caspase-3 through protein cleavage. Upstream of caspase-3, TGF- induced mitochondrial depolarization, cytochrome c release and cleavage of caspase-9, which suggests that the intrinsic apoptotic pathway mediates TGF--induced apoptosis in RIE-1/Smad3 cells.
