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HORMONES AND SIGNALING

Identification of apoptotic genes mediating TGF-/Smad3-induced cell death in intestinal epithelial cells using a genomic approach

Departments of ¹Surgery and ²Biochemistry and Molecular Biology, Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, Texas; ³Mayo Clinic, Jacksonville, Florida; and ⁴Department of Surgery, Texas A&M Health Science Center, Temple, Texas

Submitted 18 September 2005 ; accepted in final form 31 July 2006

Transforming growth factor (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 (RIE)-1 cells. However, RIE-1 cells are relatively resistant to TGF--induced apoptosis due to a low endogenous Smad3-to-Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF--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-/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.

apoptosis; Affymetrix oligonucleotide microarrays; caspases; transforming growth factor-