Identification of apoptotic genes mediating TGF-beta/Smad3-induced cell death in intestinal epithelial cells using a genomic approach.
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.
Cao
Yanna
Y
Department of Surgery, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0737, USA.
Chen
Lu
L
Zhang
Weili
W
Liu
Yan
Y
Papaconstantinou
Harry T
HT
Bush
Craig R
CR
Townsend
Courtney M
CM
Thompson
E Aubrey
EA
Ko
Tien C
TC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3 / Q000187:drug effects / Q000235:genetics; D016923:Cell Death / Q000187:drug effects*; D002460:Cell Line; D053938:DNA Fragmentation; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051900:Smad3 Protein / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D012313:RNA; D053148:Caspase 3