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Identification of apoptotic genes mediating TGF-beta/Smad3-induced cell death in intestinal epithelial cells using a
Yanna Cao1, Lu Chen, Weili Zhang
1Department of Surgery, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0737, USA.
Abstract:
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.
Insights
Transforming growth factor-beta (TGF-β) induces apoptosis in rat intestinal epithelial cells by upregulating caspase-3. Overexpressing Smad3 enhances this TGF-β-induced apoptosis, revealing key molecular players in cell death pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Transforming growth factor-beta (TGF-β) is crucial for eliminating abnormal cells via apoptosis.
- Rat intestinal epithelial (RIE)-1 cells exhibit resistance to TGF-β-induced apoptosis due to an unfavorable Smad3-to-Akt ratio.
- Overexpression of Smad3 in RIE-1 cells (RIE-1/Smad3) sensitizes them to TGF-β-induced apoptosis.
Purpose of the Study:
- To identify downstream target genes regulated by TGF-β/Smad3 using a genomic approach.
- To elucidate the molecular mechanisms underlying TGF-β-induced apoptosis in RIE-1/Smad3 cells.
Main Methods:
- Genomic analysis using Affymetrix oligonucleotide microarrays to identify TGF-β-regulated genes.
- Real-time RT-PCR to confirm the induction of specific apoptotic genes, particularly caspase-3.
- Analysis of caspase-3 activation, mitochondrial depolarization, cytochrome c release, and caspase-9 cleavage.
Main Results:
- TGF-β regulated 518 probe sets, indicating widespread gene expression changes.
- Caspase-3 was the only known apoptotic gene significantly induced by TGF-β.
- TGF-β activated caspase-3 via protein cleavage, involving the intrinsic apoptotic pathway (mitochondrial depolarization, cytochrome c release, caspase-9 cleavage).
Conclusions:
- TGF-β/Smad3 signaling induces apoptosis in RIE-1/Smad3 cells primarily through the intrinsic pathway.
- Caspase-3 is a key downstream effector of TGF-β-induced apoptosis in this cellular model.
- Understanding these pathways provides insights into targeted therapeutic strategies for diseases involving abnormal cell proliferation.
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