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Am J Physiol Gastrointest Liver Physiol 292: G28-G38, 2007. First published August 10, 2006; doi:10.1152/ajpgi.00437.2005
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HORMONES AND SIGNALING

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

Yanna Cao,1 Lu Chen,3 Weili Zhang,1 Yan Liu,3 Harry T. Papaconstantinou,4 Craig R. Bush,3 Courtney M. Townsend, Jr.,1 E. Aubrey Thompson,3 and Tien C. Ko1,2

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


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

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

THE INTESTINAL EPITHELIUM undergoes complete renewal every 3–8 days, depending on the species, making it one of the most rapidly proliferating tissues in the adult body (3, 32). Renewal of the intestinal epithelium involves a complex, dynamic cellular process that is directionally oriented along the crypt-villus axis. As intestinal epithelial cells migrate to the villus, they undergo cell cycle arrest, differentiation, and apoptosis (11, 47). In normal tissue, renewal of the intestinal epithelium is controlled by signals that limit cell division and survival, regulate motility, and control differentiation. Derangement of the gut renewal process leads to diarrhea, malabsorption, altered barrier function, and neoplastic formation (21).

Transforming growth factor (TGF)-beta is one of the important regulators of intestinal epithelial renewal (13, 25, 37, 38). TGF-beta inhibits the proliferation of crypt cells in the small intestine and colon when administered in vivo (9). Likewise, TGF-beta is an inhibitor of proliferation of intestinal epithelial cells in vitro (4, 33, 36, 53). TGF-beta stimulates the migration of intestinal epithelial cells (16, 57) and induces their differentiation (34). Recently, we (17) have shown that TGF-beta induces apoptosis in intestinal epithelial cells that overexpress Smad3, a downstream mediator of the TGF-beta signaling pathway.

Apoptosis is an active cell suicide program that is important in development, tissue homeostasis, and a wide range of diseases, including cancer (54, 56). The initiation of apoptosis can be induced by the activation of specific cytokine-mediated pathways, as a result of cell injury, or by the withdrawal of survival signals from the environment (15). The morphological characteristics of apoptosis include membrane blebbing, condensation of nucleoplasm, and degradation of chromosomal DNA at internucleosomal intervals (58). A central step in apoptosis is the activation of the caspases, a class of cysteine proteases. To date, at least 14 mammalian caspase family members have been identified (12). They are synthesized as inactive zymogens and activated by proteolysis to form an active enzyme (39).

Apoptotic caspases are classified as either initiators or effectors, depending on their point of entry into the apoptotic cascade. Initiator caspases, such as caspase-8 and -9, are the first to be activated in a particular death pathway, and they activate effector caspases, such as caspase-3 and -7, by the cleavage of linker segments (8, 49). Once activated, caspases cleave a variety of important structural proteins, enzymes, and regulatory molecules (55), which leads to DNA fragmentation (1, 22, 54). Apoptotic cascades have been categorized into either extrinsic or intrinsic pathways depending on the specific death stimuli and initiator caspases involved. The extrinsic pathway is responsible for the elimination of cells during development and immunosurveillance. It is initiated by the activation of a transmembrane death receptor of the tumor necrosis factor type 1 superfamily; Fas has become the paradigm for the study of the extrinsic pathway. Upon ligand binding, the Fas receptor recruits Fas-associated protein with a death domain, which in turn recruits and activates initiator caspase-8 (2). The intrinsic pathway eliminates cells in response to mitochondrial damage, ionizing radiation, and chemotherapeutic drugs. After the appropriate stiumulus, mitochondria become selectively permeabilized, leading to the release of cytochrome c, which orchestrates the assembly of caspase-9 and its activator, apoptotic protease-activating factor-1, into a complex known as the "apoptosome" (62).

As an important regulator of gut epithelial homeostasis, TGF-beta exerts its biological effects through its binding to a cell surface receptor complex that consists of type I and type II receptors. Upon ligand binding, the type II receptor phosphorylates the type I receptor, which subsequently phosphorylates Smad2 and Smad3. These form a heteromeric complex with Smad4, translocate into the nucleus, and regulate the transcription of target genes (25, 38, 61). Current understanding of the mechanisms by which TGF-beta induces various cellular effects is limited mostly to its effects on cell cycle arrest. Thus, although TGF-beta-induced apoptosis is a well-documented phenomenon in many different cell types, the biological mechanism responsible for mediating this death process is still poorly understood (15, 18, 46). Recently, we have shown that the cellular response to TGF-beta-induced apoptosis is dependent on the endogenous ratio of Smad3 and Akt. Cells that have a high ratio of Smad3 to Akt undergo apoptosis after TGF-beta treatment, whereas cells such as rat intestinal epithelial (RIE)-1 cells, which have a low level of Smad3/Akt, are relatively resistant to TGF-beta-induced apoptosis. RIE-1 is a nontransformed, nontumorigenic intestinal cell line (7). Unlike many other epithelial cell lines that are derived from cancers, this cell line contains intact TGF-beta receptors and Smad proteins and is sensitive to TGF-beta-induced growth inhibition (33). When the ratio of Smad3 to Akt was altered in RIE-1 cells by overexpressing Smad3, the cells became sensitive to TGF-beta-induced apoptosis. These effects were not observed with the overexpression of Smad2 or Smad4 (17). Taken together, these results indicate that Smad3 is a key mediator of TGF-beta-induced apoptosis in various cell types. The focus of the present study was to determine the downstream targets of TGF-beta/Smad3-mediated apoptosis utilizing a genomic approach. Using RIE-1 cells overexpressing Smad3, we identified caspase-3 as a downstream target of TGF-beta/Smad3-mediated apoptosis. Furthermore, upstream of caspase-3, TGF-beta induces mitochondrial depolarization, cytochrome c release, and cleavage of caspase-9. These results indicate that the intrinsic pathway is involved in TGF-beta/Smad3-mediated apoptosis in RIE-1 cells.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Cell lines. RIE-1 parental cells were maintained in DMEM (Mediatech) supplemented with 5% dialyzed FBS (Invitrogen). Cells overexpressing human Smad3 from the pBabe puro retrovirus (RIE-1/Smad3) and vector control cells (RIE-1/pBabe) were maintained in DMEM supplemented with 5% dialyzed FBS and puromycin (Calbiochem-Novabiochem) at 5 µg/ml.

Apoptosis assays. DNA fragmentation was quantified by a cell death detection ELISA assay (Roche Molecular Biochemicals) according to the manufacturer's instruction. This assay measures mononucleosomes and oligonucleosomes released into the cytoplasm of an apoptotic cell population using a specific monoclonal antibody recognizing only histone-associated DNA. The number of cells in each condition was adjusted to 5 x 105 cells/ml in lysis buffer provided by manufacturer. The absorbance (A405–490 nm), an indication of DNA fragmentation, of each sample was measured using a 96-well plate reader (Molecular Devices). Apoptotic cells were visualized by Hoechst 33258 staining as described previously (43). Briefly, cells were fixed with ice-cold acetone and acetic acid (3:1) and stained with Hoechst 33258 (Sigma-Aldrich). Photographs were taken under a fluorescence microscope at 365 nm using Kodak Ektachrome color slide film.

Microarray analyses. Confluent RIE-1 and RIE-1/Smad3 cells were incubated in DMEM supplemented with 5% FBS for 24 h and then in serum-free EMEM for 48 h. Cells were trypsinzed and split into two 100-mm tissue culture plates. In the presence of 2 mmol/l thymidine, cells were maintained in DMEM supplemented with 5% FBS for 16 h and then treated with or without 120 pmol/l TGF-beta1 (Pharmingen) for 6 h. Maximal apoptosis was observed between 40 and 200 pmol/l TGF-beta1, and120 pmol/l TGF-beta1 was used in the present study. RNA extraction, labeling, hybridization, and scanning were carried out by the University of Texas Medical Branch Genomics Core Facility as previously described (14). Insightful Splus 7 with ArrayAnalyzer 2.0 (41) was used to perform quality control and preprocessing of gene expression. Quality control of microarray chips was performed by first visually inspecting images using Affymetrix CEL files. If chip artifacts such as scratches, ghosting, or uneven liquid pooling were found, the replicates were excluded from further analysis. Variability of the data vs. the mean (Bland-Altman) plots and box plots were analyzed for chip bias and variance by a pairwise comparison. Finally, principle component analysis was used to determine how well the replicate arrays clustered relative to each other. We found very favorable clustering and low variance/bias across replicates before any data manipulation. Irizarry's GC robust multichip average (GCRMA) preprocessing method was chosen over the robust multichip average because we found much lower variance in gene expression by accounting for increased hybridization within GC-rich regions of labeled messenger (26, 60). GCRMA preprocessing resulted in a normalized dataset with exceptionally low variance across replicates and nearly uniform expression profiles across both control and treatment experimental conditions. Differential gene expression was analyzed using the local pooled error (27) and scored as the intersection of genes displaying local pooled error-adjusted P values ≤0.01 and a fold change ≥1.5. The Benjamini-Hochberg false detection rate under these circumstances was <0.1 (5).

Real-time quantitative RT-PCR. Total RNA was isolated using RNAqueous (Ambion). Real-time quantitative RT-PCR was performed using a Perkin-Elmer/Applied Biosystems Division (PE/ABD) Prism 7700 Sequence Detector as described previously (19, 24). Briefly, a probe was designed to anneal to the target sequence between the traditional forward and reverse primers. The probe was labeled at the 5'-end with a reporter fluorochrome and a quencher fluorochrome added at the 3'-end. Real-time quantitative RT-PCR was based on the detection of a fluorescent signal produced proportionally during amplification of a PCR product. A TaqMan PCR core reagent kit, murine leukemia virus reverse transcriptase, and RNase inhibitor (PE/ABD) were used for one-step RT-PCR. Total RNA (10 ng) was used for detecting caspase-3, plasminogen activation inhibitor (PAI)-1, and beta-actin transcripts. The cycling parameters for one-step RT-PCR were reverse transcription at 48°C for 30 min and activation of AmpliTag at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. For the detection of rat caspase mRNA transcripts, the following primers and probes designed from rat cDNA sequences were used: caspase-3 forward primer 5'-aattcaagggacgggtcatg-3', reverse primer 5'-gcttgtgcgcgtacagtttc-3', and probe 6-carboxyfluorescein (FAM)-5'-ttcatccagtcactttgcgccatgc-3'-6-carboxy-tetramethylrhodamine (TAMRA); caspase-8 probe 5'-ttctgttttggatgaggtgaccatc-3' (Rn00574069_m1); and caspase-9 probe 5'-atcgaggatattcagcgggcaggct-3' (Rn00581212_m1) from Applied Biosystems. For the detection of rat PAI-1 mRNA transcripts, the following primers and probes designed from rat cDNA sequences were used: forward primer 5'-accgatcctttctctttgtggtt-3', reverse primer 5'-catcagctggcccatgaag-3', and probe 5'-ccaacagagacaatcc-3' (M24067 [GenBank] ) from Applied Biosystems. For the detection of rat beta-actin mRNA transcripts, the following primers and probe were used: forward primer 5'-cgtgaaaagatgacccagatca-3' (located in exon 3), reverse primer 5'-cacagcctggatggctacgt-3' (located in exon 4), and probe FAM-5'-tgagaccttcaacaccccagccatg-3'-TAMRA (spanning exons 3 and 4). The fluorescent signals acquired from caspase or PAI-1 real-time RT-PCR were normalized to the fluorescent signals acquired from beta-actin real-time RT-PCR.

Western blot analyses. Cultured cells in 100-mm dishes were lysed in cell lysis buffer (Cell Signaling Technology). The protein concentration of cell lysates was quantified using a protein assay dye (Bio-Rad). Rabbit anti-caspase-3 (1/500 dilution) and anti-caspase-8 (1/1,000 dilution) antibodies were from Santa Cruz Biotechnology, mouse anti-caspase-9 (1/1,000 dilution) antibody was from Cell Signaling Technology, and rabbit anti-beta-actin (1/1,000 dilution) antibody was from Sigma-Aldrich. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1/5,000 dilution) and anti-mouse (1/2,500 dilution) antibodies were from Bio-Rad. Cell lysates were resolved on 10% bis-Tris gel (Invitrogen), transferred to polyvinylidene fluoride membranes (Millipore), and blocked in 5% nonfat dry milk at 4°C overnight. The blots were probed with primary antibodies. Bound primary antibodies were detected with HRP-conjugated secondary antibodies and visualized with enhanced chemiluminescent reagents (Pierce Biotechnology).

Caspase-3 enzyme activity assay. The caspase-3 enzyme activity assay was as described previously (44). Briefly, cells were seeded onto six-well plates and treated with or without TGF-beta (120 pmol/l) for 0, 6, 12, 18, and 24 h. Caspase-3 enzyme activity was determined by incubating the cell lysate with a fluorogenic peptide substrate, Ac-DEVD-AFC (Enzyme Systems Products), followed by the measurement of fluorescence using a F-4500 fluorescence spectrophotometer (Hitachi Instruments). Nonspecific caspase activity was determined by preincubating the cell lysate with a caspase-specific substrate inhibitor at a final concentration of 5 µmol/l for 15 min before the addition of the caspase substrate.

Caspase-3 gene silencing. Rat caspase-3 short interfering (si)RNA (the siGENOME SMARTpool reagent, m-080028, Dharmacon) and the transfection reagent DharmaFECT 3 (Dharmacon) were used according to the manufacturer's instruction. Nontargeting siRNA (D-001206-13, Dharmacon) was used as a nonspecific siRNA control.

Mitochondrial depolarization assay. Mitochondrial depolarization was measured by flow cytometry using JC-1 (Molecular Probes). Cells were treated with vehicle or TGF-beta1 (120 pmol/l) for 12 h, harvested, and then suspended in 1 ml DMEM containing 2.5% dialyzed FBS. JC-1 was added to a final concentration of 2 µmol/l, and cells were stained for 30 min at 37°C. FACS analyses were performed using a FACSCanto cytometer (BD Biosciences) using an excitation laser of 488 nm and emission filters of 530/30 nm and 585/42 nm according to the manufacturer's instruction. The pattern of mitochondrial membrane potential change in response to TGF-beta1 treatment was identified using confocal imaging of JC-1 fluorescence according to the manufacturer's instruction. Hoechst 33258 at 1 µg/ml was applied after JC-1 staining for nuclear counterstaining. A Zeiss LSM 510 UV meta laser scanning confocal microscope (Carl Zeiss) was used for imaging.

Cytochrome c immunostaining. Cytochrome c immunostaining was performed as previously described (28). Briefly, cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Cytochrome c was probed using an anti-cytochrome c (1/200 dilution) antibody (clone 6H.B4, Invitrogen) and subsequently Alexa Fluor 488 goat anti-mouse IgG (1/500 dilution, Molecular Probes).

Statistical analysis. Data are expressed as means ± SE. Differences between groups were analyzed by ANOVA with the Tukey-Kramer multiple comparisons test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TGF-beta induced apoptosis in RIE-1/Smad3 cells in a time- and dose-dependent fashion. Previously, we (17) have shown that RIE-1 cells are relatively resistant to TGF-beta-induced apoptosis because of a low endogenous Smad3-to-Akt ratio, and overexpression of Smad3 increases the Smad3-to-Akt ratio and sensitizes RIE-1 cells to TGF-beta-induced apoptosis. We further characterized the TGF-beta-induced apoptotic response in RIE-1/Smad3 cells using two apoptotic assays. DNA fragmentation was determined using an ELISA assay measuring mononucleosomes and oligonucleosomes released into the cytoplasm. Apoptotic nuclear condensation was detected by fluorescence microscopy using the Hoechst 33258 nuclear stain. As shown in Fig. 1A, TGF-beta induced DNA fragmentation in RIE-1/Smad3 cells in a time-dependent fashion, beginning at 18 h after TGF-beta treatment. TGF-beta also induced DNA fragmentation in a dose-dependent fashion, reaching the maximum response between 40 to 200 pmol/l TGF-beta (Fig. 1B). This apoptosis induction was confirmed by Hoescht 33258 staining, which demonstrated nuclear condensation in RIE-1/Smad3 cells treated with TGF-beta but not in RIE-1/pBabe cells (Fig. 1C).


Figure 1
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  		Fig. 1. Transforming growth factor (TGF)-beta induced apoptosis in rat intestinal epithelial (RIE)-1/Smad3 cells. RIE-1/Smad3 cells were treated with or without TGF-beta (120 pmol/l) for different lengths of time (A) or with different dosages of TGF-beta for 24 h (B). DNA fragmentation was quantified by a cell death detection assay. Results from triplicate wells are expressed as means of absorbance (A405–490 nm) ± SE. *P < 0.05 compared with the group without TGF-beta treatment. C: RIE-1/pBabe and RIE-1/Smad3 cells were treated with or without TGF-beta (120 pmol/l) for 24 h. Nuclear morphology was examined using Hoechst 33258 staining. Nuclei were viewed under a fluorescent microscope at a wavelength of 365 nm and photographed at x400 magnification. Arrows indicate apoptotic cells.

 
Genomic approach to identify downstream mediators of TGF-beta/Smad3-induced apoptosis. To identify potential downstream targets of TGF-beta/Smad3-induced apoptosis, a genomic approach was utilized to compare gene expression profiles of RIE-1/Smad3 cells with and without TGF-beta treatment. RIE-1 parental cells served as negative controls because they are resistant to TGF-beta-induced apoptosis. Before TGF-beta treatment, cells were synchronized at the G1/S interphase, by thymidine block, so as to minimize changes in gene expression that might be secondary consequences of differences in cell cycle progression in control and TGF-beta-treated cultures. We elected to profile the transcriptional response 6 h after the addition of TGF-beta. This decision was predicated upon the idea that 6 h was sufficient time to reveal primary transcriptional responses before the onset of apoptosis at 18 h (Fig. 1A), which might compromise our ability to extract intact RNA from responsive cells. Gene expression profiles were examined using Affymetrix RG-U34A, -B, and -C arrays, which included ~26,000 rat probe sets with 7,000 known genes and 19,000 expressed sequence tags. This experiment was repeated three times in its entirety using three separate cell cultures. The majority of the genes that were detected showed little or no alteration in their expression levels. TGF-beta treatment resulted in significant changes in 518 probe sets compared with untreated control in RIE-1/Smad3 cells (see Supplemental Table S1 at the American Journal of Physiology-Gastrointestinal and Liver Physiology website). The levels of most genes that have been implicated in apoptosis were not significantly altered, such as Bcl-x, Bcl-xS, Bcl10, Bad, Bak1, Bax, Bok, caspase-1, caspase-2, caspase-6, caspase-11, and cytochrome c. Interestingly, only caspase-3 was induced by TGF-beta (Table 1). These results were consistent in three independent experiments with three sets of caspase-3 probes: U49930_at, U49930_g_at, and U84410_s_at (Fig. 2A). TGF-beta had no effect on caspase-3 mRNA in parental RIE-1 cells, which are resistant to the apoptotic effects of TGF-beta (Fig. 2A). This differential effect of TGF-beta on caspase-3 mRNA was not observed in PAI-1, which is an important regulator of the extracellular matrix (6) and is strongly induced by TGF-beta (52). Microarray analyses showed that TGF-beta induced PAI-1 expression in both RIE-1 and RIE-1/Smad3 cells (Fig. 2A). To validate the results of the microarray analyses, we examined the expression of caspase-3 and PAI-1 mRNA expression by real-time RT-PCR, as shown in Fig. 2B. Consistent with the microarray results, TGF-beta induced caspase-3 mRNA expression in RIE-1/Smad3 cells but not in vector control RIE-1/pBabe cells; TGF-beta induced PAI-1 mRNA expression in both RIE-1/pBabe and RIE-1/Smad3 cells.


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  		Table 1. Microarray analysis of apoptotic genes in RIE-1/Smad3 cells treated with TGF-beta1

 

Figure 2
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  		Fig. 2. Caspase-3 mRNA expression was upregulated by TGF-beta in RIE-1/Smad3 cells. A: RIE-1 and RIE-1/Smad3 cells were treated with or without TGF-beta (120 pmol/l) for 6 h. Total RNA was extracted and applied to Affymetrix RG-U34A, -B, and -C arrays. Caspase-3 signals are expressed as means ± SE in 3 independent experiments with 3 sets of caspase-3 probes (U49930_at, U49930_g_at, and U84410_s_at). Plasminogen activation inhibitor (PAI)-1 signals from the probe set M24067_at and beta-actin signals from the probes V01217_at, rc_AI070848_f_at, and rc_AI179012_s_at are expressed as means ± SE. B: RIE-1/pBabe and RIE-1/Smad3 cells were treated with or without TGF-beta (120 pmol/l) for 24 h. Total RNA samples were extracted, and real-time RT-PCR was performed. The ratios of caspase-3 to beta-actin signals and PAI-1 to beta-actin signals are expressed as means ± SE. *P < 0.05 compared with the group without TGF-beta treatment.

 
TGF-beta induced caspase-3 expression and activation in RIE-1/Smad3 cells. RIE-1/Smad3 cells were treated with TGF-beta over a 24 h time course, and caspase-3 mRNA expression was determined by real-time RT-PCR. TGF-beta induced caspase-3 mRNA expression, with the maximum induction attained at 5 h after treatment, and persisted for 24 h (Fig. 3). The induction of caspase-3 mRNA after TGF-beta treatment preceded the onset of apoptosis (Fig. 1A). Because the probe sets used in the microarray analyses did not include caspase-8 and -9, we used real-time RT-PCR to determine whether these initiator caspases were induced by TGF-beta. In contrast to caspase-3, TGF-beta did not induce mRNA expression of either caspase-8 or -9 in RIE-1/Smad3 cells (data not shown). Taken together, we demonstrated that TGF-beta induced caspase-3 mRNA expression selectively in RIE-1/Smad3 cells, suggesting that caspase-3 is a downstream target of TGF-beta/Smad3 signaling.


Figure 3
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  		Fig. 3. Real-time RT-PCR confirmed gene array data of the upregulation of caspase-3 mRNA expression. RIE-1/Smad3 cells were treated with or without TGF-beta (120 pmol/l) for different lengths of time. Total RNA samples were extracted, and real-time RT-PCR was performed. The ratios of caspase-3 to beta-actin signals are expressed as means ± SE. *P < 0.05 compared with the group without TGF-beta treatment.

 
Caspase-3 is a central mediator of apoptosis and is normally present in the cell in an inactive form. Activation of caspase-3 occurs through proteolytic cleavage of a prodomain (59). This property of caspase-3 conveniently allowed us to measure the activation of caspase-3 by assaying for the appearance of the cleaved, active subunit. To determine whether TGF-beta induced caspase-3 protein expression and activation, Western blot analysis was performed using a specific anti-caspase-3 antibody, which recognizes both 32-kDa procaspase-3 and 17/19-kDa active caspase-3. Our results demonstrate that, in RIE-1/Smad3 cells, TGF-beta induced expression of 32-kDa procaspase-3 protein levels at all the time points examined compared with the time-matched control (Fig. 4, A and B). Furthermore, a 17-kDa cleaved caspase-3 band was first detected at 18 h after TGF-beta treatment (Fig. 4, A and B), which corresponds to the onset of DNA fragmentation shown in Fig. 1A. TGF-beta did not induce caspase-3 cleavage in RIE-1/pBabe cells (data not shown). The results of caspase-3 Western blot analyses were confirmed by caspase-3 enzyme activity assays. TGF-beta induced caspase-3 enzyme activity in RIE-1/Smad3 cells in a time-dependent fashion (Fig. 4C), which parallels the expression of 17/19-kDa cleaved caspase-3 protein (Fig. 4A). Taken together, these results demonstrate that caspase-3 is a downstream target of TGF-beta/Smad3-induced apoptosis.


Figure 4
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  		Fig. 4. TGF-beta induced caspase-3 activation. A: whole cell lysates were extracted from RIE-1/Smad3 cells with and without TGF-beta treatment for different lengths of time. Western blot analysis was performed to detect caspase-3 proteins. The cleaved, active form of caspase-3 protein was detected as the smaller bands. Equal loading of protein was confirmed by probing the blots with beta-actin antibody. B: quantification of caspase-3 protein from Western blots using densitometry analysis. C: caspase-3-enzyme activity was measured and is expressed as means ± SE. *P < 0.05 compared with the group without TGF-beta treatment.

 
Knockdown of caspase-3 gene expression blocked TGF-beta-induced apoptosis. To address the functional significance of caspase-3 expression, siRNAs for rat caspase-3 were used to knockdown caspase-3 expression. Treatment with caspase-3 siRNAs completely blocked TGF-beta-induced caspase-3 mRNA expression in RIE-1/Smad3 cells, whereas nonspecific siRNAs had no effect on caspase-3 mRNA expression (Fig. 5A). Correspondingly, TGF-beta induced DNA fragmentation in the nonspecific siRNA group, whereas TGF-beta did not induce DNA fragmentation in the caspase-3 siRNA-treated group (Fig. 5B). These results demonstrate that knockdown of caspase-3 gene induction blocked TGF-beta-induced apoptosis, indicating that caspase-3 induction was causally related to TGF-beta-induced apoptosis. Furthermore, caspase-3 siRNAs did not affect PAI-1 mRNA expression in the absence and presence of TGF-beta (Fig. 5A), which suggests that caspase-3 siRNAs specifically silenced caspase-3 induction by TGF-beta without affecting other signaling transduction through the TGF-beta pathway.


Figure 5
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  		Fig. 5. Knockdown of caspase-3 expression blocked TGF-beta-induced apoptosis. RIE-1/Smad3 cells were transfected with rat caspase-3 short interfering (si)RNA (50 nmol/l) for 24 h using DharmaFECT3. Nontarget siRNA was used as a nonspecific control. A: cells were treated with TGF-beta (120 pmol/l) for 24 h and then harvested for real-time RT-PCR analysis of caspase-3 and PAI-1 mRNA expression. B: cells were replated 24 h after siRNA transfection and treated with TGF-beta (120 pmol/l) for 24 h. DNA fragmentation was quantified by a cell death detection assay. Results from triplicate wells are expressed as means of absorbance ± SE. *P < 0.05 compared with the group without TGF-beta treatment.

 
TGF-beta induced protein cleavage of caspase-9. In the apoptotic cascade, caspase-3, as an effector caspase, is activated by upstream initiator caspases, such as caspase-8 or -9. Initiator caspases are also activated by protein cleavage. To determine whether TGF-beta induced cleavage of caspase-8 or -9, Western blot analyses were used to measure both the procaspase and cleaved forms of caspase-8 and -9. TGF-beta induced the cleaved forms of caspase-9 in RIE-1/Smad3 cells after 18 and 24 h of treatment without altering the expression of procaspase-9 (Fig. 6, A and B). The cleaved forms of caspase-9 were not detected in RIE-1/pBabe cells after TGF-beta treatment (data not shown). No cleavage of caspase-8 was detected by Western blot analyses in both RIE-1/Smad3 cells (Fig. 6C) and RIE-1/pBabe cells (data not shown) after TGF-beta treatment. These results demonstrate a selective cleavage of caspase-9 in RIE-1/Smad3 cells.


Figure 6
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  		Fig. 6. TGF-beta induced caspase-9 protein cleavage. A: whole cell lysates were extracted from RIE-1/Smad3 cells with and without TGF-beta treatment for different lengths of time. Western blot analysis was performed to detect caspase-9 proteins. The cleaved, active form of caspase-9 was detected as the smaller bands of ~36/40 kDa. Equal loading of protein was confirmed by probing the blots with beta-actin antibody. B: quantification of caspase-9 protein from Western blots using densitometry analysis. C: caspase-8 protein was measured by Western blot analysis using specific caspase-8 antibody. The positive control (PC) was Jurkat cells treated with staurosporine.

 
TGF-beta induced mitochondrial depolarization and cytochrome c release in RIE-1/Smad3 cells. The induction of the cleavage of caspase-9 and -3 by TGF-beta suggests that the intrinsic apoptotic pathway including mitochondrial damage and subsequent cytochrome c release is involved in TGF-beta-induced apoptosis in RIE-1/Smad3 cells. To assess the effects of TGF-beta on the mitochondrial membrane potential, we utilized MitoProbe JC-1 dye, which exhibits potential-dependent accumulation of JC-1 aggregates in mitochondria. Mitochondrial depolarization is indicated by a decrease in aggregated JC-1, which emits a red fluorescence (23, 30). Flow cytometric analysis demonstrated that TGF-beta induced mitochondrial depolarization in 35.1% of RIE-1/Smad3 cells compared with only 5.3% of vector control RIE-1/pBabe cells (Fig. 7A). As a depolarization control, cells were also treated with carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial membrane potential disruptor that causes a collapse of the pH gradient across the membrane and a loss of aggregated JC-1 (Fig. 7A). The flow cytometric results were confirmed by image analyses using confocal microscopy. TGF-beta dramatically decreased the formation of JC-1 aggregates in RIE-1/Smad3 cells as shown by the decrease in red fluorescence in mitochondria (Fig. 7B). These results indicated that TGF-beta induced mitochondrial depolarization in RIE-1/Smad3 cells.


Figure 7
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  		Fig. 7. TGF-beta induced mitochondrial depolarization. Cells were treated with TGF-beta (120 pmol/l) for 12 h. A: cells were harvested for JC-1 staining and analyzed by a flow cytometer. Mitochondrial depolarization is indicated by the percentage of cells with a decrease in red fluorescence. Cells treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial depolarization agent, served as the positive control. B: cells were also harvested for confocal imaging analysis. The red color shows aggregated JC-1 staining in mitochondria; the overlay shows red aggregated JC-1 staining in mitochondria with blue nuclear counterstaining using Hoechst 33258.

 
Next, we determined whether TGF-beta induced cytochrome c release in RIE-1/Smad3 cells. Immunostaining of vector control RIE-1/pBabe cells and RIE-1/Smad3 cells without TGF-beta treatment demonstrated a punctuate staining pattern of mitochondrial localization (Fig. 8A). In RIE-1/Smad3 cells, treatment with TGF-beta altered the pattern of cytochrome c staining, demonstrating the release of mitochondrial cytochrome c (Fig. 8A). Quantification of cells with an altered pattern of cytochrome c staining indicated a 16.6% increase in cytochrome c release after TGF-beta treatment in RIE-1/Smad3 cells (Fig. 8B). However, TGF-beta did not induce cytochrome c release in vector control RIE-1/pBabe cells. Taken together, these results demonstrated that TGF-beta induced mitochondrial depolarization and cytochrome c release in RIE-1/Smad3 cells.


Figure 8
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  		Fig. 8. Cytochrome c was released from mitochondria in response to TGF-beta treatment. A: RIE-1/pBabe and RIE-1/Smad3 cells were treated with TGF-beta1 (120 pmol/l) for 24 h. Cytochrome c immunostaining (green) and Hoechst 33258 staining (blue) were performed and analyzed by confocal microscopy. Cells with diffused cytochrome c staining are indicated by arrows. B: quantification of cells with diffused cytochrome c staining. Data are expressed as means ± SE of the percentage of cells with diffused cytochrome c. *P < 0.05 compared with the group without TGF-beta treatment.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Smad3 is an important regulator of TGF-beta-mediated transcription, and we (17) have shown that Smad3 plays an essential role in TGF-beta-induced apoptosis. However, the downstream target genes regulated by TGF-beta/Smad3 are largely unknown. To gain a mechanistic insight into the TGF-beta/Smad3-mediated apoptotic pathway in gut epithelial cells, we utilized a genomic approach to examine the potential involvement of key apoptotic regulators such as caspases and Bcl family members. Microarray data revealed a consistent upregulation of caspase-3 mRNA in RIE-1/Smad3 cells in response to TGF-beta treatment. However, caspase-1, caspase-2, caspase-6, caspase-11, and other apoptosis-related molecules, such as Bcl-x, Bcl-xS, Bcl10, Bad, Bak1, Bax, Bok, and cytochrome c, were not significantly altered by TGF-beta. Using microarray analysis, others (28) have reported that many apoptosis-related genes including caspase-3 were not altered by TGF-beta in hepatoma cells. However, the analysis perfumed by Jang et al. (28) was carried out 2 h after TGF-beta treatment, a time point at which the caspase-3 mRNA level was not altered by TGF-beta (Fig. 3B). In another study, using cervical cancer cell lines for gene expression profiling by microarray, Kloth et al. (31) found that most TGF-beta-induced gene expression changes occur after 6 h. In our study, in addition to the upregulation of caspase-3 mRNA expression, caspase-3 protein expression was also upregulated by TGF-beta. Furthermore, TGF-beta induced protein cleavage and activation of caspase-3, and the time course of caspase-3 activation paralleled that of DNA fragmentation. Our results demonstrate that both caspase-3 mRNA and protein were induced by TGF-beta in RIE-1/Smad3 cells, which provides a potential marker for TGF-beta-induced apoptosis in our cell model. The mechanism of this upregulation is currently under investigation. We (42, 44) have previously demonstrated the induction of caspase-3 activity in intestinal epithelial cells in response to a number of inducers of apoptosis, such as methotrexate and glutamine starvation. This observation is also in agreement with previous reports on the activation of caspase-3 associated with TGF-beta-induced apoptosis in the human hepatoma cell line Hep3B (15) and in the Ramos B-lymphoma cell line (50). Therefore, caspase-3 cleavage and activation in apoptosis induction is a common mechanism among different cell lines of both epithelial and mesenchymal origin.

Various apoptotic stimuli usually converge on a common intracellular pathway that includes the activation of caspases (15). Effector caspases (caspase-3, -6, and -7) are activated by initiator caspases when cells receive an apoptosis-inducing signal. Because caspase-3 is an effector caspase that can be activated by upstream initiator caspases from both the intrinsic and extrinsic apoptotic pathways, we determined which upstream initiator caspases were involved in TGF-beta-induced apoptosis. Our initial microarray analyses did not include probe sets for caspase-8 and -9; therefore, caspase-8 and -9 gene expression were determined by real-time RT-PCR. We found that TGF-beta did not induce the expression of caspase-8 and -9 mRNA. However, TGF-beta induced caspase-9 protein cleavage, suggesting that the activation of caspase-9 by TGF-beta is due to processing of preexisting procaspase-9. Furthermore, upstream of caspase-9/-3, we found that TGF-beta induced mitochondrial depolarization and cytochrome c release in RIE-1/Smad3 cells. These results suggest that TGF-beta activates the intrinsic apoptotic pathway via mitochondrial damage in intestinal epithelial cells, which is consistent with findings showing that TGF-beta induces apoptosis through cytochrome c release from mitochondria in rat and human hepatoma cells (20, 28, 62). However, the upstream signals leading to mitochondrial damage and cytochrome c release are still largely unknown. Bcl-2 or Bcl-xL prevents the release of cytochrome c, thereby suppressing apoptosis, whereas Bax and cleaved Bid promotes cytochrome c release and apoptosis (10, 40, 45). However, the involvement of Bax and Bcl-xL in TGF-beta-induced apoptosis is controversial (48, 51). Recent studies have suggested that Bad cleavage (29), expression of death-associated protein kinase (28), and mitochondrial septin-like protein ARTS (35) are involved in TGF-beta-induced apoptosis. However, much remains to be learned about the multiple steps by which TGF-beta signaling leads to apoptosis. Although we have selected to use microarray analyses to examine changes in mRNA abundance, other potential mechanisms, such as phosphorylation, are likely to be also involved in TGF-beta-induced apoptosis.

In conclusion, using a genomic approach, we identified caspase-3 as a downstream target of TGF-beta/Smad3 signaling in RIE-1/Smad3 cells. Furthermore, TGF-beta activates caspase-9 by inducing protein cleavage, mitochondrial membrane depolarization, and cytochrome c release. These results suggest that the intrinsic pathway is involved in TGF-beta/Smad3-mediated apoptosis. The present focus in our laboratory is to determine the roles of the genes induced by TGF-beta identified from our gene array analysis and to study the mechanisms of TGF-beta/Smad3-induced apoptosis. Understanding the molecular mechanisms by which TGF-beta activates the apoptotic machinery in gut epithelial cells may provide important information about the renewal of gut epithelium and may offer therapeutic strategies for the treatment of gastrointestinal tumors.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants RO1-DK-060105 and PO1-DK-35608 (to T.C. Ko) and RO1-CA-24347 (to E. A. Thompson). C. R. Bush is supported by a training fellowship from the Keck Center for Computational and Structural Biology of the Gulf Coast Consortium (National Library of Medicine Grant 5-T15-LM-07093).


   ACKNOWLEDGMENTS
 
The authors thank C. Deng, D. Song, and C. Xie for technical support and E. Figueroa and S. Schuenke for manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. C. Ko, Dept. of Surgery, Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0737 (e-mail: tko{at}utmb.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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