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TDAG51 deficiency promotes oxidative stress-induced apoptosis through the generation of reactive oxygen species in mouse embryonic fibroblasts

Apoptosis has an important role in maintaining tissue homeostasis in cellular stress responses such as inflammation, endoplasmic reticulum stress, and oxidative stress. T-cell death-associated gene 51 (TDAG51) is a member of the pleckstrin homology-like domain family and was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death. However, its pro-apoptotic function remains controversial. In this study, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death in mouse embryonic fibroblasts (MEFs). TDAG51 expression was highly increased by oxidative stress responses. In response to oxidative stress, the production of intracellular reactive oxygen species was significantly enhanced in TDAG51-deficient MEFs, resulting in the activation of caspase-3. Thus, TDAG51 deficiency promotes apoptotic cell death in MEFs, and these results indicate that TDAG51 has a protective role in oxidative stress-induced cell death in MEFs.


INTRODUCTION
Apoptosis has an essential role in tissue homeostasis in various organisms and is activated by a number of cellular stress responses such as oxidative stress, endoplasmic reticulum (ER) stress, and inflammatory responses. 1-4 The receptor-mediated (extrinsic) and mitochondria-mediated (intrinsic) pathways are two major distinct apoptotic pathways that are active in mammalian cells. 5 The receptor-mediated pathway is triggered by the stimulation of death receptors such as Fas/CD95 and the tumor necrosis factor receptor superfamily. Ligand interactions with cognate receptors initiate caspase-8 and methodically activate caspase-3. Activated caspase-3 directly induces apoptotic cell death. 6 The mitochondria-mediated pathway is activated by several cellular stresses, including oxidative stress, ER stress, and growth factor depletion. Cellular stress induces release of cytochrome c from mitochondria, and the released cytochrome c binds to apoptotic protease activating factor-1 and caspase-9. This complex directly induces caspase-3 activation and subsequently activates apoptotic cell death. 7,8 Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism, as well as during cellular responses to xenobiotics, cytokines, and bacterial invasion. 9 The generation and accumulation of intracellular ROS can lead to intrinsic oxidative stress in cells. 4 ROS are noxious to cells; therefore, the cell has defense mechanisms to eliminate intracellular ROS accumulation. However, an imbalance between ROS generation and elimination induces severe cellular oxidative stress. Excessive ROS derived from endogenous or exogenous routes can damage or cause complete degradation of essential molecules, including cellular lipids, proteins, and DNA, resulting in activation-induced apoptotic cell death (AICD). 4,10 Therefore, the modulation of the oxidative stress response in cells is closely related to a series of human diseases such as neurodegeneration, atherosclerosis, diabetes, aging, and even cancer. 9 T-cell death-associated gene 51 (TDAG51) was first described to have roles in the induction of CD95/Fas gene expression and AICD in response to the engagement of the T-cell receptor in a murine T-cell hybridoma. 11 This gene is also known as pleckstrin homology-like domain family A member 1 (PHLDA1), and it possesses an N-terminal pleckstrin homology-like domain. 12 In the C-terminal region, TDAG51 contains both a proline-glutamine (PQ) repeat domain and a proline-histidine (PH) repeat domain, which may be involved in its pro-apoptotic function. 11,13,14 However, we have previously shown that TDAG51 is not required for AICD in murine T cells in an in vivo study using TDAG51deficient (TDAG51 À/ À ) mice. 15 A human T-cell study further confirmed that TDAG51 expression is not correlated with AICD in human T cells. 16 Collectively, the precise role of TDAG51 in apoptotic cell death remains controversial; therefore, it is important to elucidate whether TDAG51 expression is involved in pro-apoptotic or anti-apoptotic functions.
In the present study, we investigated the role of TDAG51 in oxidative stress-induced apoptosis in TDAG51 À/ À mouse embryonic fibroblasts (MEFs). We observed that TDAG51 expression is significantly induced by oxidative stress in MEFs. Upon oxidative stress, TDAG51 deficiency results in enhanced generation of ROS in TDAG51 À/ À MEFs. Thus, TDAG51 À/ À MEFs were more sensitive to oxidative stress-induced apoptosis compared to wild-type (TDAG51 þ / þ ) MEFs. These results suggest that TDAG51 deficiency promotes oxidative stressinduced apoptosis in MEFs.

MEF isolation and culture
Embryos generated from the intercross of TDAG51 heterozygous knockout (TDAG51 þ / À ) mice were dissected out of decidua, and all maternal tissues were removed. MEFs were isolated from each mouse embryo at embryonic day (E) 13.5. The brain and visceral organs of E13.5 embryos were surgically removed, and the remainder of the embryonic body was finely minced with sterile animal operating scissors. The minced embryos were treated repeatedly with 0.05% trypsin at 37 1C for 10 min with intermittent agitation to isolate MEFs. MEFs were maintained in Dulbecco's modified Eagle's medium (Welgene, Daegu, Korea) supplemented with 15% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 1 mM L-glutamine (Welgene), and 1 Â antibiotic/antimycotic solution (Welgene) in 5% CO 2 at 37 1C. The average yield from an embryo was B3 Â 10 7 cells. To determine MEF genotypes, genomic DNA was extracted from MEFs, and the cells were genotyped by PCR as described previously. 15

Apoptotic cell death assays
Isolated MEFs were seeded in 6-well plates (2 Â 10 5 cells ml À1 ) and further cultured for 24 h. To induce apoptotic cell death, MEFs were then treated with the indicated concentration of H 2 O 2 for 6 h. Cell survival was determined using DAPI staining and PI staining as previously described. 17,18 For DAPI staining, the cells were washed twice with ice-cold 1 Â phosphate-buffered saline (Welgene), fixed with 4% paraformaldehyde for 10 min, and then stained with 0.1 mg ml À1 of DAPI (Sigma-Aldrich). Finally, the cells were observed under an inverted fluorescence microscope (DM-IRB, Leica Microsystems, Wetzlar, Germany). For PI staining, the dead cells were stained with 1 mg ml À1 of PI for 1 min at room temperature and analyzed using a flow cytometer (FACSCalibur, Becton-Dickinson, San Jose, CA, USA).

Determination of generated ROS
Intracellular ROS were measured with a flow cytometer using DCFH-DA (Sigma-Aldrich) as previously described. 19 Briefly, MEFs (2 Â 10 5 cells ml À1 ) were cultured in 6-well plates. After 24 h, MEFs were incubated in serum-free culture medium without phenol red, and the cells were stimulated with 0.5 mM H 2 O 2 in a time-dependent manner. After H 2 O 2 stimulation, the cells were washed twice with phosphatebuffered saline and then incubated with 10 mM DCFH-DA for 30 min. Finally, the cells were harvested using cell dissociation solution (Sigma-Aldrich) and resuspended in phosphate-buffered saline containing 2% fetal bovine serum. Quantification of ROS was measured by flow cytometry using the FACSCalibur.

Luciferase reporter assays
The murine TDAG51 promoter (P T51 -3 K-Luc) from À2450 to þ 551 was amplified by PCR and cloned into the pGL3-basic vector (Promega, Madison, WI, USA). A luciferase reporter assay was performed as described previously. 21 Briefly, MEFs were plated on 24-well plates (0.5 Â 10 5 cells per well), and the cells were cotransfected with the TDAG51 promoter plasmid and b-galactosidase expression plasmid. All transfections were performed in triplicate using TurboFect transfection reagent (Fermentas) following the manufacturer's instructions. At 48 h post transfection, MEFs were treated with 0.5 mM H 2 O 2 for 1 h in the presence or absence of the signaling inhibitors described above. The cells were evaluated for luciferase activity using a luciferase assay kit (Promega). All values represent luciferase activity normalized to b-galactosidase activity.

Statistical analysis
All experiments were performed at least three times. Data represent the mean±s.d. (n ¼ 3 per group). Student's t-test was used to determine the significance of differences between experimental samples.

RESULTS
The expression of TDAG51 is enhanced by oxidative stress TDAG51 is a known cellular stress-responsive gene; 11,13,14 therefore, we first examined whether TDAG51 expression is enhanced by oxidative stresses in a time-and concentrationdependent manner. First, TDAG51 þ / þ MEFs were stimulated for 8 h with H 2 O 2 in a dose-dependent manner. As a result, TDAG51 gene expression was greatly enhanced by H 2 O 2 treatment in the concentration range between 0.5-0.8 mM ( Figure 1a). Accordingly, the next study was designed to determine the relationship between time-dependent expression of TDAG51 and oxidative stresses. To determine TDAG51 expression during oxidative stress, TDAG51 þ / þ MEFs were stimulated with 0.5 mM H 2 O 2 in a time-dependent manner. We observed that TDAG51 expression was significantly enhanced by H 2 O 2 stimulation, and the cells were found to exhibit peak TDAG51 expression after 8 h of H 2 O 2 stimulation (Figure 1b). Similarly, we detected elevated TDAG51 expression by H 2 O 2 stimulation in other cell types such as Huh-7, 293T, and HeLa cells (Figure 1b). However, concordant with the results of previous reports, 13,14 TDAG51 induction showed cell type-dependent differential expression; a rapid increase in TDAG51 expression was observed in 293T cells with a peak at 2 h, whereas a gradual increase in TDAG51 expression reached a peak at 12 h in Huh-7 and HeLa cells (Figure 1b). To further confirm the effect of oxidative stress on TDAG51 expression, we used 250 mM ethanol as another oxidative stressor to treat TDAG51 þ / þ MEFs in a time-dependent manner. Similar results were obtained demonstrating that TDAG51 expression was significantly elevated by ethanol treatment, although the peak expression of TDAG51 was slightly delayed (Figure 1c). ER stress induced by the unfolded protein response is closely linked to the generation and accumulation of intracellular ROS, which can lead to intrinsic oxidative stress in cells. 4 Thus, we next examined whether TDAG51 expression is enhanced by ER stress responses. Upon treatment with ER stressors such as tunicamycin and dithiothreitol, we observed that the increased expression of TDAG51 appeared to correlate with the expression of the major ER stress marker GRP78 (Figures 1d  and e). These results suggest that TDAG51 is an oxidative stress-responsive gene product.
To further examine the mechanism of TDAG51 induction by oxidative stress, we generated TDAG51 promoter-reporter constructs harboring a 3 kb murine TDAG51 promoter fragment driving expression of the luciferase gene (Figure 2a). In the luciferase assays, we observed that the activity of the TDAG51 3 kb promoter (P T51 -3K-Luc) was induced in a dosedependent manner by H 2 O 2 stimulation (Figure 2a). We next determined whether mitogen-activated protein kinases and NF-kB signaling are involved in TDAG51 induction by H 2 O 2 stimulation. The promoter activities of P T51 -3K-Luc were reduced by treatment with inhibitors of mitogen-activated protein kinases and NF-kB signaling (Figure 2b). Coincidently, we observed that the induction of TDAG51 protein was significantly reduced by treatment with inhibitors of mitogenactivated protein kinases and NF-kB signaling (Figure 2c). These results indicate that TDAG51 expression is modulated by mitogen-activated protein kinases and NF-kB signaling in response to H 2 O 2 stimulation.

TDAG51 deficiency enhances intracellular ROS generation
Intracellular ROS production is dramatically increased in cellular responses to environmental stimuli. 9,22 TDAG51 expression is involved in the oxidative stress response as shown in Figure 1; therefore, we examined whether TDAG51 deficiency affects the level of intracellular ROS production. To evaluate the effects of intracellular ROS production caused by TDAG51 deficiency, TDAG51 À/ À MEFs were treated with 0.5 mM H 2 O 2 for several time points, incubated with DCFH-DA, and then analyzed by flow cytometry. As shown in Figure 3a, intracellular ROS production in TDAG51 À/ À MEFs was significantly enhanced by H 2 O 2 stimulation compared to TDAG51 þ / þ MEFs. The level of intracellular ROS production was quantified and summarized in Figure 3b. Intracellular ROS production was significantly enhanced in a time-dependent manner by H 2 O 2 stimulation in TDAG51 À/ À MEFs compared to TDAG51 þ / þ MEFs (Figure 3b). Taken together, these results indicate that TDAG51 deficiency enhances intracellular ROS production during the oxidative stress response.
Oxidative stress-induced apoptosis is elevated in TDAG51 À/ À MEFs Oxidative stress induces intracellular ROS accumulation in cells, which results in apoptotic cell death. 9,22 Accordingly, we next compared apoptotic cell death between TDAG51 þ / þ and TDAG51 À/ À MEFs induced by H 2 O 2 stimulation. To test this comparison, MEFs were treated with H 2 O 2 for 6 h, and cell survival was determined by DAPI staining. Cell survival was remarkably reduced 1.9-to 3.6-fold by H 2 O 2 stimulation in TDAG51 À/ À MEFs compared to TDAG51 þ / þ MEFs (Figure 4a). These results were reconfirmed by PI staining, which demonstrated that TDAG51 À/ À MEFs were more sensitive (1.3-to 2.1-fold) to H 2 O 2 -induced cell death compared to TDAG51 þ / þ MEFs (Figure 4b). Similarly, cell survival decreased in a time-dependent manner after treatment with 0.5 mM H 2 O 2 in TDAG51 À/ À MEFs compared to TDAG51 þ / þ MEFs (Figure 4c). Caspase-3 activation by the apoptosome during the oxidative stress response is a final effector phase that subsequently activates apoptotic cell death. 7,8 Oxidative stress-induced cell death was significantly enhanced in TDAG51 À/ À MEFs as shown in Figures 4a-c; therefore, we next examined whether caspase-3 activation is enhanced by H 2 O 2 stimulation in TDAG51 À/ À MEFs. To test this idea, we treated MEFs with 0.5 mM H 2 O 2 in a timedependent manner and examined the process of caspase-3 activation by western blot analysis with a specific antibody against the active form of caspase-3. We observed that the active form of caspase-3 was significantly increased in   (Figure 4d). These observations indicate that TDAG51 has a protective role in oxidative stress-induced cell death.
To further examine the anti-apoptotic function of TDAG51, we investigated whether knocking down the expression of the endogenous TDAG51 gene via RNA interference (RNAi) would affect oxidative stress-induced apoptosis in MEFs. We cloned a short hairpin RNA (shRNA) specific for TDAG51 into the pSuper-retro-puro vector (pSR-TDAG51i) and performed western blot analysis to test the effects of the TDAG51 RNAi (TDAG51i). GFPi was used as a control as described previously. 20 We observed that the expression of TDAG51 was significantly decreased in response to the introduction of TDAG51i into 293T cells (Figure 5a). Next, retroviral short hairpin RNA-infected MEFs were treated with H 2 O 2 for 6 h, and cell survival was determined by DAPI staining. Consistent with the results in TDAG51 À/ À MEFs (Figure 4), cell survival was reduced 1.5-to 2.7-fold by H 2 O 2 stimulation in TDAG51itransduced MEFs compared with GFPi-transduced MEFs (Figure 5b). Similarly, we observed that the active form of caspase-3 was significantly increased in TDAG51i-transduced MEFs compared with GFPi-transduced MEFs after H 2 O 2 stimulation. Furthermore, we observed that the levels of cleaved caspase-9 and Bax were increased in TDAG51itransduced MEFs after H 2 O 2 stimulation (Figure 5c). In summary, we conclude that oxidative stress-induced apoptosis is elevated in TDAG51 À/ À MEFs.

DISCUSSION
Apoptosis is an elaborate cellular process that removes damaged or infected cells from normal tissue in multicellular organisms. Abnormal control of apoptotic cell death is closely related to the development of several types of human diseases such as inflammation, autoimmunity, diabetes, Alzheimer's, and cancer. Thus, understanding the regulatory process of apoptotic cell death is truly important to prevent disease development and for medical intervention. Based on these aspects, we investigated the functional role of TDAG51 expression in oxidative stress-induced apoptotic cell death. TDAG51 was first identified as a modulator of T-cell apoptosis through the induction of the expression of the death receptor CD95/Fas. 11 However, to date, several reports demonstrate that TDAG51 may have both pro-and antiapoptotic functions depending on the cellular context and circumstances. Related to the pro-apoptotic function of TDAG51, several reports support the notion that the expression of TDAG51 is closely associated with enhanced apoptosis. 11,[23][24][25] Specifically, it has been reported that TDAG51 expression is highly induced by homocysteine and heat shock stress, and it promotes apoptotic cell death. 13,14 In addition, the process of tumorigenesis is enhanced by the downregulation of TDAG51, which results in the inhibition of tumor cell apoptosis. [26][27][28] However, in contrast to its proapoptotic function, TDAG51 has also been shown to have an important role in insulin-like growth factor-1-induced cell survival. 29 Similarly, our group and another group have confirmed that TDAG51 expression is not involved in AICD in human and murine T cells. 15,16 In addition, several groups reported that TDAG51 expression is highly enhanced in some tumor types such as pancreatic, colon, and intestinal tumors, indicating that TDAG51 may be involved in tumor cell proliferation but not in apoptotic cell death. 16,[30][31][32] Collectively, the exact role of TDAG51 expression in apoptotic cell death remains controversial. Thus, a better understanding of the functional role of TDAG51 in cell fate control will provide valuable insight into the regulation of tumor development and cellular stress responses.
ROS are ubiquitous, highly reactive, and short-lived derivatives of oxygen metabolism that serve as critical signaling molecules in cell proliferation and cell death. 33 ROS are generated during energy metabolism by mitochondria and from metabolic processes elsewhere in the cell that cause damage to cellular macromolecules, including nucleic acids, proteins, and lipids. 9 Therefore, ROS production is considered to be a major cause of cellular oxidative stress. 34 An imbalance between ROS generation and elimination induces severe oxidative stress in cells, which results in apoptotic cell death. 9 In this study, we demonstrated that TDAG51 is a negative modulator of apoptotic cell death induced by oxidative stress through the downregulation of ROS generation. However, an interesting question still remains involving the enhancement of ROS generation in TDAG51 À/ À MEFs. Under physiological conditions, cellular protection against oxidative damage by ROS accumulation is provided by both enzymatic (for example, superoxide dismutase and catalase) and non-enzymatic (for example, vitamins E and C) components. 33 Interestingly, TDAG51 contains both a PQ-repeat domain and a proline-histidinerepeat domain that function as a putative transcriptional activator. 11 Thus, TDAG51 may be involved in the modulation of the expression of antioxidant enzymes or non-enzymatic components to eliminate excess ROS generation in response to oxidative stress. In addition, ER stress is a major source of ROS production. 35 Specifically, numerous clues suggest that ER stress induced by the unfolded protein response is linked to the generation of intracellular ROS, which can lead to the oxidative stress response. 4,36 In another interesting observation, Hinz et al. 37 suggest that TDAG51 functions as a negative regulator of protein biosynthesis through an interaction with translation regulatory proteins. In our current study, we also observed that TDAG51 expression was significantly enhanced by treatment with ER stressors such as tunicamycin and dithiothreitol (Figure 1). Thus, we assume that the functional role of TDAG51 may also be involved in the regulation of the unfolded protein response in the ER. Based on this hypothesis, it is possible to explain the enhancement of ROS levels in TDAG51 À/ À MEFs, although intensive studies are needed to reveal the mechanisms underlying a direct link between TDAG51 expression and ROS generation regulated by the unfolded protein response.
In conclusion, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death during TDAG51 deficiency. TDAG51 expression was highly enhanced by oxidative stress responses. In response to oxidative stress, the production of intracellular ROS was significantly enhanced in TDAG51 À/ À MEFs and resulted in the activation of caspase-3. Furthermore, TDAG51 deficiency enhanced apoptotic cell death in MEFs, and these results indicated that TDAG51 has a protective role in oxidative stress-induced cell death. Further studies will need to address the regulatory mechanism of the physiological significance of TDAG51 expression in the oxidative stress response.