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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC Apr 1, 2011.
Published in final edited form as:
PMCID: PMC2848894
NIHMSID: NIHMS176199

Dual regulation by AP endonuclease-1 inhibits gastric epithelial cell apoptosis during Helicobacter pylori infection

Abstract

Human AP-endonuclease-1 (APE-1), a key enzyme involved in repair of oxidative DNA base damage, is an important transcriptional co-regulator. We previously reported that Helicobacter pylori infection induces apoptosis and increases APE-1 expression in human gastric epithelial cells (GEC). Although both the DNA repair activity and the acetylation-mediated transcriptional regulation of APE-1 are required to prevent cell death, the mechanisms of APE-1 mediated inhibition of infection-induced apoptosis are unclear. Here, we demonstrate that shRNA-mediated stable suppression of APE-1 results in increased apoptosis in GEC after H. pylori infection. We show that programmed cell death involves both the caspase 9-mediated mitochondrial pathway and the caspase 8-dependent extrinsic pathway by measuring different markers for both the pathways. Overexpression of wild type APE-1 in APE-1-suppressed GEC reduced apoptosis after infection; however, overexpression of the DNA repair mutant or the nonacetylable mutant of APE-1 alone was unable to reduce apoptosis, suggesting that both DNA repair and acetylation functions of APE-1 modulate programmed cell death. We demonstrate for the first time that the DNA repair activity of APE-1 inhibits the mitochondrial pathway while the acetylation function inhibits the extrinsic pathway during H. pylori infection. Thus, our findings establish that the two different functions of APE-1 differentially regulate the intrinsic and the extrinsic pathway of H. pylori-mediated GEC apoptosis. As pro-apoptotic and anti-apoptotic mechanisms determine the development and progression of gastritis, gastric ulceration and gastric cancer, this dual regulatory role of APE-1 represents one of the important molecular strategies by H. pylori to sustain chronic infection.

Keywords: APE-1, H. pylori, apoptosis, gastric epithelial cells, caspase

Introduction

Helicobacter pylori infects half of the world’s population and is a causative agent of gastritis, peptic ulcer, gastric cancer and lymphoma (1, 2). The host response to H. pylori provides an environment in which epithelial cells may be damaged by mediators of inflammation including cytokines (3), proteases and reactive oxygen and nitrogen species (4, 5). Infection with H. pylori results in apoptosis of gastric epithelial cells due to multiple mechanisms including the Fas/FasL system (6), MHC class II (7), the mitochondrial pathway (8) as well as the p53 protein family (9).

Apoptosis is executed by the activation of caspases which act as effector molecules (10). Caspase activation is initiated at different points including tumor necrosis factor (TNF) receptor superfamily members such as Fas/CD95 (representing the extrinsic pathway) or at the mitochondrial level (the intrinsic pathway) (11). Both the intrinsic (8, 12) and the extrinsic apoptotic pathways (13) are important in H. pylori-induced gastric epithelial cell death.

Mammalian APE-1 is a multifunctional protein that regulates apoptosis and is induced by reactive oxygen species (ROS) (14, 15). It plays a central role in the base excision repair pathway to correct DNA damaged by ROS and alkylating agents (16, 17). We have shown that APE-1 expression is increased in the gastric mucosa during H. pylori infection (18) and that APE-1 controls infection-mediated chemokine expression in GEC (19). Another distinct transcriptional regulatory role of APE-1 is mediated by the N-terminal Lys6/Lys7 acetylation of APE-1 which represses certain promoters (20, 21). Recently, we established that acetylated APE-1 represses bax expression by binding to the negative calcium response element present in the promoter of this gene (22) and inhibits H. pylori-mediated GEC apoptosis.

APE-1 downregulation inhibits cell proliferation and activate apoptosis in cell lines of diverse origin (23, 24). It has been demonstrated that both the repair activity and the acetylation-mediated transcriptional regulatory functions of APE-1 are required to prevent apoptosis (25). However, how the diverse functional properties of APE-1 differentially regulate apoptosis, including the intrinsic and extrinsic apoptotic pathways, has not been examined. Since H. pylori infection regulates apoptosis via both pathways, we sought to define the role of the different functional regions of APE-1 in controlling apoptosis. Using gastric epithelial AGS cells with stably downregulated APE-1, we observed increased apoptosis after H. pylori infection. APE-1 suppressed apoptosis through its effects on both the mitochondrial pathway and the extrinsic pathway. Furthermore, we found that the DNA repair activity of APE-1 regulated the intrinsic pathway while the acetylation function regulated the extrinsic pathway, thereby showing that APE-1 has distinct functions that differentially inhibit apoptosis during H. pylori infection which may impact the development of gastric cancer and other clinical consequences of infection.

Materials and Methods

Cell culture and bacterial strains

AGS cell line is a human gastric adenocarcinoma line obtained from the American Type Culture Collection (ATCC). Empty vector (pSIREN), APE-1 shRNA expressing (shRNA) cells or nontransfected AGS (AGS) cells were harvested and cultured as previously described (22). The effectiveness of shRNA suppression was periodically tested and on average a 60% reduction in APE-1 protein level was observed in shRNA cells compared to AGS and pSIREN cells. H. pylori 26695, a cag PAI(+) strain (ATCC) was maintained on blood agar plates (Becton Dickinson). Bacteria were cultured overnight at 37°C in Brucella broth (GIBCO-BRL) with 10% FBS under microaerophilic conditions before infecting GECs. As described in previous studies, we found that a multiplicity of infection (MOI) of 300 for 3 h was the optimum dose to induce APE-1 (18) and its acetylation (22). However, an initial dose response study demonstrated that an MOI of 100 was optimal for apoptosis assays up to 24h after infection while MOI 300 increased necrotic GEC death with time. Accordingly, a MOI of 100 was used for most of the experiments in this study unless mentioned otherwise.

Plasmids

APE-1, K6R/K7R APE-1 (lysines 6 and 7 of APE-1 cDNA replaced with arginine) and H309N APE-1 (histidine 309 replaced with asparagine) constructs were generated by cloning into pFLAG-CMV™ −5.1 Expression Vector (Sigma) between the EcoR1 and BamH1 sites.

Western analysis

Western analysis was performed as described previously (22) after cells were lysed in RIPA buffer. Primary antibodies that were used included APE-1 (36 kD, Novus Biologicals), cleaved caspase 3 (19 and 17 kD), cleaved PARP (89kD), cleaved caspase 8 (18kD), pro-caspase 8 (57, 43 kD), cleaved caspase 9 (37kD), cytochrome c (14 kD), FLIPL/ FLIPS (58/30 kD), FADD (28 kD) (all from Cell Signaling), Bcl-xS /Bcl-xL (29/21 kD, Chemicon), Bax (21 kD, BD Pharmingen) and α-tubulin (Abcam). Secondary antibodies were anti-rabbit or anti-mouse HRP-conjugated IgG (Cell Signaling Technology). An AcAPE1-specific rabbit antibody was used as previously described (26). Immunoreactions were visualized by chemiluminescence (Cell Signaling Technology). Protein loading was normalized to α-tubulin and analyzed by IMAGEQUANT software.

Caspase activity assays

1 × 106 cells were washed with PBS, centrifuged and resuspended in 50 µl of sample buffer (Caspase 3 Activity Assay Kit, QIA70, Calbiochem, Caspase 8 Fluorometric Assay kit, BF2100/ Caspase 9 Fluorometric Assay kit, BF7100, R&D systems) and caspase activity was determined according to manufacturer’s protocol. The reversible caspase 3 inhibitor DEVD-CHO provided with the kit was used according to the supplied protocol. Caspase 8 inhibitor (Z-IETD-FMK) or caspase 9 (Z-LEHD-FMK) inhibitor (BD Biosciences) was added 1 h prior to H. pylori infection. All caspase activities were measured by fluorescence (Ex 400 nm/Em 505 nm) using a Gemini plate reader.

Transfection

1 × 106 AGS, pSIREN or shRNA cells were seeded in six-well plates 18–24 h before transfection. For overexpression studies, shRNA cells were transfected with 2 µg of plasmid DNA and 6 µl of Lipofectamine™ 2000 reagent (Invitrogen) as per manufacturer’s protocol. Cells were infected with H. pylori 40 h post-transfection.

Immunoprecipitation

5 × 106 cells were plated in 10 cm plates. Following infection, cells were washed with PBS, lysed with 250 µl RIPA buffer (150 mM NaCl, 50 mM Tris Cl pH 7.4, 1% NP-40, 0.1% sodium deoxycholate, 1 mM EDTA with protease inhibitor cocktail) on ice for 30 min. Lysates of two wells treated identically were pooled and clarified at 13,000 × g for 10 min at 4° C. 500 µg of cell lysates were incubated either with 7.5 µl p300 antibody or 7.5 µl of Fas antibody (Santa Cruz, Biotechnology) at 4° C overnight. 15 ul 50% A/G plus agarose (Santa Cruz Biotechnology) were added and incubated for additional 3 h. The agarose-bound immunocomplex was washed three times with the same lysis buffer, boiled and resolved by SDS-PAGE, followed by western blot to detect associated proteins.

Mitochondrial membrane potential assay

JC-1 is a cationic dye that accumulates in negatively charged mitochondria of healthy cells in a membrane potential-dependent manner where it forms aggregates and fluoresces red. In apoptotic cells where the mitochondrial membrane potential has collapsed, JC-1 exists only as monomers throughout the cell and fluoresces green. Using a JC-1 mitochondrial membrane potential detection kit (JC 100, Cell Technology) cells were analyzed by flow cytometry and the percentage of cells shifted to green (FL-2) was calculated for control and infected groups according to the manufacturer’s protocol.

Statistical analysis

Results are expressed as the mean ± SEM. Results were compared using two-tailed Student's t test and considered significant if p values were < 0.05.

Results

APE-1 inhibits H. pylori-mediated apoptosis

To determine the effect of the level of APE-1 on infection-induced apoptosis in GECs, we used AGS, pSIREN and shRNA cells as described under ‘materials and methods’. Western analysis of whole cell lysates showed a significant time-dependent increase in the level of active caspase 3 and cleaved poly (ADP)-ribose polymerase (PARP) after H. pylori infection (MOI 100) in APE-1 suppressed shRNA cells compared to vector control (Fig. 1A). Elevated caspase 3 activity in shRNA cells compared to control or uninfected cells further corroborated these findings (Fig. 1B). These data suggest that APE-1 is important for the inhibition of cell death during H. pylori infection. Given that active caspase 3 generation requires both caspase 8 and caspase 9, their specific inhibitors were used to show partial reduction of active caspase 3 level as detected by western analysis after H. pylori infection. The magnitude of inhibition by either inhibitor was less in cells with reduced levels of APE-1 (Fig. 1C) suggesting that APE-1 is involved in both caspase 8- and caspase 9-mediated apoptotic pathways.

Figure 1
Increased apoptosis in APE-1 suppressed GEC after H. pylori infection. A, pSIREN and shRNA cells were treated with H. pylori (MOI 100) for 6, 12 or 24 h or left uninfected. Western analysis was performed for active caspase 3 (left panel) or cleaved PARP ...

APE-1 inhibits the intrinsic pathway of apoptosis

We next examined the contribution of APE-1 in modulating apoptosis resulting from the perturbation of mitochondria due to H. pylori infection. A significant increase in active caspase 9 (Fig. 2A) and enhanced release of cytochrome c (Fig. 2B) into the cytosol after infection were observed in APE-1-downregulated cells relative to the vector control. To determine if APE-1 regulates Bcl-2 family members, cell lysates of H. pylori infected GEC were assayed for expression of anti-apoptotic Bcl-xL and proapoptotic Bcl-xS. H. pylori decreased Bcl-xL and increased Bcl-xS in a time dependent manner and this effect was greater in APE-1-deficient cells (Fig. 2C). To directly measure mitochondrial damage, the loss of mitochondrial membrane potential (MMP) was analyzed at 1 h after H. pylori infection. Infection increased the loss of MMP in both cell types but the loss was greater in APE-1-deficient cells further indicating an inhibitory role of APE-1 in the mitochondrial pathway of apoptosis (Fig. 2D).

Figure 2
Reduction of APE-1 level enhances H. pylori-mediated apoptosis via the mitochondria-dependent pathway. pSIREN and shRNA cells were treated with H. pylori (MOI 100 ) for 6, 12 or 24 h or left uninfected. Western analysis was performed for (A) active caspase ...

APE-1 inhibits the extrinsic pathway of apoptosis

The regulatory role of APE-1 in the extrinsic pathway of apoptosis was assessed by measuring active caspase 8. Western analysis demonstrated an increase in active caspase 8 within 4 h of H. pylori infection in APE-1-downregulated cells relative to the control cells (Fig. 3A). Similar results were obtained by measuring activation of caspase 8 further confirming APE-1’s inhibitory role (Fig. 3B). Fas-associated death domain-like IL-1-converting enzyme-inhibitory proteins (FLIP), which counteract caspase 8 activation, were subsequently measured. The shorter protein FLIPS is exclusively a caspase inhibitor whereas the longer FLIPL has dual functions as either a caspase inhibitor or activator (27). A rapid decrease of FLIPS expression over 12 h of H. pylori infection was observed in both cell types but the decrease was greater in APE-1-deficient cells (Fig. 3C).

Figure 3
Reduction of APE-1 level enhances H. pylori-mediated apoptosis via the extrinsic pathway. pSIREN and shRNA cells were treated with H. pylori (MOI 100) for 4, 8 or 12 h or left uninfected. Western analysis was performed for (A) active caspase 8 and (B ...

Caspase 8 is an integral part of the death inducing signaling complex (DISC) along with Fas, FADD or FLIP (28). Within 4 h of H. pylori infection, DISC pulldown using Fas antibody showed enhanced binding of apoptotic pro-caspase 8 and FADD in shRNA compared to pSIREN cells and the binding of anti-apoptotic FLIPS was significantly decreased in shRNA cells (Fig. 3D). These results demonstrate that DISC association during H. pylori infection is regulated by APE-1.

Both repair and regulatory functions of APE-1 are involved in inhibiting apoptosis

To determine the relative contribution of the DNA repair versus the regulatory function of APE-1 in the inhibition of H. pylori-induced apoptosis, we employed functional mutants of APE-1. A schematic diagram of APE-1 (Fig. 4A) illustrates that the N-terminal lysines 6 and 7 are potential sites for acetylation while the C-terminal histidine 309 is a key residue required for DNA repair activity (25). Along with FLAG-tagged wild type (WT) APE-1, we constructed a DNA repair mutant by replacing histidine 309 with asparagine (H309N, middle panel) and an acetylation mutant by replacing lysines 6 and 7 with arginines (K6R/K7R, bottom panel).

Figure 4
Acetylation and DNA repair functions of APE-1 both contribute to the inhibition of apoptosis. A, schematic diagram showing WT human APE-1 (upper panel), the repair mutant (middle panel) and the acetylation mutant (bottom panel). B, shRNA cell extracts ...

shRNA cells were separately transfected with an empty vector or the WT, K6R/K7R or H309N APE-1 constructs. Subsequent western blot analyses at 12 and 24 h after H. pylori infection demonstrated significantly reduced active caspase 3 at both time points in WT APE-1 transfected cells compared to vector control (Fig. 4B). In contrast, active caspase 3 expression in cells transfected with either the DNA repair mutant or the acetylation mutant construct was not significantly different from cells not transfected with APE-1 (Fig. 4B). This finding underscores the importance of both functions of APE-1 in controlling apoptosis during H. pylori infection.

It is reported that H. pylori MOI 100 is optimal for inducing apoptosis (29, 30), whereas APE-1 acetylation occurs maximally with a MOI of 300 (22). Thus, in this study, we confirmed the induction of APE-1 acetylation by H. pylori before embarking on the role of acetylated APE-1 in regulating different pathways of apoptosis. A significant increase in acetylated APE-1 at MOI 300 and 600 compared to MOI 100 was observed (Fig. 4C). To determine the contribution of APE-1’s acetylation function, we measured cytosolic cytochrome c, Bax and active caspase 9 expression in shRNA cells as shown in Fig. 4B after infection with H. pylori MOI 100 and 300. While WT APE-1 overexpression reduced the protein level of cytochrome c and Bax, overexpression of the acetylation mutant significantly increased their expression with MOI 300 compared to MOI 100 (Fig. 4D, upper and middle panels, compare lanes 6 and 7). This suggests that the APE-1 acetylation inhibits the intrinsic apoptotic pathway when a higher dose of H. pylori infection is used. This may be due to inhibition of Bax transcription by acetylated APE-1 as previously reported (22). However, unlike Bax and cytochrome c, active caspase 9 expression was decreased in presence of the acetylation mutant compared to vector control after infection with both MOI 100 and 300 (Fig. 4D, middle panel, compare lanes 2 with 6 and 3 with 7). These data implicate an inhibitory role of the DNA repair function of APE-1 downstream of mitochondrial membrane damage in the setting of H. pylori infection.

The DNA repair function of APE-1 inhibits mitochondria-mediated apoptosis

As H. pylori MOI 300 increased necrotic death of GEC over time, we used MOI 100 to examine the relative roles of the two different functions of APE-1 in subsequent experiments. Western analysis of whole cell lysates at 6 and 12 h after H. pylori infection showed increased levels of active caspase 9 and decreased levels of anti-apoptotic Bcl-xL (Fig. 5A) in cells transfected with the DNA repair mutant. In contrast, transfection with WT APE-1 or the acetylation negative mutant showed reduced levels of active caspase 9 and increased levels of Bcl-xL. Assays of caspase 9 activity corroborated the western blot data (Fig. 5B). We also found a significant increase in MMP loss in DNA repair mutant transfected APE-1 deficient cells after H. pylori infection, however, this was not observed in APE-1 deficient cells overexpressing WT or the acetylation negative mutant of APE-1 (Fig. 5C). This suggests that APE-1’s DNA repair activity is required to inhibit the mitochondrial pathway of apoptosis.

Figure 5
DNA repair activity of APE-1 is required to inhibit mitochondria-mediated apoptosis during H. pylori infection. A, shRNA cells were transiently transfected as in Fig. 4B and infected with H. pylori for 6 or 12 h before performing western analysis with ...

Acetylation function of APE-1 inhibits the extrinsic pathway of apoptosis

Similar approaches were used to assess the relative roles of the repair and acetylation functions of APE-1 in the extrinsic pathway of apoptosis. In contrast to what was shown for the intrinsic pathway, the H. pylori mediated increase in active caspase 8 was inhibited in both WT and DNA repair mutant APE-1 transfected shRNA cells. Overexpression of the acetylation mutant of APE-1 did not inhibit active caspase 8 generation (Fig. 6A) or caspase 8 activity (Fig. 6B). Finally, reduced expression of anti-apoptotic FLIPS after infection in acetylation mutant transfected cells compared to those transfected with WT or DNA repair mutant APE-1 (Fig. 6C) confirmed the previous observations indicating that impaired acetylation of N-terminal lysines inhibits the ability of APE-1 to attenuate the caspase 8-mediated extrinsic pathway of apoptosis.

Figure 6
The acetylation function of APE-1 is required to inhibit the extrinsic pathway of apoptosis during H. pylori infection. A, shRNA cells were transiently transfected as in Fig. 4B and infected with H. pylori for 4 or 8 h before performing western analysis ...

Discussion

H. pylori pathogenesis depends on the balance between proliferation and cell death of the gastric epithelium (8, 31), and both bacterial factors and host signaling pathways determine the outcome of infection. H. pylori infection has been shown to exert anti-apoptotic effects to promote gut epithelium self-renewal in response to bacterial factor CagA (31). Our results indicate that molecular events within host cells also play a role in inhibiting apoptosis due to Cag PAI-bearing H. pylori strain, 26695. Specifically, we demonstrated that APE-1 has a key inhibitory role in programmed cell death resulting from H. pylori infection of gastric epithelial cells. We also showed that APE-1 inhibited both the intrinsic and extrinsic pathways of cell death and that the DNA repair activity and the acetylation function of APE-1 differentially inhibited these two pathways. Thus our results enhance the current understanding of programmed cell death during H. pylori infection and advance our knowledge of the multifunctional role of APE-1 in modulating apoptosis. Given the role apoptosis and alterations of cell cycle may play in carcinogenesis, our findings also have implication for understanding H. pylori-associated disease pathogenesis.

The data presented in this report indicated that suppression of endogenous APE-1 in GEC rendered the cells more susceptible to apoptosis after H. pylori infection as evidenced by increased caspase 3 activity. To further define the basis for caspase 3 activation, we first investigated the intrinsic pathway of apoptosis (32, 33). Our findings of increased cytosolic cytochrome c, together with elevated caspase 9 in APE-1-suppressed GEC compared to vector control cells provided evidence for APE-1 as a regulator of the intrinsic apoptotic pathway. The increase in the Bcl-xs/Bcl-xL ratio in shRNA cells indicated a shift towards proapoptotic Bcl-2 family members when APE-1 is reduced, presumably because of damaged mitochondrial genome due to the accumulation of ROS in GEC which we and others have shown occurs during H. pylori infection (4, 12).

Our work also established a role of APE-1 in the extrinsic pathway of apoptosis which is dependent on Fas-mediated DISC activation (34). FLIPS binding to the DISC complex prevents apoptosis by inhibiting the DISC-mediated activation of procaspase 8 (35, 36). We showed that APE-1 suppression not only resulted in a decrease in anti-apoptotic FLIPS expression but also diminished the DISC binding of FLIPS. Downregulation of APE-1 led to an increase in DISC-associated procaspase 8 and FADD, possibly due to a decrease in DISC-associated FLIPS. In our study, the increase in caspase 8 activation occurred earlier than the increase in caspase 9 similar to previously published observations(37, 38). This pattern of caspase activation was not affected by cellular levels of APE-1. Many of the effects of APE-1 in suppressing H. pylori-induced apoptosis show similar patterns as reported for epidermal growth factor receptor (EGFR) activation (39). Thus, examining the possible relationship between APE-1 and EGFR transactivation will be an important area for future research.

APE-1 exerts its many effects via DNA repair, reductive activation, and acetylation-mediated gene regulation. However, it has been difficult to establish the relative contribution of these various properties of APE-1 to regulate apoptosis. Although we showed that APE-1 separately inhibited the intrinsic and extrinsic pathways of apoptosis, links between these two pathways are known to exist at different levels. Upon death receptor signaling; activation of caspase 8 results in translocation of truncated Bid to mitochondria with subsequent activation of caspase 9 (40). We observed that the DNA repair activity of APE-1 was critical in inhibiting the intrinsic pathway, as overexpression of the acetylation mutant of APE-1 inhibited caspase 9 activation but had no effect on caspase 8. Conversely, overexpression of the DNA repair mutant affected only the intrinsic pathway including caspase 9 activation and suppression of anti-apoptotic Bcl-xL, while caspase 8 activity remained low, presumably because of functional N terminal lysine 6 and 7.

In summary, our findings demonstrate APE-1 as a critical host response molecule that may promote persistence of H. pylori infection by inhibiting apoptosis. This is the first report showing the simultaneous inhibition of mitochondrial damage and Fas-mediated apoptosis by APE-1. Moreover, the intrinsic and extrinsic pathways of apoptosis are shown to be inhibited by two different functional domains of APE-1. The role of APE-1 in H. pylori-mediated disease pathogenesis remains to be elucidated. Our findings also have implications for understanding programmed cell death in other infections and host cell populations.

Acknowledgements

We thank Prof. Sankar Mitra and Dr. Kishor Bhakat of University of Texas Medical Branch at Galveston, Texas for critical review of this manuscript and for scientific advice. Support from the DNA Sciences Core and Immunology and Cell Isolation Core of UVA is gratefully acknowledged. This work was supported by NIH grant (RO1 DK61769) and an American Gastroenterological Association Funderburg Award.

Abbreviations

APE-1
apurinic/apyrimidinic endonuclease-1
GEC
gastric epithelial cells
PARP
poly (ADP-ribose) polymerase
MMP
mitochondrial membrane potential
WT
wild type
IP
immunoprecipitation

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