Adenovirus vectors activate survival pathways in lung epithelial cells
Dawn M. Flaherty,
Sara L. Hinde,
Martha M. Monick,
Linda S. Powers,
Mary A. Bradford,
Timur Yarovinsky, and
Gary W. Hunninghake
Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine; and Veterans Affairs Medical Center, Iowa City, Iowa 52242
Submitted 16 October 2003
; accepted in final form 19 April 2004
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ABSTRACT
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Airway epithelial cells are often the sites of targeted adenovirus vector delivery. Activation of the host inflammatory response and modulation of signal transduction pathways by adenovirus vectors have been previously documented, including activation of MAP kinases and phosphatidylinositol 3-kinase (PI3-kinase). The effect of activation of these pathways by adenovirus vectors on cell survival has not been examined. Both the PI3-kinase/Akt and ERK/MAP kinase signaling pathways have been linked to cell survival. Akt has been found to play a role in cell survival and apoptosis through its downstream effects on apoptosis-related proteins. Constitutive activation of either PI3-kinase or Akt blocks apoptosis induced by c-Myc, UV radiation, transforming growth factor-
, Fas, and respiratory syncytial virus infection. We examined the effect of adenovirus vector infection on activation of these prosurvival pathways and its downstream consequences. Airway epithelial cells were transduced with replication-deficient adenoviral vectors containing a nonspecific transgene, green fluorescent protein driven by the cytomegalovirus promoter, or an empty vector with no transgene. They were then exposed to the proapoptotic stimulus actinomycin D plus TNF-
, and evidence of apoptosis was evaluated. Compared with the cells treated with actinomycin/TNF alone, the adenovirus vector-infected cells had a 50% reduction in apoptosis. When we examined induction of the prosurvival pathways, ERK and AKT, in the viral vector-infected cells, we found that there was significant activation of both Akt and ERK.
extracellular signal-regulated kinase; phosphatidylinositol 3-kinase; Akt; apoptosis
IT IS WELL DOCUMENTED that first-generation recombinant adenovirus vectors induce potent host immune responses that promote inflammation and activation of a number of cell signaling pathways. Adenovirus vectors have been shown to modulate the MAP kinase signal transduction pathway, affecting intracellular signaling. In A549 cells, adenoviral vectors induce ERK, JNK, and p38 MAP kinase phosphorylation within minutes of addition of virus, resulting in downstream NF-
B activation with induction of ICAM mRNA (54). P38 and ERK are activated in kidney-derived epithelial cells in response to adenovirus infection with subsequent increases in expression of the CXC chemokine interferon-
-inducible protein 10 (IP-10) (56). Li and colleagues (34) have shown that adenovirus serotype 2 infection in a colon adenocarcinoma cell line, SW480, results in phosphorylation of the integrin-associated focal adhesion complex protein, p125FAK and p130CAS, with downstream activation of phosphatidylinositol 3-kinase (PI3-kinase) (5, 56). The activation of PI3-kinase appears to be essential for adenovirus internalization and promotes reorganization of the actin cytoskeleton, a requirement for efficient entry of virus into the cell (32). These are early immune responses to the virus and do not appear to be dependent on viral gene expression (19, 23, 54).
Adenovirus vectors also trigger an adaptive immune response, making the cells more susceptible to host-mediated elimination that involves cytotoxic T-cell killing of the virus-infected cells (8, 27, 62, 63). This, in fact, has been one major limitation in the development of these vectors for in vivo gene therapy since rapid host elimination occurs, making persistent expression of the desired gene product difficult. Although wild-type adenovirus is well documented to have antiapoptotic effects on the host cell (17, 37, 47, 57), the only studies that have shown adenoviral vector-mediated enhancement in cell survival and inhibition of apoptosis have been of the endothelium (21, 50, 65). In these studies, human umbilical vein endothelial cells were infected with replication-deficient adenovirus vectors deleted in the E1 and E3 viral genes but expressing the E4 genes and were found to have increased viability in culture. These investigators also found that apoptosis was suppressed in these cells and that this enhanced survival phenotype was dependent on the expression of the viral E4 genes.
Because airway epithelial cells are an important site of adenovirus vector infection, we sought to determine the effects of adenovirus vectors on cell survival and resistance to apoptosis in human bronchial epithelial (HBE) and A549 cells. In the following studies, we show that adenovirus vectors are protective against TNF-
-induced apoptosis. We also show that this effect of the vectors is due to the activation of the prosurvival pathways, ERK and Akt.
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MATERIALS AND METHODS
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Reagents.
LY-294002 and U-0126 were purchased from Calbiochem. Ser473-phospho-Akt, phospho-ERK, and cleaved poly(ADP-ribose) polymerase (PARP, Asp214) antibodies were purchased from Cell Signaling Laboratories (Beverly, MA). Antibodies against total ERK and total Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The active caspase-3 antibody was purchased from Abcam (Cambridge, UK). Recombinant human TNF-
was purchased from R & D Systems (Minneapolis, MN), and actinomycin D was from Calbiochem (San Diego, CA).
Cell culture.
A549 cells, a tumor cell line with properties of normal airway epithelial cells (30, 41), were obtained from American Type Culture Collection (Manassas, VA) and incubated at 37°C in 5% CO2. The cells were cultured in Eagle's minimum essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 40 mg/ml gentamicin and were subcultured by harvesting in 0.12% trypsin no more than 20 times from stock originally designated at passage 70. HBE cells, kindly provided by Dr. Dwight Look at our institution, were obtained from human donor lungs and cultured as previously described (22). All protocols were approved by the University of Iowa Institutional Review Board.
Adenoviral vectors and wild-type adenovirus.
First-generation recombinant adenovirus was generated by the University of Iowa Gene Transfer Vector Core (2). The particle titers of the adenoviral stocks were typically 1012 DNA particles/ml; functional titers were
4 x 1010 plaque-forming U/ml. Adenovirus vectors expressing the transgene for green fluorescent protein (AdGFP) driven by the cytomegalovirus promoter or an empty vector containing no transgene (AdEV) were used to transduce the cells at a multiplicity of infection (MOI) of 100. These vectors were free of wild-type virus contamination as determined by plaque assay and PCR (64). Wild-type adenovirus serotype 5 was obtained from Advanced Technologies (Columbia, MD). The virus particle titers were 7.9 x 107, and cells were infected at an MOI of 10. Cells were plated and grown overnight and then virus was added for 2 h in serum-free media. In the A549 cells, fetal bovine serum was added back to the cultures to a final concentration of 10%. HBE cells were grown in specially supplemented media as previously described (22). The cells were incubated at 37°C for 24 h and harvested for total cellular protein. In some studies, the cells were treated for 1 h with 20 µM LY-294002 (an inhibitor of PI3-kinase), 10 µM U0126 (an inhibitor of ERK), or both inhibitors before viral infection. Efficiency of transduction was determined in each experiment by examining green fluorescence of the AdGFP-infected cells under a Leica DMIRB inverted fluorescence microscope (Wetzlar, Germany). Similar transduction efficiencies were assumed with the AdEV virus. For UV inactivation, recombinant adenovirus particles (AdGFP or AdEV) at 1 x 1012 particles/ml were placed in 250 µl of serum-free medium per well in a 24-well tissue culture plate and exposed to varying amounts of UV irradiation with a Stratalinker 1800 (Stratagene, La Jolla, CA). A dose-response curve was created, and an energy amount was selected (0.94 J) that resulted in <1/25,000 infected cells per well, as detected by green fluorescence (data not shown). For these experiments, cells were plated and treated as described above, and freshly UV-inactivated virus was added to the cells for the 24-h infection time. Lack of GFP expression in the UV-inactivated AdGFP-infected cells was confirmed by fluorescent microscopy (data not shown). To confirm that the UV-inactivated virus remained intact, we examined cells infected with untreated and UV-inactivated virus using transmission and scanning electron microscopy.
Determination of apoptosis.
Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) analysis for DNA fragmentation was carried out using the In Situ Cell Death Kit (Roche Diagnostics, Indianapolis, IN). Briefly, either HBE or A549 cells were plated on glass chamber slides at 5 x 104 cells/well and grown overnight. The following day, they were infected with the adenovirus vectors (AdGFP or AdEV) at 100 MOI as described above. After 2024 h of infection, cells were treated with TNF-
(1 ng/ml) plus actinomycin D (2.5 µg/ml) for 1218 h. After treatment, cells were fixed, washed, permeabilized, and then stained with the TUNEL reaction mix according to the manufacturer's protocol. Red fluorescent cells were counted in a blinded fashion under a fluorescent microscope. Cells (200400) were counted for each sample, and percent apoptotic cells was determined. A second method used to examine apoptosis was Western analysis with a polyclonal antibody directed against cleaved PARP.
Western analysis.
After experimental exposure, we washed cells in sterile PBS and harvested them by scraping them into lysis buffer (0.05 M Tris, pH 7.4, 0.15 M NaCl, and 1% Nonidet P-40) with added protease (EDTA-free mini-tab, Roche Diagnostics) and phosphatase (Calbiochem) inhibitors. The cell material was sonicated for 20 s on ice, allowed to sit for 20 min, and then centrifuged at 15,000 g for 10 min. The protein concentration in the lysate supernatant was measured by the Bradford assay normalized to bovine serum albumin. Equal amounts of protein (3050 µg) were mixed 1:1 with 2x sample buffer (20% glycerol, 4% SDS, 10% 2-mercaptoethanol, 0.05% bromphenol blue, and 1.25 M Tris, pH 6.8; all chemicals from Sigma Chemical), loaded onto a 10% SDS-PAGE gel, and run at 100 V for 2 h. Cell proteins were transferred to nitrocellulose (ECL; Amersham, Arlington Heights, IL), blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 for 1 h, washed, and then incubated with the primary antibody (1:1,000 for phospho-ERK, 1:200 for phospho-Akt) for 1 h. The blots were washed and incubated with a horseradish peroxidase-conjugated secondary antibody and developed with a chemiluminescent substrate, ECL Plus (Amersham). After development of phospho-Akt and phospho-ERK, we removed bound immunoglobulins from the membranes by washing them twice at 30 min each at room temperature in ImmunoPure IgG Elution Buffer (Pierce), and the membranes were reprobed for total Akt and ERK.
Statistical analysis.
One-way analysis of variance with multiple comparisons and paired t-tests were performed for all statistical parameter calculations.
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RESULTS
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Adenovirus vectors protect airway epithelial cells from TNF-
-mediated apoptosis.
TNF-
binding to its receptor can induce apoptosis by a signaling pathway that involves activation of caspases, cleavage of downstream substrates, and characteristic changes in cells, resulting in eventual cell death (44). To address the question of whether adenovirus vectors could affect the cellular response to apoptosis, we examined the extent of apoptosis in A549 and HBE cells using a known inducer of apoptosis, TNF-
plus actinomycin D. We first showed that TNF-
/actinomycin D induced apoptosis by evaluating active caspase-3 by Western blot analysis. Both exposures caused a clear upregulation of active caspase-3 (data not shown), as previously reported (38, 39, 58, 59). Using TUNEL staining and the detection of cleaved PARP, we also showed that TNF-
/actinomycin D induces apoptosis in both the A549 cell line and primary HBE cells. In Fig. 1, we show that TNF-
plus actinomycin D induces apoptosis in >20% of the cells (Fig. 1, A and B, A549 and HBE cells, respectively). When these cells are infected with a first-generation adenovirus vector containing the nonspecific transgene AdGFP or an empty vector, AdEV, 24 h before addition of the TNF-
and actinomycin D, apoptosis is reduced by >70% (P < 0.01). Another well-established method for the determination of apoptosis is detection of cleaved PARP. PARP is a nuclear poly(ADP-ribose) polymerase that is involved in DNA repair in response to environmental stress. It is one of the main targets for cleavage by caspases, and the presence of the cleaved PARP protein serves as a marker for apoptosis (48). Using this method for detection of apoptosis, we also showed that cells infected with adenovirus vectors before treatment with the proapoptotic stimuli had less cleaved PARP than cells treated with TNF/actinomycin alone. Although the vectors inhibited apoptosis induced by TNF-
/actinomycin D, the vectors themselves induced apoptosis in a small percentage of the cells.
Adenovirus vectors activate ERK at late time points after infection.
Replication-deficient adenoviral vectors have been described to activate the ERK MAP kinase signal transduction pathway (3, 6, 54). These prior studies examined the host response to adenovirus vector infection and showed early activation of this pathway that was independent of viral gene transcription and was accompanied by the activation of downstream proinflammatory cytokines. Because signaling through the ERK MAPK pathway has an important role in cell survival, we asked whether activation of ERK might be involved in the protection against apoptosis observed in the adenovirus-infected cells. We treated A549 and HBE cells with and without a specific MEK1/MEK2 inhibitor (U0126) followed by transduction with a first-generation adenoviral vector (AdGFP or AdEV) and examined activation of ERK using a phospho-specific ERK antibody after 24 h of infection. Figure 2A shows that A549 cells are transduced with 100% efficiency by the AdGFP vector at an MOI of 100. In Fig. 2B, we show that both the viral vectors significantly activated ERK above control levels (P < 0.005), and MEK1/2 inhibition blocks this effect without a change in levels of total ERK. When we performed this experiment using HBE cells, we observed similar results (Fig. 2C) using either the adenovirus vectors or the wild-type virus. In time course studies of viral infection, we observed a biphasic activation of both ERK and Akt at 1 h postinfection that quickly returned to control levels, increasing again at 12 h, with persistent activation out to 24 h (data not shown). Results were similar regardless of serum conditions (10 vs. 0.5%).

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Fig. 2. Adenovirus vectors activate ERK in airway epithelial cells. A549 or HBE cells were treated with and without inhibitors of ERK [U0126 (U) at 10 µM] and infected with replication-deficient adenovirus constructs containing no transgene (AdEV) or AdGFP for 24 h (multiplicity of infection 100). The cells were harvested for total cellular protein, and Western analysis was performed using a polyclonal antibody specific for p42 and p44 MAP kinase (ERK1 and 2) phosphorylated (pERK) at Thr202 and Tyr204. The immunoreactive bands were visualized by chemiluminescence and autoradiography. Densitometry of the phosphorylated ERK is shown as optical density (OD) units/mm2. The blot was stripped and reprobed with an antibody against total ERK to demonstrate equal loading. A: there is 100% infection of the cells with the AdGFP. B: results of a representative Western analysis in the A549 cells. C: similar results in the HBE cells infected with either the adenovirus vectors or with the wild-type (Wt) adenovirus (Ad) 5 virus.
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Adenovirus vector activation of Akt is dependent on PI3-kinase.
Others have shown that adenovirus entry into the host cell is dependent on
v-integrin binding and requires PI3-kinase activation (33). The relationship between activation of PI3-kinase in response to adenovirus infection and enhanced cell survival in epithelial cells, however, has not been previously described. To assess the activation of PI3-kinase by adenovirus, we infected A549 and HBE cells with the adenovirus vector AdGFP or the empty vector AdEV and looked at phosphorylation of the downstream kinase Akt 24 h postinfection. Figure 3 illustrates a significant increase in Akt activation with both adenoviral vectors (P < 0.005) that is blocked with the PI3-kinase inhibitor LY-294002, suggesting that this activation of Akt is PI3-kinase dependent. Figure 3A shows the results obtained in A549 cells, and Fig. 3B shows data using HBE cells. When we performed a similar experiment using the wild-type adenovirus, we observed the same results. These data demonstrate that adenovirus vector infection of lung epithelial cells leads to activation of PI3-kinase and the downstream kinase Akt.

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Fig. 3. Adenovirus vectors activate Akt via phosphatidylinositol 3-kinase (PI3-kinase). A549 cells or HBE cells were plated overnight and then treated with 20 µM LY-294002 (LY) for 1 h before infection. Replication-deficient recombinant adenovirus containing the nonspecific transgene for GFP or AdEV was added to the cells. After 24 h, cells were harvested for total cellular protein, and Western analysis was performed with an antibody specific for the Akt phosphorylated on serine 473. A: A549 cells; B: HBE cells. In a separate experiment, HBE cells were treated similarly and infected with wild-type Ad 5 virus (B).
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Adenovirus-mediated ERK and Akt activation occurs via independent pathways.
To test the hypothesis that adenovirus-mediated activation of the two survival pathways, ERK and Akt, occur independently, we treated A549 cells with and without inhibitors of these pathways and examined activation of ERK and Akt. Similar to the results shown in Figs. 2 and 3, both adenovirus vectors activated ERK and Akt at 24 h after infection. Specific inhibition of the MEK1/2 with U0126 did not affect phosphorylated Akt, nor did inhibition of PI3-kinase with LY-294002 affect ERK phosphorylation (data not shown). This suggests that activation of ERK and Akt by adenovirus occurs independently, via two distinct pathways.
ERK and Akt activation by adenovirus requires viral gene transcription.
Adenovirus has been shown to stimulate the host inflammatory response, resulting in the production of proinflammatory cytokines and chemokines and the activation of a number of signal transduction pathways including MAP kinases, focal adhesion kinase, and PI3-kinase (6, 19, 33, 34, 56). These responses occur early after virus binding and are independent of viral gene transcription. Because we noted a late increase in ERK and AKT phosphorylation, 24 h after virus infection, we asked whether or not viral gene transcription is required for this activation. In this set of experiments, the adenovirus vectors AdGFP and AdEV were inactivated by UV irradiation. The exact joules used for inactivation were determined by a dose-response experiment in which varying amounts of energy were used to inactivate the virus followed by the standard infection protocol (see MATERIALS AND METHODS for details). We used the energy level that abolished all but
0.01% of infectivity as measured by counting fluorescent cells in AdGFP-infected cells. Also, to confirm that the UV irradiation was not damaging the virus particle, we examined cells infected with either control or UV-inactivated AdGFP using scanning and transmission electron microscopy. These studies showed that the virus particles were intact and bound normally after UV inactivation (data not shown). Figure 4 shows that infection with adenovirus vectors increases ERK and Akt activation. In Fig. 4A, transduction with either AdGFP or AdEV results in increased phospho-ERK. This increase is abolished in the UV-inactivated virus-treated cells. Similarly, Fig. 4B shows activation of Akt in response to adenovirus vector transduction that disappears in the UV-inactivated virus groups. Similar results were obtained in separate experiments performed in HBE cells (data not shown). To determine that the lack of GFP fluorescence was not due to decreased amounts of virus from nonspecific binding to the plate, we performed a parallel experiment in which virus was plated but not irradiated and then added to the cells. In these cells, transduction efficiency was 100% as determined by fluorescence microscopy. This supports the hypothesis that late activation of ERK and Akt by adenovirus vectors requires transcriptionally competent virus.

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Fig. 4. ERK and Akt activation by adenovirus requires viral gene transcription. A549 cells were infected with active or UV-inactivated AdGFP and AdEV, and cells were harvested for total cell protein after 24 h. Western analysis was performed with phospho-specific antibodies to ERK (A) and Akt (B). After development of the immunoreactive bands, IgG was eluted from the membranes, and they were reprobed with antibodies against total ERK and total Akt. These blots are representative of 3 separate experiments. Identical results were obtained in similar experiments with HBE cells (results not shown).
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ERK and Akt activity is required for protection against apoptosis in adenovirus vector-infected cells.
Our data show that adenovirus vector infection is protective against TNF-mediated apoptosis in airway epithelial cells and that, as a result of virus infection, cells upregulate ERK and Akt activation. To assess the role of ERK and Akt in protection against apoptosis in adenovirus vector-infected airway epithelial cells, we examined apoptosis and cell survival after treatment of the cells with specific inhibitors of these pathways. Figure 5A shows the Western analysis for cleaved PARP from cells treated with an inhibitor of PI3-kinase activation (LY-294002) or ERK (U0126) with and without prior adenovirus vector infection. In cells treated with the inhibitors alone, there is very little apoptosis; however, if ERK or Akt activation is inhibited before adenovirus vector transduction, there is a marked increase in apoptosis. Importantly, the adenovirus vectors cause little or no apoptosis in the absence of other stimuli/inhibitors (see Fig. 1). Figure 5B shows the effect of ERK or Akt inhibition on cell survival and apoptosis in control and virus-infected cells after exposure to the proapoptotic stimulus TNF-
plus actinomycin D. In this experiment, cells were transduced with the adenovirus vector AdGFP for 2024 h and then treated with TNF/actinomycin or TNF/actinomycin plus inhibitors of ERK or Akt. Cell death measurements were obtained at 12 h posttreatment. As shown, TNF/actinomycin induces marked cell death, and prior viral infection significantly reduces this. If ERK or Akt activation is inhibited in the virus-infected cells, there is a loss of protection from apoptosis. Because the inhibitors are in a DMSO solution, we also performed a DMSO control that showed no differences in apoptosis compared with controls (data not shown).

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Fig. 5. ERK and Akt are important for cell survival in adenovirus vector-infected cells. Either A549 or HBE cells were infected with the recombinant AdGFP vector for 24 h and then treated with U0126 (20 µM), an inhibitor of ERK activation; LY-294002 (20 µM), an inhibitor of PI3-kinase; or both for 18 h. In some experiments, cells were also treated with TNF- plus actinomycin D (T/A) to induce apoptosis. A: apoptosis as measured by the presence of cleaved PARP. B: effect of ERK and Akt inhibition on cell survival (graph) and apoptosis (Western analysis) in virus-infected cells. Percent cell death was assessed with trypan blue staining of dead cells. A polyclonal antibody against cleaved PARP was used for the Western blot.
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DISCUSSION
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In these studies, we show that adenovirus vectors protect epithelial cells in vitro against TNF-
-induced cell death by activating two important prosurvival cell signaling pathways (ERK and PI3-kinase). Because the ERK and PI3-kinase signaling pathways modulate inflammation, as well as apoptosis, the observations of this study are consistent with earlier studies that evaluated the effect of adenovirus on inflammation (5, 6, 8, 11, 35, 43, 45, 46, 54, 56). Activation of inflammatory responses has been well described in utilizing first-generation recombinant adenovirus vectors. Adenovirus has also been shown to modulate a number of cell signaling pathways, including ERK and PI3-kinase, although the exact mechanisms through which the virus affects signal transduction are not well understood. Bruder and Kovesdi (6) found that HeLa cells infected with adenovirus vectors exhibited increases in Raf-1 and p42 (ERK) activation after infection. This occurred with a variety of adenovirus vectors and was correlated with increases in IL-8 expression. Tibbles and colleagues (56) also demonstrated activation of the MAP kinase pathway in response to adenovirus vector infection. In a mouse kidney cell line, they found that activation of ERK and p38 occurred early after adenovirus infection and was linked to downstream IP-10 expression. Finally, Tamanini et al. (54) have shown that adenovirus vectors stimulate MAP kinase activation and increases in NF-
B DNA binding after transduction of A549 cells and that this induces ICAM-1 gene expression. Similarly, we observed activation of ERK at early time points after viral infection. Activation occurred at 1 h postinfection, diminished to control levels by 3 h, and peaked again at 12 h with persistent activation to 24 h. This later activation coincides with late viral gene transcription and is abolished by UV inactivation of virus, suggesting that viral gene transcription is involved.
PI3-kinase has also been shown to be activated in response to adenovirus infection. Li and colleagues (34) examined mechanisms of integrin-mediated virus entry into the cell and found that adenovirus internalization and downstream activation of p130CAS were dependent on PI3-kinase. They also showed, in a separate study, that the Rho GTPases Cdc42 and Rac1 could be activated downstream of PI3-kinase by adenovirus infection, promoting endocytosis of the virus (32). Our data showed that Akt, a protein activated downstream of PI3-kinase, was minimally activated at 1 h postinfection and did not show activation again until 12 h after adenovirus transduction. As seen with ERK, activation of Akt by the adenovirus vectors was abolished if cells were infected with UV-treated, transcriptionally inactive virus.
Both Akt and ERK play important roles in cell survival. Akt, or protein kinase B, is an important effector protein for modulating various cell survival pathways. It is present as a cytoplasmic protein that is activated via phosphorylation by its upstream kinase, PI3-kinase (1). Akt is involved in the regulation of cell survival and protection from apoptosis induced by oxidant injury, Fas, UV irradiation, serum withdrawal, c-Myc, and anoikis (10, 13, 24, 25, 29, 36). It inhibits apoptosis by decreasing activation of caspase-3, caspase-9, Bad, and additional proapoptotic pathways linked to forkhead transcription factors and glycogen synthase kinase 3 (7, 28, 40). In our studies, we observed, consistent with prior studies, that TNF/actinomycin D triggered apoptosis by a process that involved caspase-3, resulting in downstream cleavage of PARP.
ERK activation via the MAP kinase signaling pathway is also important in modulating cell growth and survival (4, 15, 26). Signaling through this pathway usually involves a receptor-mediated event that may involve integrins, receptor tyrosine kinases, or Ca2+ influx via ion channels. This results in activation of Ras that allows for recruitment of Raf, downstream phosphorylation of MEK1/2, and subsequent activation of ERK. Activated ERK can then translocate to the nucleus and alter the activity of a number of transcription factors involved in cytokine gene transcription and inhibition of apoptosis (4, 14, 53). Activated ERK has also been shown to inhibit caspase-3 activation, a process that is relevant to these studies (12, 55, 61). We have shown that adenovirus vectors protect epithelial cells from TNF-induced apoptosis and that this effect is, in part, dependent on ERK and Akt activation, since inhibition of these pathways using specific chemical inhibitors results in loss of protection from apoptosis in adenovirus vector-transduced cells. Although wild-type adenovirus is well documented to have antiapoptotic effects on the host cell (17, 37, 47, 57), the only other studies that have shown adenovirus vector-mediated enhancement in cell survival and inhibition of apoptosis have been of the endothelium. Ramalingam and colleagues (51) found that cells infected with adenovirus vectors deleted in the E1 and E3 regions but containing the E4 gene (E1-E4+) exhibited a phenotype different than that of "usual" cultured endothelium and had prolonged viability in culture. They showed that the virus-infected cells had increased Bcl2, an important antiapoptotic protein, and decreased Bax, an initiator of apoptosis, which they hypothesized was involved in the observed changes in cell survival. They subsequently found that this effect was mediated by the adenovirus E4 region (65).
In wild-type adenovirus infections, a number of viral gene products are involved in modulating cellular metabolism, making the host cell more susceptible to viral replication and preventing host cell apoptosis (52). The E1B 19K protein is analogous to Bcl2 and blocks apoptosis by inactivating Bax, a protein involved in the induction of caspases in the death receptor pathway (17, 47). The E1A 12S protein also enables cells to overcome apoptosis and promotes oncogenesis (49). The E2 gene products are not known to be involved in cell survival, and their primary function is to provide the machinery for viral gene replication (18). E3 genes are involved in the inhibition of Fas-Fas ligand mediated apoptosis (37) and the cellular immune response to viral infection (20, 60). The adenovirus vectors used in these studies do not have fully functional E1 and E3 regions; therefore, the effects of these viral gene products cannot explain the observations of this study. The E4 genes, which are present in the adenovirus vectors, regulate viral DNA replication and downregulate host protein synthesis (16, 31). They may also block apoptosis by interfering with p53 transcriptional activation (9, 42). As suggested by Ramalingam and colleagues (51), any of the E4 gene products could also be linked to protection against apoptosis. Our studies show that adenovirus vector infection induces late activation of two important prosurvival pathways, ERK and Akt, a process that is dependent on viral gene transcription, although which viral genes are involved has yet to be defined. Furthermore, activation of these pathways contributes to cell survival in the face of apoptotic signals. The use of adenovirus vectors as a gene delivery system via the airways increases the importance of understanding epithelial biology subsequent to vector infection. This is the first study to demonstrate a role for adenovirus vectors in preventing cell death in lung epithelial cells. It will be interesting to determine whether this observation alters the normal response of the lung epithelium to viral or bacterial infections.
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GRANTS
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This manuscript was supported by a Veterans Affairs Merit Review grant and National Heart, Lung, and Blood Institute Grants HL-60316 (to G. W. Hunninghake) and HL-04428-01 (to D. M. Flaherty).
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FOOTNOTES
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Address for reprint requests and other correspondence: D. M. Flaherty, Div. of Pulmonary, Critical Care, and Occupational Medicine, Univ. of Iowa Hospitals and Clinics, C-33 GH, Iowa City, Iowa 52242 (E-mail: flahertydm{at}mail.medicine.uiowa.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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REFERENCES
|
---|
- Anderson RA, Boronenkov IV, Doughman SD, Kunz J, and Loijens JC. Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes. J Biol Chem 274: 99079910, 1999.[Free Full Text]
- Anderson RD, Haskell RE, Xia H, Roessler BJ, and Davidson BL. A simple method for the rapid generation of recombinant adenovirus vectors. Gene Ther 7: 10341038, 2000.[CrossRef][ISI][Medline]
- Bhat NR and Fan F. Adenovirus infection induces microglial activation: involvement of mitogen-activated protein kinase pathways. Brain Res 948: 93101, 2002.[CrossRef][ISI][Medline]
- Bonni A, Brunet A, West AE, Datta SR, Takasu MA, and Greenberg ME. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286: 13581362, 1999.[Abstract/Free Full Text]
- Bowen GP, Borgland SL, Lam M, Libermann TA, Wong NC, and Muruve DA. Adenovirus vector-induced inflammation: capsid-dependent induction of the C-C chemokine RANTES requires NF-kappa B. Hum Gene Ther 13: 367379, 2002.[CrossRef][ISI][Medline]
- Bruder JT and Kovesdi I. Adenovirus infection stimulates the Raf/MAPK signaling pathway and induces interleukin-8 expression. J Virol 71: 398404, 1997.[Abstract]
- Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, and Reed JC. Regulation of cell death protease caspase-9 by phosphorylation. Science 282: 13181321, 1998.[Abstract/Free Full Text]
- Dai Y, Schwarz EM, Gu D, Zhang WW, Sarvetnick N, and Verma IM. Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc Natl Acad Sci USA 92: 14011405, 1995.[Abstract]
- Dobner T, Horikoshi N, Rubenwolf S, and Shenk T. Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor. Science 272: 14701473, 1996.[Abstract]
- Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DR, and Greenberg ME. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275: 661665, 1997.[Abstract/Free Full Text]
- Engelhardt JF, Ye X, Doranz B, and Wilson JM. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci USA 91: 61966200, 1994.[Abstract]
- Erhardt P, Schremser EJ, and Cooper GM. B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/Erk pathway. Mol Cell Biol 19: 53085315, 1999.[Abstract/Free Full Text]
- Eves EM, Xiong W, Bellacosa A, Kennedy SG, Tsichlis PN, Rosner MR, and Hay N. Akt, a target of phosphatidylinositol 3-kinase, inhibits apoptosis in a differentiating neuronal cell line. Mol Cell Biol 18: 21432152, 1998.[Abstract/Free Full Text]
- Garrington TP and Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol 11: 211218, 1999.[CrossRef][ISI][Medline]
- Gutkind JS. The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 273: 18391842, 1998.[Free Full Text]
- Halbert DN, Cutt JR, and Shenk T. Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff. J Virol 56: 250257, 1985.[ISI][Medline]
- Han J, Sabbatini P, Perez D, Rao L, Modha D, and White E. The E1B 19K protein blocks apoptosis by interacting with and inhibiting the p53-inducible and death-promoting Bax protein. Genes Dev 10: 461477, 1996.[Abstract]
- Hay RT, Freeman A, Leith I, Monaghan A, and Webster A. Molecular interactions during adenovirus DNA replication. Curr Top Microbiol Immunol 199: 3148, 1995.[Medline]
- Higginbotham JN, Seth P, Blaese RM, and Ramsey WJ. The release of inflammatory cytokines from human peripheral blood mononuclear cells in vitro following exposure to adenovirus variants and capsid. Hum Gene Ther 13: 129141, 2002.[CrossRef][ISI][Medline]
- Ilan Y, Droguett G, Chowdhury NR, Li Y, Sengupta K, Thummala NR, Davidson A, Chowdhury JR, and Horwitz MS. Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits long-term gene expression. Proc Natl Acad Sci USA 94: 25872592, 1997.[Abstract/Free Full Text]
- Jornot L, Petersen H, Lusky M, Pavirani A, Moix I, Morris, and Rochat T. Effects of first generation E1E3-deleted and second generation E1E3E4-deleted/modified adenovirus vectors on human endothelial cell death. Endothelium 8: 167179, 2001.[Medline]
- Joseph TD and Look DC. Specific inhibition of interferon signal transduction pathways by adenoviral infection. J Biol Chem 276: 4713647142, 2001.[Abstract/Free Full Text]
- Kafri T, Morgan D, Krahl T, Sarvetnick N, Sherman L, and Verma I. Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy. Proc Natl Acad Sci USA 95: 1137711382, 1998.[Abstract/Free Full Text]
- Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C, Coffer P, Downward J, and Evan G. Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 385: 544548, 1997.[CrossRef][ISI][Medline]
- Khwaja A, Rodriguez-Viciana P, Wennstrom S, Warne PH, and Downward J. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J 16: 27832793, 1997.[Abstract/Free Full Text]
- King WG, Mattaliano MD, Chan TO, Tsichlis PN, and Brugge JS. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation. Mol Cell Biol 17: 44064418, 1997.[Abstract]
- Knowles MR, Hohneker KW, Zhou Z, Olsen JC, Noah TL, Hu PC, Leigh MW, Engelhardt JF, Edwards LJ, Jones KR, Grossman M, Wilson JM, Johnson LG, and Boucher RC. A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N Engl J Med 333: 823831, 1995.[Abstract/Free Full Text]
- Kops GJ, de Ruiter ND, De Vries-Smits AM, Powell DR, Bos JL, and Burgering BM. Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398: 630634, 1999.[CrossRef][ISI][Medline]
- Kulik G and Weber MJ. Akt-dependent and -independent survival signaling pathways utilized by insulin-like growth factor I. Mol Cell Biol 18: 67116718, 1998.[Abstract/Free Full Text]
- Lazrak A, Samanta A, and Matalon S. Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells. Am J Physiol Lung Cell Mol Physiol 278: L848L857, 2000.[Abstract/Free Full Text]
- Leppard KN. E4 gene function in adenovirus, adenovirus vector and adeno-associated virus infections. J Gen Virol 78: 21312138, 1997.[Free Full Text]
- Li E, Stupack D, Bokoch GM, and Nemerow GR. Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases. J Virol 72: 88068812, 1998.[Abstract/Free Full Text]
- Li E, Stupack D, Klemke R, Cheresh DA, and Nemerow GR. Adenovirus endocytosis via alpha(v) integrins requires phosphoinositide-3-OH kinase. J Virol 72: 20552061, 1998.[Abstract/Free Full Text]
- Li E, Stupack DG, Brown SL, Klemke R, Schlaepfer DD, and Nemerow GR. Association of p130CAS with phosphatidylinositol-3-OH kinase mediates adenovirus cell entry. J Biol Chem 275: 1472914735, 2000.[Abstract/Free Full Text]
- Liu Q and Muruve DA. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther 10: 935940, 2003.[CrossRef][ISI][Medline]
- Lu Y, Parkyn L, Otterbein LE, Kureishi Y, Walsh K, Ray A, and Ray P. Activated Akt protects the lung from oxidant-induced injury and delays death of mice. J Exp Med 193: 545549, 2001.[Abstract/Free Full Text]
- Lukashok SA, Tarassishin L, Li Y, and Horwitz MS. An adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis complexes with dynein and a small GTPase. J Virol 74: 47054709, 2000.[Abstract/Free Full Text]
- MacFarlane M, Merrison W, Dinsdale D, and Cohen GM. Active caspases and cleaved cytokeratins are sequestered into cytoplasmic inclusions in TRAIL-induced apoptosis. J Cell Biol 148: 12391254, 2000.[Abstract/Free Full Text]
- Maeyama T, Kuwano K, Kawasaki M, Kunitake R, Hagimoto N, Matsuba T, Yoshimi M, Inoshima I, Yoshida K, and Hara N. Upregulation of Fas-signalling molecules in lung epithelial cells from patients with idiopathic pulmonary fibrosis. Eur Respir J 17: 180189, 2001.[Abstract/Free Full Text]
- Maiti D, Bhattacharyya A, and Basu J. Lipoarabinomannan from Mycobacterium tuberculosis promotes macrophage survival by phosphorylating Bad through a phosphatidylinositol 3-kinase/Akt pathway. J Biol Chem 276: 329333, 2001.[Abstract/Free Full Text]
- Mastronarde JG, He B, Monick MM, Mukaida N, Matsushima K, and Hunninghake GW. Induction of interleukin (IL)-8 gene expression by respiratory syncytial virus involves activation of nuclear factor (NF)-kappa B and NF-IL-6. J Infect Dis 174: 262267, 1996.[ISI][Medline]
- Moore M, Horikoshi N, and Shenk T. Oncogenic potential of the adenovirus E4orf6 protein. Proc Natl Acad Sci USA 93: 1129511301, 1996.[Abstract/Free Full Text]
- Muruve DA, Barnes MJ, Stillman IE, and Libermann TA. Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo. Hum Gene Ther 10: 965976, 1999.[CrossRef][ISI][Medline]
- Nagata S. Apoptosis by death factor. Cell 88: 355365, 1997.[ISI][Medline]
- Nicolis E, Tamanini A, Melotti P, Rolfini R, Berton G, Cassatella MA, Bout A, Pavirani A, and Cabrini G. ICAM-1 induction in respiratory cells exposed to a replication-deficient recombinant adenovirus in vitro and in vivo. Gene Ther 5: 131136, 1998.[CrossRef][ISI][Medline]
- Noah TL, Wortman IA, Hu PC, Leigh MW, and Boucher RC. Cytokine production by cultured human bronchial epithelial cells infected with a replication-deficient adenoviral gene transfer vector or wild-type adenovirus type 5. Am J Respir Cell Mol Biol 14: 417424, 1996.[Abstract]
- Ohi N, Tokunaga A, Tsunoda H, Nakano K, Haraguchi K, Oda K, Motoyama N, and Nakajima T. A novel adenovirus E1B19K-binding protein B5 inhibits apoptosis induced by Nip3 by forming a heterodimer through the C-terminal hydrophobic region. Cell Death Differ 6: 314325, 1999.[CrossRef][ISI][Medline]
- Oliver FJ, de la Rubia G, Rolli V, Ruiz-Ruiz MC, de Murcia G, and Murcia JM. Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. J Biol Chem 273: 3353333539, 1998.[Abstract/Free Full Text]
- Quinlan MP. E1A 12S in the absence of E1B or other cooperating oncogenes enables cells to overcome apoptosis. Oncogene 8: 32893296, 1993.[ISI][Medline]
- Ramalingam R, Rafii S, Worgall S, Brough DE, and Crystal RG. E1(-)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells. Blood 93: 29362944, 1999.[Abstract/Free Full Text]
- Ramalingam R, Rafii S, Worgall S, Hackett NR, and Crystal RG. Induction of endogenous genes following infection of human endothelial cells with an E1(-) E4(+) adenovirus gene transfer vector. J Virol 73: 1018310190, 1999.[Abstract/Free Full Text]
- Russell WC. Update on adenovirus and its vectors. J Gen Virol 81: 25732604, 2000.[Free Full Text]
- Schaeffer HJ and Weber MJ. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 19: 24352444, 1999.[Free Full Text]
- Tamanini A, Rolfini R, Nicolis E, Melotti P, and Cabrini G. MAP kinases and NF-kappaB collaborate to induce ICAM-1 gene expression in the early phase of adenovirus infection. Virology 307: 228242, 2003.[CrossRef][ISI][Medline]
- Tashker JS, Olson M, and Kornbluth S. Post-cytochrome C protection from apoptosis conferred by a MAPK pathway in Xenopus egg extracts. Mol Biol Cell 13: 393401, 2002.[Abstract/Free Full Text]
- Tibbles LA, Spurrell JC, Bowen GP, Liu Q, Lam M, Zaiss AK, Robbins SM, Hollenberg MD, Wickham TJ, and Muruve DA. Activation of p38 and ERK signaling during adenovirus vector cell entry lead to expression of the C-X-C chemokine IP-10. J Virol 76: 15591568, 2002.[Abstract/Free Full Text]
- Tollefson AE, Hermiston TW, Lichtenstein DL, Colle CF, Tripp RA, Dimitrov T, Toth K, Wells CE, Doherty PC, and Wold WS. Forced degradation of Fas inhibits apoptosis in adenovirus-infected cells. Nature 392: 726730, 1998.[CrossRef][ISI][Medline]
- Wang R, Alam G, Zagariya A, Gidea C, Pinillos H, Lalude O, Choudhary G, Oezatalay D, and Uhal BD. Apoptosis of lung epithelial cells in response to TNF-alpha requires angiotensin II generation de novo. J Cell Physiol 185: 253259, 2000.[CrossRef][ISI][Medline]
- Watanabe N, Dickinson DA, Krzywanski DM, Iles KE, Zhang H, Venglarik CJ, and Forman HJ. A549 subclones demonstrate heterogeneity in toxicological sensitivity and antioxidant profile. Am J Physiol Lung Cell Mol Physiol 283: L726L736, 2002.[Abstract/Free Full Text]
- Wold WS and Gooding LR. Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions. Virology 184: 18, 1991.[ISI][Medline]
- Xia Z, Dickens M, Raingeaud J, Davis RJ, and Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 13261331, 1995.[Abstract]
- Yang Y, Ertl HC, and Wilson JM. MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity 1: 433442, 1994.[ISI][Medline]
- Yang Y, Li Q, Ertl HC, and Wilson JM. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 69: 20042015, 1995.[Abstract]
- Yarovinsky TO, Powers LS, Butler NS, Bradford MA, Monick MM, and Hunninghake GW. Adenoviral infection decreases mortality from lipopolysaccharide-induced liver failure via induction of TNF-alpha tolerance. J Immunol 171: 24532460, 2003.[Abstract/Free Full Text]
- Zhang F, Cheng J, Hackett NR, Lam G, Shido K, Pergolizzi R, Jin DK, Crystal RG, and Rafii S. Adenovirus E4 gene promotes selective endothelial cell survival and angiogenesis via activation of the vascular endothelial-cadherin/Akt signaling pathway. J Biol Chem 279: 1176011766, 2004.[Abstract/Free Full Text]