Scott and White Clinic, Texas A&M University System Health Science Center, College of Medicine, Temple, Texas 76508
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ABSTRACT |
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Double-stranded RNA (dsRNA) is produced during replicative viral infection or genotoxic stress. Thus knowledge of the cellular response to dsRNA is necessary to understand the effects of DNA damage or viral infection in biliary epithelia. We assessed the effect of dsRNA on biliary epithelial cell proliferation and apoptosis and the role of the stress-activated p38 MAPK signaling pathway in these responses. dsRNA did not induce apoptosis or proliferation in Mz-ChA-1 human malignant cholangiocytes, but decreased cytotoxicity induced by camptothecin or tumor necrosis factor-related apoptosis inducing ligand and decreased activity of caspases 3, 8, and 9. Furthermore, dsRNA increased p38 MAPK and JNK kinase active site phosphorylation but had no effect on either MAPK kinase (MEK)1/2 or protein kinase R phosphorylation. Inhibition of p38 MAPK with SB-203580 increased basal caspase activity. Thus dsRNA stimulates a p38 MAPK-dependent cell-survival pathway in biliary epithelial cells that may modulate the response of the biliary epithelia to dsRNA produced during genotoxic injury or virus infection.
cholangiocyte; protein kinase R; caspase; apoptosis; mitogen-activated protein kinase
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INTRODUCTION |
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DOUBLE-STRANDED RNA (dsRNA) molecules are not a major component of mammalian cells but are actively formed intracellularly during viral infection and genotoxic stress (9). Minute quantities of dsRNA can profoundly alter the cellular physiology, and dsRNA can induce several genes involved in diverse cellular processes including apoptosis, RNA synthesis, protein synthesis, cell metabolism, transport, and maintenance of cell structure (4). During viral replication, dsRNA may be produced as an essential replicative intermediate for RNA synthesis or as a by-product generated by annealing of complementary RNAs encoded by the opposite strands of a DNA virus genome (9). Host cellular response to dsRNA produced during viral infection involves the activation of antiviral responses that result in host cell apoptosis or promote survival of noninfected cells. Induction of apoptosis by dsRNA thus serves as a powerful mechanism to limit viral infection by the elimination of virally infected cells (10).
Several kinase signaling pathways involved in cellular responses to stress have been identified. These include JNK, p38 MAPK, and extracellularly regulated p44/p42 MAPK. Although p38 MAPK is a classical stress-activated protein kinase, the role of p38 MAPK signaling in the cellular response to dsRNA remains poorly understood. We have shown constitutive expression of the p38 MAPK signaling pathway in malignant cholangiocytes (18). Furthermore, p38 MAPK signaling maintains a transformed cell phenotype in malignant human cholangiocytes (19). These observations suggest a role for aberrant cellular stress-mediated signaling in maintaining the malignant phenotype in cholangiocytes.
Some of the cellular effects of dsRNA are mediated through the
dsRNA-dependent protein kinase R (PKR), a 65- to 68-kDa
serine-threonine kinase activated by binding to dsRNA (reviewed in Ref.
23). PKR is involved in several signaling pathways
mediating stress responses and antiproliferative and apoptotic
responses (20). In addition to mediating virally induced
apoptosis, PKR has been found to be associated with STAT1,
phosphorylate p53, and mediate NF-B signaling (14, 25).
Although PKR has been postulated as having tumor suppressor effects,
this remains to be proven. However, PKR is expressed in proliferating
cholangiocytes and in malignant cholangiocarcinoma (21).
These suggest that cholangiocyte responses to dsRNA may be altered
during cellular proliferation or carcinogenesis.
Although dsRNA can modulate the expression of a wide variety of genes with profound physiological consequences, the cellular responses of biliary epithelia to dsRNA are unknown. Knowledge of these responses is central to understanding the effects of cellular stress related to DNA damage or viral infection in biliary epithelia. Thus the aims of our study were to study the cellular response to dsRNA and, in particular, to assess the role of the stress-activated p38 MAPK signaling pathway in these responses. We therefore asked the following questions. Can dsRNA elicit a biologically relevant response in biliary epithelia? Is apoptosis or proliferation perturbed in response to dsRNA? Are stress-activated protein kinases activated by dsRNA? Does activation of p38 MAPK signaling mediate the cellular response to dsRNA?
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EXPERIMENTAL PROCEDURES |
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Cell lines and culture. Mz-ChA-1 cells, malignant human cholangiocytes, (kindly provided by Dr. J. G. Fitz, University of Colorado, Denver, CO) were used for these studies. The cells were cultured in CMRL 1066 media with 10% fetal bovine serum, 1% L-glutamine, and 1% antimycotic antibiotic mix. H69 cells (nonmalignant human cholangiocytes) and KMCH-1 cells (malignant human cholangiocytes) were obtained and cultured as previously described (17, 18).
Cytokine gene expression. Macroarray experiments were performed by using the GE Array Expression Array kit (SuperArray, Bethesda, MD) following the manufacturer's instructions without any modifications. In brief, total RNA was isolated from cells incubated with or without dsRNA for 24 h. Biotinylated cDNA probes were synthesized by using MMLV reverse transcriptase and biotin-16-dUTP. The probes were then hybridized to nylon membranes containing cDNA fragments from cytokine genes (original series human common cytokine array). Membranes were then incubated with alkaline phosphatase-conjugated streptavidin. Gene expression was detected by chemiluminescence using the alkaline phosphatase substrate CDP-Star provided in the kit. The array image was recorded and analyzed by using a charge-coupled device (CCD) camera-based imaging station (MultiImager; Alpha Innotech, San Leandro, CA). Expression analysis was performed by using the GE ArrayAnalyzer software (SuperArray). Background subtraction was performed by subtracting the readings from plasmid DNA (PUC 18) negative controls. Cytokine gene expression was normalized to GAPDH expression and expressed as a ratio of expression in dsRNA-treated cells compared with untreated controls. A ratio of <0.5 or >2.0 was considered to represent a meaningful (at least twofold) difference in expression.
Proliferation assay. Cells were seeded into 96-well plates (10,000 cells/well) and incubated in a final volume of 200 µl of medium. Cell proliferation was assessed as previously described by using a commercially available colorimetric cell proliferation assay (CellTiter 96 AQueous; Promega, Madison, WI) (19).
Apoptosis assays. Morphological changes indicative of cell death by apoptosis were identified and quantitated by fluorescence microscopy and the use of acridine orange as previously described (18). Fluorescence was visualized by using an upright fluorescence microscope (model BX40; Olympus America, Melville, NY). Apoptotic nuclei were identified by condensed chromatin as well as nuclear fragmentation. At least 300 nuclei in four high-power fields were counted. Biochemical changes of apoptosis resulting in DNA fragmentation were assessed by using a cell death colorimetric enzyme immunoassay detection ELISA kit to quantitate cytoplasmic histone-associated DNA fragments (Cell Death Detection ELISA; Roche Biochemicals, Indianapolis, IN). Cells were incubated for 24 h and assayed following the manufacturer's instructions.
Viability assay. Cells were seeded into 96-well plates (10,000 cells/well) and incubated in a final volume of 200 µl of medium. For the kinase inhibitor studies, cells were preincubated with the inhibitor for 1 h. The kinase inhibitors were stored as concentrated stock solutions in DMSO. Controls contained equivalent volumes of DMSO to those used in the inhibitors. Cells were then incubated with varying concentrations of dsRNA for 24 h before the addition of either camptothecin or TNF-related apoptosis-inducing ligand (TRAIL). Cell viability was assessed by using a commercial tetrazolium bioreduction assay for viable cells and was expressed as a percentage of control (CellTiter 96 Aqueous; Promega).
Generation of stably transfected cell lines. Stably transfected cell lines were generated from parental Mz-ChA-1 malignant human cholangiocytes. Cells transfected with pRc/RSV-Flag MKK3 (Ala) (encoding a dominant interfering upstream activator of p38 MAPK with double-point mutations in Ser189 and Thr193 replaced by Ala) had decreased constitutively and stimulated p38 MAPK activity compared with control cells transfected with pRc/RSV-Flag MKK3. Expression plasmids were kindly provided by Dr. Roger Davis (Howard Hughes Medical Institute, Worcester, MA). Plasmids were purified by using the Plasmid Midi Kit (Qiagen, Valencia, CA) and linearized by restriction enzyme digestion before transfection by using Trans-IT (Panvera, Madison, WI). After 48 h, the media were replaced with media containing G418. Stable transfection was confirmed after 3 wk by immunostaining with the M2 monoclonal antibody to the Flag epitope, by using a fluorescein-conjugated goat anti-mouse secondary antibody and fluorescence microscopy to visualize stable transfectants.
Measurement of cytosolic caspase activity. Cells were washed with PBS, and lysed with 1 ml of a hypotonic buffer containing (in mM) 25 HEPES, 5 MgCl2, 1 EGTA, and freshly added 0.5 PMSF plus 2 µg/ml pepstatin and 2 µg/ml leupeptin. Cells were then homogenized by 20 strokes by using a Tissue Tearor (Biospec, Bartlesville, OK). The homogenate was centrifuged at 21,000 g for 45 min at 4°C by using a Microfuge R centrifuge (Beckman Instruments, Palo Alto, CA). The protein content in the supernatant cytosolic fraction was measured by using the Bradford reagent. Caspase activity was assayed by adding 50 µl of cytosolic protein to 0.45 ml of buffer containing (in mM) 25 HEPES (pH 7.5), 10 dithiothreitol, 0.5 PMSF, plus 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 2 µl/ml aprotinin, and 20 µM fluorogenic substrate. The substrates used were Ile-Glu-Thr-Asp (IETD)-7-amino-4-trifluoromethylcoumarin (-AFC), Leu-Glu-His-Asp (LEHD)-7-amino-4-methylcoumarin (-AMC) and Asp-Glu-Val-Asp (DEVD)-AMC for caspase 8-, 9- and 3-like activities, respectively. After incubation at 37°C for 30 min, 1 ml of dH2O was added. The change in fluorescence intensity was measured over 30 min by using a fluorometer (model TD700; Turner Designs, Mountain View, CA) with excitation and emission wavelengths of 360 and 460 nm. With each experiment, standard curves were generated with AMC or AFC, and caspase activity was expressed as picomole AMC or AFC per milligram protein per minute.
Immunoblot analysis. Confluent cells in culture were trypsinized and sonicated for 20 s at 4°C (Sonic dismembrator; Fisher Scientific, Pittsburgh, PA) in a lysis buffer containing (in mM) 50 Tris base, 2 EDTA, 100 NaCl, plus 1% NP-40 and one miniprotease inhibitor cocktail tablet. Protein content was determined by the Bradford assay. Protein samples were separated on 4-12% gradient polyacrylamide gels (Novex, San Diego, CA) under reducing conditions and electroblotted to positively charged 0.45 µM nitrocellulose membrane (Millipore, Bedford, CA). Membranes were soaked for 5 min in transfer buffer (13.4 mM Tris, pH 8.3, 20% methanol, 108 mM glycine). Blots were preblocked in 20 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk, for 3-4 h or overnight at 4°C. Kinase expression levels were assessed by using phosphorylation state-independent polyclonal rabbit antibodies, and the presence of phosphorylated (activated) kinase was measured by using phosphorylation state-specific antibodies to PKR, p38 MAPK, MEK1/2, or JNK as previously described (19). Membranes were incubated overnight at 4°C with the respective anti-human primary antibody used at a 1:1,000 dilution. Primary antibodies were diluted in a solution containing 20 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk. Membranes were washed three times for 10 min with 20 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (TTBS) and then incubated with the secondary antibody, a polyclonal goat anti-rabbit immunoglobulin-peroxidase conjugate (Zymed, San Francisco, CA) at a 1:5,000 dilution for 60 min at 4°C. The secondary antibody was diluted in TTBS buffer. For all immunoblots, membranes were washed three times for 10 min with TTBS and were then visualized by using an enhanced chemiluminescence kit (ECL plus; Amersham Biosciences, Piscataway, NJ) following the manufacturer's directions. The relative activity of phosphorylated to total kinase expression was determined by densitometry by using a CCD camera-based image analyzer (MultiImager; Alpha Innotech).
NF-B activation assay.
Activation of NF-
B was determined with the use of TransAM assay
(Active Motif, Carlsbad, CA) following the manufacturer's instructions. Briefly, cells cultured in 35-mm dishes were washed with
ice-cold PBS and removed by trypsinization. The cells were centrifuged
for 10 min at 1,000 rpm at 4°C and resuspended in 100 µl of lysis
buffer at 4°C. Lysates (5 µg of total protein) were incubated in
96-well dishes containing immobilized oligonucleotides containing the
NF-
B consensus DNA-binding site (5'-GGGACTTTCC-3') for 1 h at
room temperature. Wells were then washed three times, and 100 µl of
p65 subunit monoclonal antibody (1:1,000 dilution) were added to
each well for 1 h at room temperature. Wells were washed three
times, and 100 µl of horseradish peroxidase-conjugated secondary
antibody (1:1,000 dilution) then were added to each well for 1 h
at room temperature. Wells were washed four times, and 100 µl of
developing solution were added to each well for 10 min at room
temperature. Stop solution (100 µl) was added to each well and the
absorbance at 450 nm was determined by using a Versamax plate
spectrophotometer. Specificity of binding was determined with the use
of 200-fold excess wild-type and mutated NF-
B oligonucleotides.
Extracts from HeLa cells treated with TNF-
were used as positive controls.
Materials. Polyinosinic-polycytidylic acid [poly(dI-dC)], fetal bovine serum, Bradford reagent, and camptothecin were obtained from Sigma (St. Louis, MO). CMRL 1066 media, L-glutamine, and antibiotic-antimycotic mix were from GIBCO-BRL (Grand Island, New York). P38 MAPK-, JNK-, and MEK1/2-specific antibodies were obtained from Cell Signaling (Beverly, MA). PKR antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and phosphospecific PKR antibodies were obtained from Biosource (Camarillo, CA). Kinase inhibitors and cycloheximide were obtained from Calbiochem-Novabiochem (San Diego, CA). Protease inhibitor cocktail tablets were obtained from Roche Molecular Biochemicals (Indianapolis, IN). All other reagents were of analytical grade from the usual commercial sources. Soluble, recombinant human TRAIL (Killer TRAIL) was obtained from Alexis Biochemicals (Carlsbad, CA). Monoclonal anti-human IL-6R antibody was from R&D Systems (Minneapolis, MN).
Statistical analysis. Data are expressed as the means ± SD from at least three separate experiments performed in triplicate, unless otherwise noted. The differences between groups were analyzed by using a double-sided Student's t-test when only two groups were present. For repeated measures among multiple groups, analysis was performed by using ANOVA with a post hoc Bonferroni test to correct for multiple comparisons. Statistical significance was considered as P < 0.05. Statistical analyses were performed with the GB-STAT statistical software program (Dynamic Microsystems, Silver Spring, MD)
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RESULTS |
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dsRNA elicits a cellular response in malignant human
cholangiocytes.
We began by first determining whether a biologically relevant cellular
response could be elicited in human Mz-ChA-1 cells in response to
incubation with dsRNA. For our studies, we used poly(dI-dC), a
synthetic dsRNA molecule that is not virus specific. At the
concentrations used for these studies, dsRNA was found to be nontoxic
by using a viable cell assay and did not significantly alter cell
proliferation. Expression of proinflammatory cytokines is
commonly observed in infected epithelial cells. Thus we assessed the
effect of incubation with this dsRNA molecule on cytokine gene
expression in cholangiocytes by using a membrane-based cDNA hybridization macroarray. Incubation with dsRNA (5 µg/ml) for 24 h resulted in altered gene expression of several cytokines that are
important mediators of inflammation. The most prominent changes
observed included alterations in interferon-, IL-6, and transforming
growth factor
-1 (TGF-
) (Fig. 1).
Thus incubation with dsRNA elicits biologically relevant cellular
responses in Mz-ChA-1 cells.
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dsRNA does not induce apoptosis in malignant
cholangiocytes.
Apoptosis is a common response to virally infected cells, and
can be induced by dsRNA in several cell types, including hepatocytes (3). However, the lack of toxicity observed during
incubation with dsRNA prompted an assessment of whether or not dsRNA
could induce apoptosis in Mz-ChA-1 human cholangiocytes. Cells
were incubated with varying concentrations of dsRNA, and the occurrence of apoptotic cell death was assessed by using both
morphological and biochemical criteria. First, cells demonstrating
characteristic nuclear morphological changes of apoptosis were
quantitated (Fig. 2A). We then
used an ELISA-based assay for the identification of histone-bound DNA
(Fig. 2B). In support of these observations, we were also
unable to demonstrate cleavage of poly(ADP-ribose) polymerase
by Western blot analysis of protein extracts from cells incubated with dsRNA (data not shown). These complementary assays thus
showed that incubation with dsRNA does not induce apoptosis in
Mz-ChA-1 human cholangiocytes. Similar results (using morphological assays) were observed with both nonmalignant (H69) and malignant (KMCH-1) human cholangiocyte cells (data not shown). Thus incubation with dsRNA does not induce cellular apoptosis in human
cholangiocytes.
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dsRNA decreases toxin or death receptor-mediated cytotoxicity in
Mz-ChA-1 human cholangiocytes.
Cytoprotective responses may be elicited in response to viral infection
or stress to limit intracellular damage. Therefore, we studied whether
dsRNA was cytoprotective. Endogenous inducers of apoptosis
include death receptor ligands as well as chemotherapeutic agents. To
assess whether dsRNA could protect against apoptosis, we
assessed cytotoxicity in response to the death receptor ligand TRAIL or
the chemotherapeutic agent camptothecin. In preliminary studies, we
established the concentration dependency of cytotoxicity in response to
these agents. For our next studies, we chose concentrations of these
agents that resulted in 40-60% cytotoxicity after 24 h.
Cells were preincubated with dsRNA for 24 h before
incubation with TRAIL or camptothecin at these concentrations, and
toxicity was assessed after 24 h (Fig.
3). Incubation with dsRNA decreased toxicity due to either camptothecin or TRAIL. Because dsRNA increases the expression of IL-6, we assessed the role of IL-6-mediated signaling
on the cellular response to dsRNA. Incubation with neutralizing antibodies for human IL-6 did not alter dsRNA cytoprotection from camptothecin cytotoxicity. Cell viability in cells treated for 24 h with camptothecin (100 nM) was 85 ± 8% in cells preincubated with 5 µg/ml dsRNA alone compared with 82 ± 7% in cells
preincubated with dsRNA and anti-IL-6 (1 µg/ml). Thus indirect
effects through IL-6 signaling are unlikely to contribute to the cell
survival observed in response to dsRNA. These findings suggested that
dsRNA might preferentially activate a mechanism that protects against apoptosis. We thus postulated that cytoprotection by dsRNA may represent a mechanism to limit cell death during genotoxic stress or
viral infection in vivo.
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dsRNA decreases basal caspase activity in Mz-ChA-1 human
cholangiocytes.
Activation of caspases represents a common intracellular mechanism
culminating in apoptosis. We therefore assessed the effect of
dsRNA on activation of intracellular caspases assessed fluorometrically by using peptide aminomethyl or trifluoromethyl coumarin substrates. Caspase-3-like activity was increased during incubation with
camptothecin. However, the increased caspase-3-like activity
was decreased in cells preincubated with dsRNA for 24 h before
incubation with camptothecin (Fig. 4).
Unexpectedly, basal levels of caspase-3-like activity were decreased in
the presence of dsRNA alone. Furthermore, incubation with
dsRNA decreased the hydrolytic activity of caspase-3-, -8-, and -9-like activity under basal conditions (Fig.
5). Preincubation with the protein
synthesis inhibitor cycloheximide (100 µg/ml) inhibited the effect of
dsRNA on caspase-3-like activity. Thus the protective effect of dsRNA
is likely to be mediated by altered expression of genes or proteins
that act upstream of caspase activation and are effective under basal
conditions in the absence of signaling pathways inducing
apoptosis.
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Differential activation of intracellular protein kinases by dsRNA.
Because protein kinase activation participates in multiple cellular
responses to external stress, such as infection, we assessed the role
of several stress-activated protein kinases. First, kinase activation
was assessed by the use of phosphorylation state-specific antibodies to
p38 MAPK, MEK1/2, and JNK. Incubation of Mz-ChA-1 cells with dsRNA
increased active site phosphorylation of the p38 MAPK as well as the
JNK but not MEK1/2, which resulted in activation of the p44/p42 MAPK
(Fig. 6). The differential activation of
kinases with selective activation of the stress-associated protein
kinase signaling pathways indicates that the cellular response to dsRNA
is similar to that in response to exposure to bacterial products or
inflammatory cytokines, such as IL-1 and TNF-. Similar to our
previous observations with serum stimulation (19), we did
not detect an increase in p38 MAPK activation in H69 nonmalignant human
cholangiocytes in response to incubation with 5 µg/ml dsRNA. p38 MAPK
activity was not increased during incubation with camptothecin (100 nM)
or TRAIL 200 ng/ml for 24 h.
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dsRNA-dependent kinase PKR is not involved in dsRNA-mediated cell
survival in malignant human cholangiocytes.
The dsRNA-dependent protein kinase PKR plays a fundamental role in
limiting viral replication and inhibiting protein synthesis and is an
important mediator of the cellular response to dsRNA. Because this
molecule participates in a variety of cell- signaling pathways involved
in induction or inhibition of apoptosis, we assessed PKR
expression and activation in Mz-ChA-1 cholangiocytes. Indeed, PKR was
constitutively expressed. However, phosphorylation of dsRNA was not
increased by dsRNA (Fig. 8). Furthermore,
basal caspase-3-like activity was not altered by the use of the PKR inhibitor 2-aminopurine (Fig. 9).
Likewise, cell viability was not altered in cells incubated with 5 µg/ml dsRNA by pretreatment with 1 mM 2-aminopurine for 30 min. PKR
activation has been linked to activation of the transcription factor
NF-B (14). However, NF-
B is not activated in
cholangiocytes during incubation with dsRNA (Fig.
10). Thus neither PKR nor NF-
B are
involved in the cellular response to dsRNA. Although PKR can induce
apoptosis in response to diverse stimuli, such as serum
deprivation and viral infection in other cell types (i.e.,
hepatocytes), the resistance to apoptosis in the setting of PKR
expression suggests that PKR apoptosis pathways are being
circumvented in human cholangiocytes.
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DISCUSSION |
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These studies emphasize the cell-type specificity of the cellular response to dsRNA. In contrast to several other epithelial cell types, malignant human cholangiocytes respond to dsRNA by the stimulation of a cytoprotective response. dsRNA was not toxic and did not alter cellular apoptosis or proliferation. However, dsRNA activated a cell survival program that suppressed the caspase effector machinery and inhibited cytotoxicity due to camptothecin or to TRAIL death receptor ligation. These cell type-specific effects on intracellular processes mediating cell survival are relevant in understanding viral infections affecting the liver. Furthermore, these observations may have implications for the use of sequence-specific dsRNA for the functional ablation of specific genes, an approach that is gaining increasing importance as a strategy for the manipulation of selective gene expression in eukaryotic cells (2).
The cell survival program initiated by dsRNA involves phosphorylation of the p38 MAPK, but not the p44/p42 MAPK or the dsRNA-dependent PKR. Activation of p38 MAPK has been shown to occur in response to dsRNA in fibroblasts and HeLa cells via a PKR- independent mechanism (8). PKR is a potent inducer of apoptosis and negative regulator of epithelial cell growth in response to dsRNA (reviewed in Ref. 5). However, PKR expression increases in proliferating cholangiocytes, and PKR is overexpressed in cholangiocarcinoma (21). The lack of an apoptotic response to PKR in malignant cholangiocytes may be explained by the presence of cellular mechanisms that override growth inhibition and apoptosis induced by PKR activation. Although our observations do not address the important question of whether the cellular responses to dsRNA are a cause or consequence of malignant transformation, these studies support the presence of a dominant protective effect mediated by p38 MAPK over apoptotic signaling in response to dsRNA in malignant cholangiocytes.
Several cellular modulators of the response to dsRNA have been identified in uninfected cells. These include p58, a member of the tetratricopeptide family that includes many regulators of cell cycle activity, such as cdc23 and cdc16. Interestingly, overexpression of p58 results in malignant transformation of cells in vitro, presumably by inhibiting endogenous PKR (1). Other modulators of PKR function have also been identified, including the eIF-2-associated glycoprotein p67 and the Tar RNA-binding protein (16, 24). We speculate that p38 MAPK may alter the expression of endogenous inhibitors of PKR function and that additional study to ascertain these relationships is warranted.
We (19) have previously shown the involvement of p38 MAPK signaling in transformed growth of malignant cholangiocytes. Aberrant p38 MAPK signaling in response to stress therefore represents a potential mechanism by which carcinogenesis is promoted. Indeed, the failure of dsRNA to initiate apoptosis despite constitutive expression of PKR emphasizes a mechanism by which carcinogenesis is promoted independently of viral infection. dsRNA is produced within cells as a result of direct perturbations to cellular RNA by genotoxic agents or as a secondary effect resulting from aberrant transcription of damaged DNA. The activation of stress-associated protein kinases (p38 MAPK and JNK) in response to dsRNA provides additional support of the hypothesis that dsRNA may function as an intracellular stress sensor or signal during genotoxic stress (7, 22). Failure to mount an apoptotic response in these situations may predispose to malignant transformation by allowing the persistence of cells that have sustained genotoxic damage and allowing for inheritable genomic damage.
Cholangiocytes are exposed to and are susceptible to virus infection
during acute or chronic viral hepatitis (6, 11, 15).
Cellular response to exogenous dsRNA mimics that of dsRNA produced
during replicative viral infection, and many of the genes induced are
also stimulated by viral infection or interferon. Prominent changes
were observed in expression of inflammatory cytokines that can
stimulate (e.g., IL-6) or inhibit (e.g., TGF-) cholangiocyte
proliferation. Host cellular responses prevent viral spread by inducing
apoptosis in infected cells or by acting in a paracrine manner
to protect surrounding cells. The cellular responses involve both
dsRNA- and interferon-induced cytoprotective and cytotoxic mechanisms
to promote host survival. Many viruses have evolved mechanisms to
modulate host cell responses and thus the specific cellular response
elicited is virus specific.
Recent studies (12) have demonstrated an association between chronic hepatitis C virus (HCV) infection and biliary tract malignancies. In hepatocytes, HCV activates PKR, which subsequently plays a fundamental role in the regulation of apoptosis. Evasion of host apoptosis is an important mechanism by which viruses maintain persistent infection. Disruption of PKR-dependent apoptosis is associated with the interferon-resistant phenotype of HCV and with persistence of viral HCV infection (13). Indeed, the HCV nonstructural 5A protein from interferon-resistant HCV can disrupt dsRNA-induced host apoptotic signaling by inhibiting PKR and contribute to tumor formation (3). Although PKR expression is also increased in cholangiocytes during chronic HCV infection, cholangiocyte death is infrequently observed, and viral persistence occurs in biliary epithelia. Abrogation of intracellular apoptotic signaling in response to dsRNA therefore represents a potential mechanism by which cholangiocytes may escape cell death during viral infection. We speculate that these aberrant responses to dsRNA contribute to persistent viral infection and the establishment of a reservoir in the biliary tract during infection with chronic HCV infection. Thus further study is warranted to ascertain the specific responses of nontransformed human biliary epithelia to dsRNA during infection with hepatotrophic viruses such as hepatitis C.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Patel, Associate Professor of Medicine, Division of Gastroenterology, Scott and White Clinic, Texas A&M Univ. System Health Science Center, College of Medicine, 2401 South 31st St., Temple, TX 76508 (E-mail: tpatel{at}medicine.tamu.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.
First published January 22, 2003;10.1152/ajpgi.00355.2002
Received 22 August 2002; accepted in final form 21 January 2003.
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