1Department of Internal Medicine I and 2Institute of Pathology, University of Regensburg, D-93042 Regensburg, Germany; and 3University of North Carolina at Chapel Hill, North Carolina 27599
Submitted 7 August 2003 ; accepted in final form 8 January 2004
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ABSTRACT |
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intestinal epithelial cells; colitis; inflammation; cancer
Intestinal epithelial cells are exposed to various concentrations and compositions of BAs throughout the fecal stream. Several effects of BAs on colonic epithelial cells (CECs) have been described, including induction of proliferation and apoptosis (30, 37). Furthermore, epidemiological and animal studies have demonstrated that BAs may be endogenous colon tumor promoters (2, 3, 42).
The mechanism by which BAs induce gene expression is not fully understood. BAs utilize multiple signaling cascades such as p53 (29), the mitogen-activated protein kinases ERK and p38 (28, 30), or PKC (27), phosphatidylinositol 3-kinase (PI3-kinase) (36), and the activator protein-1 (AP-1) transcription factor (28) to modify gene expression.
A critical signaling cascade controlling inducible gene expression is the NF-B transcriptional system. Many inducers such as cytokines, growth factors, and bacteria activate the NF-
B signaling cascade that then triggers transcription of a wide array of proinflammatory and immune system-modulating genes. The main integrator of signal-induced NF-
B activation is the I
B kinase (IKK) complex. The subunit IKK
is responsible for I
B
serine residues 32 and 36 phosphorylation, a process that marks the protein for ubiquitination and subsequent proteolytic degradation through the proteasome pathway (20). Destruction of the NF-
B inhibitor releases the transcription factor that migrates to the nucleus, binds to
B-dependent gene promoters, and induces transcription.
Numerous studies have linked the NF-B signaling cascade to important biological processes in the gut, such as carcinogenesis, as well as innate and immune responses. For example, systemic or local delivery of RelA oligonucleotide antisense has been shown to prevent and reverse experimental colitis without apparent damage to the intestinal barrier (25). Also, selective deletion of IKK
in intestinal epithelial cells prevented ischemia-reperfusion-induced TNF secretion and lung injury (6). Therefore, an inducer of NF-
B signal transduction may impact the state of intestinal homeostasis. Because BAs induce various proinflammatory gene expressions in different cell types, it is important to resolve the molecular mechanism responsible for this effect.
In this study, we characterized molecular mechanisms underlying the BA-induced gene expression in CECs. We report that BAs induce IL-8 gene expression through activation of the transcription factor NF-B. It is interesting that deoxycholic acid (DCA) signaled to NF-
B through proteasome-mediated I
B degradation, whereas taurodeoxycholic acid (TDCA) mainly enhanced RelA phosphorylation.
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MATERIALS AND METHODS |
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DCA and TDCA were obtained as sodium salts (Sigma, St. Louis, MO). Stock solutions of BAs (10 mM) were prepared in water and stored at 4°C. Before starting stimulation experiments, they were incubated in a water bath at 37°C and brought to the final working solution with medium without FCS plus penicillin/streptomycin.
TNF served as control as a strong inducer of NF-B activation and was obtained from R&D Systems (Minneapolis, MN). Pharmacological blockade of the proteasome was performed by preincubation with MG132 (Peptides International, Minoh-Shi Osaka, Japan) at a concentration of 10 µg/ml.
Cells and Cell Culture
Isolation of CECs. CECs were isolated as previously described (34). Briefly, intestinal mucosa was stripped from the submucosa within 30 min after bowel resection and rinsed several times with PBS at room temperature. The mucus was removed by rotating the resection two times in 1 mM DTT (Sigma) for 15 min at 37°C. After being washed with PBS to remove DTT, the mucosa was rotated in 2 mM EDTA in Hanks' balanced salt solution without calcium and magnesium (PAA, Linz, Austria) for 10 min at 37°C. The resulting supernatant-containing debris and mainly villus cells was discarded. The remaining mucosa was vortexed in PBS, and the supernatant containing complete crypts and some single cells was collected into a 15-ml tube. Vortexing was repeated until the supernatant was almost clear. To separate crypts from single cells (partially nonepithelial cells), the suspension was allowed to settle down for 35 min. These steps were all carried out at room temperature. The sedimented crypts were collected and washed with PBS, and cell viability was assessed by 0.1% trypan blue exclusion and flow cytometry.
Culture of CECs. The 5 x 105 cells were resuspended in 400 µl MEM supplemented with Earle's salts, 5% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml gentamycin, and 2.5 µg/ml fungizone (JRH Bioscience, Lenexa, KS). Media were purchased from Biochrom (Berlin, Germany), and supplements were obtained from Sigma. Approximately 1 x 106 CECs were seeded into Millicell-CM culture plate inserts (Millipore, Eschborn, Germany) suitable for six-well plates, coated with 0.5 mg/ml collagen A (Biochrom, Berlin, Germany). The medium containing the isolated cells was placed inside the filter inserts. The cells were rapidly forced to contact the collagen coating of the membranes as the medium passed the filters. Just enough medium was added to cover the cells with a liquid film. The cells were incubated at 37°C in 10% CO2.
HT-29 cells. Human colon carcinoma HT-29 cells were obtained from American Type Culture Collection (Manassas, VA). These cell cultures were maintained in Dulbecco's high glucose medium supplemented with 10% heat-inactivated FCS and 5% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2 in air.
Cell stimulation. Before stimulation in individual experiments, cells were starved for 24 h to avoid the confounding variable of serum-induced signaling. TNF at a concentration of 10 ng/ml served as control.
RNA Isolation and IL-8 mRNA Quantification
RNA was isolated by using the RNeasy Mini Kit including a RNase-free DNase step following the manufacturer's instructions (Qiagen, Hilden, Germany). RNA amounts were analyzed by using a fluorescence microplate reader following the instructions of the RiboGreen RNA Quantitation Reagent and kit (MoBiTec, Göttingen, Germany). Integrity of the RNA was verified by agarose gel electrophoresis, and ribosomal bands were visualized with ethidium bromide staining. First-strand cDNA was synthesized by using 1 µg of total RNA and the AMV-reverse transcription reaction (Promega, Madison, WI) in a total volume of 25 µl utilizing oligo(dT) primers.
To quantify the expression of cDNAs, the real-time PCR LightCycler system (Roche, Mannheim, Germany) was used. For PCR, 13 µl cDNA preparation, 0.52.4 µl 25 mM MgCl2, 0.5 µM forward and reverse primer, and 2 µl of SYBRgreen LightCycler mix in a total volume of 20 µl were applied.
The following sets of primers were used: -actin forward, 5'-cta cgt cgc cct gga ctt cga gc-3';
-actin reverse, 5'-gat gga gcc gcc gat cca cac g-3'; IL-8 forward, 5'-tct gca gct ctg tgt gaa ggt gca gtt-3'; and IL-8 reverse, 5'-aac cct ctg cac cca gtt ttc ct-3'. The following PCR program was performed: 600 s at 95°C (initial denaturation); 20°C/s temperature transition rate up to 95°C for 15 s, 10 s at 5868°C, 22 s at 72°C, and 10 s at 82°C acquisition mode single, repeated 40 times (amplification). MgCl2 concentration and annealing temperature were optimized. The PCR reaction was evaluated by melting curve analysis following the manufacturer's instructions and checking the PCR products on 1.8% agarose gels. Each quantitative PCR was performed at least in duplicate for two sets of RNA preparations.
IL-8 ELISA
Cells were plated in 24-well plates and serum starved for 24 h before stimulation. Subsequently, supernatants were collected and centrifuged to remove cellular debris, and IL-8 concentration was analyzed by a sandwich ELISA following the instructions (Biosource, Camarillo, CA).
LDH Release
The release of cytosolic lactate dehydrogenase (LDH) was measured to investigate significant membrane disruption using the cytotoxicity detection kit (LDH) (Boehringer-Mannheim, Mannheim, Germany) following the manufacturer's instructions. LDH release is given as the percentage of possible maximum release by induction of total cell lysis with Triton X 2%.
Additionally, cytotoxicity was analyzed morphologically and by trypan blue exclusion dye test, using a phase contrast microscope (Leitz, Wetzlar, Germany)
Nuclear Extracts
Nuclear extracts were prepared as previously described (4). Protease inhibitors in the form of the complete minitablets (Roche, Mannheim, Germany) were used.
EMSA.
Nuclear extracts (5 µg) were incubated with a radiolabeled, double-stranded oligonucleotide-containing class I myosin heavy chain B binding site (GGCTGGGATTCCCCATCT), separated by electrophoresis, and analyzed by autoradiography as described previously (13). Specificity of the probe was evaluated by incubating the nuclear extracts with an excess (100x) of unlabeled oligonucleotide.
Quantification of activated nuclear NF-B concentration.
Activated NF-
B was quantified in nuclear extracts with the ELISA-based kit TransAm from Active Motif (Rixensart, Belgium) according to the manufacturer's instructions as described previously (33). ELISA plates were coated with oligonucleotide (5'-GGGACTTTCC-3') coding for an NF-
B consensus site. Plates were preincubated with a binding buffer containing DTT and herring sperm DNA. Twenty micrograms of nuclear extracts, solved in 20 µl lysis buffer containing DTT and protease inhibitors, were added per well and incubated at room temperature for 1 h. After completion of a washing step, an anti-RelA antibody was added and incubated for another hour at room temperature. After an additional washing step, a horseradish peroxidase-conjugated secondary antibody was added, followed by an additional hour of incubation at room temperature. After a last washing step, developing solution was added, and the absorption was measured at 450 nm.
RelA Immunofluorescence
Cellular RelA distribution was analyzed by immunofluorescence as described previously (13). Briefly, HT-29 cells grown on chamber slides (Nunc, Naperville, IL) or primary CECs were stimulated for various time intervals (30 min to 3 h) with DCA, TDCA, or TNF- (10 ng/ml), briefly washed with PBS, and then fixed with ice-cold 100% methanol for 10 min. After being blocked for 30 min with 10% nonimmune goat serum (NGS; Sigma), cells were incubated with rabbit anti-RelA antibody (Rockland, Gilbertsville, PA) diluted 1:200 in 10% NGS/PBS. Slides were washed three times with PBS before 30-min incubation with rhodamine isothiocyanate-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA) diluted 1:100 in 10% NGS/PBS. The RelA localization was visualized with a fluorescence light microscope (Leica, Bensheim, Germany). Double staining with Hoechst dye for detecting nuclear DNA was performed as described previously (35).
Adenoviruses
HT-29 cells were infected overnight with adenoviral dominant-negative (dn) IKK (Ad5dnIKK
) or Ad5I
B
AA in serum-free media (Opti-MEM; GIBCO, Grand Island, NY) at 50 multiplicity of infection. The dnIKK
construct cloned in adenoviral vector consisted of a point mutation in the kinase domain (K44A) as described previously (35) and contained an extra 24-bp DNA nucleotides coding for the FLAG peptide (DYLDDDDL). The Ad5I
BAA virus has been characterized and described previously (19). Ad5GFP containing green fluorescent protein was used as negative control. After a washing step, fresh medium containing serum without antibiotics was added, and cells were used for subsequent experiments.
Western Blot Analysis
HT-29 cells were stimulated for various times (03 h) with DCA or TDCA. The cells were lysed in 1x Laemmli buffer, and 20 µg of protein was subjected to electrophoresis on 10% SDS-polyacryl-amide gels. Anti-phosphoserine IB
(Cell Signaling, Beverly, MA) and anti-phosphoserine RelA (S536; Cell Signaling) antibodies were used to detect immunoreactive phospho-I
B
and phospho-RelA, respectively, using enhanced chemiluminescence light-detecting kit (Amersham, Arlinghton Heights, IL) as previously described (11). To proof equal loading, the blots were additionally analyzed for actin expression using anti-actin antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). Furthermore, anti-flag antibodies (Kodak Eastman) were applied to demonstrate efficient gene expression of the FLAG-tagged dnIKK
in HT-29 cells after adenoviral transfection with Ad5dnIKK
.
Statistical Analysis
Statistical significance was evaluated by the two-tailed Student's t-test for paired data. P < 0.05 was considered statistically significant.
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RESULTS |
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HT-29 cells were stimulated with different concentrations of DCA and TDCA, and IL-8 secretion was analyzed in the supernatant 24 h after stimulation. Both BA induced a dose-dependant increase of IL-8 secretion with maximum induction varying from fivefold for DCA to eightfold for TDCA, respectively (Fig. 1, A and B). Optimal doses inducing IL-8 secretion varied considerably between DCA and TDCA. DCA (300 µM) compared with 2 mM TDCA resulted in maximal IL-8 secretion. However, also at lower concentrations of individual BA, as occurring in the aqueous phase of human feces, effects on IL-8 secretion of HT-29 cells were observed.
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Time Course of DCA- and TDCA-Induced IL-8 mRNA Expression and IL-8 Secretion
DCA- and TDCA-induced IL-8 secretion was completely blocked by preincubation of HT-29 cells with actinomycin D, indicating that the IL-8 induction was dependent on transcription (data not shown). This was further confirmed by analyzing the effects of DCA and TDCA on IL-8 mRNA expression. HT-29 cells were stimulated with DCA and TDCA for different time intervals, and IL-8 mRNA accumulation was quantified by using real-time PCR. Both BA induced IL-8 mRNA after 1 h of stimulation that peaked at 6 h (Fig. 2, A and B). This induction differed significantly from TNF-induced IL-8 mRNA induction that started earlier, peaked at 3 h, and sharply dropped at 6 h (Fig. 2C).
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Effects of BAs on NF-B Signal Transduction
To dissect the molecular mechanism of BA-induced IL-8 gene expression in CECs, we investigated the effect of BA on the activation of the transcription factor NF-B. The first step of NF-
B activation is signal-induced I
B
phosphorylation/ degradation and nuclear translocation of the transcription factor. It is interesting that DCA induced I
B
serine 32 phosphorylation in HT-29 cells, whereas TDCA revealed only minimal effects (Fig. 3).
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To further confirm interaction of NF-B with gene promoter elements, we analyzed nuclear extracts derived from HT-29 cells stimulated with either DCA or TDCA with EMSA. Weak induction of NF-
B binding activity was observed 1 h after stimulation with DCA. However, binding activity increased with time, reaching a maximum 3 h after stimulation with DCA in accordance with the time course of RelA translocation. In contrast, NF-
B DNA binding activity was not significantly induced in TDCA-stimulated cells (Fig. 5C, top). On the other hand, TDCA strongly induced AP-1 DNA binding activity (Fig. 5C, bottom), suggesting a selective effect of this BA on different signal transduction pathways in HT-29 cells.
Functional Role of NF-B in BA-Induced IL-8 Gene Expression
We have previously shown that cytokine-induced IL-8 gene expression is mediated by NF-B in intestinal epithelial cells (18, 19). To verify the role of NF-
B in BA-induced IL-8 gene expression, we used MG132, a pharmacological inhibitor of the proteasome. Whereas MG132 completely blocked DCA-induced IL-8 mRNA expression and secretion, only moderate effects were seen with TDCA-stimulated cells (Fig. 6).
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To further analyze the physiological impact of BA on proinflammatory gene expression, we investigated primary human colonic CECs ex vivo in a cell-culture model recently established in our laboratory (34). DCA and TDCA induced IL-8 secretion in primary CECs, although induction was lower than seen in HT-29 cells (Fig. 8). Similarly, TNF-induced IL-8 secretion was lower in CECs than in HT-29 cells (twofold compared with tenfold, respectively).
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DISCUSSION |
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Cytokine-mediated IL-8 expression in CECs has been shown to be regulated by the transcription factor NF-B (19). Therefore, to dissect the molecular mechanism of BA-induced IL-8 gene expression, we investigated the effects of DCA and TDCA on NF-
B activation. It is interesting that we found differences between DCA and TDCA in their potential to induce the classical NF-
B pathway. DCA induced I
B
serine 36 phosphorylation, RelA nuclear translocation, and increased nuclear NF-
B-binding activity, whereas TDCA revealed no significant effects.
RelA translocation has been reported (26) previously in response to DCA in another colon tumor cell line (HCT-116). However, other authors (15) were unable to demonstrate increased NF-B binding activity in LoVo adenocarcinoma cells on stimulation with chenodeoxycholic acid. Moreover, a recent study (40) using the rat small intestinal cell line (IEC-6) found NF-
B activation also in response to TDCA stimulation. It is unclear whether these discrepancies are due to the use of different bile salts, different concentrations of BA, or the different cell lines used. To exclude that the BA effects on IL-8 gene expression and the NF-
B signal transduction were specific for certain cell lines, we performed key experiments with primary human CECs. As in HT-29 cells, DCA and TDCA were capable of inducing IL-8 secretion in primary CECs, whereas only DCA induced RelA translocation. This confirms that there seems to be a classical NF-
B-dependent and an independent pathway in BA-induced IL-8 expression in CECs.
To elucidate the mechanism of action of TDCA on gene expression in CECs, we analyzed RelA phosphorylation, because we recently demonstrated that it plays a critical role in bacteria-induced gene expression in CECs (10, 11). It is interesting that TDCA induced RelA serine 536 phosphorylation, and this effect was blocked by molecular inhibition of IKK. It has been shown previously (10, 17, 44) that NF-
B can be activated by cytokines or bacterial products through phosphorylation of the RelA/p65 subunit at serine 536 without altering the level of the phosphorylation of I
B and nuclear localization of RelA. However, this mechanism has not been described for BA so far.
The functional relevance of these different mechanisms of action on the NF-B signaling cascade between DCA and TDCA was confirmed by experiments investigating BA-induced IL-8 expression after inhibition of the proteasome or the I
B kinase IKK
. Although the pharmacological inhibitor MG132 completely blocked DCA-induced IL-8 expression, only moderate effects were seen in cells stimulated with TDCA. However, in contrast, adenoviral delivery of a dominant-negative form of IKK
completely blocked TDCA-induced IL-8 expression. These data suggest that TDCA induces IL-8 gene expression primarily through RelA phosphorylation, whereas DCA utilizes mainly signal-induced I
B degradation and RelA nuclear translocation.
Currently, we can only speculate on the potential molecular mechanisms causing the differences between DCA and TDCA on NF-B signal transduction. It seems likely that they are attributable to the decreased permeability of the cytoplasmatic membrane of the taurine-conjugated BA TDCA compared with the lipophilic, unconjugated BA DCA. In line with this hypothesis, TDCA is capable of inducing classic NF-
B activation in hepatoma cells (36). In contrast to CECs, these cells express a transporter for taurine-conjugated BA. As an alternative mechanism, recent studies (32, 45) indicate that BA-mediated signal transduction can be mediated through receptors located on the cell membrane such as the EGF receptor. Further studies are required to elucidate what mechanisms cause the different effects between DCA and TDCA on the NF-
B signaling cascade and whether these mechanisms are specific for CECs.
Constitutive oxidant defense levels are relatively low in human CECs compared with the liver (9). Thus in vivo activation of NF-B by DCA, which represents the major fraction of BA in the colon, may be needed to protect CECs from oxidative stress caused by bile salts (7, 22) that would otherwise lead to toxic effects and apoptosis. It has been shown before in a hepatoma cell line that some BA attenuate their inherited cytotoxic effects by activating a PI3-kinase-dependent survival signal that is mediated by PKC
and NF-
B (36). Payne et al. (26) previously found that apoptosis induced by DCA is potentiated by inhibition of NF-
B, and there is further evidence that NF-
B is involved in the regulation or prevention of apoptosis. However, this seems to be a delicate balance, because an increase in the number of cells with activated NF-
B in the normal-appearing colonic mucosa of a patient with cancer has been found, leading Payne et al. (26) to suggest the hypothesis of induction of apoptosis-resistant cells in colon carcinogenesis. Furthermore, the increased activation of NF-
B has been shown to lead to cellular transformation (21), tumorgenicity (14, 38), or a metastatic phenotype (5).
Our results could indicate that, in contrast to TDCA, DCA may prevent its inherited cytotoxicity by simultaneously activating intrinsic cell survival signals in CECs, eventually, however, reducing their resistance to carcinogenesis.
The different effects of DCA and TDCA on the NF-B signaling cascade may have further implications for dietary or pharmaceutical strategies to change BA concentrations and composition. Such therapeutic strategies have been designed for treatment or prevention of relapse of irritable bowel disease or for chemo prevention of cancer. A Western-type diet, with its high meat and fat content, will increase the intestinal BA concentration and, by virtue of its high taurine content, will also increase the percentage of taurine-conjugated BA (8, 16). It is estimated that diets rich in taurine-containing food, such as meat and seafood, can raise the amount of taurine-conjugated BA that passes daily through the colon >10-fold (12). However, it is possible to alter the BA concentrations and composition in fecal water through dietary intervention, with food rich in fibers (1, 31). Our results may provide a possible mechanism by which such diets could interfere with signaling pathways differentially affected by individual BA and influence carcinogenesis and inflammation.
In summary, in this study we demonstrated differences between DCA and TDCA in their molecular mechanisms of proinflammatory gene expression in a colonic tumor cell line as well as in primary CECs. The relevance of these in vitro findings under physiological and pathophysiological conditions is currently unknown and has to be further evaluated. Insights into the mechanisms responsible for the differences between BAs in their effects on the NF-B signal transduction pathway may improve our understanding of the pathophysiology of intestinal inflammation and carcinogenesis.
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FOOTNOTES |
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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 |
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