Taurodeoxycholate Stimulates Intestinal Cell Proliferation and Protects Against Apoptotic Cell Death Through Activation of NF-κB

Digestive Diseases and Sciences, Vol. 49, No. 10 (October 2004), pp. 1664–1671 (C© 2004)

Taurodeoxycholate Stimulates Intestinal CellProliferation and Protects Against Apoptotic Cell

Death Through Activation of NF-κB

ALEXANDER TOLEDO, MD, JON YAMAGUCHI, MD, JIAN-YING WANG, MD, PhD,BARBARA L. BASS, MD, DOUGLAS J. TURNER, MD, and ERIC D. STRAUCH, MD

We hypothesized that the NF-κB pathway would be operative in the proliferative effect of bile saltson enterocytes. To determine this, we studied the effect of the bile salt taurodeoxycholate on culturedrat enterocyte proliferation and apoptosis and examined the role of NF-κB activation in these growthregulatory processes. Intestinal epithelial cells were grown for 6 days with or without taurodeoxy-cholate. Proliferation was measured. The cells were exposed to a known apoptotic stimulus, TNF-αand cyclohexamide. Apoptosis was quantified using cell number and the TUNEL stain. NF-κB ac-tivation was determined by an electrophoretic mobility shift assay. NF-κB activation was inhibitedby an IκB superrepressor. Taurodeoxycholate stimulated cell proliferation (P < 0.01) and inducedresistance to TNF-α induced apoptosis (P < 0.01). Taurodeoxycholate induced NF-κB activation.Inhibition of NF-κB prevented taurodeoxycholate-induced IEC-6 cell proliferation and renderedcells sensitive to TNF-α-induced apoptosis. Taurodeoxycholate stimulates intestinal epithelial cellproliferation and protects intestinal epithelial cells from TNF-α-induced apoptosis through NF-κB.These data support an important beneficial role of bile salts in regulation of mucosal growth andrepair. Decreased enterocyte exposure to luminal bile salts, as occurs during starvation and parenteralnutrition, may have a detrimental effect on mucosal integrity.

KEY WORDS: enterocyte; NF-κB; bile salts; apoptosis; proliferation.

Small intestinal digestive and secretory function requires,an intact mucosa. The mucosa additionally serves as abarrier to a broad spectrum of noxious substances withinthe intestinal lumen. Mucosal integrity is governed by thebalance between cell loss and cell regeneration. Largemucosal injuries require cell proliferation for mucosalrenewal, and increased proliferation is necessary for adap-tive changes after intestinal loss. Decreased intestinal ep-ithelial cell death will speed the repair or adaptive processas well. Mucosal renewal is dependent on metabolic hor-

Manuscript received December 8, 2003; accepted February 16, 2004.From the University of Maryland Hospital, Baltimore, Maryland,

USA.Address for reprint requests: Eric D. Strauch, MD, University

of Maryland Hospital, N4E37, 22 South Greene Street, Baltimore,Maryland 21201, USA; [email protected].

mones such as insulin and growth hormone. The growthof the mucosa is also affected by a plethora of agents andnutritional factors found within the intestinal lumen. Fac-tors regulating the balance between cell proliferation andloss will affect mucosal healing, integrity, and adaptiveresponse and can potentially cause mucosal injury.

Nuclear factor (NF)-κB is a ubiquitous transcriptionfactor that regulates activation of a number of genes in-volved in proinflammatory responses, differentiation andgrowth (1–3). NF-κB is found in the cytoplasm boundto the endogenous inhibitors, known as IκBs. Activationof NF-κB occurs when IκB is phosphorylated, resultingin IκB degradation and cytosolic release of NF-κB. NF-κB then translocates into the nucleus and induces tran-scription of specific genes (4–8). The NF-κB signalingcascade has been shown to be activated in the intestinal

1664 Digestive Diseases and Sciences, Vol. 49, No. 10 (October 2004)0163-2116/04/1000-1664/0C© 2004 Springer Science+Business Media, Inc.

TAURODEOXYCHOLATE STIMULATES INTESTINAL RENEWAL

epithelium by tumor necrosis factor-α (TNF-α), bacte-rial lipopolysacharides, interleukin-1 (IL-1), bacteria, andother agents. Once activated, the NF-κB signaling path-way transcriptionally regulates many cellular genes im-plicated in early immune, acute phase, and inflammatoryresponses including TNF-α, IL-2, IL-6, IL-8, IL-12, in-ducible nitric oxide synthase, cyclooxygenase-2, and in-tercellular adhesion molecule-1 (3). Thus, the NF-κB sig-naling pathway is highly relevant to intestinal injury andrepair.

NF-κB can also have a proapoptotic or antiapoptoticfunction depending on the cell type and death stimulus(9–13). NF-κB activation appears to be necessary for theactivation of genes that suppress apoptosis. For example,inhibition of NF-κB activity in immature B cells or in liverduring development markedly enhances apoptotic death(14, 15). Antiapoptotic genes that are regulated by NF-κB include manganese superoxide dismutase (16) and thezinc finger protein (17). Li (13) has shown that polyaminedepletion in intestinal epithelial cells results in an increasein NF-κB activation which protects the cells against TNF-α-induced apoptosis.

Our previous studies have shown that bile salts such astaurodeoxycholate (TDCA) have a beneficial affect on in-testinal mucosal integrity by stimulating intestinal epithe-lial cell migration through NF-κB activation (18). Othershave shown that a decrease in luminal bile salts exacer-bates the mucosal atrophy seen in the absence of entericnutrients (19–23). Animals with biliary diversion aftersmall bowel resection demonstrate an impaired adaptiveresponse (21–23). Thus bile salts can also have potentiallybeneficial affects during mucosal renewal.

The present studies asked whether bile salt-inducedNF-κB activation can have a postitive affect on mucosalrenewal. First, we determined if bile salts changed thesusceptibility to TNF-α-induced apoptosis in intestinalepithelial cells. Second, we examined whether TDCA-induced NF-κB activity played a role in the observed in-crease in intestinal proliferation in the presence of bilesalts and the changed susceptibility of intestinal epithelialcells in the presence of bile salts.

MATERIALS AND METHODS

Materials. Disposable culture were was purchased fromCorning Glass Works (Corning, NY) and Becton Dickinson(Franklin Lakes, NJ). Tissue culture media and fetal bovineserum (FBS) were from GIBCO (Grand Island, NY). Biochem-icals were purchased from Sigma (St. Louis, MO).

Cell Culture and Experimental Protocol. The IEC-6 cellline was purchased from ATCC at passage 13. The IEC-6 cellline was derived from normal rat intestine and was developedand characterized by Quaroniet al. (24). IEC-6 cells, from je-

junal crypt cells, were maintained in T-175 flasks in Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated FBS, 1% antibiotic, and 0.1 U/ml insulin. They arenontumorigenic and retain the undifferentiated character of ep-ithelial stem cells. Flasks were incubated at 37◦C in a humidifiedatmosphere of 95% air and 5% CO2. Stock cells were subcul-tured once a week at 1:3; the medium was changed three timesweekly. Passages 15–20 were used in the experiments. Therewere no significant changes of biologic function and character-izations from those passages. Cells were restarted from frozenstock.

Cell Proliferation. IEC-6 cells were plated at 2×104 cells/cm2 in supplemented DMEM on 100-mm plates andgrown for 24 hr. Medium was then changed to contain vary-ing concentrations of TDCA (0–1 mM). Medium was changedevery 2 days. Cells were harvested with 0.4% trypsin, and cellnumber was determined at days 1, 2, 4, and 6 by counting usinga hemocytometer and light microscopy. Results are reported ascells per plate. All experiments were performed with ann of 6and repeated in triplicate.

Apoptosis. Apoptosis was quantified by counting the numberof dead cells and using terminal deoxynucleotide transferase la-beling (TUNEL). Apoptosis identification was performed usingthe DeadEnd IEC-6 cells Florometric TUNEL system (Promega,Madison, WI). Cells were fixed in freshly prepared 4% methanol-free formaldehyde for 25 min, centrifuged at 300g for 10 min at4◦C, washed with PBS, and then resuspended in PBS at a con-centration of 2× 107 cells/ml. One hundred microliters of thecell suspension was smeared onto poly-L-lysine-coated slides.The TUNEL regent was prepared as directed (Promega). Thereactions were terminated by immersing the slides in 2× SSCfor 15 min at room temperature. The samples were washed threetimes in PBS to remove unincorporated fluorescein-12-dUTP.The samples were stained in 0.1 mg/ml propidium iodide buffer.The samples were analyzed using a fluorescent microscope.

Preparation of Nuclear Protein and Electrophoretic ShiftAssays.Nuclear proteins were prepared by the procedure de-scribed previously (25), and the protein contents in nuclearpreparations were determined by the method described byBradford (26). Using the same cell culture methods, cells wereharvested at 6 hr after wounding for nuclear protein extrac-tions. The double-stranded oligonucleotides used in these ex-periments included 5′-AGTTGAGGGGACTTTCCCAGGC-3′,which contains a consensus NF-κB binding site that is boldfaced.These oligonucleotides were radioactively end-labeled with [γ -32P]ATP and T4 polynucleotide kinase. For mobility shift assays,0.035 pmol of32P-labeled oligonucleotides (∼30,000 cpm) and10 µg of nuclear protein were incubated in a total volume of25 µl in the presence of 10 mM Tris·HCL (pH 7.5), 50 mMNaCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and 1µgof poly (dI–dC). The binding reactions were allowed to proceedat room temperature for 20 min. Thereafter, 2µl of bromphenolblue (0.1% in water) was added, and protein–DNA complexeswere resolved by electrophoresis on nondenaturing 5% polyacry-lamide gels and visualized by autoradiography. The specificityof binding interactions was assessed by competition with an ex-cess of unlabeled double-stranded oligonucleotide of identicalsequence. All experiments were repeated in triplicate.

Western Blotting Analysis. Ten micrograms of cytoplasmicprotein extracts was dissolved in SDS sample buffer, boiled for5 min, and then subjected to electrophoresis on acrylamide gelsaccording to Laemmli (27). After SDS-PAGE, the gels were

Digestive Diseases and Sciences, Vol. 49, No. 10 (October 2004) 1665

TOLEDO ET AL.

transferred to nitrocellulose membranes for 1 hr at 4◦C. The blotswere blocked with 5% nonfat dry milk in PBS–0.1% Tween 20(PBS-T) overnight at 4◦C. Immunological evaluation was per-formed for 1 hr in PBS-T containing 0.2µg/ml affinity-purifiedpolyclonal antibodies IκBα. The blots were washed with PBS-Tand incubated for 1 hr with goat anti-rabbit IgG antibody con-jugated to peroxidase at a dilution of 1:3000 in PBS-T. Afterextensive washing in PBS-T, the blots were developed for 30 to60 sec with enhanced chemiluminescence reagents.

IκB Superrepressor. The recombinant replication-deficientadenovirus Ad5IκB was constructed by the methods of Grahamand Bett (28, 29) as performed by Iimuro (30). In brief,the plasmid pCMV-IκBαM, which contains a human super-repressor of NF-κB, was subcloned into theXbaI site ofthe pACCMV.PLPASR(+) plasmid to construct the plasmidpACCMV/IκB, in which IκB is driven by the CMV pro-moter/enhancer. The plasmid DNA was prepared by the alkalinelysis method and purified by CsCl–tethidium bromide densitygradient centrifugation. The recombinant adenovirus IκB wasconstructed by cotransfection of the 293 embryonic human kid-ney cell line with the pACCMV/IκB plasmid plus the purifiedfragment of theClaI-digested DNA from E1-deleted adenovirus

Fig 1. Effect of various concentrations of TDCA on IEC-6 cell proliferation. Cells were grown for 24 hrand exposed to 0.05–1 mM TDCA for 1–6 days. Cells were counted using 0.4% tryphan blue staining and ahemocytometer. Values are mean± SE. **P < 0.01 and ***P < 0.001 versus control.

type 5 (Ad5). The presence of the mutant IκB sequence pack-aged into the recombinant Ad5 virus (Ad5IκB) was confirmedby PCR and Western blotting. Ad5IκB was grown in 293 cellsand purified by banding twice on CsCl gradients. Viral titer wasdetermined by optical densitometry (particle per milliliter) andby plaque assay, and recombinant virus was stored in 10% glyc-erol at−20◦C.

Statistics. Values are means± SE from six dishes. Autora-diographed results were repeated. The significance of the differ-ence between means was determined by ANOVA.

RESULTS

The Effect of Long-Term Exposure to TDCA on IntestinalEpithelial Cell Proliferation. Exposure of 0.5 and 1 mMTDCA to growing IEC-6 cells for up to 6 days signif-icantly stimulated intestinal epithelial cell proliferationin a dose-dependent manner (Figure 1). There were nomorphological changes identified after 6 days of exposureto the TDCA.

1666 Digestive Diseases and Sciences, Vol. 49, No. 10 (October 2004)


The Effect of Bile Salt on Tumor Necrosis Factor-α/Cyclohexamide (TNF-α/C)-Induced Apoptosis. Ex-posure to TNF-α, a widely accepted agent for inducingapoptosis (9, 13), was used in this study. Within 4 hr, TNF-α/C induced 75% of the IEC-6 cells to undergo cell death.The TUNEL stain was used to show nuclear fragmenta-tion confirming that the cell death visualized in the pres-ence of the TNF-α/C was apoptosis (Figure 2A). Exposingthe IEC-6 cells to increasing concentrations of TDCA for6 days had no effect on the morphology of the IEC-6 cells.The presence of TDCA did significantly inhibit the num-ber and percentage of cells that underwent apoptosis inthe presence of TNF-α/C. Figure 2B shows that the cellsexposed to 0.5 mM TDCA had a significantly smallerpercentage of dead cells. The TUNEL stain confirms thatthere is significantly less DNA fragmentation. These re-sults indicate that TDCA promotes the resistance of IEC-6cells to apoptotic cell death induced by TNF-α/C.

TDCA-Induced Changes in NF-κB Sequence-Specific Binding Activity. Previously, we showed thatconcentrations of TDCA that induced intestinal epithelialcell migration increased specific NF-κB binding activityand decreased IκB expression (18). We wanted to deter-mine if these concentrations of TDCA that affected in-testinal epithelial cell proliferation and susceptibility toTNF-α/C-induced apoptosis also increased NF-κB bind-ing activity. Figure 3A shows that 0.5 and 1 mM TDCAincreased NF-κB binding. To confirm that the measuredNF-κB binding activity was sequence-specific, compet-itive inhibition experiments were performed. As showin Figure 3B, NF-κB binding activity in wounded cellsand cells exposed to TDCA was dose-dependently inhib-ited when graded concentrations of the unlabeled NF-κBoligonucleotide were added to the binding reaction mix-ture. We also examined the effect of the labeled oligonu-cleotide containing a mutated NF-κB binding site onNF-κB binding activity and found that the NF-κB-labeled,mutated oligonucleotide did not bind to the wounded IEC-6 cells or the IEC-6 cells exposed to TDCA. Further,Figure 3D shows that supershift of NF-κB using specificantibody against the p65 subunit confirms NF-κB bindingspecificity.

The Effect of NF-κB Inhibition on TDCA-InducedProliferation and Protection Against TNF-α/C-Indu-ced Apoptosis.To confirm the specificity of these effectsto NF-κB activation, an IκB superrepressor was used toinhibit NF-κB activity. IκBαM contains two mutations, atresidues 32 and 36, of serine to alanine that prevent phos-phorylation of IκBα. Therefore ubiquination and degra-dation of IκBα are inhibited, which prevents activationof the NF-κB pathway. We confirmed that transfection ofIEC-6 cells resulted in an increase in expression of the

IκBSR and an inhibition of unbound NF-κB (Figure 4).We determined the effect that the IκB superrepressor hadon NF-κB activation. Figure 5A shows that the IκB su-perrepressor transfected into the IEC-6 cells significantlyinhibited NF-κB activity in the presence of TDCA versuscontrol or transfection of the vector alone. Next we studiedwhat effect the presence of the IκB superrepressor had onTDCA induced cell proliferation. TDCA-induced prolif-eration was significantly inhibited in the presence of theIκB superrepressor versus the control or the vector alone(Figure 5B).

Second, we studied the effect of the presence of the IκBsuperrepressor on TNF-α/C-induced apoptosis in the pres-ence of TDCA. The protective effect against TNF-α/C-induced apoptosis established by the presence of TDCAon the intestinal epithelium is eliminated after transfectionwith the IκB superrepressor (Figure 6).

DISCUSSION

Mucosal integrity is governed by the balance betweencell loss and cell regeneration. Factors that increase cellloss or inhibit cell regeneration will result in a decreaseof mucosal integrity. Previously, we have shown that bilesalts can exert a beneficial affect on the intestinal mucosathrough activation of NF-κB (18). These studies show thatthe bile salt TDCA has a beneficial affect on intestinalmucosal integrity by stimulating intestinal epithelial cellproliferation and protecting against TNF-α-induced apop-tosis through a NF-κB dependent pathway.

The observation that bile salts at physiologic concen-trations have a beneficial effect on mucosal integrity is notsurprising given that bile salts are a normal component ofthe intestinal luminal contents. It is difficult to determinewhat physiologic concentration of soluble bile salts theintestinal mucosa actually is exposed to, as most bile saltsare in micellar form and their concentrations very widelyduring the digestive process. Micellar concentrations ofbile salts may reach 10 mM during digestion, while freebile salts reach soluble concentrations of up to 2–3 mM(31). Preprandial newborns, where there is minimal lipidto form micelles, have intestinal concentrations of bilesalts of up to 4 mM (32). Thus, these concentrations ofbile salts that can stimulate mucosal renewal are withinthe physiologic range of mucosal exposure.

The intestinal mucosa is known to require the presenceof luminal contents and pancreaticobiliary secretions tomaximize the adaptive response after resection (19–23).Animals with biliary diversion after small bowel resectiondemonstrate an impaired adaptive response (21–23). Oralsodium taurocholate improved anastomotic healing afterbile duct ligation (33). Thus bile has been shown to have a


TOLEDO ET AL.

Fig 2. Apoptotic response of IEC-6 cells to 20 ng/ml TNF-α and 25µg/ml cyclohexamide(TNF-α/C) after 6 days of exposure to TDCA. (A) TNF-α/C-induced apoptosis photomicro-graphs (top) and the TUNEL stain (bottom) in (a) control, (b) cells exposed to 0.2 mM TDCA,(c) cells exposed to 0.5 mM TDCA, (d) cells exposed to TNF-α/C for 4 hr (e) cells exposed toTNF-α/C for 4 hr in the presence of 0.2 mM TDCA, and (f) cells exposed to TNF-α/C for 4 hrin the presence of 0.5 mM TDCA. (B) The effect of TDCA on TNF-α/C-induced IEC-6 cellapoptosis expressed as the percentage of viable cells. IEC-6 cells were grown in TDCA for 6days and exposed to 20 ng/ml TNF-α and 25µg/ml cyclohexamide. Apoptosis was quantifiedby cell count with trypan blue and the TUNEL stain. *P < 0.01 versus TNF-α/C alone.



Fig 3. Changes in sequence-specific NF-κB binding activity in IEC-6 cells exposed to TDCA. (A) Representative audiograms of NF-κB binding.Cells were grown in DMEM with 5% dialyzed FBS with and without TDCA for 6 days. Nuclear extracts were prepared, and electrophoreticmobility shift assay (EMSA) was performed by using 10µg of nuclear proteins and 0.035 pmol of32P-end-labeld oligonucleotides containinga single NF-κB binding site. Position of specifically bound DNA-protein complex is indicated. (B) Effects of unlabeled NF-κB oligonucleotideas a cold competitor on NF-κB binding activity in the presence of 0.5 mM TDCA as measured by EMSA. (C) NF-κB supershift using the p65antibody. Supershift against Control, 0.05, and 0.5 mM TDCA with (+) and without (−) p65 supershift antibody.

beneficial role after small intestinal injury, and the absenceof bile salts can be detrimental to the intestinal mucosa af-ter injury. Further bile salts exert their beneficial affectsthrough known transcription factors, such as NF-κB, ac-tivated after mucosal injury.

The finding that activation of NF-κB protects againstapoptosis induced by TNF-α/C is consistent with those ofother investigators. Li (13) has shown that polyamine de-pletion activates NF-κB, which decreases susceptibility ofIEC-6 cells to TNF-α and cyclohexamide-induced apop-tosis. Others (9, 12, 15) have shown that NF-κB is a cellsurvival factor, which protects cells from cell death stim-uli. In general, NF-κB has been believed to inhibit apop-tosis through the caspase-dependent pathways. Numerousreports document the antiapoptotic action of NF-κB andapoptosis induced by inactivation of NF-κB by a varietyof cell types (9, 12, 15, 34)

Fig 4. IκBSR expression. (A) Representative Western immunoblots of IκBα-SR. IEC-6 cells were infected with the recombinanentadenoviral vector encoding human IκBα-SR cDNA (AdIκBSR) or an adenoviral vector lacking AdIκBSR cDNA at a multiplicity ofinfection of 1-50 plaque forming units per cell (pfu/cell). Levels of IκBα-SR protein were analyzed by Western blot analysis using thespecific monoclonal anti-IκBα antibody 48 h after the infection. Actin immunoblotting was performed as an internal control for equalloading. (B) Effect of overexpression of IκBα-SR on levels of nuclear NF-κB protein (p65) in IEC-6 cells treated with TNF-α. a: nuclearNF-κB protein (p65) levels; b: IκBα-SR levels. Cells were infected with either the AdIκBSR or control vector (50 pfu/cell) for 48 h andthen exposed to TNF-α (20 ng/ml). Total and nuclear proteins were prepared 4 h after the treatment with TNF-α, and levels of cellularIκBα-SR protein and nuclear NF-αB (p65) protein were assseyed by Western immunoblot analysis. Actin immunoblotting was performedas an internal control for equal loading.

Our data cannot address the mechanism of signal trans-duction through which TDCA initiates NF-κB activation.Hsaet al.have characterized TNF-α-induced NF-κB ac-tivation in enterocytes. They identified scaffolding andadapter proteins which transmit extracellular signals in-side the cells. TNF-α stimulates the TNF receptor-1(TNFR1) to trimerize and recruit the TNF receptor-associated factor (TRAF) and the receptor interactingprotein to the cytoplasmic portion of the TNF receptor-1(35, 36). The signal is transmitted to the NF-κB-inducingkinase which activates the IKK, resulting in degradation ofthe NF-κB-inhibitor IκB allowing NF-κB activation. Sim-ilarly, IL-1β activates NF-κB through a signaling cascadethat requires the participation of IL-1 receptor accessoryprotein, MyD88, and IL-1 receptor-associated kinase toassociate with and activate TRAF-6, where the signal istransmitted to NIK, which activates the IKK complex


TOLEDO ET AL.

Fig 5. (A) Changes in NF-κB sequence-specific binding activity inIEC-6 cells exposed to TDCA in the presence of the IκBSR or con-trol vector. Representative autoradiograms from control cells and cells6 hr after exposure to 0.5 and 1 mM TDCA. EMSA was performedas described above. Positions of the specifically bound DNA–proteinbinding complex are indicated. (B) The effect of the IκB superrepressoron TDCA-induced prolifeation. *P < 0.05 versus control; **P < 0.05versus TDCA alone.

(37, 40). However, we have determined that TDCA ac-tivates by inducing degradation of IκB and cytosolic re-lease of NF-κB, which then translocates into the nucleusand binds to the DNA, resulting in regulation of gene tran-scription (18).

Bile acids have been shown to have many physiologiceffects besides their primary role of facilitating lipid andlipid-soluble vitamin absorption. Deoxycholate has beenshown to stimulate prostaglandin E2 synthesis by causingabrupt transient increases in cytosolic calcium (41). Othershave shown that there exist bile acid response elements,

Fig 6. The effect of the IκBSR (I) on TNF-α/C-induced apoptosisin the presence of 0.5 mM TDCA. IEC-6 cells transfected with theIκBSR were grown in the presence and absence of 0.5 mM TDCA for6 days. Apoptosis was induced using TNF-α/C as described above. Dis-played on theY-axis is the percentage of viable cells. *P < 0.01 versusTNF-α/C alone; **P < 0.01, TDCA+ TNF-α/C versus TNF-α/C +TNF-α + I.

DNA sequences that contain AGGTCA direct repeats sim-ilar to the elements recognized by nuclear receptors, whichregulate transcription of target genes (42, 43). Bile acidsregulate many functions within cells by activating signal-ing pathways and transcription factors. Clearly they havemany purposes besides just absorbing fat. The presenceof bile salts are important to regulate these functions; theabsence of bile salts may be quite detrimental to intestinalfunction. The mucosal atrophy of starvation and criticalillness, while multifactorial in etiology, may also be re-lated to cholestasis and diminished luminal bile. Bile saltsmay play a critical role during necrotizing enterocolitis, is-chemia, inflammatory bowel disease, and other conditionswhere there is mucosal injury. Further characterization ofthe beneficial functions of bile salts on the small intestinalmucosal are warranted.

REFERENCES

1. Baeuerle PA, Henkle P: Function and activation of NF-κB in theimmune system. Annu Rev Immunol 12:141–179, 1994

2. Barnes PJ, Karin M: Nuclear factor-κB, a pivotal transcription factorin chronic inflammatory diseases. N Engl J Med 336:1066–1071,1997

3. Jobin C, Sartor RB: The IκB/NF-κB system: A key determinant ofmucosal inflammation and protection. Cell Physiol 278:451–462,2000



4. Brown KS, Gerstberger L, Carlson G,et al.: Control of IκBαproteolysis by site-specific, signal-induced phosphrylation. Science267:1485–1488, 1995

5. Beg AA, Finco TS, Nantermet PV,et al.: Tumor necrosis factor andinterleukin-1 lead to phosphorylation and loss of IκBα: A mecha-nism for NF-κB activation. Mol Cell Biol 13:3301–3310, 1993

6. Lee FS, Hagler J, Chen ZJ,et al.: Activation of the IκBα kinasecomplex by MEKK1, a kinase of the JNK pathway. Cell 88:213–222, 1997

7. Mercurio FH, Zhu BW, Murray A,et al.; IKK-1 and IKK-2:Cytokine-activated IκB kinases essential for NF-κB activation.Science 278:860–866, 1997

8. Thompson JE, Phillips RJ, Erdjument-Bromage H,et al.: IκBβ reg-ulates the persistent response in a biphasic activation of NF-κB. Cell80:573–582, 1995

9. Beg AA, Baltimore D: An essential role for NF-κB in preventingTNF-α-induced cell death. Science 274:782–784, 1996

10. Lin K-I, DiDonato JA, Hoffman A, Hardwick JM, Ratan RR: Sup-perssion of steady-state, but not stimulus induced, NF-κB activityinhibits alphavirus-induced apoptosis. J Cell Biol 141:1479–1487,1998

11. Qin ZH, Wang Y, Nakai M, Chase TN: Nuclear factor-κB contributesto excitotoxin-induced apoptosis in rat striatum. Mol Pharmacol53:33–42, 1998

12. Van Antwerp DJ, Martin SJ, Kafri TD, Green R, Verma LM: Sup-pression of TNF-α-induced apoptosis by NF-κB. Science 274:787–789, 1996

13. Li L, Rao JN, Bass BL, and Wang J-Y: NF-κB activation and suscep-tibility to apoptosis after polyamine depletion in intestinal epitelialcells. Am J Physiol Gastrointest Liver Phsyiol 280:G992–G1004,2001

14. Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D: Embryoniclethality and liver degeneration in mice lacking the RelA componentof NF-κB. Nature 376:167–170, 1995

15. Wu M, Lee H, Bellas RE, Schauer SL, Arsura DM, Fitzgerald MJ,Rothstein TL, Sherr DH, Sonenshein GE: Inhibition of NF-κB/Relinduces apoptosis of murine B cells. EMBO J 15:4682–4690,1996

16. Wang, J-Y, McCormack SA, Viar MJ,et al.: Decreased expressionof protooncogenes c-fos, c-myc and c-jun following polyamine de-pletion in IEC-6 cells. Am J Physiol 265 (Gastrointest Liver Physiol30):G331–G338, 1993

17. Opipari AW jr, Hu HM, Yabknoitz R, Dixit VM: The A20 zincfinger protein protects cells from tumor necrosis factor cytotoxicity.J Biol Chem 267:12424–12427, 1992

18. Strauch ED, Bass BL, Rao JN, Vann JA, Wang J-Y: NF-κB regulatesintestinal epithelial cell and bile salt induced migration after injury.Ann Surg 237:494–501, 2003

19. Ford WD, de Vries JE, Ross JS, Malt RA: Effect of luminal contentson postresectional longtiduninal and mucosal growth in the ileumof suckling rats. Surgery 98:935–941, 1985

20. Thompson JS, Ferguson DC: Effect of the distal remnant on ilealadaptation. J Gastrointest Surg 4:430–434, 2000

21. Roy CC, Laurendeau G, Doyon G, Chartran L, Rivest MR: The effectof bile and of sodium taurocholate on the epithelial cell dynamicsof the rat small intestine. Proc Soc Exp Biol Med 149:1000–1004,1975

22. Williamson RC, Bauer FL, Ross JS, Malt RA: Contributions of bileand pancreatic juice to cell proliferation in ileal mucosa. Surgery83:570–576, 1978

23. Al-Mukhtar MY, Sagor GR, Ghatei MA, Bloom SR, Wright NA:The role of pancreatico-biliary secretions in intestinal adaptation

after resection, and its relationship to plasma enteroglucagon. Br JSurg 70:398–400, 1983

24. Quaroni A, Wands J, Trelstad RL, Isselbacher KJ: Epithelial cellcultures from rat small intestine. J Cell Biol 80:248–265, 1979

25. Ye ZS, Samuels HH: Cell- and sequence-specific binding of nuclearprotein to 5′-flanking DNA of the rat growth hormone gene. J BiolChem 262:6313–6317, 1987

26. Bradford MM: A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the priciple of protein-dyebinding. Anal Biochem 72:248–254, 1976

27. Laemmli UK: Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature 227:680–685, 1970

28. Graham FL, Prevec L: Manipulation of adenovirus vectors.In Meth-ods in Molecular Biology: Gene Transfer and Expression Protocols.EJ Murray (ed). Humana Press, Clifton, NJ, 1991, pp 109–128

29. Bett AJ, Haddara L, Prevec L, Graham FL: An efficient and flexiblesystem for construction of adenovirus vectors with insertion of dele-tions in regions 1 and 3. Proc Natl Acad Sci USA 91:8802–8806,1994

30. limuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhaven R,Grisham JW, Brenner DA: NF-κB prevents apoptosis and liver dys-function during liver regeneration. J Clin Invest 101(4):802–811,1998

31. Hoffman AF: Intestinal absorption of bile acids and biliary con-stituents Physiol Gastrointest Tract 2:1845–1865, 1994

32. Boehm G, Braun W, Moro G, Minoli I: Bile acid concentrations inserum and duodenal aspirates of healthy and preterm infants: Effectsof gestational and postnatal age. Biol Neonate 71:207–214, 1997

33. Sayan M, Alponat A, Yavus N, Altinkaya E, Goksel S, Sariyar M:The effect of oral sodium taurocholate on endotoxemia and intestinalanastomotic wound healing in rats with obstructive jaundice. SurgToday 27:953–957, 1997

34. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin DV Jr:NF-κB antiapoptosis; Induction of TRAF1 and TRAF2 and c-IAP2to suppress caspase-8 activation. Science 281:1680–1683, 1998

35. Hsu H, Huang J, Shu HB,et al.: TNF-dependent recruitment ofthe protein kinase RIP to the TNF receptor I signaling complex.Immunity 4:387–396, 1996

36. Hsu H, Shu HB, Pan MG,et al.: TRADD-TRAF-2 and TRAD-FADD interactions define two distinct TNF receptor signal trans-duction pathways. Cell 84:299–308, 1996

37. Burns K, Martinon F, Esslinger C,et al.: MYD88, and adaptor pro-tein involved in interleukin-1 signaling. J Biol Chem 273:12203–12209, 1998

38. Cao Z, Henzel WJ, Gao X: IRAK: A kinase associated with theinterleukin-1 receptor. Science 271:1128–1131, 1996

39. Cao Z, Xiong J, Takeuchi M,et al.: TRAF6 is a signal transductorfor interleukin-1. Nature 383:443–446, 1996

40. Wesche H, Henzel WJ, Shillinglaw W,et al.: MYD88: An adaptorthat recruits IRAK to the IL-1 receptor complex. Immunity 7:837–847, 1997

41. Zhu Y, Hua P, Rafiq S, Waffner EJ, Duffey ME, Lance P: Ca2+and PKC-dependent stimulation of PGE2 synthesis by deoxycholicacid in human colonic fibroblasts. Am J Physiol Gastrointest LiverPhysiol 283:G503–G510, 2002

42. Chiang JYL, Kimmel R, Weinberger C, Stroup D: Farnesoid Xreceptor responds to bile Acids and represses cholesterol 7α-hydroxylase gene (CYP7A1) transcription. J Biol Chem 275:10918–10924, 2000

43. Chen W, Owsley E, Yang Y, Stroup D, Chian JYL: Nuclear receptor-mediated repression of human cholesterol 7α-hydroxylase gene tran-scription by bile acids. J Lipid Res 42:1402–1412, 2001


Documents

Taurodeoxycholate Stimulates Intestinal Cell Proliferation and Protects Against Apoptotic Cell Death Through Activation of NF-κB