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Am J Physiol Gastrointest Liver Physiol 293: G568-G576, 2007. First published June 28, 2007; doi:10.1152/ajpgi.00201.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

Polyamines are required for expression of Toll-like receptor 2 modulating intestinal epithelial barrier integrity

Jie Chen,1,3 Jaladanki N. Rao,1,3 Tongtong Zou,1,3 Lan Liu,1,3 Bernard S. Marasa,1,2 Lan Xiao,1,3 Xing Zeng,1 Douglas J. Turner,1,3 and Jian-Ying Wang1,2,3

1Cell Biology Group, Department of Surgery; 2Department of Pathology, University of Maryland School of Medicine; and 3Baltimore Veterans Affairs Medical Center, Baltimore, Maryland

Submitted 4 May 2007 ; accepted in final form 27 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Toll-like receptors (TLRs) allow mammalian intestinal epithelium to detect various microbes and activate innate immunity after infection. TLR2 and TLR4 have been identified in intestinal epithelial cells (IECs) as fundamental components of the innate immune response to bacterial pathogens, but the exact mechanism involved in control of TLR expression remains unclear. Polyamines are implicated in a wide variety of biological functions, and regulation of cellular polyamines is a central convergence point for the multiple signaling pathways driving different epithelial cell functions. The current study determined whether polyamines regulate TLR expression, thereby modulating intestinal epithelial barrier function. Depletion of cellular polyamines by inhibiting ornithine decarboxylase (ODC) with {alpha}-difluoromethylornithine decreased levels of TLR2 mRNA and protein, whereas increased polyamines by ectopic overexpression of the ODC gene enhanced TLR2 expression. Neither intervention changed basal levels of TLR4. Exposure of normal IECs to low-dose (5 µg/ml) LPS increased ODC enzyme activity and stimulated expression of TLR2 but not TLR4, while polyamine depletion prevented this LPS-induced TLR2 expression. Decreased TLR2 in polyamine-deficient cells was associated with epithelial barrier dysfunction. In contrast, increased TLR2 by the low dose of LPS enhanced epithelial barrier function, which was abolished by inhibition of TLR2 expression with specific, small interfering RNA. These results indicate that polyamines are necessary for TLR2 expression and that polyamine-induced TLR2 activation plays an important role in regulating epithelial barrier function.

innate immunity; ornithine decarboxylase; epithelial paracellular permeability; lipopolysaccharide; intestinal epithelial integrity

EPITHELIAL CELLS LINE THE intestinal mucosa and form an important barrier that protects the subepithelial tissue against a wide array of noxious substances, allergens, and luminal microbial pathogens. Instead of a passive barrier, intestinal epithelium acts as an active sensor of the luminal environment and participates in the mucosal immune response through expression of proinflammatory genes, release of inflammatory cytokines, and recruitment of inflammatory cells in response to pathogenic bacteria and their products (35, 43). Intestinal epithelial cells (IECs) express a variety of potential sensing receptors, including Toll-like receptors (TLRs), which allow mammalian intestinal epithelium to detect various microbes and different pathogens (1, 20). An increasing body of evidence indicates that TLRs are fundamental components of the innate immune response and recognize structures that are common to different bacterial pathogens and to some endogenous molecules (2). To date, at least 10 human TLRs have been identified, which are type I transmembrane proteins containing an extracellular domain with leucine-rich repeats and cytoplasmic domain with sequence homology to the IL-1 receptor and several plant disease-resistance proteins (9, 12). Although IECs express TLRs at their apical poles in vivo (3, 33), the intestinal mucosa nevertheless coexists with the commensal microflora without chronic inflammation (30). Transformed IECs that highly express TLRs in vitro are shown to be slightly responsive to TLR ligands (1, 34), suggesting that sensing of commensal bacteria through TLRs in vivo does not initiate inflammatory cascades but rather is required for the maintenance of the epithelial barrier (5) and TLR-dependent intestinal homeostasis (39). However, the exact mechanisms involved in control of TLR expression in IECs remain unknown.

The natural polyamines spermidine and spermine and their precursor putrescine are organic cations found in all eukaryotic cells and have distinct regulatory roles in IECs (31). Since high intracellular polyamine levels are detrimental to cells, polyamine content in IECs needs to be critically controlled under physiological conditions (13). Ornithine decarboxylase (ODC) catalyzes the conversion of ornithine into putrescine, which is the first rate-limiting step in the synthesis of endogenous polyamines (13, 31). Induced ODC by treatment with various growth factors or ectopic expression of the ODC gene increases cellular polyamines (29, 48), whereas inhibition of ODC by its active-site inhibitor, D,L-{alpha}-difluoromethylornithine (DFMO), leads to a decrease in intracellular polyamines (4). Polyamines modulate expression of various genes involved in cell proliferation (24, 28), growth arrest (26, 41), and apoptosis (25, 54) and are absolutely required for maintenance of intestinal epithelial integrity (15, 50). Moreover, polyamines are not involved only in regulating cell growth and death. For example, polyamines are shown to play a role in the inflammatory response in vitro because exposure to the endotoxin LPS increases ODC mRNA expression in monocytes (32). Furthermore, spermine represses expression of the inducible isoform of nitric oxide synthase in LPS-treated J774.2 macrophages (46) and decreases production of IL-12 p40 and IFN-{gamma}, but it significantly increases IL-10 synthesis in LPS-treated macrophages (18). Recently, Soulet and Rivest (44) have reported that polyamines are crucial for the innate immune response in the mouse central nervous system and have a major impact on the neuronal integrity and cerebral homeostasis during immune insults.

Several studies have shown that IECs highly express TLR2 and TLR4 in vitro as well as in vivo (3, 5, 21, 43) and that activation of TLR2-signaling pathways enhances zonula occludens-1 (ZO-1)-associated epithelial barrier integrity (5). Our recent studies (15, 16, 50) have demonstrated that polyamines are necessary for expression of various intercellular junction proteins such as occludin, ZO-1, and E-cadherin and that polyamine depletion by inhibiting ODC with DFMO disrupts the intestinal epithelial barrier function. The current study further tested the hypothesis that polyamines regulate expression of TLR2- and TLR4-modulating intestinal epithelial barrier function. First, we examined changes in basal levels of TLR2 and TLR4 mRNAs and proteins following decreased or increased levels of cellular polyamines. Second, we determined the response of TLR expression to the specific TLR ligand, LPS, in the presence or absence of cellular polyamines. Finally, we examined the involvement of polyamine-modulated TLR expression in the regulation of the epithelial barrier function. Some of these results have been published previously in abstract form (7).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and supplies. Disposable cultureware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum (dFBS) were obtained from Invitrogen (Carlsbad, CA), and biochemicals were from Sigma (St. Louis, MO). The primary antibody, monoclonal anti-ODC antibody (0-1136), and the secondary antibody, anti-mouse IgG conjugated to horseradish peroxidase (A-8924), were purchased from Sigma. The affinity-purified rabbit polyclonal antibodies against TLR2 and TLR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and DFMO was from Ilex Oncology (San Antonio, TX). L-[1-14C]ornithine (sp act 51.6 Ci/mmol) was purchased from NEN (Boston, MA), and 14C-labeled mannitol was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). The 12-mm Transwell filters (0.4-µm pore size, clear polyester) were obtained from Costar (Cambridge, MA).

Cell culture. The IEC-6 cell line was purchased from the American Type Culture Collection (ATCC) at passage 13. The cell line was derived from normal rat small intestine and was developed and characterized by Quaroni et al. (38). IEC-6 cells originated from intestinal crypt cells, as judged by morphological and immunologic criteria. They are nontumorigenic and retain undifferentiated characteristics of small intestinal crypt cells. Stock cells were maintained in T-150 flasks in DMEM supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2, and passages 15–20 were used in experiments. IEC-6 cells at passages 15–20 exhibit a stable phenotype (23, 27).

ODC-overexpressing IEC-6 (ODC-IEC) cells were developed as described (29, 57) and expressed a more stable ODC variant with full enzyme activity (14). Replication-defective retroviral vector for ODC gene expression (pLOSN) was a gift from Dr. Susan K. Gilmour (Lankenau Institute for Medical Research, Wynnewood, PA) (8). The pLOSN vector encoded the mouse ODC cDNA containing an introduced stop codon at position 425, which resulted in a truncated and more stable ODC protein with full enzyme activity (14, 29). Cells were infected with pLOSN and control retroviral vector lacking ODC cDNA (pLXSN) for 4 h with 4 µg/ml polybrene. After 2 days, the cells were selected with medium containing G418 (600 µg/ml) for 3–7 days. Three days after refeeding with serum-containing medium, cells were assayed for levels of ODC protein and its enzyme activity.

The Caco-2 cells (a human colon carcinoma cell line) also were obtained from ATCC at passage 16. They were maintained similarly to the IEC-6 cells except that they were maintained in an atmosphere of 95% air-5% CO2. The medium used was Eagle's minimum essential medium with 10% heat-inactivated FBS, and passages 18–23 were used for the experiments as described in our previous studies (26).

RNA interference. The small interfering (si) RNA specifically targeting the coding region of TLR2 mRNA (siTLR2) was purchased from Dharmacon (Chicago, IL). Scrambled control siRNA (C-siRNA), which had no sequence homology to any known genes, was used as the control. The siTLR2 and C-siRNA were transfected into cells as described previously (56, 57). Briefly, for each 60-mm cell culture dish, 15 µl of the 20 µM stock siTLR2 or C-siRNA was mixed with 300 µl of Opti-MEM medium (Invitrogen). This mixture was gently added to a solution containing 15 µl of LipofectAMINE 2000 (Invitrogen) in 300 µl of Opti-MEM. The solution was incubated for 20 min at room temperature and gently overlaid onto monolayers of cells in 3 ml of medium, and cells were harvested for various assays after 48 and 72 h incubation.

RNA isolation and real-time RT-PCR analysis. Total RNA was extracted with the RNAeasy mini kit from Qiagen by following the instructions provided by the manufacturer. Final RNA was dissolved in water and estimated from its UV absorbance at 260 nm using a conversion factor of 40 units. Real-time quantitative PCR (Q-PCR) was performed using an Applied Biosystems instrument (Applied Biosystems, Foster City, CA) using specific primers, probes, and software (57) (Applied Biosystems). Briefly, RNA at the amount of 200 ng was placed into a 20-µl reaction volume containing 1 µl of specific primers and probe for TLR2 or TLR4 and 10 µl of 2x TaqMan Fast Universal PCR Master mixture. Detection of the fluorescent product was performed at the end of the extension period at 60°C for 1 min. To confirm amplification specificity, the PCR products were subjected to a standard curve analysis. The level of TLR2 and TLR4 mRNAs was quantified by real-time PCR analysis and normalized GAPDH levels with the Applied Biosystems analysis software.

Western blot analysis. Cell samples, placed in SDS sample buffer (250 mM Tris·HCl, pH 6.8, 2% SDS, 20% glycerol, and 5% mercaptoethanol), were sonicated, and then centrifuged (10,000 g) at 4°C for 15 min. The supernatant from cell samples was boiled for 5 min and then subjected to electrophoresis on 10% acrylamide gels according to the method of Laemmli (22). After the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 1x phosphate-buffered saline/Tween 20 [PBS-T: 15 mM NaH2PO4, 80 mM Na2HPO4, 1.5 M NaCl, pH 7.5, and 0.5% (vol/vol) Tween 20]. Immunological evaluation was then performed for 1 h in 1% BSA/PBS-T buffer containing 1 µg/ml of the specific antibody against ODC, TLR2, or TLR4 proteins. The filters were subsequently washed with 1x PBS-T and incubated for 1 h with the second antibody conjugated to peroxidase by protein cross-linking with 0.2% glutaraldehyde. After extensive washing with 1x PBS-T, the immunocomplexes on the filters were reacted for 1 min with Chemiluminescence Reagent (NEL-100; NEN, Boston, MA). Finally, the filters were placed in a plastic sheet protector and exposed to autoradiography film for 30 or 60 s.

Paracellular tracer flux assay. Flux assays were performed on the 12-mm Transwell as described in our previous publications (15, 16). Briefly, cells were grown in control cultures or cultures containing 5 mM DFMO or DFMO plus 10 µM putrescine for 2 days, then trypsinized and plated at confluent density of 4 x 104 cells/cm2 on the insert, and maintained at same culture conditions for additional 48 h to establish tight monolayers. [14C]mannitol (mol wt 184) is a membrane-impermeable molecule and served as the paracellular tracer in this experiment. At the beginning of the flux assay, both sides of the bathing wells of Transwell filters were replaced with fresh medium containing 5 mM unlabeled mannitol. The tracer was added to a final concentration of 3.6 nM in the apical bathing wells that contained 0.5 ml of medium. The basal bathing well had no added tracers and contained 1.5 ml of the same flux assay medium as in the apical compartment. All flux assays were performed at 37°C, and the basal medium was collected 2 h after addition of [14C]mannitol for a Beckman liquid scintillation counter. The results were expressed as percentage of total count values of tracer.

Assay for ODC enzyme activity. ODC activity was determined by radiometric technique in which the amount of 14CO2 liberated from L-[1-14C]ornithine was estimated (48). Sample collection and the assay procedure were carried out as described in our previous publications (29, 49). Enzymatic activity was expressed as picomoles of CO2 per milligram of protein per hour.

Polyamine analysis. The cellular polyamines content was analyzed by high-performance liquid chromatography (HPLC) as previously described (48). Briefly, after 0.5 M perchloric acid was added, the cells were frozen at –80°C until ready for extraction, dansylation, and HPLC analysis. The standard curve encompassed 0.31–10 µM. Values that fell >25% below the curve were considered undetectable. The results are expressed as nanomoles of polyamines per milligram of protein.

Statistical analysis. All data are expressed as means ± SE from six samples. PCR and immunoblotting results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined by Dunnett's multiple range test (17).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of cellular polyamines on expression of TLR2 and TLR4 in IECs. Since polyamines are implicated in the regulation of different epithelial functions through distinct signaling pathways, we sought to further investigate whether polyamines regulate expression of TLRs in IECs. First, we examined changes in expression of TLR2 and TLR4 following decreased levels of cellular polyamines. Consistent with our previous studies (40, 49), levels of cellular polyamines were depleted by inhibiting ODC with its specific inhibitor DFMO (4). Exposure of IEC-6 cells to 5 mM DFMO for 4 days completely inhibited ODC enzyme activity and substantially decreased cellular polyamines. Levels of putrescine and spermidine were undetectable on day 4 after treatment with DFMO, and spermine had decreased by ~60% (data not shown). Similar results have been published in our previous studies (23, 49). As shown in Fig. 1, A and B, depletion of cellular polyamines by DFMO significantly inhibited TLR2 expression but had no effect on levels of TLR4 mRNA and protein. Level of TLR2 mRNA in cells exposed to DFMO for 4 days was decreased by ~70%, which was associated with a decrease in TLR2 protein (Fig. 1B). In the presence of DFMO, addition of exogenous putrescine (10 µM) to cultures not only prevented the decreased level of TLR2 mRNA but also restored TLR2 protein level to near normal. Spermidine (5 µM) had an effect equal to putrescine on TLR2 expression when it was added to cultures that contained DFMO (data not shown). On the other hand, polyamine depletion did not alter expression of TLR4 in IEC-6 cells. There were no significant differences in levels of TLR4 mRNA (Fig. 1Ab) and protein (Fig. 1B, bottom) between control cells and cells exposed to DFMO alone or DFMO plus putrescine for 4 days. These results indicate that decreased levels of cellular polyamines repress expression of TLR2 but not TLR4 in IECs.


Figure 1
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  		Fig. 1. Changes in expression of TLR2 and TLR4 after decreased or increased levels of cellular polyamines in IEC-6 cells. A: levels of TLR mRNAs. a, TLR2 mRNA; b, TLR4 mRNA. IEC-6 cells were grown in the control cultures, cultures in which ornithine decarboxylase activity (ODC) was inhibited with 5 mM {alpha}-difluoromethylornithine (DFMO), and cultures inhibited with DFMO and supplemented with 10 µM putrescine (PUT) for 4 days. Total cellular RNA was harvested, and levels of TLR2 and TLR4 mRNAs were measured by quantitative real-time RT-PCR analysis. Data were normalized to amount of GAPDH (optical quantitative of TLR2 or TLR4 mRNA/optical quantitative of GADPH mRNA). Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with controls and cells exposed to DFMO plus PUT. B: representative immunoblots of Western analysis for TLR2 and TLR4 proteins in cells described in A. Fifty micrograms of total protein were applied to each lane, and immunoblots were hybridized with the specific antibody against TLR2 (~90 kDa) or TLR4 (~89 kDa). After the blots were stripped, actin (~42 kDa) immunoblotting was performed as an internal control for equal loading. Three separate experiments were performed that showed similar results. C: representative immunoblots for TLR2 and TLR4 in stable ODC-IEC cells. IEC-6 cells were infected with either the retroviral vector containing the sequence encoding mouse ODC cDNA or control retroviral vector lacking ODC cDNA, and stable clones highly expressing ODC were isolated and characterized. Whole cell lysates from clonal (C1 and C2) populations of stable ODC-IEC cells and cells infected with the control vector were harvested. Levels of TLR2 and TLR4 proteins were measured by Western blot analysis, and equal loading was monitored by immunoblotting of actin.

 
Second, the effect of increasing cellular polyamines by overexpression of the ODC gene on TLR expression was examined by using stable ODC-expressing cells (ODC-IEC) recently developed in our laboratory (29, 57). These stable ODC-IEC cells highly expressed ODC protein and exhibited greater than 50-fold increase in ODC enzyme activity. Levels of putrescine, spermidine, and spermine in stable ODC-IEC cells were increased by ~12-fold, ~2-fold, and ~25% when compared with cells transfected with the control vector lacking ODC cDNA (data not shown). As shown in Fig. 1C, expression of TLR2, but not TLR4, was substantially increased in stable ODC-IEC cells. Levels of TLR2 protein were increased by ~60% in stable ODC-IEC cells, while levels of TLR4 protein in stable ODC-IEC cells were indistinguishable from that observed in cells transfected with the control vector. The effect of ODC overexpression on expression of TLR2 was not due simply to clonal variation, since two stable clones, ODC-IEC-C1 and ODC-IEC-C2, showed similar responses. These results indicate that increasing cellular polyamines stimulates TLR2 expression in intestinal epithelial cells.

Effect of LPS on TLR expression in the presence or absence of cellular polyamines. To determine the role of cellular polyamines in the stimulation of TLR expression in IECs, we chose LPS, a specific TLR ligand (53), as the TLR activator in this study. Cells were grown in the absence or presence of 5 mM DFMO for 4 days and then treated with low-dose LPS (5 µg/ml) after 24-h serum deprivation. Exposure of normal quiescent IEC-6 cells (without DFMO) to this low dose of LPS stimulated polyamine biosynthesis as indicated by a significant increase in levels of ODC protein (Fig. 2Aa) and its enzyme activity (Fig. 2Ab). Levels of ODC protein and enzyme activity increased 1 h after LPS was added, peaked at 2–6 h after addition, and then began to decrease. Increased ODC protein and its enzyme activity by LPS were paralleled by an increase in levels of TLR2 mRNA (Fig. 2Ba) and protein (Fig. 2Bb). The marginal increases in TLR2 mRNA and protein were observed at 1 h after administration of the low dose of LPS, and the maximum increases occurred at 2–6 h thereafter. Levels of TLR2 protein were increased by ~2.2-fold at 2 h, ~2.7-fold at 4 h, and ~2.9-fold at 6 h after exposure to LPS. On the other hand, ODC activity in DFMO-treated cells was inhibited totally and cellular polyamines were depleted regardless with or without LPS treatment (data not shown). Polyamine depletion almost completely prevented this LPS-induced TLR2 expression, since there were only slight changes in levels of TLR2 mRNA (Fig. 3A) and protein (Fig. 3B) after exposure to LPS. However, treatment with LPS failed to alter expression of TLR4 in control and polyamine-deficient cells. There were no significant differences in levels of TLR4 mRNA and protein between control cells and DFMO-treated cells after exposure to LPS.


Figure 2
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  		Fig. 2. Effect of exposure to low-dose lipopolysaccharide (LPS) on ODC activity and TLR expression in IEC-6 cells. A: changes in ODC activity. Aa: representative immunoblots of Western analysis for ODC protein. Cells were cultured in control medium for 4 days and serum deprived for 24 h before experiments. Whole cell lysates were harvested at different times after treatment with LPS at the concentration of 5 µg/ml, and levels of ODC protein were measured by Western blot analysis using specific antibody against ODC (~53 kDa). Equal loading was monitored by immunoblotting of actin, and experiments were repeated three times. Ab: ODC enzyme activity in cells described in Aa. Values are means ± SE from 6 dishes. *P < 0.05 compared with groups at 0 h post LPS. B: changes in expression of TLR2 and TLR4 in cells described in A. Ba: levels of TLR2 and TLR4 mRNAs as measured by quantitative real-time RT-PCR analysis. Data were normalized to amount of GAPDH, and values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with groups at 0 h post LPS. Bb: representative immunoblots of Western analysis for TLR2 and TLR4 proteins. Three separate experiments were performed that showed similar results.

 

Figure 3
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  		Fig. 3. Changes in expression of TLR2 and TLR4 after exposure to LPS in polyamine-deficient IEC-6 cells. A: levels of TLR2 and TLR4 mRNAs. Cells were grown in the cultures containing 5 mM DFMO for 4 days, and total cellular RNA was harvested at different times after administration of LPS (5 µg/ml) in the presence of DFMO. Levels of TLR2 and TLR4 mRNAs were measured by quantitative real-time RT-PCR analysis. Data were normalized to amount of GAPDH, and values are means ± SE of data from 3 separate experiments. B: representative immunoblots of Western analysis for TLR2 and TLR4 proteins in cells described in A. Levels of TLR proteins were measured by Western blot analysis, and equal loading was monitored by immunoblotting of actin. Three experiments were performed that showed similar results.

 
To extend these findings obtained from IEC-6 cells, we further determined the effect of cellular polyamines in the stimulation of TLR2 expression in Caco-2 cells (a line of human colon epithelial cells) after exposure to LPS. Consistent with observations from IEC-6 cells, depletion of cellular polyamines by DFMO decreased the basal level of TLR2 protein but had no effect on expression of TLR4 in Caco-2 cells. Results presented in Fig. 4A also showed that exposure of Caco-2 cells to the low dose of LPS (5 µg/ml) increased expression of TLR2 protein, which was almost completely prevented by polyamine depletion (Fig. 4B). In addition, treatment with LPS did not affect expression of TLR4 protein regardless of presence or absence of cellular polyamines. These results indicate that induced polyamines are essential for the stimulation of TLR2 expression in intestinal epithelial cells.


Figure 4
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  		Fig. 4. Changes in expression of TLR2 and TLR4 proteins after exposure to LPS in the presence or absence of cellular polyamines in Caco-2 cells. A: representative immunoblots for TLR2 and TLR4 proteins in normal Caco-2 cells. Cells were grown in the standard growth medium for 4 days, and whole cell lysates were harvested at different times after treatment with LPS at the concentration of 5 µg/ml. Levels of TLR2 and TLR4 proteins were measured by Western blot analysis by using specific antibody against TLR2 or TLR4, and equal loading was monitored by actin immunoblotting. Three separate experiments were performed that showed similar results. B: representative immunoblots TLR2 and TLR4 proteins in polyamine-deficient Caco-2 cells. Cells were cultured in the presence of 5 mM DFMO for 4 days and then exposed to low-dose LPS. Levels of TLR2 and TLR4 proteins were measured at indicated times after administration of LPS.

 
Effect of polyamine-modulated TLR2 expression on the epithelial barrier function. To determine the involvement of polyamine-modulated TLR2 expression in the regulation of intestinal epithelial barrier function, the following three experiments were performed. In the first study, we assessed changes in the permeability as measured by the paracellular flux of membrane-impermeable tracer across the confluent monolayer in controls and polyamine-deficient cells with or without the challenge by the high dose of LPS (50 µg/ml). IEC-6 cells were grown in control cultures or cultures containing DFMO or DFMO plus putrescine for 2 days, then plated at confluent density on the insert, and maintained for additional 48 h to establish a tight monolayer in the same culture conditions. A widely accepted hydrophilic paracellular tracer molecule, [14C]mannitol (mol wt 184), was used in this study. To verify the system used for paracellular permeability assays, the effect of removal of extracellular Ca2+ on the paracellular flux of [14C]mannitol was tested, serving as a positive control. As expected, exposure to the Ca2+-free medium for 2 h remarkably increased paracellular permeability (data not shown). Interestingly, decreased TLR2 expression following polyamine depletion was associated with an increase in paracellular permeability (Fig. 5A). In DFMO-treated cells, level of paracellular flux of [14C]mannitol was about twofold the control value. Putrescine given together with DFMO restored the permeability to normal levels. Levels of paracellular flux of [14C]mannitol in cells exposed to DFMO plus putrescine were similar to those observed in control cells. Results presented in Fig. 5B further showed that epithelial barrier in polyamine-deficient cells was more vulnerable than that observed in control cells when they were challenged by high dose of LPS. Levels of paracellular flux of [14C]mannitol were increased from 9.8 ± 0.4 in control cells to 14.6 ± 0.6 in polyamine-deficient cells after challenge with high dose of LPS.


Figure 5
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  		Fig. 5. Changes in levels of paracellular permeability in control and polyamine-deficient IEC-6 cells with or without challenge with high-dose LPS. A: basal levels of paracellular permeability. After cells were grown in control cultures or cultures containing DFMO or DFMO plus PUT for 2 days, they were then trypsinized, plated at confluent density on the insert, and maintained under the same culture conditions for additional 48 h. Membrane-impermeable tracer molecule, [14C]mannitol, was added to the insert medium, and the entire basal medium was collected 2 h thereafter for paracellular tracer flux assays. Values are means ± SE of data from 6 samples. *P < 0.05 compared with control cells and cells treated with DFMO plus PUT. B: paracellular permeability in controls and polyamine-deficient cells after challenge with high-dose LPS (50 µg/ml) for 2 h. Values are means ± SE of data from 6 samples. *P < 0.05 compared with corresponding groups without the challenge with high-dose LPS. #P < 0.05 compared with control cells exposed to high-dose LPS. C: effect of pretreatment with low-dose LPS (5 µg/ml) on paracellular permeability after exposure to high-dose LPS (50 µg/ml) in control (left) and polyamine-deficient cells (right). Cells were grown in control medium or medium containing 5 mM DFMO for 2 days and then cultured on the insert for 48 h before experiments. Cells were initially treated with low-dose LPS for 4 h and then exposed to high-dose LPS. Levels of [14C]mannitol were measured 2 h after administration of high-dose LPS. Values are means ± SE of data from 6 samples. *P < 0.05 compared with control cells and cells exposed to low-dose LPS alone. +P < 0.05 compared with cells exposed to high-dose LPS alone.

 
In the second study, we examined effect of activation of TLR2 expression by treatment with low-dose LPS on the epithelial barrier function in the presence or absence of cellular polyamines. Initially, control and polyamine-deficient cells were pretreated with low-dose LPS (5 µg/ml) for 4 h and then exposed to high-dose LPS (50 µg/ml) for 2 h. Activation of TLR2 expression by pretreatment with low-dose LPS (as shown in Fig. 2) significantly protected the epithelial barrier from the challenge with high-dose LPS in control cells (Fig. 5C, left) but not in polyamine-deficient cells (Fig. 5C, right). After exposure to high-dose LPS, levels of paracellular flux of [14C]mannitol were decreased by ~50% in control cells pretreated with low-dose LPS. However, this protective effect of low-dose LPS on the epithelial barrier was completely prevented by polyamine depletion. There were no significant differences in paracellular flux of [14C]mannitol between cells exposed to DFMO alone and DFMO-treated cells pretreated with low-dose LPS after exposure to high-dose LPS. In fact, the levels of paracellular flux of [14C]mannitol in DFMO-treated cells exposed to both the low dose and high dose of LPS were slightly higher than those observed in DFMO-treated cells exposed to the high dose of LPS alone, although the difference was not statistically significant.

In the third study, we examined changes in paracellular permeability after specific inhibition of TLR2 expression by siTLR2. These specific siTLR2 nucleotides were designed to cleave rat TLR2 mRNA by activating endogenous RNase H and to have a unique combination of specificity, efficiency, and reduced toxicity (47). Initially, we determined the transfection efficiency of the siRNA nucleotides in IEC-6 cells and demonstrated that >95% of cells were positive when they were transfected with a fluorescent FITC-conjugated siRNA for 24 h (data not shown). As shown in Fig. 6A, transfection with the siTLR2 dramatically inhibited expression of TLR2 in IEC-6 cells. Levels of TLR2 protein were decreased by ~35% at 48 h and ~90% at 72 h after the transfection with siTLR2 compared with those observed in control cells and cells transfected with C-siRNA. Results presented in Fig. 6B further showed that inhibition of TLR2 expression by siTLR2 totally prevented the protective effect of the low dose of LPS on the epithelial barrier function. There were no significant differences in levels of paracellular flux of [14C]mannitol between control cells exposed to the high dose of LPS alone and siTLR2-transfected cells pretreated with the low dose of LPS and then exposed to the high doses of LPS. In contrast, levels of paracellular flux of [14C]mannitol were decreased by ~55% in C-siRNA-transfected cells that were pretreated with the low dose of LPS for 4 h and then challenged with the high dose of LPS as compared with control cells exposed to the high dose of LPS alone. In addition, neither siTLR2 nor C-siRNA affected cell viability as measured by Trypan blue staining (data not shown). These findings indicate that polyamine-induced TLR2 expression enhances the epithelial barrier function.


Figure 6
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  		Fig. 6. Effect of inhibition of TLR2 expression by specific siRNA targeting TLR2 (siTLR2) on paracellular permeability after exposure to high-dose LPS in IEC-6 cells. A: representative immunoblots of Western analysis for TLR2 protein. Cells were transfected with siTLR2 or control siRNA (C-siRNA), levels of TLR2 protein were measured 48 and 72 h after transfection, and equal loading was monitored by immunoblotting of actin. Three separate experiments were performed that showed similar results. B: changes in levels of paracellular permeability in TLR2-knockdown cells exposed to high-dose LPS. After cells were transfected with siTLR2 or C-siRNA for 24 h, they were transferred into transwells for 48 h before experiments. Cells were pretreated with low-dose LPS (5 µg/ml) for 4 h and then exposed to high-dose LPS (50 µg/ml). Levels of paracelluar permeability were measured 2 h after exposure to high-dose LPS. Values are means ± SE of data from 6 wells. *P < 0.05 compared with control cells; +P < 0.05 compared with cells treated with high-dose LPS alone; and #P < 0.05 compared with cells treated with C-siRNA and then exposed to low-dose LPS plus high-dose LPS.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Cellular polyamines are shown to play a critical role in maintenance of the intestinal epithelial integrity under physiological and various pathological conditions (16, 50, 51, 52), but few specific functions of polyamines at cellular and molecular levels have been defined to date. The present study provides new evidence showing that polyamines are required for expression of TLR2 in IECs and may be involved in the control of the innate immune system in the intestinal mucosa. Our results show that decreased levels of cellular polyamines by inhibiting ODC reduced basal levels of TLR2 mRNA and protein, while increased polyamines by ectopic ODC overexpression stimulated TLR2 expression, but neither intervention changed levels of TLR4. Exposure of IECs to LPS at the low dose stimulated the synthesis of endogenous polyamines as indicated by robust increases in ODC protein and its enzyme activity, which was associated with an increase in TLR2 expression. On the other hand, polyamine depletion almost completely prevented the increased TLR2 expression induced by treatment with the low dose of LPS. This polyamine-mediated TLR2 expression has a functional consequence, since TLR2 activation by the low dose of LPS enhanced the epithelial barrier function. Furthermore, this protective effect on the epithelial barrier was abolished when endogenous TLR2 was specifically knocked down by TLR2 siRNA. These findings strongly suggest that TLR2 expression requires polyamines and plays an important role in regulating intestinal epithelial barrier function.

The results reported herein clearly show that cellular polyamines regulate expression of TLR2, but not TLR4, in IECs. To provide insight into the molecular basis for TLR2 expression by polyamines, the results presented in Fig. 1 indicate that levels of TLR2 mRNA decreased significantly in cells treated with DFMO, which was paralleled by a reduction of TLR2 protein. The specificity of these effects was examined by the addition of exogenous putrescine, which completely prevented the decreases in TLR2 mRNA and protein, indicating that the observed changes in TLR2 expression in DFMO-treated cells must be related to polyamine depletion rather than to the nonspecific effect of DFMO. This inhibitory effect of polyamine depletion on TLR2 expression is specific, because there were no significant differences in levels of TLR4 mRNA and protein between control cells and cells exposed to DFMO alone or DFMO plus putrescine. Consistently, increased levels of cellular polyamines by ODC overexpression failed to induce TLR4 expression. The exact reasons for the different responses of expression of TLR2 and TLR4 to changes in levels of cellular polyamines remain unknown, but they may be related to the following facts and possibilities. First, expression of TLR2 is much more sensitive than that of TLR4 in response to TLR ligands and stress in general (5, 11, 30, 35). Consistent with previous observations in other cell types (6, 11), results presented in Fig. 2 show that exposure to the low dose of LPS increased levels of TLR2 mRNA and protein but did not alter expression of TLR4 in IEC-6 and Caco-2 cells. Second, regulation of the TLR4 activity occurs at the levels of its protein-protein interactions, phosphorylation, and translocation (10, 19) but not at the level of its protein expression. In support of this possibility, our results indicate that basal level of TLR4 mRNA in IEC-6 cells was much lower than that of TLR2 mRNA (Fig. 2Ba), although the level of TLR4 protein was higher than that of TLR2 (Fig. 2Bb), suggesting that TLR4 protein is stable and has a low turnover rate. Third, polyamines may have distinct regulatory effects on expression of different types of TLRs, and expression of TLR4 in IECs is regulated through a distinct mechanism that is independent of cellular polyamines.

Although the exact mechanism by which cellular polyamines regulate expression of TLR2 in IECs remains unknown, the current study and previous findings (28, 29, 52) suggest that this stimulatory effect of polyamines on TLR2 expression appears to occur at the transcriptional level. A series of studies from our previous works (55–57) and work of Stephenson et al. (45) have demonstrated that polyamines are implicated in both transcription and posttranscription of various genes encoding different cellular signaling proteins and that decreases in mRNAs following polyamine depletion result predominantly from the inhibition of their gene transcription. In contrast, decreasing polyamines increases cellular signaling factors primarily by stabilizing their mRNAs and proteins (26, 57). For example, increasing polyamines results in an increase in levels of c-myc and c-jun mRNAs by stimulating mRNA synthesis without effect on their degradation (28, 49), whereas polyamine depletion decreases c-myc and c-jun mRNAs by repressing their gene transcription but not by affecting their mRNA stability (36, 49). On the other hand, decreasing cellular polyamines increases levels of p53 (24, 56), TGF-beta (41), nucleophosmin (56, 57), and JunD (26, 37) through stabilization of their mRNAs without effect on their gene transcription. Clearly, further studies are needed to define the molecular process by which polyamines regulate transcription of the TLR2 gene in IECs.

The data from the present study also show that polyamine-modulated TLR2 expression plays a critical role in the regulation of epithelial barrier function. Decreased levels of TLR2 in polyamine-deficient cells were associated with a significant increase in epithelial paracellular permeability (Fig. 5A), while activated TLR2 expression by increased polyamines through pretreatment with the low dose of LPS enhanced epithelial barrier function (Fig. 5C), which was prevented by inhibition of TLR2 expression with specific TLR2 siRNA (Fig. 6). These findings are consistent with observations from Cario et al. (5) who have demonstrated that activation of TLR2 signaling triggers a Ca2+-dependent pathway that regulates the complex tight junction-associated interactions of membrane signaling patterns leading to enhancement of intestinal epithelial barrier integrity. Our previous studies also show that cellular polyamines are implicated in regulating expression of various intercellular junction proteins through distinct cellular signaling pathways in IECs (15, 50). Polyamine depletion decreases levels of tight junction proteins occludin, ZO-1, and ZO-2 without affecting their mRNAs but inhibits expression of both mRNAs and proteins of claudin-2 and claudin-3 (16). In addition, polyamines regulate expression of the adherens junction protein E-cadherin at the transcriptional level, and depletion of cellular polyamines decreases E-cadherin mRNA primarily through inhibition of transcription of the E-cadherin gene (50). Polyamine depletion also destabilizes E-cadherin protein by decreasing intracellular Ca2+ concentration via decreasing membrane potential through inhibition of voltage-gated K+ channel expression (42). These different mechanisms involved in regulation of adherens junctions and tight junctions by polyamines are not surprising, because polyamines have been involved in multiple signaling pathways in the expression of various genes in IECs. It is unclear at present how polyamine-modulated TLR2 activation mediates changes in expression of adherens and tight junction proteins in IECs.

In summary, these results indicate that polyamines are necessary for TLR2 expression and implicated in the control of the innate immune system in intestinal epithelial cells. The regulatory effect of polyamines on TLR2 expression is specific because neither increased nor decreased levels of cellular polyamines alter expression of TLR4. In addition, polyamine depletion not only reduces the basal level of TLR2 expression but also prevents the induced TLR2 activation induced by exposure to the low dose of LPS. Our results have further demonstrated that polyamine-modulated TLR2 activation plays an important role in regulation of the epithelial barrier function. These findings suggest that polyamines function as the biological regulators for TLR2 expression and are crucial for the maintenance of the epithelial barrier integrity and mucosal homeostasis under physiological conditions.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported by Merit Review Grant from the Department of Veterans Affairs and by National Institutes of Health Grants DK-57819, DK-61972, and DK-68491. J.-Y. Wang is a Research Career Scientist, Medical Research Service, US Department of Veterans Affairs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Dr. Jian-Ying Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene Street, Baltimore, MD 21201 (e-mail: jwang{at}smail.umaryland.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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  1. Abreu MT, Fusajuki M, Arbiti M. TLR signaling in the gut in health and disease. J Immunol 174: 4453–4460, 2005.[Abstract/Free Full Text]
  2. Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol 12: 13–19, 2000.[CrossRef][Web of Science][Medline]
  3. Bambou JC, Giraud A, Menard S, Begue B, Rakotobe S, Heyman M, Taddei F, Cerf-Bensussan N, Gaboriau-Routhiau V. In vitro and ex vivo activation of the TLR5 signaling pathway in intestinal epithelial cells by a commensal Escherichia coli strain. J Biol Chem 279: 42984–42992, 2004.[Abstract/Free Full Text]
  4. Bey P, Danzin C, Van Dorsselaer V, Mamont P, Jung M, Tardif C. Analogues of ornithine as inhibitors of ornithine decarboxylase. New deductions concerning the topography of the enzyme's active site. J Med Chem 21: 50–55, 1978.[CrossRef][Web of Science][Medline]
  5. Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterology 127: 224–238, 2004.[CrossRef][Web of Science][Medline]
  6. Chabot S, Wagner JS, Farrant S, Neutra MR. TLRs regulate the gate-keeping functions of the intestinal follicle-associated epithelium. J Immunol 176: 4275–4283, 2006.[Abstract/Free Full Text]
  7. Chen J, Rao JN, Zou TT, Liu L, Zhang AH, Turner DJ, Wang JY. Polyamines are required for expression of Toll-like receptors in intestinal epithelial cells and involved in regulation of innate immune response (Abstract). Gastroenterology 130: A232, 2006.
  8. Clifford A, Morgan D, Yuspa SH, Soler AP, Gilmour S. Role of ornithine decarboxylase in epidermal tumorigenesis. Cancer Res 55: 1680–1686, 1995.[Abstract/Free Full Text]
  9. Dangl J, Holub E. La dolce vita: a molecular feast in plant-pathogen interactions. Cell 91: 17–24, 1997.[CrossRef][Web of Science][Medline]
  10. Fan H, Peck OM, Tempel GE, Halushka PV, Cook JA. Toll-like receptor 4 coupled GI protein signaling pathways regulate extracellular signal-regulated kinase phosphorylation and AP-1 activation independent of NF-{kappa}B activation. Shock 22: 57–62, 2004.[CrossRef][Web of Science][Medline]
  11. Faure E, Thomas L, Xu H, Medyedev A, Equils O, Arditi M. Bacterial lipopolysaccharide and IFN-{gamma} induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-{kappa}B activation. J Immunol 166: 2018–2024, 2001.[Abstract/Free Full Text]
  12. Gay NJ, Keith FJ. Drosophila Toll and IL-1 receptor. Nature 351: 355–356, 1991.
  13. Gerner EW, Meyskens FL Jr. Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 4: 781–792, 2004.[CrossRef][Web of Science][Medline]
  14. Ghoda L, van Daalen Wetters T, Macrae M, Ascherman D, Coffino P. Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation. Science 243: 1493–1495, 1989.[Abstract/Free Full Text]
  15. Guo X, Rao JN, Liu L, Zou TT, Turner DJ, Bass BL, Wang JY. Regulation of adherens junctions and epithelial paracellular permeability: a novel function for polyamines. Am J Physiol Cell Physiol 285: C1174–C1187, 2003.[Abstract/Free Full Text]
  16. Guo X, Rao JN, Liu L, Zou T, Keledjian KM, Boneva D, Marasa D, Wang JY. Polyamines are necessary for synthesis and stability of occludin protein in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 288: G1159–G1169, 2005.[Abstract/Free Full Text]
  17. Harter JL. Critical values for Duncan's new multiple range test. Biometrics 16: 671–685, 1960.[CrossRef][Web of Science]
  18. Hasko G, Kubel DG, Marton A, Nemeth ZH, Deitch EA, Szabo C. Spermine differentially regulates the production of interleukin-12 p40 and interleukin-10 and suppresses the release of the T helper 1 cytokine interferon-gamma. Shock 14: 144–149, 2000.[Web of Science][Medline]
  19. Hazeki K, Masuda N, Funami K, Sukenobu N, Matsumoto M, Akira S, Takeda K, Seya T, Hazeki O. Toll-like receptor-mediated tyrosine phosphorylation of paxillin via MyD88-dependent and -independent pathways. Eur J Immunol 33: 740–747, 2003.[CrossRef][Web of Science][Medline]
  20. Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol 20: 197–216, 2002.[CrossRef][Web of Science][Medline]
  21. Kojima K, Musch MW, Ropeleski MJ, Boone Dl, Chang EB. Escherichia coli LPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation. Am J Physiol Gastrointest Liver Physiol 286: G645–G652, 2004.[Abstract/Free Full Text]
  22. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1990.
  23. Li L, Li J, Rao JN, Li M, Bass BL, Wang JY. Inhibition of polyamine synthesis induces p53 gene expression but not apoptosis. Am J Physiol Cell Physiol 276: C946–C954, 1999.[Abstract/Free Full Text]
  24. Li L, Rao JN, Gou X, Liu L, Santora R, Bass BL, Wang JY. Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation. Am J Physiol Cell Physiol 281: C941–C953, 2001.[Abstract/Free Full Text]
  25. Li L, Rao JN, Bass BL, Wang JY. NF-{kappa}B activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 280: G992–G1004, 2001.[Abstract/Free Full Text]
  26. Li L, Liu L, Rao JN, Esmaili A, Strauch ED, Bass BL, Wang JY. JunD stabilization results in inhibition of normal intestinal epithelial cell growth through P21 after polyamine depletion. Gastroenterology 123: 764–779, 2002.[CrossRef][Web of Science][Medline]
  27. Liu L, Santora R, Rao JN, Gou X, Zou T, Zhang HM, Turner DJ, Wang JY. Activation of TGF-beta-Smad signaling pathway following polyamine depletion in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 285: G1056–G1067, 2003.[Abstract/Free Full Text]
  28. Liu L, Li L, Rao JN, Zou T, Zhang HM, Boneva D, Bernard MS, Wang JY. Polyamine-modulated expression of c-myc plays a critical role in stimulation of normal intestinal epithelial cell proliferation. Am J Physiol Cell Physiol 288: C89–C99, 2005.[Abstract/Free Full Text]
  29. Liu L, Guo X, Rao JN, Zou T, Marasa BS, Chen J, Greenspon J, Casero RA Jr, Wang JY. Polyamine-modulated c-Myc expression in normal intestinal epithelial cells regulates p21Cip1 transcription through a proximal promoter region. Biochem J 398: 257–267, 2006.[CrossRef][Web of Science][Medline]
  30. Macdonald TT, Monteleone G. Immunity, inflammation, and allergy in the gut. Science 307: 1920–1925, 2005.[Abstract/Free Full Text]
  31. McCormack SA, Johnson LR. Role of polyamines in gastrointestinal mucosal growth. Am J Physiol Gastrointest Liver Physiol 260: G795–G806, 1991.[Abstract/Free Full Text]
  32. Messina L, Arcidiacono A, Spampinato G, Malaguarnera L, Berton G, Kaczmarek LL, Messina A. Accumulation of ornithine decarboxylase mRNA accompanies activation of human and mouse monocytes/macrophages. FEBS Lett 268: 32–34, 1990.[CrossRef][Web of Science][Medline]
  33. Ortega-Cava CF, Ishihara S, Rumi MA, Kawashima K, Ishimura N, Kazumori H, Udagawa J, Kadowadi Y, Kinoshita Y. Strategic compartmentalization of Toll-like receptor 4 in the mouse gut. J Immunol 170: 3977–3985, 2003.[Abstract/Free Full Text]
  34. Otte JM, Cario E, Podolsky DK. Mechanisms of cross hyporesponsiveness to Toll-like receptor bacterial ligands in intestinal epithelial cells. Gastroenterology 126: 1054–1070, 2004.[CrossRef][Web of Science][Medline]
  35. Panja A, Goldberg S, Eckmann L, Krishen P, Mayer L. The regulation and functional consequence of proinflammatory cytokine binding on human intestinal epithelial cells. J Immunol 161: 3675–3684, 1998.[Abstract/Free Full Text]
  36. Patel AR, Wang JY. Polyamines modulate transcription but not posttranscription of c-myc and c-jun in IEC-6 cells. Am J Physiol Cell Physiol 273: C1020–C1029, 1997.[Abstract/Free Full Text]
  37. Patel AR, Wang JY. Polyamine depletion is associated with an increase in JunD/AP-1 activity in small intestinal crypt cells. Am J Physiol Gastrointest Liver Physiol 276: G441–G450, 1999.[Abstract/Free Full Text]
  38. Quaroni A, Wands J, Trelstad RL, Isselbacher KJ. Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell Biol 80: 248–265, 1979.[Abstract/Free Full Text]
  39. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitoy R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118: 229–241, 2004.[CrossRef][Web of Science][Medline]
  40. Rao JN, Li J, Li L, Bass BL, Wang JY. Differentiated intestinal epithelial cells exhibit increased migration through polyamines and myosin II. Am J Physiol Gastrointest Liver Physiol 277: G1149–G1158, 1999.[Abstract/Free Full Text]
  41. Rao JN, Li L, Bass BL, Wang JY. Expression of the TGF-beta receptor gene and sensitivity to growth inhibition following polyamine depletion. Am J Physiol Cell Physiol 279: C1034–C1044, 2000.[Abstract/Free Full Text]
  42. Rao JN, Li L, Golovina VA, Platoshyn O, Strauch ED, Yuan JX, Wang JY. Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration. Am J Physiol Cell Physiol 280: C993–C1007, 2001.[Abstract/Free Full Text]
  43. Sitaraman SV, Merlin D, Wang L, Wong M, Gewirtz AT, Si-Tahar M, Madara JL. Neutrophil-epithelial crosstalk at the intestinal luminal surface mediated by reciprocal secretion of adenosine and IL-6. J Clin Invest 107: 861–869, 2001.[CrossRef][Web of Science][Medline]
  44. Soulet D, Rivest S. Polyamines play a critical role in the control of the innate immune response in the mouse central nervous system. J Cell Biol 162: 257–268, 2003.[Abstract/Free Full Text]
  45. Stephenson AH, Christian JF, Seidel ER. Polyamines regulate eukaryotic initiation factor 4E-binding protein 1 gene transcription. Biochem Biophys Res Commun 323: 204–212, 2004.[CrossRef][Web of Science][Medline]
  46. Szabo C, Southan GJ, Thiemermann C, Vane JR. The mechanism of the inhibitory effect of polyamines on the induction of nitric oxide synthase: role of aldehyde metabolites. Br J Pharmacol 113: 757–766, 1994.[Web of Science][Medline]
  47. Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF. Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem 278: 7108–7118, 2003.[Abstract/Free Full Text]
  48. Wang JY, Johnson LR. Polyamines and ornithine decarboxylase during repair of duodenal mucosa after stress in rats. Gastroenterology 100: 333–343, 1991.[Web of Science][Medline]
  49. Wang JY, McCormack SA, Viar MJ, Wang H, Tzen CY, Scott RE, Johnson LR. Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells. Am J Physiol Gastrointest Liver Physiol 265: G331–G338, 1993.[Abstract/Free Full Text]
  50. Wang JY. Polyamines regulate expression of E-cadherin and play an important role in control of intestinal epithelial barrier function. Inflammopharmacology 13: 91–101, 2005.[CrossRef][Medline]
  51. Wang JY. Polyamines and mRNA stability in regulation of intestinal mucosal growth. Amino Acids. In Press.
  52. Xiao L, Rao JN, Zou T, Liu L, Marasa BS, Chen J, Turner DJ, Passaniti A, Wang JY. Induced JunD in intestinal epithelial cells represses CDK4 transcription through its proximal promoter region following polyamine depletion. Biochem J 403: 573–581, 2007.[CrossRef][Web of Science][Medline]
  53. Yang RB, Mark MR, Gray A, Huang A, Xie MH, Zhang M, Goddard A, Wood WI, Gurney AL, Godowski PJ. Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signaling. Nature 395: 284–288, 1998.[CrossRef][Medline]
  54. Zhang HM, Rao JN, Guo X, Liu L, Zou T, Turner DJ, Wang JY. Akt kinase activation blocks apoptosis in intestinal epithelial cells by inhibiting caspase-3 after polyamine depletion. J Biol Chem 279: 22539–22547, 2004.[Abstract/Free Full Text]
  55. Zhang AH, Rao JN, Zou T, Liu L, Marasa BS, Xiao L, Chen J, Turner DJ, Wang JY. p53-dependent NDRG1 expression induces inhibition of intestinal epithelial cell proliferation but not apoptosis after polyamine depletion. Am J Physiol Cell Physiol 293: C379–C389, 2007.[Abstract/Free Full Text]
  56. Zou T, Rao JN, Liu L, Marasa BS, Keledjian KM, Zhang AH, Xiao L, Bass BL, Wang JY. Polyamine depletion induces nucleophosmin modulating stability and transcriptional activity of p53 in intestinal epithelial cells. Am J Physiol Cell Physiol 289: C686–C696, 2005.[Abstract/Free Full Text]
  57. Zou T, Mazan-Mamczarz K, Rao JN, Liu L, Marasa BS, Zhang AH, Xiao L, Pullmann R, Gorospe M, Wang JY. Polyamine depletion increases cytoplasmic levels of RNA-binding protein HuR leading to stabilization of nucleophosmin and p53 mRNAs. J Biol Chem 281: 19387–19394, 2006.[Abstract/Free Full Text]



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