HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 49, 45909-45913, December 7, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/49/45909    most recent M108097200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pfeffer, L. M. Articles by Johnson, L. R. Search for Related Content PubMed PubMed Citation Articles by Pfeffer, L. M. Articles by Johnson, L. R. Social Bookmarking                      What's this?

Polyamine Depletion Induces Rapid NF-B Activation in IEC-6 Cells^*

Lawrence M. Pfeffer^§, Chuan He Yang, Aruna Murti, Shirley A. McCormack^¶, Mary Jane Viar^¶, Ramesh M. Ray^¶, and Leonard R. Johnson^¶

From the Departments of  Pathology and ^¶ Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, August 22, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The proliferation of the rat intestinal mucosal IEC-6 cell line requires polyamines, whose synthesis is catalyzed by the enzyme ornithine decarboxylase (ODC). ODC inhibition leads to polyamine depletion, as well as inhibition of both cell proliferation and apoptosis by regulating gene expression. The NF-B transcription factor regulates genes involved in apoptotic, immune, and inflammatory responses. In the present study we tested the hypothesis that NF-B is activated following ODC inhibition. We found that the inhibition of ODC by -difluoromethylornithine (DFMO) resulted in a ~50% decrease in intracellular putrescine levels within 1 h. NF-B is activated by DFMO through the degradation of the inhibitory protein IB that sequesters NF-B in the cytoplasm. The DFMO-induced NF-B complexes contain the p65 and p50 members of the Rel protein family. DFMO-induced NF-B activation was accompanied by the translocation of p65 from the cytoplasm into the nucleus. DFMO selectively inhibited a gene reporter construct dependent on the B site present in the HLA-B7 gene. In contrast, DFMO had no effect on a gene reporter construct dependent on the B site present in the interleukin-8 gene. Thus, we report that ODC inhibition activates the NF-B transcription factor, which may mediate the altered physiological state of intestinal cells that occurs following polyamine depletion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The polyamines, spermidine and spermine, and their precursor putrescine are intimately required for cell growth and proliferation (1). Intracellular polyamine levels are highly regulated by ornithine decarboxylase (ODC),¹ which catalyzes the first rate-limiting step in polyamine biosynthesis. Therefore, specific inhibitors of ODC such as -difluoromethylornithine (DFMO) have been used to define the role of polyamines in cellular processes. The role of polyamines in the growth and repair of gastrointestinal mucosa has been examined extensively in the cultured intestinal epithelial cell line IEC-6, a nontransformed line derived from adult rat crypt cells. Previous studies have established that inhibition of ODC activity in IEC-6 cells leads to alterations in gene expression (2). Although polyamines are critical for optimal cell growth, excessive accumulation may interfere directly with normal cell function. Polyamines have been implicated recently in the control of the apoptotic response. For example, polyamine depletion by DFMO treatment in IEC-6 cells delays the onset of apoptosis by tumor necrosis factor- and the DNA topoisomerase inhibitor camptothecin (3). In contrast, in cells overexpressing ODC excessive accumulation of polyamines triggers apoptosis (4, 5).

Nuclear factor B (NF-B) is an inducible and ubiquitously expressed transcription factor. NF-B is a central regulator of the transcription of genes involved in cell survival, as well as genes involved in cell adhesion, immune and inflammatory responses, differentiation, and growth (6-10). Active NF-B complexes are dimers of various combinations of the Rel/NF-B family of polypeptides, which includes p50, p52, c-Rel, v-Rel, RelA (p65), and RelB (reviewed in Refs. 11 and 12). NF-B is sequestered in the cytoplasm by binding to inhibitory IB proteins, which block the nuclear localization sequences of NF-B. NF-B is activated by a variety of stimuli, including phorbol esters, cytokines (IL-1, interferon-/, and tumor necrosis factor), lipopolysaccharide, double-stranded RNA, bacteria, and viral transactivators. NF-B-inducing stimuli promote dissociation of the inactive NF-B/IB complexes via the serine phosphorylation and degradation of IB. These events lead to the unmasking of the nuclear localization sequence of NF-B, thereby allowing NF-B to enter the nucleus and bind B-regulatory elements.

In this study we tested the hypothesis that NF-B is activated following inhibition of ODC in intestinal cells. We found that inhibition of ODC by DFMO treatment results in depletion of cellular putrescine levels by 1 h. The depletion of putrescine levels was accompanied by rapid induction of NF-B activation as determined by its presence in B-dependent DNA-protein complexes. Several distinct complexes were detected that differ in Rel protein composition. In response to polyamine depletion, NF-B translocated from the cytoplasm into the nucleus. The DFMO-induced NF-B complexes selectively inhibited B-dependent reporter constructs. Thus, ODC inhibition by DFMO depletes cellular polyamine levels and activates the NF-B transcription factor, which may repress the expression of important cellular genes in intestinal cells. The regulation of gene expression mediated by NF-B activation may manifest itself in alterations in cell physiology, such as the intrinsic resistance of polyamine-depleted cells to apoptosis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cells-- The normal rat intestinal epithelial IEC-6 cell line (13) was obtained from the American Type Culture Collection (CRL-1592). IEC-6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% dialyzed fetal bovine serum, 10 µg of insulin/ml and 0.05 mg of gentamicin/ml (sDMEM). Stock cultures were subcultured once a week at 1:20, and sDMEM was replenished three times weekly. For experiments, the cells were plated at 4 × 10⁴ cells/cm² in sDMEM containing 5 mM D,L--difluoromethylornithine (DFMO). This dose of DFMO markedly inhibits ODC activity (95%) and entirely depletes putrescine and spermidine from IEC-6 cells by 6 and 48 h, respectively (2). In addition, this dose partially (60%) depletes spermine by 4 days. Control cultures received no DFMO. In some experiments 10 µM putrescine was added simultaneously with DFMO to demonstrate that exogenous polyamines can prevent the effects of DFMO. The inhibition of growth and migration resulting from polyamine depletion in IEC-6 cells can be prevented by adding 5 µM spermidine or 10 µM putrescine to DFMO-containing medium (2). Thus, the effects of DFMO treatment are caused by the absence of polyamines and not by DFMO itself.

Nuclear Extracts and Gel Shift Assays-- For preparation of nuclear extracts, the cultures were washed with ice-cold phosphate-buffered saline and harvested with a rubber policeman. Nuclei were extracted with buffer (20 mM Tris-HCl, pH 7.85, 250 mM sucrose, 0.4 M KCl, 1.1 mM MgCl₂, 5 mM -mercaptoethanol, 1 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin and 1.75 µg/ml benzamidine), and extracts were frozen and stored at 80 °C (14). For EMSA, the nuclear extracts were incubated with a ³²P-labeled B probe (5'-AGTTGAGGGGACTTTCCCAGG-3') derived from an NF-B binding sequence in the immunoglobulin gene promoter (15). To define the presence of specific Rel proteins, nuclear extracts were preincubated with a 1:25 dilution of anti-Rel antibodies at 25 °C for 20 min and then subjected to EMSA. The gels were quantitated by PhosphorImager autoradiography (Molecular Dynamics).

Assay of Intracellular Putrescine-- In brief, IEC-6 cells were rinsed three times with ice-cold PBS, 0.5 M perchloric acid was added, and the cells were frozen at 80 °C. The extracts were subjected to dansylation, and the intracellular level of putrescine in the range from 0.3 to 10 nmol/mg protein was determined by HPLC as described previously (2). The protein concentrations in extracts were determined by the Bradford method.

Immunocytochemistry-- IEC-6 cells were rinsed with PBS without Ca⁺² or Mg⁺² (PBS), fixed for 10 min in 4% paraformaldehyde in PBS, permeabilized for 5 min in 0.2% Triton X-100, and blocked with 3% bovine serum albumin in PBS. The cells were successively stained at room temperature for 1 h with rabbit polyclonal antibody to RelA (p65) and fluorescein isothiocyanate-conjugated anti-rabbit IgG. The cells were washed extensively after each incubation with PBS and mounted with VectaShield. The Images captured on a Bio-Rad MRC-1024 LaserSharp confocal laser scanning microscope were processed using Adobe Photoshop.

IB Degradation-- At various times after DFMO treatment, 1 × 10⁸ cells were lysed directly in Laemmli buffer, and equivalent amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes, immunoblotted with specific affinity-purified rabbit anti-IB antibody, and visualized by chemiluminescence with the ECL reagent (Amersham Pharmacia Biotech).

Transcriptional Assays-- IEC-6 cells were transfected by electroporation with either the pUXCAT promoter-less chloramphenicol acetyltransferase (CAT) construct; the pUXCAT 3XHLAB construct, which contains three tandemly repeated copies of the NF-B site from the HLA-B7 gene; or pUXCAT 3XIL8B, which contains three tandemly repeated copies of the NF-B site from the IL-8 gene (16). At 24-48 h after transfection, the cells were treated with DFMO for the indicated time or with both putrescine and DFMO and assayed for CAT activity. Acetylated and unacetylated [¹⁴C]chloramphenicol were separated by thin layer chromatography, and the radioactivity was measured by PhosphorImager autoradiography.

Statistics-- All data are expressed as the means ± S.E. from representative experiments. All experiments were repeated three times, in triplicate. Analysis of variance and appropriate post hoc testing determined the significance of the differences between means. Values of p < 0.05 were regarded as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Polyamine Depletion Induces the Activation of NF-B DNA Binding Activity-- NF-B proteins are present in the cytoplasm as latent transcription factors. To determine whether polyamine depletion induced NF-B activation, IEC-6 cells were treated with DFMO in the presence or absence of putresine. Nuclear extracts were prepared from the treated cells and incubated with a labeled B oligonucleotide probe, and the resultant DNA-protein complexes were analyzed by EMSA. Nuclear extracts from untreated IEC-6 cells show little detectable constitutive binding to a consensus B oligonucleotide probe. However, DFMO induced B binding within 1 h (noted by the arrows in Fig. 1A). NF-B binding persisted for 24 h after DFMO treatment (Fig. 1A). The DFMO-induced NF-B complexes at all times examined specifically reflected the effects of ODC inhibition and blockage of polyamine synthesis because their induction was prevented by the addition of putrescine.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   DFMO treatment induces the activation of NF-B DNA binding activity. A, nuclear extracts were prepared from control IEC-6 cells or cells treated with 5 mM DFMO in the absence or presence of 10 µM putrescine (put) for various time periods and subjected to EMSA with a 32P-labeled NF-B probe. The DFMO-induced complexes are denoted by arrows. B, to determine the specificity of the DFMO-induced DNA-protein complexes, nuclear extracts from DFMO-treated cells (5 h) were subjected to EMSA in the absence () or presence of a 50-fold excess of unlabeled NF-B oligonucleotide (B) probe or the unrelated sis-inducible element (SIE) oligonucleotide probe. To detect the presence of specific Rel proteins in DNA-protein complexes, nuclear extracts from DFMO-treated cells (5 h) were preincubated with anti-p50 or p65 Abs prior to EMSA analysis. The dotted arrow indicates the p65 supershifted band, whereas the solid arrow indicates the p50 supershifted band. The results shown are representative of four experiments.

To determine whether the binding to the B probe was specific, nuclear extracts prepared from DFMO-treated cells were incubated in the presence of a 50-fold excess of unlabeled B or an unrelated oligonucleotide probe. No DNA binding to the B probe was detected in the presence of excess unlabeled B oligonucleotide, and binding was not competed for by an excess of unrelated sis-inducible element oligonucleotide probe corresponding to a STAT-dependent DNA element (Fig. 1B). Taken together these results indicate that the binding to the B probe was specific.

As shown in Fig. 1, two distinct complexes were found to bind the B probe. These DFMO-induced complexes were detectable within 1 h of DFMO treatment and persisted for at least 24 h. Because active NF-B complexes are dimers of various combinations of the Rel/NF-B family of polypeptides, we defined the composition of the DFMO-induced NF-B complexes. To determine their composition we performed supershift assays with antisera directed against specific Rel proteins. As shown in Fig. 1B, the slowest migrating DFMO-induced complexes (denoted by the dotted arrow) contained p65 (RelA), because antisera to p65 supershifted the complex. In contrast, the faster migrating DFMO-induced complex (denoted by the solid arrow) was supershifted by antisera to p50. Thus, the DFMO-induced complexes of NF-B are composed of the p50 and p65 members of the Rel/NF-B family of polypeptides

ODC Inhibition Results in Depletion of Intracellular Polyamines-- The effect of DFMO could be reversed by the addition of exogenous putrescine, suggesting that the primary effect of ODC inhibition was depletion of intracellular polyamine levels as shown previously. To demonstrate directly that ODC inhibition by DFMO resulted in depletion of intracellular polyamine levels, the effect of DFMO treatment on the intracellular levels of putrescine was determined in IEC-6 cells. Perchloric acid extracts of IEC-6 cells were prepared at varying times after DFMO addition and subjected to dansylation, and the intracellular levels of putrescine were determined by HPLC. As shown in Fig. 2, the intracellular level of putrescine in control IEC-6 cells was relatively stable throughout the 6-h time course of the experiment (0.21 ± 0.03 nmol/mg protein). In contrast, DFMO treatment for 1 h resulted in ~50% decrease in intracellular putrescine levels. The intracellular putrescine levels continued to decline, reaching undetectable levels 6 h after addition of DFMO. These results confirm the previous finding that putrescine is rapidly depleted in IEC-6 cells upon ODC inhibition by DFMO (2).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   DFMO treatment depletes intracellular putrescine. Intracellular putrescine levels in perchloric extracts of IEC-6 cells 0-6 h after DFMO addition was determined by HPLC as described previously. The data shown are the averages of two experiments done in triplicate ± S.E. and normalized to the protein concentrations in the extracts.

Effects of Polyamine Depletion on the Distribution of p65-- In unstimulated cells, NF-B is localized in the cytoplasm because of the binding of inhibitory IB proteins, which block the nuclear localization sequences present in NF-B proteins. Upon stimulation, the inactive NF-B/IB complexes dissociate, the nuclear localization sequences of NF-B proteins are unmasked, and NF-B complexes enter the nucleus. As illustrated in Fig. 3, p65 (RelA) is distributed diffusely in the cytoplasm of control IEC-6 cells with no nuclear staining. After 1 h of treatment with DFMO, there was a dramatic redistribution of p65 into the nucleus, so that p65 was nearly exclusively in the nucleus with little cytoplasmic staining (Fig. 3). The translocation of p65 into the nucleus induced by DFMO treatment persisted for at least 5 h and specifically reflected the effects of ODC inhibition and blockage of polyamine synthesis because nuclear translocation was prevented by the addition of putrescine. Thus, p65 was present nearly exclusively in the cytoplasm of IEC-6 cells incubated for 5 h with DFMO in the presence of putrescine and resembled the distribution in control, untreated IEC-6 cells. The immunofluorescent studies on p65 confirmed the findings obtained by gel shift analysis, i.e. p65 translocates into the nucleus of IEC-6 cells upon DFMO addition.

View larger version (162K): [in this window] [in a new window]   Fig. 3.   DMFO induces the redistribution of p65 from the cytoplasm to the nucleus. IEC-6 cells were incubated with 5 mM DFMO for 0, 1, or 5 h, or with DFMO and 10 µM putrescine (put) for 5 h. The cells were fixed, permeabilized with Triton X-100, and immunostained for the presence of p65 (RelA). Magnification 40×. The results shown are representative of two experiments.

Polyamine Depletion Promotes the Degradation of IB-- The activity of NF-B is tightly controlled by inhibitory IB proteins that bind to NF-B complexes and sequester NF-B in the cytoplasm (11, 12). Viruses, cytokines, lipopolysaccharides, and other stimulating agents promote NF-B activation by the serine phosphorylation and subsequent degradation of IB. To determine whether NF-B activation observed after DFMO treatment reflects IB degradation, IB levels were determined at various times after DFMO addition by immunoblotting with anti-IB antisera. As shown in Fig. 4, DFMO induced a progressive decrease in cellular levels of IB, indicating that DFMO induced NF-B activation by promoting IB degradation. IB degradation was observable within 1 h of DFMO addition, and a dramatic decrease in IB levels was observed at 5 h after treatment. Quantitation of IB levels by PhosphorImager analysis of the data demonstrated a 15% decrease in IB levels at 1 h and a 70% decrease at 5 h. The kinetics of IB degradation are consistent with that of NF-B activation, i.e. a detectable decrease in IB at 1 h, at which time NF-B activation is observed. DFMO-induced degradation of IB specifically reflected the effects of blockage of polyamine synthesis because it was prevented by the addition of putrescine (Fig. 4).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   DFMO promotes the degradation of IB. IEC-6 cells were incubated with 5 mM DFMO for various times or with DFMO and 10 µM putrescine (Put) for 5 h. Cell lysates were prepared, resolved by SDS-polyacrylamide gel electrophoresis, blotted onto polyvinylidene difluoride membranes, probed with anti-IB, and visualized by enhanced chemiluminescence. The results shown are representative of three experiments.

Effects of Polyamine Depletion on NF-B-dependent Reporter Constructs-- To determine the functional consequences of NF-B activation induced by inhibition of ODC, we examined the effect of DFMO treatment on the transcriptional activity of CAT reporter constructs driven by NF-B-dependent promoters. IEC-6 cells were transfected with either the pUXCAT promoter-less CAT construct; the pUXCAT 3XHLAB construct, which contains three tandemly repeated copies of the NF-B site from the HLA-B7 gene; or pUXCAT 3XIL8B, which contains three tandemly repeated copies of the NF-B site from the IL-8 gene (16). The transfected cells were treated for either 1 or 24 h with DFMO and then assayed for CAT activity. As shown in Fig. 5A, there was significant basal activity of the promotorless CAT construct as determined by the formation of acetylated chloramphenicol. The basal activity of the promoter-less construct was not affected by DFMO treatment. In contrast, DFMO treatment for either 1 or 24 h resulted in a marked decrease (~90% inhibition) in the transcriptional activity of the 3XHLAB construct, which contains three tandemly repeated copies of the NF-B site from the HLA-B7 gene. In addition, DFMO treatment had no effect on a reporter construct driven by the NF-B site from the IL-8 gene (3XIL8B CAT).

View larger version (43K): [in this window] [in a new window]   Fig. 5.   DMFO effects on an NF-B-dependent reporter construct. A, IEC-6 cells were transiently transfected with pUXCAT, 3XHLAB, or 3XIL8B, treated with 5 mM DFMO, and assayed for CAT activity. After thin layer chromatography, radioactive acetylated products were visualized by PhosphorImager autoradiography. B, IEC-6 cells were transiently transfected with 3XHLAB or 3XIL8B, treated with DFMO in the absence or presence of 10 µM putrescine (put), and assayed for CAT activity. The data shown are from one of three experiments with quantitatively similar results.

A time course of the effect of DFMO on the CAT reporter activity driven by the HLA B site demonstrated that the inhibitory effect was first detected at 1 h of DFMO addition (Fig. 5). The inhibitory effect of DFMO was not observed in a CAT reporter activity driven by the IL-8 B site. The inhibitory effect of DFMO on the HLA B-dependent promoter persisted for at least 3 days after DFMO addition (data not shown). The decreased transcriptional activity of the HLA B-dependent reporter construct was specific for polyamine depletion, because putrescine addition blocked the DFMO-induced decrease in CAT reporter activity (Fig. 5B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The polyamines, spermidine and spermine, and their precursor putrescine are found in virtually all cells of higher eukaryotes and are intimately involved in and are required for cell growth and proliferation (1). An important mechanism of action of the polyamines concerns their control of growth-regulated genes. Increased ODC activity occurs concomitantly with increases in the mRNA of several proto-oncogenes in growth-stimulated cells (17). ODC is the enzyme responsible for catalyzing the first rate-limiting step in polyamine biosynthesis. Inhibition of ODC activity by DFMO decreases mRNA levels for the c-fos, c-myc, and c-jun proto-oncogenes in IEC-6 cells (18). In the present report we investigated the potential role of the NF-B transcription factor family in the regulation of gene expression by polyamines. We report that inhibition of ODC induces the activation of the NF-B transcription factor as measured by its presence in DNA-binding complexes and its translocation from the cytoplasm into the nucleus. DFMO-induced activation of NF-B specifically reflected the effects of ODC inhibition and blockage of polyamine synthesis because it was prevented by the addition of putrescine, which is the end product of the reaction catalyzed by ODC.

Distinct DFMO-induced NF-B complexes were detected that differ in Rel protein composition as well as in their time course of activation. Two DFMO-induced complexes were detectable within 1 h of DFMO treatment and persisted for at least 24 h. The results of supershift analysis with specific Rel antisera indicate that these complexes are comprised of p50 and p65. The dimeric forms of NF-B differ in their preference for certain B sites on DNA, transactivation potentials, kinetics of nuclear translocation, and levels of tissue expression (11). The p50 homodimer is generally thought to act as an inhibitor of B-dependent transcription (19).

Of particular interest was the rapidity of NF-B activation in response to DFMO. In Fig. 2 we show that in IEC-6 cells putrescine levels decreased by 50% within 1 h of exposure to DFMO and are at undetectable levels by 6 h. These data are nearly identical to those previously reported for IEC-6 cells (2). However, the levels of the polyamines, spermidine and spermine, did not decline at this time. In fact, spermidine does not decline to undetectable levels until 48 h of DFMO addition, and significant spermine remains after 6 days (2). Therefore, these results demonstrate that NF-B activation is clearly not caused by a general "polyamine depletion." Instead, we believe that there is only a small pool of free polyamines within the cell. This pool, consisting largely of putrescine, is rapidly depleted by conversion to spermidine upon inhibition of new putrescine synthesis. Because almost all intracellular polyamines must be bound and unavailable for biological processes, a slight decrease in putrescine levels is sensed by the cell and activates a NF-B-dependent response pathway. This view of maintaining cellular polyamine equilibrium is supported by the often reported finding that ODC activity peaks within 3-4 h of cellular exposure to serum or other growth stimuli (1). It is obvious that cellular polyamines are not free to take part in the proliferative response and that new polyamines must be synthesized.

We next investigated the effect of ODC inhibition on transcriptional activity using B-dependent reporter gene assays. DFMO selectively inhibited a gene reporter construct dependent on the B site present in the HLA-B7 gene but had no effect on a construct dependent on the B site present in the IL-8 gene. The inhibitory effect of DFMO on the B site present in the HLA-B7 gene was detectable within 1 h and persisted for at least 24 h after DFMO treatment. These results correlate with the time course of DFMO induction of NF-B activation, activation within 1 h that persists up to 24 h after DFMO addition. This is of particular importance, as noted above, because the p50 homodimer apparently inhibits NF-B-dependent transcription. Moreover, the results on the inhibitory effect of DFMO on the B site in the HLA-B7 gene versus the IL-8 gene highlight the selective effects of decreases in polyamine levels on the activated NF-B complexes that bind to B regulatory elements. A similar selectivity for B sites has been described for the interferon-induced expression of a CAT reporter construct driven by the B site in HLA-B7 gene, but interferon had no effect on a construct driven by the B site in the IL-8 gene (20).

The polyamine spermine activates NF-B in human breast cancer cells (21, 22). These findings appear to be in conflict with the results reported herein. One possible explanation for this discrepancy is the differential responses of normal cells (IEC-6 cells) versus cancer cells to polyamine effects. Alternatively, both inhibition of ODC activity by DFMO (Fig. 2) and exogenous spermine addition (21) result in depletion in putrescine levels. Because a slight decrease in putrescine levels may be sensed by the cell to activate a NF-B-dependent response pathway, these apparently contradictory reports may be entirely consistent with one another.

An emerging area of research in intestinal homeostasis and inflammation is the role of NF-B in regulating intestinal epithelial cell (IEC) gene expression (23, 24). IECs form a single layer of cells that isolate the host from the hostile gut luminal environment. Aside from their classical absorptive and physical barrier roles, an emerging concept views IECs as immunological sentinels of the intestinal mucosa. IECs are capable of responding to a wide array of biologically active agents commonly found in the lumen, including bacterial products, adherent and invasive bacteria, cytokines, and short chain fatty acids. NF-B regulates the transcription of a number of proinflammatory molecules, including IL-1, tumor necrosis factor-, IL-6, IL-8, IL-12, inducible nitric-oxide synthase, ICAM-1, VCAM-1, and major histocompatibility complex class II molecules, involved in acute responses to injury and in chronic intestinal inflammation (23, 24). NF-B activation has been documented in the intestine of patients with various forms of inflammatory bowel disease, such as Crohn's disease, ulcerative colitis, and self-limited colitis (25-27). Immunohistochemistry performed on tissue sections isolated from patients with inflammatory bowel disease demonstrates the presence of activated NF-B in IECs located at the crypts but not at the surface region (26). Thus, it is clearly important that we have found that polyamine depletion of IEC-6 cells results in NF-B activation.

In many cell types NF-B plays a protective role against apoptosis, mediated by death signals such as tumor necrosis factor- and radiation (6-9). Therefore, NF-B activation may have an impact on intestinal hyperplasia through cell removal by apoptosis in a manner similar to experimental rheumatoid arthritis (28). We report that ODC inhibition by DFMO treatment rapidly induces NF-B activation. It has been recently shown that apoptosis induced by DNA damaging agents and tumor necrosis factor- is delayed in polyamine-depleted cells (3). Taken together, these results implicate NF-B in the protective action of DFMO against apoptotic agents in IEC-6 cells.

In summary, we have shown that polyamine depletion activates the NF-B transcription factor. NF-B activation resulted in the inhibition of selective NF-B-dependent reporter constructs. Therefore, our results show that polyamine depletion in intestinal epithelial cells activates the NF-B signal transduction pathway, which is a pathway with important physiological consequences.

	ACKNOWLEDGEMENTS

We thank Dr. Nancy Rice for generously providing anti-NF-B/Rel Abs and Dr. Jan Vilcek for generously providing CAT reporter constructs.

	FOOTNOTES

^* This work was supported by National Institute of Health Grants CA73753 (to L. M. P.) and DK-16505 (to L. R. J.) and by the Thomas A. Gerwin Endowment.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.

^§ To whom correspondence should be addressed: Dept. of Pathology, University of Tennessee Health Science Center, 899 Madison Ave., Memphis, TN 38163. Tel.: 901-448-7020; Fax: 901-448-1876; E-mail: lpfeffer@utmem.edu.

Published, JBC Papers in Press, October 5, 2001, DOI 10.1074/jbc.M108097200

	ABBREVIATIONS

The abbreviations used are: ODC, ornithine decarboxylase; DFMO, -difluoromethylornithine; NF-B, nuclear factor B; IL, interleukin; sDMEM, Dulbecco's modified Eagle's medium supplemented with 5% dialyzed fetal bovine serum, 10 µg of insulin/ml and 0.05 mg of gentamicin/ml; EMSA, electrophoretic mobility shift assay(s); PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; CAT, chloramphenicol acetyltransferase; IEC, intestinal epithelial cell.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				B. S. Marasa, L. Xiao, J. N. Rao, T. Zou, L. Liu, J. Wang, E. Bellavance, D. J. Turner, and J.-Y. Wang Induced TRPC1 expression increases protein phosphatase 2A sensitizing intestinal epithelial cells to apoptosis through inhibition of NF-{kappa}B activation Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1277 - C1287. [Abstract] [Full Text] [PDF]

				A.-H. Zhang, J. N. Rao, T. Zou, L. Liu, B. S. Marasa, L. Xiao, J. Chen, D. J. Turner, and J.-Y. Wang p53-Dependent NDRG1 expression induces inhibition of intestinal epithelial cell proliferation but not apoptosis after polyamine depletion Am J Physiol Cell Physiol, July 1, 2007; 293(1): C379 - C389. [Abstract] [Full Text] [PDF]

				M. J. Cowan, T. Coll, and J. H. Shelhamer Polyamine-mediated reduction in human airway epithelial migration in response to wounding is PGE2 dependent through decreases in COX-2 and cPLA2 protein levels J Appl Physiol, October 1, 2006; 101(4): 1127 - 1135. [Abstract] [Full Text] [PDF]

				N. Babbar, A. Hacker, Y. Huang, and R. A. Casero Jr. Tumor Necrosis Factor {alpha} Induces Spermidine/Spermine N1-Acetyltransferase through Nuclear Factor {kappa}Bin Non-small Cell Lung Cancer Cells J. Biol. Chem., August 25, 2006; 281(34): 24182 - 24192. [Abstract] [Full Text] [PDF]

				T. Zou, K. Mazan-Mamczarz, J. N. Rao, L. Liu, B. S. Marasa, A.-H. Zhang, L. Xiao, R. Pullmann, M. Gorospe, and J.-Y. Wang Polyamine Depletion Increases Cytoplasmic Levels of RNA-binding Protein HuR Leading to Stabilization of Nucleophosmin and p53 mRNAs J. Biol. Chem., July 14, 2006; 281(28): 19387 - 19394. [Abstract] [Full Text] [PDF]

				H. M. Zhang, K. M. Keledjian, J. N. Rao, T. Zou, L. Liu, B. S. Marasa, S. R. Wang, L. Ru, E. D. Strauch, and J.-Y. Wang Induced focal adhesion kinase expression suppresses apoptosis by activating NF-{kappa}B signaling in intestinal epithelial cells Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1310 - C1320. [Abstract] [Full Text] [PDF]

				C. Muscari, F. Bonafe, I. Stanic, F. Flamigni, C. Stefanelli, G. Farruggia, C. Guarnieri, and C. M. Caldarera Polyamine Depletion Reduces TNF{alpha}/MG132-Induced Apoptosis in Bone Marrow Stromal Cells Stem Cells, August 1, 2005; 23(7): 983 - 991. [Abstract] [Full Text] [PDF]

				H. M. Zhang, J. N. Rao, X. Guo, L. Liu, T. Zou, D. J. Turner, and J.-Y. Wang Akt Kinase Activation Blocks Apoptosis in Intestinal Epithelial Cells by Inhibiting Caspase-3 after Polyamine Depletion J. Biol. Chem., May 21, 2004; 279(21): 22539 - 22547. [Abstract] [Full Text] [PDF]

				T. Zou, J. N. Rao, X. Guo, L. Liu, H. M. Zhang, E. D. Strauch, B. L. Bass, and J.-Y. Wang NF-{kappa}B-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion Am J Physiol Cell Physiol, May 1, 2004; 286(5): C1009 - C1018. [Abstract] [Full Text] [PDF]

				S. Bhattacharya, R. M. Ray, M. J. Viar, and L. R. Johnson Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-{alpha} in IEC-6 cells Am J Physiol Gastrointest Liver Physiol, November 1, 2003; 285(5): G980 - G991. [Abstract] [Full Text] [PDF]

				R. M. Ray, S. A. McCormack, C. Covington, M. J. Viar, Y. Zheng, and L. R. Johnson The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1 J. Biol. Chem., April 4, 2003; 278(15): 13039 - 13046. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/49/45909    most recent M108097200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed