Inhibition of ornithine decarboxylase induces STAT3 tyrosine phosphorylation and DNA binding in IEC-6 cells

Lawrence M. Pfeffer1, Chuan He Yang1, Susan R. Pfeffer1, Aruna Murti1, Shirley A. McCormack2, and Leonard R. Johnson2

Departments of 1 Pathology and 2 Physiology and Biophysics, University of Tennessee Health Science Center, Memphis, Tennessee 38163


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Polyamines are required for the proliferation of the rat intestinal mucosal IEC-6 cell line. Ornithine decarboxylase (ODC) is the enzyme that catalyzes the first step in polyamine synthesis. ODC inhibition not only leads to polyamine depletion but also leads to inhibition of cell proliferation and regulates the expression of the immediate-early genes c-fos, c-myc, and c-jun. Members of the signal transducers and activators of transcription (STAT) transcription factor family bind to the sis-inducible element (SIE) present in the promoters to regulate the expression of a variety of important genes. In the present study, we tested the hypothesis that the STAT3 transcription factor, which is responsible for activation of the acute phase response genes, is activated after inhibition of ODC. We found that inhibition of ODC rapidly induces STAT3 activation as determined by STAT3 tyrosine phosphorylation, translocation of STAT3 from the cytoplasm into the nucleus, and the presence of STAT3 in SIE-dependent DNA-protein complexes. STAT3 activation upon inhibition of ODC was accompanied by the activation of a STAT3-dependent reporter construct. Moreover, prolonged polyamine depletion resulted in downregulation of cellular STAT3 levels.

polyamines; signal transducers and activators of transcription proteins; growth; DL-alpha -difluoromethylornithine; c-fos; sis-inducible element


    INTRODUCTION
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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. Intracellular polyamine levels are highly regulated through the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting step in polyamine biosynthesis. Therefore, specific inhibitors of ODC such as DL-alpha -difluoromethylornithine (DFMO) have been used to define the role of polyamines in cellular processes. Previous studies have established that inhibition of ODC activity in the rat intestinal mucosal IEC-6 cell line leads to transient induction of the immediate-early genes c-fos, c-myc, and c-jun (11, 16). These genes are activated by polypeptide growth factors, and their promoters contain binding sites for growth factor-responsive elements. The sis-inducible element (SIE) is an epidermal growth factor (EGF)- and platelet-derived growth factor (PDGF)-responsive element in the c-fos promoter, which contributes to c-fos transcription in vivo (15).

Signal transducers and activators of transcription (STAT) proteins are a family of latent transcription factors that undergo ligand-dependent tyrosine phosphorylation and activation (4, 6). Multiple distinct forms of STAT proteins have been described to date. Many cytokines and growth factors signal through activation of the STAT transcription factors, a pathway that has been analyzed in detail for interferon (IFN)-regulated gene expression (4, 6). STAT3 is the transcription factor responsible for activation of the acute phase response genes and binds to the SIE found in the c-fos promoter regulated by PDGF (3, 19). We recently showed that IFN-alpha /beta , an important family of antiviral and antiproliferative cytokines, induces STAT3 activation in a wide variety of cells (13, 18). In addition, STAT3 also appears to play an important role in the antiproliferative action of some cytokines (12, 17).

Because we have shown that STAT3 is activated during the antiproliferative response and because polyamine depletion leads to inhibition of cell proliferation, in this study we tested the hypothesis that STAT3 is also activated following inhibition of ODC. We show that inhibition of ODC resulted in STAT3 activation as determined by STAT3 tyrosine phosphorylation and the presence of STAT3 in SIE-dependent complexes. STAT3 activation by inhibition of ODC is accompanied by the activation of a chloramphenicol acetyltransferase (CAT) construct driven by the STAT3 binding site present in the IFN response factor (IRF)-1 gene. Furthermore, inhibition of ODC resulted in the translocation of STAT3 from the cytoplasm into the nucleus in IEC-6 cells. In addition, prolonged inhibition of ODC resulted in STAT3 downregulation.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Cells. The normal rat intestinal epithelial cell line IEC-6 was maintained in DMEM supplemented with 5% dialyzed fetal bovine serum, 10 µg/ml insulin, and 0.05 mg/ml gentamicin (sDMEM). Stock cultures were subcultured once a week at 1:20, and sDMEM was replenished three times per week. For experiments, cells were plated at 4 × 104 cells/cm2 in sDMEM containing 5 mM 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 (11). In addition, this dose partially (60%) depletes spermine by 4 days. Control cultures received no DFMO. In some experiments, spermidine (5 µM) was added simultaneously with DFMO to determine whether exogenous polyamines could reverse DFMO effects. The inhibition of growth and migration resulting from polyamine depletion in IEC-6 cells can be prevented by adding 5 µM spermidine to DFMO-containing medium (11).

Preparation of nuclear and cytoplasmic extracts for immunoblotting. Cultures of IEC-6 cells were washed with ice-cold PBS and harvested in nuclear homogenization buffer (20 mM Tris · HCl, pH 7.4, 10 mM NaCl, and 3 mM MgCl2) as previously described (14). After the addition of NP-40 to a final concentration of 0.15%, the cells were subjected to Dounce homogenization and centrifugation (1,500 g, 5 min). The supernatant corresponding to the cytosolic extract was saved, and the nuclear pellets were suspended in nuclear homogenization buffer and centrifuged again. The remaining nuclear pellet was resuspended in a buffer containing 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol and extracted for 30 min at 4°C and 10 min at 22°C. The nuclear extract was incubated for 10 min at 22°C after the addition of DNase I (200 units) and clarified by centrifugation (12,000 g, 30 min) at 4°C.

Immunoprecipitations and immunoblot analysis. Cultures (1 × 108 cells) were washed with ice-cold PBS and lysed for 20 min in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 15% glycerol) containing 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin, and 1.75 µg/ml benzamidine (5). Samples were centrifuged (12,000 g, 15 min) at 4°C, and supernatants were immunoprecipitated with rabbit polyclonal anti-STAT3 antibody (Santa Cruz Laboratories) overnight at 4°C. Immune complexes were collected using protein A-Sepharose beads (Pharmacia) and eluted in sample buffer. Samples were run on SDS-7.5% polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Millipore), and probed with anti-STAT3 antibody or anti-phosphotyrosine antibody (anti-pTyr, Oncogene Sciences Ab-2; dilution 1:1,000), followed by anti-mouse IgG coupled with horseradish peroxidase (Amersham). Blots were developed using enhanced chemiluminescence (ECL, Amersham).

Nuclear extracts and gel shift assays. For preparation of nuclear extracts, cultures were washed with ice-cold PBS and harvested with a rubber policeman. Nuclei were extracted with buffer containing 20 mM Tris · HCl (pH 7.85), 250 mM sucrose, 0.4 M KCl, 1.1 mM MgCl2, 5 mM beta -mercaptoethanol, and 0.4 mM PMSF, and extracts were frozen on dry ice and stored at -80°C (7, 8, 10). For gel shift analysis of STAT3 DNA binding activity, the nuclear extracts were incubated with a 32P-end-labeled promoter probe for the high-affinity SIE from the c-fos gene (5'-AGCTTCATTTCCCGTAATCCCTAAAGCT-3') (15, 18) at 25°C for 30 min, and the free probe was separated from protein-DNA complexes on 5% polyacrylamide gels. For supershift assays, nuclear extracts were preincubated with a 1:50 dilution of normal rabbit serum or anti-STAT3 antibody (Santa Cruz Laboratories) at 25°C for 0.5 h. Gels were quantitated by phosphorimaging (Molecular Dynamics).

Transcriptional assays. IEC-6 cells were transfected by electroporation with the promoterless CAT construct or with the IRF-1 3X GAS-CAT reporter plasmid containing three copies of the SIE (equivalent to GAS) site from the IRF-1 gene (provided by Drs. V. Viggiano and H. Young, National Cancer Institute-Frederick Research Cancer and Development Center, Frederick, MD). After being allowed to attach for 5 h, transiently transfected cells were treated for 18 h with DFMO or with both spermidine and DFMO and were assayed for CAT activity. Acetylated and unacetylated [14C]chloramphenicol were separated by TLC and quantified by phosphorimaging.


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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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DFMO treatment induces the tyrosine phosphorylation of STAT3 and STAT3 nuclear translocation. STAT proteins are present in the cytoplasm as latent transcription factors. Tyrosine phosphorylation of STATs is essential for their DNA binding activity and their translocation from the cytoplasm into the nucleus (4). To determine the effects of inhibition of ODC on the translocation and tyrosine phosphorylation of STAT3, nuclear and cytosolic extracts prepared from IEC-6 cells were analyzed by blotting with antibodies directed against STAT3 or tyrosine-phosphorylated STAT3. As shown in Fig. 1A, low levels of tyrosine-phosphorylated STAT3 (of both the 91- and 84-kDa forms) were observed in untreated IEC-6 cells, which probably reflects endogenous STAT3 activation by growth factors present in serum-containing medium. Although there was no increase in tyrosine-phosphorylated STAT3 in nuclear extracts 1 or 2 h after DFMO addition, 4 h after DFMO addition there was a significant increase of tyrosine-phosphorylated STAT3 in nuclear extracts of IEC-6 cells. An increase in nuclear tyrosine-phosphorylated STAT3 was observed in IEC-6 cells 24 h after DFMO addition. The appearance of tyrosine-phosphorylated STAT3 in nuclear extracts directly correlated with the disappearance of STAT3 from cytosolic extracts prepared from IEC-6 cells 4 and 24 h after DFMO addition (Fig. 1A). Moreover, DFMO did not affect the tyrosine phosphorylation of other STAT proteins (STAT1 and STAT2) we examined (data not shown). The appearance of tyrosine-phosphorylated STAT3 specifically reflected the effects of ODC inhibition and blockage of polyamine synthesis, since it was prevented by the addition of spermidine (Fig. 1B).


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Fig. 1.   Effects of inhibition of ornithine decarboxylase (ODC) on tyrosine phosphorylation and nuclear translocation of STAT3 in IEC-6 cells. A: nuclear and cytosolic extracts prepared from control or DL-alpha -difluoromethylornithine (DFMO)-treated IEC-6 cells were resolved by SDS-PAGE, blotted onto polyvinylidene difluoride (PVDF) membranes, and probed with antibodies directed against either STAT3 or tyrosine-phosphorylated (pTyr) STAT3. B: lysates prepared from control, DFMO-treated (1 day), or spermidine (SPD)- and DFMO-cotreated (1 day) IEC-6 cells were immunoprecipitated with anti-STAT3. Proteins were resolved by SDS-PAGE, blotted onto PVDF membranes, and probed with either anti-STAT3 or anti-pTyr.

DFMO induces gel shift activity bound to an SIE probe. The tyrosine phosphorylation of STAT3 is essential for its DNA binding activity to SIE-dependent responsive genes. Nuclear extracts prepared from control or DFMO-treated (24 h) IEC-6 cells were incubated with a labeled probe for the high-affinity SIE, and the resultant DNA-protein complexes were analyzed by electrophoretic mobility shift assays. Figure 2 shows that DFMO treatment induced DNA binding to the SIE. No DNA binding to the SIE was detected in the presence of excess unlabeled SIE oligonucleotide, and binding was not competed for by an excess of unrelated oligonucleotide corresponding to a kappa B element. Taken together, these results indicate that the binding to the SIE probe was specific.


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Fig. 2.   Presence of STAT3 in sis-inducible element (SIE)-dependent complexes induced by inhibition of ODC. Left: nuclear extracts were prepared from control or DFMO-treated (1 day) IEC-6 cells or cells treated with both spermidine and DFMO (2 days). Extracts were subjected to electrophoretic mobility shift assay (EMSA) with a 32P-labeled SIE probe in absence or presence of unlabeled SIE oligonucleotide probe. Right: to detect presence of STAT3 in DNA-protein complexes, nuclear extracts from DFMO-treated cells were preincubated with or without (no Ab) anti-STAT3 before EMSA analysis.

To determine whether the effect of DFMO on induction of an SIE-dependent gel shift complex was specific for inhibition of polyamine synthesis, we also treated IEC-6 cells with spermidine in the presence of DFMO to prevent polyamine depletion. As shown in Fig. 2, addition of exogenous spermidine completely blocked the appearance of SIE-dependent gel shift activity. Thus the induction of this complex required inhibition of polyamine synthesis and was not due to an indirect effect of DFMO.

Previous studies have demonstrated that STAT3 can bind as homodimers as well as heterodimers with STAT1 to the SIE oligonucleotide probes, as determined by gel supershift assays. To detect the presence of STAT3 in DNA-protein complexes formed upon polyamine depletion, we performed gel supershift assays with STAT3-specific antisera. As shown in Fig. 2, anti-STAT3 supershifted the complex formed with the SIE in DFMO-treated cells. Neither control normal rabbit serum nor anti-STAT2 shifted the DFMO-induced SIE DNA-protein complexes (data not shown). Thus inhibition of ODC in IEC-6 cells induced DNA binding activity attributable to STAT3.

DFMO effects on an SIE-dependent reporter construct. To determine the functional consequences of STAT3 activation induced by inhibition of ODC, we examined the effect of DFMO treatment on the transcriptional activity of an SIE-dependent CAT reporter construct. IEC-6 cells were transfected with the IRF-1 3X GAS-CAT reporter construct containing three copies of the SIE site from the IRF-1 gene, were treated with DFMO, and at 1 day after transfection were assayed for CAT activity. As shown in Fig. 3, whereas DFMO treatment had no effect on the control CAT reporter construct (PBL), DFMO treatment resulted in a marked enhancement (~10-fold increase) in the transcriptional activity of the IRF-1 3X GAS-CAT reporter construct. The increased transcriptional activity of the SIE-dependent reporter construct was specific for polyamine depletion, since spermidine addition blocked the DFMO-induced increase in CAT reporter activity. Moreover, overexpression of STAT3 increased SIE-dependent CAT reporter activity (data not shown).


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Fig. 3.   SIE-dependent reporter gene activity in DFMO-treated IEC-6 cells. IEC-6 cells were transiently transfected with interferon response factor (IRF)-1 3X GAS-chloramphenicol acetyltransferase (CAT) construct or promoterless CAT construct (PBL). They were treated for 1 day with DFMO, with both spermidine and DFMO, or with neither, as a control (con), and were assayed for CAT activity. Data are from 1 of 3 experiments with quantitatively similar results and are expressed relative to CAT activity in cells transfected with empty PBL vector as determined by phosphorimaging.

Time course of DFMO-induced tyrosine phosphorylation of STAT3 and its downregulation. Our results demonstrate that inhibition of ODC induced the tyrosine phosphorylation of STAT3, STAT3 downregulation, and STAT3-containing gel shift activity. However, it was unclear how long the tyrosine phosphorylation of STAT3 persisted and whether this event affected cellular STAT3 levels. Lysates prepared from control and DFMO-treated IEC-6 cells were precipitated with anti-STAT3, and analyzed by blotting with anti-STAT3 or anti-pTyr. As shown in Fig. 4, tyrosine-phosphorylated STAT3 was observed from 24 to 72 h after DFMO addition. In contrast, although the cellular level of STAT3 was partially downregulated (50% by densitometric analysis of autoradiograms) at 24 h after DFMO treatment, it decreased 80% by 72 h after DFMO addition. These results demonstrate that inhibition of ODC resulted in the prolonged tyrosine phosphorylation of STAT3 and in the downregulation of cellular STAT3 levels.


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Fig. 4.   Time course of DFMO effect on tyrosine phosphorylation of STAT3 and cellular content of STAT3. Lysates prepared from IEC-6 cells at times indicated after DFMO addition were immunoprecipitated with anti-STAT3. Proteins were resolved by SDS-PAGE, blotted onto PVDF membranes, and probed with either anti-STAT3 or anti-pTyr.


    DISCUSSION
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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. An important mechanism of action of the polyamines is their control of growth-regulated genes. Increased activity of ODC, the enzyme responsible for catalyzing the first rate-limiting step in polyamine biosynthesis, occurs concomitantly with increases in the mRNA of several proto-oncogenes in growth-stimulated cells. Furthermore, inhibition of ODC and polyamine depletion by DFMO results in a rapid but transient increase in mRNA levels for the c-fos, c-myc, and c-jun proto-oncogenes in cultured small intestinal crypt cells (16). Moreover, long-term polyamine depletion decreases mRNA levels for the c-fos, c-myc, and c-jun proto-oncogenes. In the present report, we investigate the potential role of the STAT transcription factor family in the regulation of gene expression by polyamines. We report that inhibition of ODC induces the activation of the STAT3 transcription factor as measured by its tyrosine phosphorylation, by its translocation from the cytoplasm into the nucleus, and by its presence in DNA binding complexes. STAT3 activation was rapid (within 4 h of DFMO addition) and was specific for the inhibition of polyamine synthesis, since exogenous spermidine prevented the effect of DFMO. Moreover, inhibition of ODC also resulted in increased gene transcription of a STAT3-dependent reporter construct. However, we also found that long-term polyamine depletion resulted in persistent STAT3 activation but downregulation of cellular STAT3 levels.

STAT3, or acute phase response factor (APRF), is a transcription factor that binds to the interleukin-6 (IL-6) responsive elements within the promoters of genes for various acute phase response proteins (1, 19). During the acute phase response, significant increases occur in the serum levels of the acute phase proteins, such as haptoglobin, hemopexin, C-reactive protein, T-kininogen, and alpha 2-macroglobulin (1). The induction of the acute phase proteins is regulated primarily by the alteration of the transcription rates of acute phase genes.

Numerous cytokines (e.g., IL-1, -6, and -11; tumor necrosis factor; leukemia inhibitory factor; oncostatin M; ciliary neurotrophic factor) directly induce genes for acute phase proteins. The molecular cloning of APRF demonstrated that it was highly homologous with STAT proteins (1). In fact, the cDNA for APRF was independently cloned by low-stringency hybridization with a STAT1 probe (19). Cytokines, such as growth hormone, IFN-alpha , IL-6, oncostatin M, and EGF induce the tyrosine phosphorylation of STAT3 and its presence in DNA-protein complexes (2, 3, 18). Thus the regulation of STAT3 by various cytokines may integrate diverse signals into common transcriptional responses.

In addition, we also found that prolonged polyamine depletion resulted in a marked downregulation of STAT3. These findings are consistent with the finding that IFN-gamma downregulates STAT1 by targeting tyrosine-phosphorylated STAT1 for degradation by a ubiquitin-proteasome pathway (9). Therefore, our results indicate that a ubiquitin-proteasome pathway targets STAT3 for degradation upon its tyrosine phosphorylation in a similar manner. Moreover, because STAT3 is activated in response to a variety of growth factors, its downregulation in response to prolonged DFMO treatment may be a mechanism whereby polyamine depletion attenuates the cellular response to growth factor stimulation.

In summary, we report that inhibition of ODC results in the tyrosine phosphorylation of STAT3, the induction of STAT3-dependent DNA binding activity, the nuclear translocation of STAT3, and, subsequently, the downregulation of this isoform. We hypothesize that, through STAT3, polyamine depletion provides important nuclear signals to mediate its dramatic cellular effects.


    ACKNOWLEDGEMENTS

We thank Drs. James Ihle and James E. Darnell, Jr., for generously providing anti-STAT3 antibodies and Dr. Howard Young for generously providing the IRF-1 3X GAS-CAT reporter construct.


    FOOTNOTES

This work was supported by funds from the Department of Pathology, University of Tennessee Health Science Center, and by National Institutes of Health Grants CA-73753 (to L. M. Pfeffer) and DK-16505 (to L. R. Johnson).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. M. Pfeffer, Dept. of Pathology, University of Tennessee Health Science Center, 899 Madison Ave., Memphis, TN 38163 (E-mail: lpfeffer{at}utmem.edu).

Received 3 May 1999; accepted in final form 12 October 1999.


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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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