Our previous studies have shown that polyamines are required for normal intestinal mucosal growth and that decreased levels of polyamines inhibit intestinal epithelial cell (IEC) proliferation by stabilizing p53 and other growth-inhibiting proteins. Nucleophosmin (NPM) is a multifunctional protein that recently has been shown to regulate p53 activity. In the present study, we sought to determine whether polyamine depletion increases NPM modulating the stability and transcriptional activity of p53 in a normal IEC-6 intestinal epithelial cell line. Depletion of cellular polyamines by α-difluoromethylornithine, the specific inhibitor of polyamine biosynthesis, stimulated expression of the NPM gene and induced nuclear translocation of NPM protein. Polyamine depletion stimulated NPM expression primarily by increasing NPM gene transcription and its mRNA stability, and it induced NPM nuclear translocation through activation of phosphorylation of mitogen-activated protein kinase kinase. Increased NPM interacted with p53 and formed a NPM/p53 complex in polyamine-deficient cells. Inhibition of NPM expression by small interfering RNA targeting NPM (siNPM) not only destabilized p53 as indicated by a decrease in its protein half-life but also prevented the increased p53-dependent transactivation as shown by suppression of the p21 promoter activity. Decreased expression of NPM by siNPM also promoted cell growth in polyamine-deficient cells. These results indicate that 1) polyamine depletion increases expression of the NPM gene and enhances NPM nuclear translocation and 2) increased NPM interacts with and stabilizes p53, leading to inhibition of IEC-6 cell proliferation.
- ornithine decarboxylase
- stability of mRNA and protein
- gene transcription
- growth arrest
- intestinal epithelium
the epithelium of the intestinal mucosa has the most rapid turnover rate of any tissue in the body, and maintenance of its integrity requires epithelial cell decisions that regulate signaling networks controlling expression of various genes involved in cell proliferation, differentiation, migration, and apoptosis (17, 18). Undifferentiated epithelial cells continuously replicate in the proliferative zone within crypts and differentiate as they migrate up the luminal surface of the colon and villous tips in the small intestine under physiological conditions (39, 40). Mature differentiated cells at the luminal surface and villous tips are quickly lost through the process of apoptotic cell death and replaced by new cells. This rapid dynamic turnover rate of intestinal epithelial cells (IECs) is highly regulated and critically controlled by numerous factors, including the cellular polyamines spermidine and spermine and their precursor, putrescine (13, 18, 30). Polyamines have been implicated in a wide variety of biological functions, and the regulation of cellular polyamines has been recognized to be a central convergence point for the multiple signaling pathways driving different epithelial cell functions (11, 49). We (23, 24, 26, 35, 54) and others (18, 30) have shown that normal IEC proliferation in the intestinal mucosa depends on a supply of polyamines to the dividing cells in the crypts and that decreasing cellular polyamines inhibits cell renewal. We recently identified a novel mechanism through which decreased levels of cellular polyamines inhibit IEC proliferation by stabilizing p53 and other growth-inhibiting proteins (24, 26, 29, 35). However, the exact mechanisms by which polyamines modulate p53 stability in normal IECs remain to be demonstrated.
Nucleophosmin (NPM) is a phosphoprotein that was originally identified as a nucleolar protein involved in ribosome biogenesis (6, 43). Since then, a number of cellular activities associated with NPM indicate that NPM has multiple cellular functions, especially in the regulation of cell proliferation. NPM acts as a ribosomal assembly and transport protein (3, 59), binds to proteins containing nuclear localization signals for their import (47), and functions as a molecular chaperone (34, 57). The NPM gene is implicated in several tumor-associated chromosomal translocations, which lead to the formation of fusion proteins that retain the amino terminus of NPM (57). Recently, NPM was shown to bind to different cellular and viral proteins and plays a critical role in the regulation of subcellular localization and activities of these proteins. NPM physically interacts with p53 (7, 20), Arf tumor suppressor protein (2), Pololike kinase (61), NF-κB (8), p120 (51), nucleolin (22), and several viral proteins such as Rex of human T-cell leukemia virus (1) and hepatitis δ-virus antigen (16). In addition, NPM also prevents protein aggregation, protects enzymes from thermal denaturation, and facilitates renaturation of chemically denatured proteins (48).
Activation of p53 in response to polyamine depletion is an important event that results in growth inhibition (39, 40) and is associated with changes in the sensitivity to apoptosis in IECs (25, 60, 62). Because p53 triggers cell cycle arrest or apoptosis (45), its potent activity demands tight control of its functions. Although NPM is able to form a complex with p53 in various types of cells, the regulatory effect of the NPM-p53 interaction on p53 activity is controversial, depending on the cellular context and type of activating agents (7, 19, 20, 27, 31, 56). On the one hand, ectopic expression of the NPM gene enhances the formation of nuclear NPM/p53 complex, stabilizes p53, and increases its transcriptional activity in human W138 fibroblasts (7, 19). In support of this finding, a recent study further showed that NPM interacts with MDM2 and protects p53 from MDM2-mediated degradation (20). These results strongly suggest that NPM is an enhancer of p53. On the other hand, induced NPM after exposure to UV radiation binds to the p53 NH2-terminal end and sets a high threshold for p53 response to stress (31, 56). Hypoxia-induced NPM protects cell death through inhibition of p53 (27), suggesting that NPM acts as a negative regulator for p53. In the present study, we sought to determine whether polyamines modulate NPM expression in normal IECs and further define the exact role of induced NPM in regulation of p53 activity after polyamine depletion. The data presented herein demonstrate that polyamines downregulate NPM expression and its nuclear translocation and that increased NPM after polyamine depletion interacts with and stabilizes p53, leading to inhibition of IEC proliferation. Some of these data have been published previously in abstract form (62).
MATERIALS AND METHODS
Chemicals and supplies.
Disposable culture ware 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 obtained from Sigma (St. Louis, MO). Antibodies against NPM, phosphorylated mitogen-activated protein kinase kinase (p-MEK), total MEK (T-MEK), and U0126 were purchased from Cell Signaling Technology (Beverly, MA). Antibody against p53 protein was obtained from BD Biosciences Clontech (Palo Alto, CA), and d,l-α-difluoromethylornithine (DFMO) was purchased from Ilex Oncology (San Antonio, TX).
Reporter plasmids and transient transfection.
The NPM promoter reporter construct was a gift from Dr. Qishen Pang (University of Cincinnati School of Medicine, Cincinnati, OH) (27). A nucleotide fragment (−587 to +4 nucleotide of human NPM sequence) encompassing the basal elements of human NPM promoter was cloned into the NcoI and SmaI sites of a PGL-3 vector (Promega, Madison, WI) containing the luciferase reporter. The construct of the p21 promoter luciferase reporter was a gift from Dr. Wafik S. El-Deiry (University of Pennsylvania School of Medicine, Philadelphia, PA), which contains the −2,326 to −16 sequence of the human p21 promoter with two p53-binding sites (46). The p53 promoter luciferase reporter construct was purchased from BD Biosciences Clontech. Transient transfection was performed using the LipofectAMINE kit and performed as recommended by the manufacturer (GIBCO-BRL, Gaithersburg, MD). The pRSV-β-galactosidase was cotransfected in every experiment to monitor transfection efficiency.
The silencing RNA duplexes that were designed specifically to cleave NPM mRNA were synthesized and transfected into cells as described previously (7, 27). The small interfering RNA (siRNA) sequence-targeting NPM gene (siNPM) corresponded to nt 103–125 of the coding region relative to the first nucleotide of the start codon (sense, 5′-UGAUGAAAAUGAGCACCAGTT-3′; antisense, 5′-CUGGUGCUCAUUUUCAUCATT-3′). As a control siRNA (C-siRNA), we used the inverted sequence (sense, 5′-GACCACGAGUAAAAGUAGUTT-3′; antisense, 5′-ACUACUUUUACUCGUGGUCTT-3′). For each 60-mm cell culture dish, 15 μl of the 20 μM stock duplex siNPM 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 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-h incubation.
The IEC-6 cell line was purchased from the American Type Culture Collection (Manassas, VA) at passage 13. The cell line was derived from normal rat intestinal crypt cells and was developed and characterized by Quaroni et al. (41). Stock cells were maintained in T-150 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heated-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. There were no significant changes of biological function and characterization of IEC-6 cells at passages 15–20 (24, 29).
In the first series of studies, we examined the effect of polyamine depletion on expression and cellular distribution of NPM in IEC-6 cells. Cells were grown in control cultures or in cultures containing 5 mM DFMO or DFMO plus 10 μM putrescine for 4 and 6 days. The monolayers of cells were washed three times with ice-cold Dulbecco's phosphate-buffered saline (D-PBS), and different solutions were added according to the assays to be conducted. Cytoplasmic and nuclear proteins were isolated, and levels of NPM were determined using Western blot analysis. In a separate study, the association of NPM with p53 and their interaction were also examined after polyamine depletion.
In the second series of studies, we examined the mechanisms by which polyamines modulate the expression of the NPM gene in IEC-6 cells. Transcriptional regulation of the NPM gene by polyamines was determined by measuring NPM promoter activity, while posttranscriptional regulation of the NPM gene was examined by determining the stability of NPM mRNA. Actinomycin D (5 μg/ml) was added to cultures to completely inhibit RNA synthesis, and the levels of NPM mRNA were assayed at different times after administration of actinomycin D. The half-life of NPM mRNA was measured in the presence or absence of cellular polyamines.
In the third series of studies, we investigated whether the observed increase in nuclear NPM regulates p53 activity after polyamine depletion. Functions of increased NPM in polyamine-deficient cells were elucidated by treatment with siNPM, and p53 stability and its transcriptional activity were measured. After cells were exposed to DFMO alone or DFMO plus putrescine for 4 days, they were transfected with either siNPM or C-siRNA. The activity of luciferase reporter of p53-dependent promoter (p21 promoter) and the stability of p53 protein were examined 48 h after transfection.
Reverse transcription and PCR.
Total RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA). Equal amounts of total RNA (2 μg) were transcribed to synthesize single-stranded cDNA with a RT-PCR kit (Invitrogen Life Technologies, Carlsbad, CA). The specific sense and antisense primers for NPM included 5′-CGATGGACATGGACATGAGC-3′ and 5′-TTCCTCTACAGCTACTAGGT-3′, and the expected size of NPM fragments was 238 bp. These particular sequences were chosen on the basis of the specificity established in previous publications by other investigators (3, 27). Reverse transcription and PCR were performed as described in our earlier publications (12, 42). To quantify the PCR products (the amounts of mRNA) of NPM, an invariant mRNA of β-actin was used as an internal control. The optical density (OD) values for each band on the gel were measured using a gel documentation system (UVP, Upland, CA), and their signals were normalized to the OD values in the β-actin signals.
Immunoprecipitation and immunoblotting analysis.
Cell samples dissolved in ice-cold radioimmunoassay precipitation assay buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 2 μg/ml leupeptin, and 2 mM sodium orthovanadate) were sonicated and centrifuged at 4°C, and the supernatants were collected for immunoprecipitation. Equal amounts of proteins (300 μg) for each sample were incubated with the specific antibody against p53 (2 μg) at 4°C for 3 h, and protein G-PLUS-Agarose was added and incubated overnight at 4°C. The precipitates were washed five times with ice-cold D-PBS, and the beads were resuspended in SDS sample buffer for subsequent Western blot analysis. For immunoblotting, samples were subjected to electrophoresis on 10% acrylamide gels according to the method of Laemmli (21). Briefly, after the transfer of protein onto nitrocellulose membranes, the membranes were incubated for 1 h in 5% nonfat dry milk in 1× TBST buffer (Tris-buffered saline, pH 7.4, with 0.1% Tween 20). Immunological evaluation was then performed overnight at 4°C in 5% nonfat dry milk-TBST buffer containing specific antibodies against NPM, p53, p-MEK, and T-MEK proteins. The membranes were subsequently washed with 1× TBST and incubated with the secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. The immunocomplexes on the membranes were reacted for 1 min with enhanced chemiluminescence reagent (NEL-100; DuPont NEN).
The immunofluorescence staining procedure was performed according to the method of Vielkind and Swierenga (52) with minor changes (12, 29). After the monolayers of control and polyamine-deficient cells were fixed and rehydrated, they were incubated with the primary antibody against NPM or p53 at 4°C overnight and then incubated with secondary antibody conjugated with FITC for 2 h at room temperature. After the slides were rinsed three times, they were mounted and viewed through a Zeiss confocal microscope (model LSM410). Images were processed using PhotoShop software (Adobe, San Jose, CA).
The cells were collected at 48 h after transfection, and luciferase activity was assayed with a commercial kit (Promega). The luciferase activity from individual transfections was normalized according to β-galactosidase activity from cotransfected plasmid pRSV2 β-galactosidase. The experiments were performed in triplicate and are reported in mean relative light units/β-galactosidase.
Values are means ± SE of three to nine samples. Autoradiographic results were repeated three times. The significance of the differences between means was determined using ANOVA. The level of significance was determined using Duncan's multiple-range test (14).
Effect of polyamine depletion on expression and cellular distribution of NPM and p53.
Exposure of IEC-6 cells to 5 mM DFMO for 4 and 6 days completely inhibited ornithine decarboxylase (ODC) activity and almost totally depleted cellular polyamines. The levels of putrescine and spermidine were undetectable at 4 and 6 days after treatment with DFMO, and spermine was decreased by ∼60% (data not shown). Similar results have been reported in our previous publications (23, 55). The results presented in Fig. 1 show that depletion of cellular polyamines by DFMO increased levels of nuclear NPM, which was associated with a significant increase in nuclear p53. The induction of nuclear NPM occurred as early as day 4 and remained elevated on day 6 after exposure to DFMO, although a significant increase in cytoplasmic NPM occurred at 6 days. Levels of nuclear NPM in cells treated with DFMO for 4 and 6 days were ∼2.7 and ∼3 times the control value, respectively. On the other hand, there was just a slight induction of cytoplasmic NPM on day 4 after DFMO treatment, but the level of cytoplasmic NPM increased remarkably on day 6 and was more than twice the normal value.
Interestingly, levels of nuclear p53 increased dramatically after induced accumulation of nuclear NPM in polyamine-deficient cells (Fig. 1, Ab and B). Levels of nuclear p53 in DFMO-treated cells for 4 and 6 days were ∼1.7 and ∼3.5 times the control value, respectively. Putrescine (10 μM) administered together with DFMO not only prevented the increased nuclear accumulation of NPM but also blocked the induced levels of nuclear p53. Spermidine (5 μM) had an effect equal to that of putrescine on levels of NPM and p53 when it was added to cultures that contained DFMO (data not shown). These results indicate that polyamines are implicated in the regulation of NPM activity and that induced levels of nuclear NPM after polyamine depletion is associated with an increase in nuclear p53 in IECs.
To extend the findings of association of induced NPM with p53 after polyamine depletion, we examined the cellular distribution and physical interaction of NPM and p53 in the cells grown in the presence or absence of DFMO for 6 days. In control cells, the slight immunostaining levels of NPM and p53 were visible and present in the cytoplasm and nuclei (Fig. 2 Aa). These results were consistent with our Western blot analysis data that revealed a basal expression level of NPM and p53. In the DFMO-treated cells, however, these nuclear immunostaining levels of NPM and p53 significantly increased, as expected (Fig. 2Ab). Increased immunostaining for NPM and p53 proteins in polyamine-deficient cells were present just inside a defined nuclear area, and identification was facilitated for every experiment by heavily staining nuclei. In the presence of DFMO, putrescine prevented the increased immunostaining levels for nuclear NPM and p53, and the cellular distribution of NPM and p53 in cells exposed to DFMO plus putrescine were indistinguishable from that observed in control cells (data not shown).
To evaluate the relationship between NPM and p53 in normal IECs, we examined whether induced NPM forms a specific complex with p53 after polyamine depletion. After cells were grown in the presence of DFMO for 6 days, whole cell lysates were harvested and immunoprecipitated with the specific anti-p53 antibody, and then these precipitates were examined using Western blot analysis with the anti-NPM antibody. As shown in Fig. 2B, NPM was able to form a complex with p53 in IEC-6 cells, and this NPM/p53 complex was increased after polyamine depletion. These results indicate that induced NPM physically interacts with p53 in normal IECs.
Effect of polyamine depletion on NPM gene transcription and NPM mRNA stability.
To determine the mechanisms by which polyamine depletion increases levels of NPM protein, we examined changes in transcription and posttranscription of the NPM gene in the presence or absence of cellular polyamines. Although there were no changes in the levels of NPM mRNA in cells exposed to DFMO for 4 days, the levels of NPM mRNA increased significantly in cells exposed to DFMO for 6 days and were ∼3.9 times the control value, which was completely prevented by putrescine administered together with DFMO (Fig. 3, A and B). To assess the possibility that the increase in NPM mRNA levels in polyamine-deficient cells results from an increase in the NPM gene transcription, changes in the NPM promoter activity were examined by luciferase reporter gene assays. Inhibition of polyamine synthesis by DFMO significantly increased the activity of NPM promoter activity in IEC-6 cells (Fig. 3C). The level of NPM promoter luciferase reporter activity was ∼2.8 times the control values in cells treated with DFMO for 6 days. In the presence of DFMO, the addition of putrescine prevented the increase in NPM promoter activity. In addition, we also examined the effect of treatment with DFMO on transfection efficiency by using either the pRSV-β-galactosidase construct or the empty vector in IEC-6 cells and demonstrated that there were no significant differences in transfection rates between controls and cells treated with DFMO for 6 days (data not shown), showing that the induction of NPM promoter activity in polyamine-deficient cells does not result from nonspecific effects of DFMO. Consistent with the observations regarding NPM mRNA levels, treatment with DFMO for 4 days failed to induce NPM promoter activity (data not shown). These results indicate that induced levels of NPM mRNA after polyamine depletion are due at least partially to activation of NPM gene transcription.
To test the implication of posttranscriptional regulation in this process, we determined the effect of polyamine depletion on NPM mRNA stability by measurement of mRNA half-life. As shown in Fig. 4, depletion of cellular polyamines by DFMO increased the stability of NPM mRNA in IEC-6 cells. In control cells, mRNA levels declined rapidly after the inhibition of gene transcription by the addition of actinomycin D. The half-life of NPM mRNA was ∼60 min. However, the stability of NPM mRNA was increased by polyamine depletion with a half-life of >360 min, which was prevented by exogenous putrescine. The half-life of NPM mRNA in cells exposed to DFMO plus putrescine was ∼65 min, similar to that of controls (without DFMO). These findings suggest that polyamines also regulate NPM gene expression posttranscriptionally and that polyamine depletion induces NPM mRNA levels partially through the increase in mRNA stability.
Involvement of MEK in NPM nuclear translocation after polyamine depletion.
Polyamine depletion also promoted nuclear translocation of NPM protein in IEC-6 cells because of induction of nuclear NPM in cells exposed to DFMO for 4 days without significant increases in NPM mRNA (Fig. 3A) and cytoplasmic protein (Fig. 1). To determine whether MEK is involved in the stimulation of NPM nuclear translocation after polyamine depletion, we examined changes in the levels of T-MEK and p-MEK proteins. As shown in Fig. 5A, levels of T-MEK and p-MEK increased after polyamine depletion, which was associated with an increase in nuclear NPM (Fig. 1A). In cells exposed to DFMO for 4 days, the level of p-MEK increased remarkably and was approximately three times the control value, although there was a slight increase in T-MEK. Levels of both T-MEK and p-MEK were increased significantly in cells treated with DFMO for 6 days. Exposure of cells treated with DFMO for 4 days to U0126, a specific MEK inhibitor (15), decreased levels of p-MEK but had no effect on T-MEK protein (Fig. 5B). When various doses of U0126 were tested, MEK phosphorylation was inhibited dose dependently, with concentrations ranging from 10 to 40 μM. Maximum inhibition of p-MEK occurred at 40 μM, at which the levels of p-MEK were decreased by ∼80%. Consistent with the effect on MEK phosphorylation, exposure to U0126 also prevented the induced accumulation of nuclear NPM dose dependently (Fig. 5, B and C). When various doses of U0126 were added to the cultures, levels of nuclear NPM were decreased by ∼15%, ∼65%, and ∼75% at 10, 20, and 40 μM, respectively. In addition, inhibition of MEK phosphorylation by treatment with U0126 had no significant effect on levels of NPM mRNA in cells exposed to DFMO for 6 days (data not shown). Furthermore, there was no apparent loss of cell viability in cells treated with DFMO alone or with DFMO plus U0126 (data not shown). These results suggest that polyamine depletion induces NPM nuclear translocation through a mechanism involving MEK activation in IECs.
Effect of inhibition of NPM by siRNA on levels of nuclear p53 and its transcriptional activity in polyamine-deficient cells.
In this study, we designed siRNA to target a specific site of an NPM mRNA-coding region (siNPM) as a means of preventing NPM induction (7) and to examine the role of induced NPM in the regulation of p53 after polyamine depletion. These specific siRNA were designed to cleave rat NPM mRNA by activating endogenous RNase H and to have a unique combination of specificity, efficacy, and reduced toxicity (7, 27, 31). 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 control siRNA for 24 h (data not shown). The results presented in Fig. 6 show that transfection with siNPM prevented the increased NPM in polyamine-deficient cells. Levels of NPM protein were decreased by ∼75% when polyamine-deficient cells were exposed to siNPM for 48 h. Transfection with control siRNA (C-siRNA) at the same concentrations for 48 h showed no inhibitory effects. Inhibition of NPM expression by siNPM also prevented the increased levels of nuclear p53 protein (Fig. 6, A and B) in polyamine-deficient cells. Nuclear p53 protein was decreased by ∼70% after inhibition of NPM by treatment with siNPM for 48 h. Consistently, decreased p53 by siNPM was associated with an inhibition of p53-dependent transcriptional activity as indicated by a decrease in p21 promoter activity (Fig. 6C). Level of p21 promoter activity in DFMO-treated cells transfected with siNPM was identical to that obtained from controls. Treatment with C-siRNA had no effect on levels of nuclear p53 protein and p53 transcriptional activity in polyamine-deficient cells. These findings strongly suggest that induced NPM after polyamine depletion plays a critical role in the regulation of p53 activity in normal IECs.
Effect of NPM inhibition by siNPM on p53 stability.
To define levels at which the effects of induced NPM on p53 activity occur, changes in p53 protein stability and transcription of the p53 gene were examined in polyamine-deficient cells treated with or without specific siNPM. Consistent with our previous studies (26), depletion of cellular polyamines by DFMO increased p53 stability in IEC-6 cells (Fig. 7, A and B). The half-life of p53 protein in control cells was ∼25 min. However, the stability of p53 protein was increased by polyamine depletion, with a half-life of >80 min. This increase in p53 half-life in polyamine-deficient cells was prevented by decreasing NPM with siNPM. When polyamine-deficient cells were transfected with siNPM, levels of p53 protein decreased at a rate similar to that observed in control, with a half-life of ∼28 min. In contrast, transfection with C-siRNA at the same concentrations had no effect on p53 stability. On the other hand, polyamine depletion did not increase transcription of the p53 gene in IEC-6 cells as reported in our previous publication (26). Furthermore, decreased levels of NPM by siNPM did not alter p53 promoter activity in polyamine-deficient cells (Fig. 7C). There were no significant differences in p53 promoter activity between controls and DFMO-treated cells exposed to siNPM or C-siRNA. These findings indicate that induced NPM after polyamine depletion stabilizes p53 but plays little role in the regulation of p53 gene transcription in normal IECs.
Effect of inhibition of NPM expression on cell growth in polyamine-deficient cells.
To determine whether increased NPM plays a role in growth inhibition after polyamine depletion, we examined the influence of inhibition of NPM expression by siNPM on cell proliferation in IEC-6 cells. Increased NPM in DFMO-treated cells was associated with a significant decrease in cell growth, which was completely prevented by putrescine (Fig. 8A). In studies in which we used siNPM, cells were initially grown in the medium containing 5 mM DFMO for 4 days and then transfected with siNPM or C-siRNA. Cell numbers were assayed 48 h after transfection in the presence of DFMO and minimal putrescine (0.5 μM). As shown in Fig. 8B, inhibition of NPM by siNPM significantly promoted cell proliferation in polyamine-deficient cells. Cell numbers were increased by ∼35% in the DFMO-treated cells exposed to the siNPM for 48 h. Neither C-siRNA nor 0.5 μM putrescine alone showed significant effects on NPM level and cell growth in DFMO-treated cells. The results suggest, for the first time, that NPM is involved in negative control of IEC growth after polyamine depletion.
As pointed out in the introduction, polyamines are absolutely required for normal intestinal mucosal growth. Although few specific functions of polyamines at the molecular level are identified, there is little doubt that polyamines regulate IEC proliferation by virtue of their ability to modulate the expression of various growth-related genes (4, 23, 25, 36, 37, 55). Our previous studies have demonstrated that decreased levels of cellular polyamines inhibit intestinal mucosal growth in vivo as well as in vitro, primarily by stabilizing p53 and other growth-inhibiting proteins (23, 24, 26). The present studies advance our previous observations by demonstrating that NPM is a critical regulator of p53 stability in IECs. Polyamine depletion by treatment with DFMO resulted in a significant accumulation of nuclear NPM in IEC-6 cells (Fig. 1), increased NPM interaction directly with p53 (Fig. 2), and regulated the increase in stability and transcriptional activation of p53. Induced NPM formed a complex with p53 in polyamine-deficient cells, and specific inhibition of NPM expression by transfection with siNPM destabilized p53 protein and decreased the p53-dependent transactivation as indicated by suppression of p21 promoter activity (Figs. 6 and 7). Furthermore, decreased expression of NPM by transfection with siNPM also promoted cell growth in polyamine-deficient cells.
The findings reported herein clearly show that polyamine depletion increases levels of NPM protein partially by stimulating transcription of the NPM gene in normal IECs. To provide insight into the molecular basis for NPM induction after polyamine depletion, the results presented in Fig. 3 indicate that there was a significant elevation of NPM promoter activity in cells treated with DFMO for 6 days, which was paralleled by an increase in NPM mRNA. These increases in NPM transcripts in DFMO-treated cells were completely prevented by the addition of exogenous putrescine, indicating that the observed changes in the transcriptional regulation of the NPM gene must be related to polyamine depletion rather than to the nonspecific effect of DFMO. These results are consistent with studies by others who have demonstrated that NPM is an immediate early response gene in mammalian cells (56) and that its induction is commonly mediated at the transcriptional level in response to environmental stress. It was recently reported that exposure to hypoxia (27) or UV radiation (56) induces expression of the NPM gene primarily through activation of NPM gene transcription. However, the current studies provide new evidence suggesting a role for transcriptional control in the induction of NPM by depletion of cellular polyamines.
The data from the current studies also show that polyamines negatively regulate posttranscription of the NPM gene. As noted in Fig. 4, administration of DFMO for 6 days dramatically increased the half-life of NPM mRNA in IEC-6 cells. This prolonged half-life also contributes to the induction of NPM mRNA after polyamine depletion, which is paralleled by an increase in NPM protein. It is not surprising that polyamines are implicated in the regulation of NPM gene expression at both the transcriptional and posttranscriptional levels, because polyamines have been shown to play distinct regulatory roles in gene expression and their effects are mediated by multiple signaling pathways (11, 29, 35, 36, 37, 53). Polyamines not only modulate transcription of c-myc and c-jun genes (4, 28, 36, 55) but also downregulate posttranscription, especially mRNA stability, of transforming growth factor-β (TGF-β), p53, and junD genes in normal IECs (24, 26, 35). Although the exact mechanisms involved in mRNA turnover rate are unclear, increased stability of NPM mRNA after polyamine depletion may be related to the specific cis-elements that are located within NPM 3′-untranslated regions. It has been shown that many labile mRNAs contain UA-rich elements (AREs) in their 3′-untranslated regions and that deletion of the ARE region enhances mRNA stability (10, 38, 44, 58). Although the 3′-UTR of NPM mRNA contains several ARE sequences (6), it is not clear at present whether these AREs are involved in the stabilization of NPM mRNA after polyamine depletion.
Treatment with DFMO for 4 days did not increase levels of cytoplasmic NPM protein (Fig. 1Aa) and its mRNA (Fig. 3A), but it significantly induced the nuclear accumulation of NPM protein (Fig. 1Ab), indicating that this early increase in nuclear NPM after polyamine depletion results primarily from the stimulation of NPM nuclear translocation but not from the activation of NPM gene expression. The results presented in Fig. 5 further show that activation of MEK activity is necessary for the stimulation of NPM nuclear translocation, because there was a remarkable increase in MEK phosphorylation in cells exposed to DFMO for 4 days and inhibition of MEK activity by treatment with U0126 prevented the induced levels of nuclear NPM. Nuclear translocation of NPM is highly regulated by numerous factors, and cellular NPM is redistributed in response to cytotoxic drugs and genotoxic stress (32, 50). It has been shown that NPM phosphorylation regulates its subcellular localization and biological activities (32, 33, 50). For example, NPM binds to centrosomes, and this interaction is dissociated by its phosphorylation on Thr199, which initiates centrosomal duplication (32, 50). Phosphorylation of NPM by cyclin-dependent kinase 2/cyclin E also decreases its RNA-binding activity (32). MEK is a multiple functional kinase that colocalizes with NPM to similar intracellular regions after exposure to different stresses and plays a role in regulation of NPM phosphorylation (5). The current studies suggest that increased MEK phosphorylation is implicated in the induction of NPM nuclear translocation after polyamine depletion.
The most significant of the new findings reported in this study is that induced NPM interacts with and stabilizes p53 after polyamine depletion in normal IECs. As shown in Fig. 2, there were physical association and colocalization of induced NPM and p53 after polyamine depletion, suggesting that NPM might regulate p53 activity directly. The results presented in Figs. 6 and 7 further indicate that specific inhibition of NPM expression by transfection with siNPM induced a marked decrease in nuclear p53 by preventing p53 stabilization in polyamine-deficient cells, which was associated with significant decrease in p53-dependent transcription as indicated by an inhibition of p21 promoter activity. Consistent with our current findings, Colombo et al. (7) reported that inhibition of NPM expression by siNPM results in a reduction of p53 stabilization induced by exposure to ionizing radiation and doxorubicin. Although the exact mechanisms by which induced NPM stabilizes p53 remains unclear, a recent study has shown that NPM protects p53 through its interaction with MDM2 protein (19, 20). It has been shown that MDM2 is a major regulator of p53 and controls the levels of p53 by acting as an E3 ubiquitin ligase initiating p53 degradation (9). NPM functions as a negative regulator for p53/MDM2 interaction in cells exposed to ionizing radiation and doxorubicin, leading to the stabilization of p53. Clearly, further studies are needed to define the role of MDM2 in NPM-induced stabilization of p53 after polyamine depletion in normal IECs.
Induced levels of nuclear NPM after polyamine depletion play an important role in growth inhibition of IECs and are of biological significance. Although intestinal epithelial integrity depends on a dynamic balance between cell proliferation, growth arrest, and apoptosis, studies of negative growth control have not attracted considerable interest until recently. Inhibition of polyamine synthesis by DFMO increased NPM, which was associated with p53 stabilization in IEC-6 cells. As reported in our previous studies (23, 25, 60), polyamine depletion also induces G1 phase growth arrest and alters susceptibility to apoptotic stimuli in IECs. The results presented in Fig. 8 show that inhibition of NPM expression by transfection with siNPM partially but significantly promoted cell proliferation in polyamine-deficient cells. Because reduction of NPM decreases p53 in DFMO-treated cells, these findings provide direct evidence to support the possibility that polyamine depletion-induced NPM stabilizes p53, resulting in the inhibition of normal intestinal mucosal growth.
In summary, our results indicate that polyamine depletion-induced NPM is implicated in the regulation of p53 stabilization and activation in IEC-6 cells. Polyamines downregulate NPM expression at both transcriptional and posttranscriptional levels, although the exact mechanisms involved in this process remain unclear. Polyamine depletion also promotes nuclear translocation of NPM, probably through activation of MEK activity. Increased NPM physically interacts with and stabilizes p53 protein and regulates p53-dependent transcriptional activity after polyamine depletion. Resultant induction of p53 by interacting with NPM in polyamine-deficient cells activates transcription of p53-targeted genes such as p21, thus blocking the G1-to-S phase transition during the cell cycle and inhibiting the proliferation of IECs. These findings suggest that NPM is a biological inhibitor for normal intestinal mucosal growth and is implicated in the negative control of epithelial cell renewal under physiological and pathological conditions.
This work was supported by a Merit Review Grant from the Department of Veterans Affairs and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-57819, DK-61972, and DK-68491. J-Y. Wang is a Research Career Scientist, Medical Research Service, Department of Veterans Affairs.
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