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Am J Physiol Cell Physiol 294: C1198-C1205, 2008. First published March 26, 2008; doi:10.1152/ajpcell.00325.2007
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VASCULAR BIOLOGY

IL-4 and IL-13 upregulate ornithine decarboxylase expression by PI3K and MAP kinase pathways in vascular smooth muscle cells

Liu Hua Wei,1 Yang Yang,2 Guoyao Wu,3 and Louis J. Ignarro1

1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California; 2California Institute of Technology, Pasadena, California; and 3Cardiovascular Research Institute and Department of Medical Physiology, Texas A & M University System Health Science Center, College Station, Texas

Submitted 24 July 2007 ; accepted in final form 25 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ornithine decarboxylase (ODC) is the first and rate-controlling enzyme in the synthesis of polyamines, which are essential for normal cell growth. We have previously demonstrated that IL-4 and IL-13 can stimulate rat aortic smooth muscle cell (RASMC) proliferation. The objective of this study was to determine whether IL-4 and IL-13 induce cell proliferation by upregulating ODC expression in RASMC. The results revealed that incubation of RASMC with IL-4 and IL-13 for 24 h caused four- to fivefold induction of ODC catalytic activity. The increased ODC catalytic activity was attributed to the increased expression of ODC mRNA. Moreover, these observations were paralleled by increased production of polyamines. We further investigated the signal transduction pathways responsible for ODC induction by IL-4 and IL-13. The data illustrated that PD-98059, a MEK (MAPK kinase) inhibitor, LY-294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and H-89, a protein kinase A (PKA) inhibitor, substantially decreased the induction of ODC catalytic activity and ODC mRNA expression induced by IL-4 and IL-13, suggesting positive regulation of the ODC gene by ERK, PI3K, and PKA pathways. Interestingly, dexamethasone, a known inhibitor of cell proliferation, completely abrogated the response of RASMC to IL-4 and IL-13. Furthermore, the inhibition of ODC by these inhibitors led to the reduced production of polyamines and decreased DNA synthesis as monitored by [3H]thymidine incorporation. Our data indicate that upregulation of ODC by IL-4 and IL-13 might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.

cytokines; rat aortic smooth muscle cells; cell proliferation; polyamines; ornithine decarboxylase

VASCULAR SMOOTH MUSCLE CELL (VSMC) proliferation is a key event in the development of atherosclerosis and angioplasty-induced restenosis (55). However, the mechanisms responsible for smooth muscle cell proliferation are not fully elucidated. The polyamines putrescine, spermidine, and spermine have been shown to be essential for cell growth and differentiation. Putrescine is generated from the cationic amino acid L-ornithine via a decarboxylation reaction catalyzed by the enzyme ornithine decarboxylase (ODC), whereas spermidine and spermine are formed by the sequential transfer of an aminopropyl group from S-adenosylmethionine (33). Smooth muscle cell proliferation induced by arterial injury, platelet-derived growth factor, thrombin, insulin, basic fibroblast growth factor, transforming growth factor-β1 (TGF-β1), and serum are all associated with a striking increase in ODC activity (10, 14, 18, 26). The ODC gene is now recognized as a proto-oncogene required for cell-cycle progression and transformation. ODC is one of the most highly regulated enzymes known (25). ODC activity is tightly regulated at four levels, namely gene transcription, mRNA degradation, mRNA translation, and protein degradation (44). An elevated cellular concentration of polyamines induces upregulated expression of antizyme, an ODC-inhibitory protein that binds ODC and induces its rapid degradation (29). This degradation leads to a fast turnover of ODC, which has a half-life of only 10–20 min. The cellular concentration of ODC is very low, estimated to comprise 0.0003–0.01% of total cellular proteins (19).

IL-4 is a multifunctional cytokine that plays a critical role in the regulation of immune responses. IL-13 is a cytokine that elicits biological responses similar to IL-4. IL-4 mRNA has been found in human and mouse atherosclerotic lesions (58). IL-4 induces transcription of the 15-lipoxygenase-I gene in human endothelial cells, which plays an important role in atherogenesis (24). More recently, IL-4 has been linked to cigarette smoke-induced atherosclerotic lesion formation (31). At present, relatively little is known about how increasing the production of IL-4 affects atherosclerotic plaque formation. In our previous study (53), we found that both IL-4 and IL-13 can upregulate arginase I expression and stimulate cell proliferation in rat aortic smooth muscle cells (RASMC). Arginase catalyzes the conversion of arginine to ornithine plus urea. Ornithine is, in turn, converted to putrescine by ODC. We have also found that elevated expression of arginase I in RASMC increases polyamine production (54). Since ODC is the first and rate-limiting enzyme in polyamine synthesis, we reasoned that IL-4 and IL-13 may upregulate ODC expression and subsequently generate more polyamines available to stimulate cell growth. The objective of the present study was to determine whether ODC expression can be regulated by IL-4 and IL-13, and whether ODC activity correlates with RASMC proliferation. In the present study, IL-4 and IL-13 were shown to significantly increase ODC activity. The signal transduction pathways regulating ODC induction were investigated. MAPK kinase (MEK)/ERK, phosphatidylinositol 3-kinase (PI3K), and protein kinase A (PKA) pathways were all shown to modulate ODC expression and activity. Dexamethasone completely abolished the response of RASMC to IL-4 and IL-13. To the best of our knowledge this is the first study to investigate the effects of IL-4 and IL-13 on the regulation of ODC expression in any cell line. Our data suggest that IL-4 and IL-13 may play an important role in VSMC growth and atherosclerotic plaque formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Dexamethasone was purchased from Sigma. H-89, LY-294002, and PD-98059 were purchased from Calbiochem. Rat recombinant IL-4 and IL-13 were purchased from Peprotech.

Cell culture of RASMC. RASMC were a generous gift from Dr. Steven Gross (Weill Medical College of Cornell University). Cells were plated in high-glucose DMEM-HEPES supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 mg/ml amphotericin B, grown until confluent, and then subcultured by trypsinization. Cell cultures were performed at 37°C in a humidified atmosphere of 5% CO2-95% air. Subculture strains were used between passages 20 and 28. Cells were plated at a density of 106 cells/100-mm dish. When cells reached 80% confluence, the culture medium was replaced with fresh DMEM-HEPES and experiments were started.

ODC assay. ODC activity was determined by monitoring the formation of [14C]CO2 from L-[1-14C]ornithine by a modification of procedures described previously (5). SMC (4 x 106) were harvested in ice-cold Tris buffer (50 mmol/l Tris, 0.1 mmol/l EDTA, 2.5 mmol/l DTT, and 40 µmol/l pyridoxal-5-phosphate, pH 7.4), sonicated, and centrifuged at 14,000 g for 20 min at 4°C. The supernatants (soluble fraction) were collected for enzyme assay. The reaction mixture (250 µl) contained 250 µmol/l [14C]L-ornithine (0.25 µCi) and 0.2 mg soluble protein in Tris buffer. Reactions were conducted in sealed tubes designed to trap CO2 on filter paper saturated with 10% KOH. Enzyme reactions were initiated by addition of enzyme source and terminated by addition of 300 µl of 6 N HCl. Tubes were maintained at 37°C for 1 h, and the filters were removed and placed in 5 ml of EcoLite scintillation cocktail at 25°C for 60 min, after which time samples were counted in a Beckman liquid scintillation spectrometer.

Determination of polyamine concentrations in cells. The concentrations of putrescine, spermidine, and spermine in RASMC were determined by a sensitive HPLC procedure (56). Briefly, RASMC were plated at a density of 106 cells/100-mm dish and grown to 80% confluence before the start of experiments. RASMC (107 cells) were rinsed with PBS and then incubated at 37°C for 24 h in complete DMEM containing 0.5% FBS and 0.4 mM L-arginine. After 24 h, the cells were rapidly washed twice in ice-cold PBS and then lysed in 0.5 ml of 1.5 M HClO4, and the solution was neutralized by the addition of 0.25 ml of 2 M K2CO3. The neutralized extracts were used for the determination of polyamines.

Northern blot analysis of ODC mRNA level. RNA was isolated by using commercially available kits (QIAshredder and RNeasy Total RNA; Qiagen). Northern blot analysis was performed by standard techniques for formaldehyde-containing agarose gels (21). Mouse cDNA probe (American Type Culture Collection) for ODC was labeled by random priming (Ambion) to a specific activity of >109 disintegrations·min–1·mg–1 with [{alpha}-32P]dCTP and added (106 disintegrations·min–1·ml–1) to the prehybridization solution (5x SSPE containing 50% formamide, 5x Denhardt's solution, 0.1% SDS, 100 µg/ml heat-chilled salmon sperm DNA). Membranes were washed to a final stringency of 15 mM NaCl at 37°C and exposed overnight to Hyperfilm MP X-ray film (Amersham) by using intensifying screens at –70°C. To normalize hybridization signals for variations in loading and/or transfer, membranes were probed with GAPDH (Ambion). Densitometry of the bands was performed on the autoradiography film using a Hewlett-Packard flatbed scanner and NIH Image densitometry software.

Measurements of cell proliferation. Cell proliferation was assayed by monitoring rates of DNA synthesis as determined by the incorporation of [3H]thymidine into DNA. Previous studies from this laboratory indicated that three different methods yielded virtually identical data in the measurement of cell proliferation (7, 22, 53). These methods are [3H]thymidine incorporation into DNA, cell protein assay, and microscopic cell counting. We elected to employ DNA thymidine incorporation in the present study. RASMC were seeded in six-well plates at a density of 25,000 cells/cm2 in DMEM-HEPES containing 10% FBS and incubated for 4 h to allow cells to adhere to the plates. Cells were synchronized in serum-free DMEM-HEPES for 48 h. The effects of IL-4 and IL-13 on cell proliferation were examined in DMEM-HEPES containing 10% FBS. IL-4 or IL-13 was added to cell cultures and incubated for 24 h at 37°C. After this incubation period, 0.1 µCi [3H]thymidine was added to each well and incubated for an additional 24 h. The cells were collected for determining rates of DNA synthesis according to procedures described previously (53).

Statistical analyses. Data are presented as means ± SE. Differences between the groups were analyzed by ANOVA. Probability values of <0.05 were taken to indicate statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ERK and PI3K signal transduction pathways regulate ODC gene expression and activity in RASMC. Figure 1A illustrates that RASMC contain basal ODC activity, and incubation of cells with IL-4 (10 ng/ml) or IL-13 (10 ng/ml) significantly increased ODC activity (four- to fivefold). Interleukin concentrations of 10 ng/ml were selected on the basis of pilot experiments on ODC activity and expression, which indicated that such concentrations elicited maximal or near maximal effects. IL-4 and IL-13 concentrations of 1–3 ng/ml showed less activity, and concentrations less than 1 ng/ml were inactive. We then wanted to evaluate whether ERK and PI3K signaling pathways were involved in the stimulation of ODC expression by IL-4 and IL-13. RASMC were incubated with PD-98059, a MEK inhibitor, or LY-294002, a PI3K inhibitor, for 24 h. Results are shown in Fig. 1A. Basal ODC activity was reduced by incubation of RASMC with LY-294002 (10 µM) but not by PD-98059 (30 µM), suggesting that the PI3K pathway regulates basal ODC expression in RASMC. Inhibitor concentrations were selected on the basis of pilot experiments conducted to ascertain those concentrations that elicited 70–90% of maximal effect without causing cell death, assessed by Trypan blue exclusion. Preincubation of RASMC with either PD-98059 or LY-294002 for 30 min almost completely blocked the increased ODC activity caused by IL-4 and IL-13. To identify whether the increased ODC activity by IL-4 and IL-13 was attributed to increased ODC gene transcription, Northern blot analysis was employed to monitor ODC mRNA expression. Figure 1B shows that both IL-4 and IL-13 markedly enhanced ODC mRNA expression compared with the basal level. Incubation of cells with LY-294002 inhibited basal ODC mRNA expression. Preincubation of cells with PD-98059 or LY-294002 for 30 min markedly attenuated IL-4- and IL-13-induced ODC mRNA levels. Relative densitometric values are given in Fig. 1C. These observations were consistent with the observed changes in ODC activity. These data implicate ERK and PI3K pathways in the regulation of IL-4- and IL-13-induced ODC expression in RASMC.


Figure 1
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  		Fig. 1. Effects of IL-4 (10 ng/ml), IL-13 (10 ng/ml), LY-294002 (LY, 10 µM), and PD-98059 (PD, 30 µM) on the induction of ornithine decarboxylase (ODC) activity (A) and mRNA expression in rat aortic smooth muscle cells (RASMC) (B). A: cells (4 x 106/dish) were left untreated (control) or treated with the indicated agents for 24 h. LY-294002 and PD-98059 were added 30 min before the addition of IL-4 or IL-13. Cells were harvested, and cell lysates were assayed for ODC activity by monitoring the formation of [14C]CO2 from L-[14C]ornithine (100 µM) as described in the text. B: cells (4 x 106/dish) were left untreated (control) or treated with the indicated agents for 18 h. Total RNA (30 µg) was isolated, and Northern blot hybridization for ODC and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (loading control) mRNA was performed as described in the text. C: relative densitometric values for Northern blot analysis. Data represent means ± SE of duplicate determinations from 3 to 4 separate experiments. *Significantly different from control (P < 0.05). **Significantly different from IL-4 alone or IL-13 alone (P < 0.05).

 
Dexamethasone and PKA signal transduction pathways regulate ODC expression and activity in RASMC. The glucocorticoid dexamethasone has been well documented to inhibit the proliferation of VSMC (38, 39). The mechanisms of this cytostatic action have not been completely defined. Therefore, the effect of dexamethasone on ODC induction was examined. Dexamethasone (1 µM) alone inhibited basal ODC activity and expression in RASMC (Fig. 2A). Preincubation of cells with dexamethasone for 30 min completely abolished the stimulatory effect of IL-4 and IL-13 on ODC induction, indicating that blocking ODC expression contributed to the cytostatic effects of dexamethasone in RASMC. We then assessed the PKA pathway in the regulation of ODC expression. H-89, a PKA inhibitor, had little effect on basal ODC activity. However, preincubation of RASMC with H-89 for 30 min markedly attenuated the increase in ODC activity caused by IL-4 and IL-13. Northern blot data (Fig. 2B) were consistent with the results observed on ODC activity. Relative densitometric values are given in Fig. 2C. Inhibitor concentrations were selected on the basis of pilot experiments conducted to ascertain those concentrations that elicited 70–90% of maximal effect without causing cell death, assessed by Trypan blue exclusion. The data suggest that the PKA pathway is involved in the regulation of ODC expression in RASMC caused by IL-4 and IL-13.


Figure 2
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  		Fig. 2. Effects of IL-4 (10 ng/ml), IL-13 (10 ng/ml), H-89 (10 µM), and Dex (1 µM) on the induction of ODC activity (A) and mRNA expression (B) in RASMC. A: cells (4 x 106/dish) were left untreated (control) or treated with the indicated agents for 24 h. H-89 and Dex were added 30 min before the addition of IL-4 or IL-13. Cells were harvested, and cell lysates were assayed for ODC activity by monitoring the formation of [14C]CO2 from L-[14C]ornithine (100 µM) as described in the text. Data represent means ± SE of duplicate determinations from 3 to 4 separate experiments. B: cells (4 x 106/dish) were left untreated (control) or treated with the indicated agents for 18 h. Total RNA (30 µg) was isolated, and Northern blot hybridization for ODC and G3PDH (loading control) mRNA was performed as described in the text. C: relative densitometric values for Northern blot analysis. Data represent means ± SE of duplicate determinations from 3 to 4 separate experiments. *Significantly different from control (P < 0.05). **Significantly different from IL-4 alone or IL-13 alone (P < 0.05).

 
Induction of ODC by IL-4 and IL-13 correlates with increased polyamine production and cell proliferation. The basal levels of putrescine, spermidine, and spermine in RASMC were 0.48 ± 0.1, 12.35 ± 2.9, and 25 ± 5 nmol/ml per 107 cells, respectively (Fig. 3). After incubation of cells with IL-4 or IL-13 for 24 h, putrescine increased four- to fivefold, spermidine increased three- to fourfold, and spermine increased twofold compared with basal levels. The increased production of polyamines correlated with the increase in cell proliferation. Treatment with IL-4 or IL-13 for 24 h showed over a 50% increase in the rate of cell proliferation when compared with the basal level as determined by thymidine incorporation into DNA (Fig. 4). PD-98059, LY-294002, H-89, and dexamethasone each markedly diminished the increase in polyamine production and cell proliferation caused by IL-4 and IL-13. Moreover, {alpha}-difluoromethylornithine (1 mM, well-known ODC inhibitor) and DETA-NO [100 µM, covalent nitric oxide-donor inhibitor of ODC (4)] caused a 40–70% reduction in polyamine production and RASMC proliferation stimulated by IL-4 (data not shown). LY-294002 and dexamethasone, but not PD-98059 or H-89, inhibited (40–50%) both polyamine production and cell proliferation (data not shown). These data further support the view that ODC plays an essential role in controlling polyamine production and cell proliferation in RASMC.


Figure 3
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  		Fig. 3. Effects of IL-4 (10 ng/ml) and IL-13 (10 ng/ml) on putrescine (A), spermidine, and spermine (B) production in RASMC and the inhibitory effects of PD (30 µM), LY (10 µM), H-89 (10 µM), and Dex (1 µM). Test agents were added to RASMC 24 h before determination of putrescine, spermidine, and spermine concentrations. Cells were washed, lysed, and extracted as described in the text. Data represent means ± SE of duplicate determinations from 3 to 4 separate experiments. *Significantly different from control (P < 0.05). **Significantly different from IL-4 alone or IL-13 alone (P < 0.05).

 

Figure 4
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  		Fig. 4. Stimulation of RASMC proliferation by IL-4 (10 ng/ml) and IL-13 (10 ng/ml) and the inhibitory influence LY (10 µM), PD (30 µM), and Dex (1 µM). Cell proliferation was assessed by thymidine incorporation into DNA during the second 24-h interval of a 48-h growth period in medium containing the indicated test agents. Data are expressed as percentage of control, which represent means ± SE of duplicate determinations from 3 separate experiments. *Significantly different from control (P < 0.05). **Significantly different from IL-4 alone or IL-13 alone (P < 0.05).

 
FBS, IL-4, and IL-13 stimulate ODC mRNA expression. RASMC were incubated in medium containing 10% FBS alone (control) or in the presence of IL-4 or IL-13 for 1, 6, 18, and 24 h. At the end of incubation, cells were collected and RNA was extracted for Northern blot analysis. Figure 5A reveals that serum enhanced ODC mRNA expression. The ODC mRNA level reached a maximum at 6 h after serum stimulation, decreased dramatically at 18 h, and returned to basal levels at 24 h. Therefore, 10% FBS-stimulated ODC mRNA expression was transient. In the presence of IL-4 or IL-13, however, ODC mRNA reached a maximum at 6 h and remained constant until 24 h (Fig. 5A and Fig. 6A). Relative densitometry data (Figs. 5B and 6B) illustrated that significant increase of ODC mRNA expression was observed at 18 and 24 h compared with control group (P < 0.05). The data suggest that two possible mechanisms might be involved in the regulation of ODC mRNA accumulation in RASMC by IL-4 and IL-13, first, increasing ODC mRNA transcription and second, inhibiting ODC mRNA degradation.


Figure 5
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  		Fig. 5. Time course of induction of ODC mRNA expression by FBS and IL-4. A: cells (4 x 106/dish) were treated with either 10% FBS alone (control) or in the presence of IL-4 (10 ng/ml) for 0–24 h as indicated. Total RNA (30 µg) was isolated, and Northern blot hybridization for ODC and G3PDH (loading control) mRNA was performed as described in the text. B: relative densitometric values for Northern blot analysis. *Significantly different from control time points (P < 0.05).

 

Figure 6
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  		Fig. 6. Time course of induction of ODC mRNA expression by FBS and IL-13. A: cells (4 x 106/dish) were treated with either 10% FBS alone (control) or in the presence of IL-13 (10 ng/ml) for 0–24 h as indicated. Total RNA (30 µg) was isolated, and Northern blot hybridization for ODC and G3PDH (loading control) mRNA was performed as described in the text. B: relative densitometric values for Northern blot analysis. *Significantly different from control time points (P < 0.05).

 
ODC mRNA stability is not affected by IL-4 and IL-13 treatment. To determine whether the IL-4- or IL-13-induced increase in ODC mRNA accumulation was attributed to the inhibition of ODC mRNA degradation, RASMC were incubated with 10% FBS alone (control) or in the presence of IL-4 or IL-13 for 12 h, and at the end of incubation actinomycin D (10 µg/ml) was added to cells to stop ODC mRNA transcription. Cells were then incubated with actinomycin D for another 2, 6, and 12 h; RNA was then extracted, and Northern blot analysis was used to asses ODC mRNA levels. Figure 7A illustrates that ODC mRNA levels decreased markedly at 6 h and even further at 12 h after actinomycin treatment. Relative densitometric values (Fig. 7B) for Northern blot analysis revealed that no significant changes in ODC degradation rate were observed among FBS-, IL-4-, and IL-13-treated cells (P > 0.05). These data further support the view that IL-4 and IL-13 increase ODC mRNA accumulation by increasing ODC mRNA transcription.


Figure 7
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  		Fig. 7. Effects of IL-4 and IL-13 on ODC mRNA stability. A: RASMC were incubated with 10% FBS alone (control) or in the presence of either IL-4 (10 ng/ml) or IL-13 (10 ng/ml) for 12 h; thereafter, actinomycin D (10 µg/ml) was added, and RNA was harvested at the indicated time points. Total RNA (30 µg) was isolated, and Northern blot hybridization for ODC and G3PDH (loading control) mRNA was performed as described in the text. B: relative densitometric values for Northern blot analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study demonstrates that IL-4 and IL-13 both significantly upregulate ODC activity in RASMC and that this is attributed to an increase in ODC mRNA expression. The increase in ODC activity was associated with an increase in polyamine production and cell proliferation. We further demonstrated that the PI3K inhibitor PD-98059, the MAPK inhibitor LY-294002, and the PKA inhibitor H-89 nearly completely blocked the upregulation of ODC expression induced by IL-4 and IL-13, and these effects were paralleled by a decrease in both polyamine production and cell proliferation. Dexamethasone, which is a known inhibitor of cell proliferation (38, 39), reduced both basal ODC expression and induced ODC expression by IL-4 and IL-13. This is the first report indicating that ODC expression and activity can be increased by IL-4 and IL-13 and that this effect accounts for the capacity of these two cytokines to promote cell proliferation.

IL-4 regulates a wide variety of biological functions. Other than its well-studied immune response function as a Th2 cytokine, IL-4 acts as a survival factor in a number of cell types, including T cells, B cells, myeloid cells, synoviocytes, endothelial cells, fibroblasts, and cancer cells (9, 16, 17, 40, 49, 50, 57). Its effects depend upon binding to and signaling through a receptor complex consisting of the IL-4 R-{alpha} chain and the common {gamma} chain ({gamma}-c), resulting in a series of phosphorylation events mediated by receptor-associated kinases (28). Upon IL-4 binding to IL-4 R-{alpha}, the receptor dimerizes with either the {gamma}-c chain to form the type I IL-4 receptor or with the IL-13-R-{alpha}I chain to form the type II IL-4 receptor. The IL-4R-{alpha} chain cytoplasmic region appears to have three functionally distinct domains, one that acts as an interaction site for the Janus kinase (Jak), one required for activation of proliferation pathways, and a third involved in the activation of pathways leading to induction of gene expression (30). In vitro experiments have indicated that Jak1, Jak2, and Jak3 are capable of directly phosphorylating insulin receptor substrate (IRS)-1 and IRS-II (47, 52). Phosphorylated IRS-1/2 then interacts with the regulatory subunit of PI3K and the adapter molecule, Grb2. These interactions lead to the activation of the PI3K and Raf/MEK/ERK signaling pathways, respectively, and both pathways play an important role in cell proliferation (12, 13, 46).

The Raf/MEK/ERK signaling cascade is the best-defined pathway involved in cell proliferation. In this pathway, a central role is played by ERK. ERK is activated through phosphorylation by a single type of dual-specificity MEK1/2 (8). IL-4 has also been reported to upregulate the expression of MEK-1, augmenting the signaling-induced phosphorylation of ERK (2). A recent study showed that IL-4 induces activation of ERK and Akt in Jurkat T cells (45). In the present study, we demonstrate that PD-98059, a cell-permeable selective inhibitor of MEK1/2, markedly reduced ODC catalytic activity and gene expression, indicating that MEK/ERK pathway was involved in the induction of ODC by IL-4 and IL-13. This observation is consistent with the finding that the Raf/MEK/ERK pathway is involved in ODC regulation in cancer cells (35). These observations, in addition to the present study, suggest that ERK is an important kinase in ODC regulation for both normal and cancer cells. PI3K plays an important role in mitogenesis, cytoskeletal rearrangement, and vesicle transportation. PI3K can generate 3-phosphorylated phosphoinositides, such as phosphatidylinositol 3, 4, 5-trisphosphate, or phosphatidylinositol 3, 4-bisphosphate, which may act as second messengers (1, 11). Studies have shown that IL-4 binding induces the activation of the PI3K pathway (6, 51). PI3K signaling contributes to proliferation signaling in part by inducing the activation of the Akt and mammalian target of rapamycin (mTOR) pathways. We found that LY-294002, a PI3K inhibitor, markedly reduced or nearly abolished the basal and IL-4- and IL-13-induced ODC activity, gene expression, and cell proliferation, suggesting that the PI3K pathway is involved in the regulation of basal and inducible ODC levels in RASMC. The inhibitory effect of LY-294002 on basal ODC activity/expression and polyamine production and cell growth suggests that the PI3K signaling pathway is involved in regulating basal ODC and distal pathways. Our results are in agreement with the observation that the PI3K/Akt/mTOR pathway is involved in the regulation of ODC expression in cancer cells (35), supporting the critical role of PI3K/Akt/mTOR in ODC regulation for both normal and cancer cells. Furthermore, the data indicate that the PKA inhibitor, H-89, significantly decreased IL-4- and IL-13-induced ODC expression. Interestingly, the ODC gene contains response elements for several transacting factors, including a cAMP response element, a possible insulin response element, and several Sp1 binding sites (23). A study in a human breast cancer cell line revealed that estrogen upregulates ODC expression through the cAMP/PKA pathway (36). We believe that the PKA signaling pathway is involved in the upregulation of ODC expression induced by IL-4 and IL-13 in RASMC. However, the cross talk among PI3K, PKA, and MAPK pathways in regulating ODC expression by IL-4 and IL-13 is not entirely clear. Recent studies suggested that PKA is a versatile kinase, which can enhance the activation of the ERK and Akt pathways in epithelial cells and cardiomyocytes, respectively (20, 27). In view of this, we suggest that PI3K, MAPK, and PKA pathways in RASMC act synergistically in the upregulation of ODC expression induced by IL-4 and IL-13 and that this sequence of events leads to an increase in cell proliferation.

Polyamines are aliphatic cations with multiple functions and are essential for life. Investigations using polyamine biosynthetic inhibitors indicate that alterations in cellular polyamine levels modulate normal and cancer cell growth. Direct binding of polyamines to DNA and their ability to modulate DNA-protein interactions appear to be important in the molecular mechanisms of polyamine action in cell proliferation (48). Mammalian cells contain three natural polyamines, putrescine, spermidine, and spermine (34). In the present study, we found that in RASMC the cellular levels of putrescine, spermidine, and spermine were all increased markedly by incubation of cells with IL-4 and IL-13. The increase in polyamines levels was correlated with the increase in cell proliferation. PD-98059, LY-294002, and H-89 each significantly inhibited polyamine production and cell proliferation mediated by IL-4 and IL-13 in RASMC. These results further confirmed that polyamines play an important role in controlling cell proliferation. Our data indicate that the stimulatory effects of IL-4 and IL-13 on RASMC proliferation are at least partly attributed to ODC induction, which subsequently makes more polyamines available for RASMC to grow.

Dexamethasone is a synthetic glucocorticosteroid that elicits a myriad of pharmacological effects including inhibition of SMC proliferation and DNA synthesis (38, 39). Dexamethasone was reported to inhibit ODC activity in lymphoid tissues (spleen and thymus) (23). Dexamethasone also reduced ODC expression, intracellular polyamine content, and cell proliferation in basophilic cells caused by phorbol ester (PMA) (15). From these reported inhibitory effects of dexamethasone on cell growth, we examined the influence of dexamethasone on cytokine-induced ODC expression in RASMC. The present study reveals that dexamethasone markedly inhibits basal and IL-4- and IL-13-induced ODC activity and gene expression in RASMC. The inhibition of ODC expression was consistent with the reduced cellular polyamine levels and decreased cell proliferation rate. Our data support the view that downregulation of ODC expression is the mechanism of the cytostatic effect of dexamethasone on VSMC.

The present study was conducted to elucidate the mechanism(s) by which IL-4 and IL-13 stimulate VSMC proliferation. In a previous study (53), we found that both cytokines could increase arginase I expression, which is consistent with increased polyamine production and cell growth (54). However, the present study reveals that IL-4 and IL-13 also upregulate ODC expression and activity and cause an increase in polyamine production that is associated with increased RASMC proliferation. By employing classical inhibitor experiments, we found that the PI3K, MAPK, and PKA signaling pathways are involved in the effects of IL-4 and IL-13. We were interested in this approach as a possible explanation for the increased proliferation of VSMC and other cell types that characterize atherosclerosis (42). The present study, as well as a previous study (54), suggests that vascular smooth muscle proliferation in atherosclerosis, a complex inflammatory disease characterized by increased cytokine production, may be at least partially attributed to arginase I induction, ODC induction, and consequent increased polyamine production. A possible scenario is that, during atherogenesis, cytokines such as IL-4 and IL-13 produced by local lymphocytes (41, 43), natural killer cells (32), and vascular endothelial cells (3, 37) upregulate both arginase I and ODC expression in nearby VSMC and stimulate their growth.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by the National Heart, Lung, and Blood Institute Grants HL-35014 and HL-58433 (to L. J. Ignarro).


   ACKNOWLEDGMENTS
 
We thank Dr. Steven Gross for the kind gift of rat aortic smooth muscle cells. We also thank Dr. Georgette M. Buga for providing expert technical support on the ornithine decarboxylase assay.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. J. Ignarro, Dept. of Molecular and Medical Pharmacology, David Geffen School of Medicine, Univ. of California, Los Angeles, CA 90095-1735 (e-mail: lignarro{at}mednet.ucla.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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