IL-4 and IL-13 upregulate arginase I expression by cAMP and JAK/STAT6 pathways in vascular smooth muscle cells

Liu Hua Wei1, Aaron T. Jacobs1, Sidney M. Morris Jr.2, and Louis J. Ignarro1

1 Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095; and 2 Department of Biochemistry and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The objectives of this study were to determine whether rat aortic smooth muscle cells (RASMC) express arginase and to elucidate the possible mechanisms involved in the regulation of arginase expression. The results show that RASMC contain basal arginase I (AI) activity, which is significantly enhanced by stimulating the cells with either interleukin (IL)-4 or IL-13, but arginase II (AII) expression was not detected under any condition studied here. We further investigated the signal transduction pathways responsible for AI induction. AI mRNA and protein levels were enhanced by addition of forskolin (1 µM) and inhibited by H-89 (30 µM), suggesting positive regulation of AI by a protein kinase A pathway. Genistein (10 µg/ml) and sodium orthovanadate (Na3VO4; 10 µM) were used to investigate the role of tyrosine phosphorylation in the control of AI expression. Genistein inhibited, whereas Na3VO4 enhanced the induction of AI by IL-4 or IL-13. Along with immunoprecipitation and immunoblot analyses, these data implicate the JAK/STAT6 pathway in AI regulation. Dexamethasone (Dex) and interferon (IFN)-gamma were investigated for their effects on AI induction. Dex (1 µM) and IFN-gamma (100 U/ml) alone had no effect on basal AI expression in RASMC, but both reduced AI induction by IL-4 and IL-13. In combination, Dex and IFN-gamma abolished AI induction by IL-4 and IL-13. Finally, both IL-4 and IL-13 significantly increased RASMC DNA synthesis as monitored by [3H]thymidine incorporation, demonstrating that upregulation of AI is correlated with an increase in cell proliferation. Blockade of AI induction by IFN-gamma , H-89, or genistein also blocked the increase in cell proliferation. These observations are consistent with the possibility that upregulation of AI might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.

interleukin; rat aortic smooth muscle cells; cell proliferation; cytokines


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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THERE ARE TWO ISOFORMS of arginase in vertebrates, both of which catalyze the conversion of arginine to ornithine and urea. They differ with regard to subcellular localization, tissue distribution, and certain enzymatic properties, reflecting the fact that they are encoded by different genes (reviewed in Ref. 20). Arginase I (AI) is expressed almost exclusively in the cytosol of liver cells where it serves as the fifth and final enzyme of the urea cycle. Expression of AI in liver is regulated by dietary protein (33) and by hormones such as glucagon and glucocorticoids (15, 36). Recently, however, AI has been found to be induced also by lipopolysaccharide (LPS), interleukin (IL)-4, cAMP, and hypoxia in macrophages (26, 27, 32, 58) and by LPS in various tissues of rats and mice (43, 47). In contrast to AI, however, arginase II (AII) is located within the mitochondrial matrix and is expressed at low levels in many tissues (13, 31). It has recently been demonstrated that AII expression can be induced by cAMP in murine macrophages (32) and by LPS in macrophages and various tissues of mice (27, 32, 43), but LPS induction of AII has not been observed for rat tissue (47). Unlike AI in liver, the physiological role of AII has not been established.

Cloned cDNA for AI and AII have been isolated for rat, mouse, human, and Xenopus laevis (summarized in Ref. 39), and the genes encoding rat and human AI (37, 52) and murine AII (45) also have been cloned. The genes for AI and AII are structurally similar and consist of eight exons, reflecting the fact that the two genes are probably the consequence of an ancient gene duplication event. Some promoter and enhancer elements in the rat AI gene that are required for expression in hepatocytes have been characterized (6, 12, 51).

With the exception of enterocytes (59), extrahepatic organs lack a complete urea cycle, and it has been suggested that arginase in tissues other than liver may be involved in the regulation of cell growth and tissue repair (1, 4, 26, 43). L-Ornithine, the enzymatic product of arginase, is a precursor for the synthesis of polyamines (putrescine, spermidine, and spermine). Polyamines have been shown to stimulate DNA synthesis, enhance transcription of growth-related genes, and are essential for cell proliferation (19, 50). Furthermore, activity of ornithine decarboxylase, which is required for polyamine synthesis, parallels the intensity of the repair processes in injured tissues (11).

IL-4 is a pleiotropic cytokine that plays an important role in immune-inflammatory reactions by modulating growth, differentiation, cytokine production, and major histocompatibility complex class II expression (23). IL-4 has also been reported to induce morphological changes in human umbilical vein endothelial cells, which may be important in angiogenesis (24). IL-4 induces arginase activity in cultured macrophages (7, 29), and this has been identified as the AI isoform (26). IL-13 is a cytokine that elicits biological responses similar to IL-4 (2, 22). The purpose for the present study was to determine whether rat aortic smooth muscle cells (RASMC) express arginase, if arginase expression can be modulated by IL-4 and IL-13, and if arginase activity correlates with RASMC proliferation. In the present study, IL-4 and IL-13 were shown to significantly increase AI activity in RASMC. The signal transduction pathways regulating this induction were investigated. Janus kinases (JAK) and signal transducer and activator of transcription (STAT) and cAMP-dependent pathways were all shown to modulate AI expression and activity. Dexamethasone and interferon (IFN)-gamma abrogated the response of RASMC to IL-4 and IL-13. Upregulation of AI was accompanied by, and may have caused, enhanced RASMC proliferation. These results suggest that elevated arginase activity may have physiological and/or pathophysiological roles in the cardiovascular system.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Reagents. Forskolin, dexamethasone, and genistein were purchased from Sigma. Sodium orthovanadate (Na3VO4) and H-89 were purchased from Calbiochem. Recombinant rat IL-4 and recombinant human IL-13 were purchased from R & D Systems. Rat recombinant IFN-gamma was purchased from GIBCO. Rabbit anti-STAT6 polyclonal antibody, mouse anti-p-Tyr (epitope, phospho-Y99) monoclonal antibody, and protein A-coupled agarose beads were purchased from Santa Cruz Biotechnology.

Cell culture of RASMC. RASMC was a generous gift from Dr. Steven Gross. 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 and were grown until confluent at which time cells were subcultured by trypsinization. Cell cultures were performed at 37°C in a humidified atmosphere of 5% CO2-95% air. Subcultured 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.

Arginase assay. Arginase activity was determined by methods that we have previously described for endothelial cells (4). Briefly, RASMC (4 × 106 cells/sample) were washed twice with PBS, harvested, pelleted by centrifugation, then lysed. Supernatant fractions were assayed for arginase activity under optimal conditions of pH (9.6) and arginase concentration (20 mM) by monitoring the conversion of L-[guanido-14C]arginine to [14C]urea during 10-min incubation.

Measurements of cell proliferation. Cell proliferation was assessed by monitoring rates of DNA synthesis as determined by the incorporation of [3H]thymidine into DNA. RASMC were seeded in six-well plates at a density of ~3,000 cells/cm2 in DMEM-HEPES containing 10% FBS and incubated for 4 h to allow cells to adhere to the plates. Cell synchronization was achieved by arresting cell growth by incubation in serum- and arginine-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 (4).

Immunoprecipitation and immunoblot analysis. For STAT6 activation assays, RASMC (107 cells/sample) were incubated with IL-4 or IL-13 at 37°C for 10 min. Cells were washed twice with PBS containing 100 µM Na3VO4, followed by addition of 1.5 ml lysis buffer (50 mM Tris·HCl, pH 7.5, 1% Triton X-100, 50 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 5 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). After 10 min on ice, lysates were cleared of insoluble materials by centrifugation for 10 min at 12,000 g. The cleared lysates were immunoprecipitated with STAT6 antiserum (1:500) plus 40 µl of protein A-coupled agarose beads overnight at 4°C. Immunoprecipitates were collected by centrifugation at 2,500 rpm for 5 min at 4°C. Immunoprecipitates were then washed four times with lysis buffer, solubilized with Laemmli buffer, boiled 5 min, and resolved by 8% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 25 mM Tris·HCl, pH 7.2, 150 mM NaCl, and 0.2% (wt/vol) Tween (TBST) buffer supplemented with 2% BSA. The membranes were subsequently probed with the following antiserum diluted in TBST: anti-p-Tyr (PY 99, 1:1,000) and anti-STAT6 (1:2,000). The membranes were then washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody. AI protein expression was determined in the same cell lysates used for the arginase activity assays. Equal samples (20 µg of total protein from each sample) were subjected to SDS-PAGE (10%) then transferred to a nitrocellulose membrane. The membrane was probed with chicken anti-rat AI antibody (1:50,000 dilution) (32). The secondary antibody for AI immunoblots was peroxidase-conjugated rabbit anti-chicken IgG (Jackson ImmunoResearch Laboratories). The immunoblots were developed with enhanced chemiluminescence Western blotting detection kits (Amersham) according to the manufacturer's directions.

Isolation and analysis of RNA. RNA was isolated using a commercially available kit (QIAshredder and RNeasy total RNA; Qiagen). Northern blot analysis was performed according to a standardized protocol (44). Full-length cDNA probes encoding rat AI (21) or murine AII (31) were used for Northern blots. A human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probe was used as an internal control (Ambion). Densitometry of the bands was performed on Fuji RX autoradiography film using a Hewlett-Packard flatbed scanner and NIH Image densitometry software.

Statistical analyses. Data were statistically analyzed using the Student's t-test for unpaired values. Probability values of <0.05 were taken to indicate statistical significance.


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The protein kinase A signal transduction pathway regulates arginase expression and activity in RASMC.

RASMC contain a basal arginase activity that is markedly increased (4- to 5-fold) by incubation of the cells with IL-4 (10 ng/ml) or IL-13 (10 ng/ml). A time course for the induction of arginase activity by IL-4 and IL-13 is illustrated in Fig. 1. Arginase activity was elevated at 12 h, peaked at 24-48 h, and started to decline at 72 h. These data are consistent with the results of a Western blot analysis of AI protein (Fig. 1), indicating that the induced arginase protein and catalytic activity are maintained through at least 72 h after cytokine induction. The 24-h time point was selected for further experiments involving test agents. Basal arginase activity is slightly increased by incubation cells with the adenylate cyclase activator forskolin (1 µM) (Fig. 1). Forskolin potentiates arginase induction by IL-4 and IL-13 about twofold. In contrast, basal arginase activity is reduced by incubating cells with the protein kinase A inhibitor H-89 (30 µM) (Fig. 2). Preincubation of RASMC with H-89 for 1 h markedly attenuates the increase in arginase activity caused by IL-4 and IL-13.


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Fig. 1.   Time course of induction of arginase activity and arginase I protein in rat aortic smooth muscle cells (RASMC). Cells (4 × 106/dish) were treated with either interleukin (IL)-4 (10 ng/ml; A) or IL-13 (10 ng/ml; B) for 0-72 h as indicated. Top: cells were harvested and cell lysates were assayed for arginase activity by monitoring the conversion of L-[guanido-14C]arginine to [14C]urea as described in text. Data represent means ± SE of duplicate determinations from 3 separate experiments. * Significantly different from control (P < 0.05). Bottom: immunoreactive arginase I was detected by Western blot analysis as described in text. Western blots were performed with the same cell lysate used for determination of arginase activity above. Data illustrated are from a single experiment and are representative of a total of 3 separate experiments.



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Fig. 2.   Effects of IL-4 (10 ng/ml), IL-13 (10 ng/ml), forskolin (1 µM) (A), and H-89 (30 µM) (B) on induction of arginase activity in RASMC. Cells (4 × 106/dish) were left untreated (control) or treated with the indicated agents for 24 h. H-89 was added 1 h before the addition of IL-4 (10 ng/ml) or IL-13 (10 ng/ml). Cells were harvested and cell lysates were assayed for arginase activity by monitoring the conversion of L-[guanido-14C]arginine to [14C]urea as described in text. Data represent means ± SE of duplicate determinations from 5 separate experiments. * Significantly different from control (P < 0.05). ** Significantly different from IL-4 or IL-13 alone (P < 0.05).

To identify the arginase isoform(s) expressed in RASMC, Northern and Western blot analyses were employed to monitor mRNA and protein expression, respectively. Untreated RASMC express AI mRNA and protein (Fig. 3). Both IL-4 and IL-13 markedly enhance AI mRNA and protein expression in comparison with the basal level. In contrast, mRNA for the AII isoform was not detected in control cells, and neither IL-4 nor IL-13 induced AII mRNA (data not shown). Coincubation of cells with forskolin enhances both the basal and IL-4- and IL-13-induced AI protein and mRNA levels. Incubation of RASMC with H-89 inhibits basal AI mRNA and protein expression. Moreover, preincubation with H-89 for 1 h significantly diminishes IL-4- and IL-13-induced AI protein and mRNA levels. There is a direct correlation between the changes in AI protein and mRNA levels with changes observed in arginase activity. These data implicate protein kinase A in the regulation of both basal and induced AI expression in RASMC.


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Fig. 3.   Effects of forskolin (1 µM), H-89 (30 µM), IL-4 (10 ng/ml), and IL-13 (10 ng/ml) on induction of arginase I (AI) mRNA (A) and protein (B and C) expression in RASMC. H-89 was added 1 h before the addition of IL-4 or IL-13. A: cells (4 × 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 AI and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (loading control) mRNA was performed as described in text. B and C: RASMC (4 × 106/dish) were left untreated (control) or treated with the indicated agents for 24 h. Immunoreactive AI was detected by Western blotting as described in text. Western blots were performed with the same cell lysate used for determination of arginase activity. Data illustrated are from a single experiment and are representative of a total 3 separate experiments.

Tyrosine phosphorylation may be involved in the regulation of basal and induced arginase activity in RASMC. To determine if tyrosine phosphorylation may be involved in the regulation of arginase, RASMC were incubated with either genistein, an inhibitor of protein tyrosine kinases, or Na3VO4, a tyrosine phosphatase inhibitor. Incubation of cells with genistein (10 µg/ml) significantly reduces basal arginase activity in RASMC (Fig. 4). Furthermore, preincubation of cells with genistein for 1 h diminishes IL-4- and IL-13-induced arginase activity by ~50%. In contrast, incubation of cells with Na3VO4 (10 µM) slightly increases basal arginase activity. In combination with either IL-4 or IL-13, Na3VO4 enhances arginase activity two- to threefold. These data for arginase activity closely agree with the changes observed in mRNA and protein expression. These data suggest that a signal transduction pathway involving phosphotyrosine formation is involved in the regulation of AI expression in RASMC.


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Fig. 4.   Top: effects of Na3VO4 (10 µM), genistein (10 µg/ml), IL-4 (10 ng/ml), and IL-13 (10 ng/ml) on induction of arginase activity in RASMC. Na3VO4 and genistein were added 1 h before the addition of IL-4 or IL-13. Cells (4 × 106/dish) were left untreated (control) or treated with the indicated agents for 24 h. Cells were harvested, and cell lysates were assayed for arginase activity by monitoring the conversion of L-[guanido-14C]arginine to [14C]urea as described in text. Data represent means ± SE of duplicate determinations from 5 separate experiments. * Significantly different from control (P < 0.05). ** Significantly different from IL-4 or IL-13 alone (P < 0.05). Bottom: influence of Na3VO4 (10 µM), genistein (10 µg/ml), IL-4 (10 ng/ml), and IL-13 (10 ng/ml) on induction of AI mRNA (A) and protein (B and C) expression in RASMC. Na3VO4 and genistein were added 1 h before the addition of IL-4 or IL-13. A: cells (4 × 106/dish) were left untreated (control) or treated with the indicated agents for 18 h. Total RNA of 40 µg was isolated, and Northern blot hybridization for AI and G3PDH (loading control) mRNA was performed as described in text. B and C: RASMC (4 × 106/dish) were left untreated (control) or treated with the indicated agents for 24 h. Immunoreactive AI was detected by Western blotting as described in text. Western blots were performed with the same cell lysate used for determination of arginase activity. Data illustrated are from a single experiment and are representative of a total 3 separate experiments.

STAT6 activation by IL-4 and IL-13 correlates with upregulation of arginase I expression. STAT6 is a cytoplasmic transcription factor regulated by its phosphorylation state and is activated in response to IL-4 and IL-13 (8). After phosphorylation of a critical tyrosine residue, STAT6 dimerizes, translocates from the cytoplasm to the nucleus, and binds to its cognate DNA, leading to a specific transcriptional upregulation of target genes (18). Because we have demonstrated in this report that phosphotyrosine is essential for the regulation of AI by IL-4 and IL-13, we investigated the status of STAT6 phosphorylation following stimulation with either IL-4 or IL-13. Unstimulated RASMC have no detectable phosphorylated STAT6 (p-STAT6) protein (Fig. 5). Addition of IL-4 or IL-13 promotes a rapid and intense phosphorylation of STAT6. The appearance of p-STAT6 correlates with the upregulation of AI expression by IL-4 and IL-13 and supports the hypothesis for a cytokine-JAK/STAT pathway for AI regulation. Studies are in progress to test the hypothesis that activation of the JAK/STAT6 pathway is involved in the induction of AI by IL-4 and IL-13 in RASMC.


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Fig. 5.   IL-4 and IL-13 induce STAT6 tyrosine phosphorylation in RASMC. Cells were untreated (control) or treated with IL-4 (10 ng/ml) or IL-13 (10 ng/ml) for 10 min at 37°C. Cell lysates were immunoprecipitated (IP) with anti-STAT6 antibody, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane. Membranes were subsequently immunoblotted with anti-phosphotyrosine (anti-p-Tyr) (A) or anti-STAT6 antibody (loading control) (B). Data shown are representative of 3 separate experiments.

Dexamethasone and IFN-gamma abrogate the stimulatory effect of IL-4 and IL-13 on AI. The glucocorticoid dexamethasone and the cytokine IFN-gamma have been well documented to inhibit the proliferation of vascular smooth muscle (25). The mechanism of this cytostatic action has not been completely defined for either agent. Therefore, the effects of dexamethasone and IFN-gamma on arginase induction were examined. Incubation of RASMC with either dexamethasone (1 µM) or IFN-gamma (100 U/ml) alone has no measurable influence on basal arginase activity (Figs. 6 and 7). However, dexamethasone or IFN-gamma each moderately attenuates arginase activity induced by IL-4 or IL-13. Moreover, the combination of dexamethasone plus IFN-gamma abolishes the stimulatory effect of IL-4 and IL-13 on arginase induction. In all cases, changes in arginase activity were correlated with relative changes in AI mRNA and protein expression (Fig. 8).


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Fig. 6.   Inhibitory effects of dexamethasone (Dex; 1 µM) and interferon (IFN)-gamma (100 U/ml) on constitutive and IL-4- or IL-13-inducible arginase activity in RASMC. Cells (4 × 106/dish) were left untreated (control) or treated with the indicated agents for 24 h. Cells were harvested and cell lysates were assayed for arginase activity by monitoring the conversion of L-[guanido-14C]arginine to [14C]urea as described in text. Data represent means ± SE of duplicate determinations from 5 separate experiments. * Significantly different from control (P < 0.05). ** Significantly different from IL-4 or IL-13 alone (P < 0.05).



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Fig. 7.   Inhibitory effects of dexamethasone (1 µM) and IFN-gamma (100 U/ml) on induction of AI mRNA (A) and protein (B and C) expression in RASMC caused by IL-4 (10 ng/ml) and IL-13 (10 ng/ml). A: cells (4 × 106/dish) were left untreated (control) or treated with the indicated agents for 18 h. Total RNA of 30 µg was isolated, and Northern blot hybridization for AI and G3PDH (loading control) mRNA was performed as described in text. B and C: RASMC (4 × 106/dish) were left untreated (control) or treated with the indicated agents for 24 h. Immunoreactive AI was detected by Western blotting as described in text. Western blots were performed with the same cell lysate used for determination of arginase activity. Data illustrated are from a single experiment and are representative of a total 3 separate experiments.



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Fig. 8.   Stimulation of RASMC proliferation by IL-4 (10 ng/ml) and IL-13 (10 ng/ml) and the inhibitory influence of IFN-gamma (100 U/ml), H-89 (10 µM), and genistein (Gen; 3 µg/ml). 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 represents cells grown in the absence of added IL-4 or IL-13. Data 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).

Upregulation of AI expression by IL-4 and IL-13 correlates with cytokine-stimulated RASMC proliferation. A recent study from this laboratory revealed that inhibiting arginase activity in the human adenocarcinoma Caco-2 cell line resulted in the inhibition of cell proliferation, thereby implicating arginase in the regulation of cell growth (4). In the present study, we investigated the relationship between AI induction and cell proliferation. DNA synthesis, as monitored by [3H]thymidine incorporation, increases by ~50% in RASMC exposed to IL-4 (10 ng/ml) or IL-13 (10 ng/ml) for 48 h (Fig. 7). This increase in cell proliferation correlates with the increase in AI expression and activity observed in response to IL-4 and IL-13, supporting our hypothesis that AI is involved in regulating the growth of RASMC. Moreover, IFN-gamma , H-89, and genistein each markedly inhibited the increase in cell proliferation caused by IL-4 or IL-13. Addition of these test agents alone to cells in the absence of added cytokine caused only little or no significant decrease in cell proliferation (data not shown). Although dexamethasone markedly inhibited the increase in cell proliferation caused by IL-4 and IL-13, dexamethasone alone significantly inhibited RASMC proliferation, and additional experiments are required to elucidate the mechanism of this effect.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

For many years, AI had been thought to be expressed exclusively in the liver. However, it was recently reported that rat endothelial cells (4) and human neutrophils (56) express AI. Of relevance to the present study are reports that AI can be induced in macrophages (26, 32) and mesangial cells (57) by either 8-bromo-cAMP or IL-4. This study provides the first demonstration that RASMC express AI; that IL-4 and IL-13 both significantly upregulate AI mRNA, protein, and activity; and that cAMP is involved in AI expression in those cells.

Our results demonstrate that the PKA inhibitor H-89 inhibits both basal and IL-4- and IL-13-induced AI expression in RASMC. Moreover, forskolin, an activator of adenylate cyclase, significantly enhances IL-4- and IL-13-induced AI levels and activity. These data indicate that the cAMP signal transduction pathway is involved in the regulation of basal and induced AI expression in RASMC. Interestingly, the AI promoter region contains a putative cAMP response element (CRE) (52), although a functional CRE in the AI has yet to be identified.

IL-4, a pleiotropic modulator of the immune system, exerts its activity on target cells through IL-4 receptor (IL-4R), which consists of the IL-4R alpha -chain (IL-4Ralpha ) and the common gamma -receptor chain (gamma c), also called the type I receptor (22). IL-4Ralpha also forms a functional IL-4R in conjunction with the IL-13 receptor (IL-13Ralpha ) instead of gamma c. This type of IL-4R, called the type II receptor, is found particularly in nonimmune cells (5). Treatment of cells with IL-4 activates cytoplasmic tyrosine kinases (8, 17). The present study reveals that genistein, an inhibitor of tyrosine kinases, inhibits both basal and IL-4- and IL-13-induced AI in RASMC. Also, Na3VO4, a protein tyrosine phosphatase inhibitor, markedly increases both basal and IL-4- and IL-13-induced AI expression in RASMC. These data illustrate, albeit indirectly, that a tyrosine kinase pathway may be involved in the regulation of basal and induced AI expression in RASMC.

Activation of the IL-4R triggers the function of intracellular signal mediators, such as the JAK/STAT pathway, which involves the IL-4- and IL-13-specific transcription factor STAT6 (8, 40). STAT proteins are phosphorylated in response to ligand stimulation through receptor-associated JAK proteins. Tyrosine phosphorylation of STAT proteins induce dimerization. The dimerized STAT then translocate to the nucleus and modulate transcription of target genes (18). We demonstrate that untreated RASMC contain no detectable phosphorylated STAT6 protein. Phosphorylated STAT6 was readily detected, however, after stimulating the cells with IL-4 or IL-13 for only 10 min at 37°C. The correlation of AI induction with STAT6 phosphorylation suggests that the JAK-STAT6 pathway may be involved in the regulation of AI expression in RASMC. Alternative pathways, however, cannot be ruled out.

Dexamethasone induces arginase in hepatocytes and hepatoma cells (9, 15, 36). In contrast, our results show that dexamethasone alone has no effect on basal AI expression in RASMC but can reduce the AI induction by IL-4 and IL-13. This observation is similar to that in a report showing that dexamethasone blocks AI induction caused by 8-bromo-cAMP in RAW 264.7 cells (32). We also found that dexamethasone plus IFN-gamma completely blocks AI induction by IL-4 and IL-13. Why dexamethasone elicits opposite effects on arginase expression in different cell type is unknown.

In the present study, IFN-gamma alone had no effect on basal AI expression in RASMC but significantly inhibited the induction of AI mediated by IL-4 and IL-13, and the combination of dexamethasone with IFN-gamma inhibited the induction of AI even more. These results support the general impression that IFN-gamma inhibits induction of arginase. For example, IFN-gamma completely blocks the induction of AI expression by 8-bromo-cAMP in RAW 264.7 cells (32). Moreover, IFN-gamma inhibits LPS-mediated induction of AII in RAW 264.7 cells (58) and may inhibit the same response in rat aorta endothelial cells (4). In contrast to these inhibitory effects, however, is the finding that IFN-gamma synergizes with IL-4 in induction of AI expression in RAW 264.7 cells (46). Thus, like dexamethasone, IFN-gamma has varying effects on AI expression in different cell types.

A family of cytokine-inducible SH2 proteins (CIS) has recently been identified (54, 61). Most CIS appear to be induced by several cytokines, and at least three of them [CIS1, CIS3 and JAB (JAK-binding protein)] negatively regulate cytokine signal transduction (10, 34, 49). CIS3 and JAB directly bind to the kinase domain of JAK, thereby inhibiting kinase activity (28). These CIS family members appear to function in a classical negative-feedback loop of cytokine signaling. Because IFN-gamma is the most potent inducer of JAB in a wide variety of cell lines (60), it is possible that IFN-gamma -activated JAB could be a part of the mechanism of preventing the AI induction by IL-4 and IL-13.

Another novel aspect of this study is that the upregulation of AI by IL-4 and IL-13 correlates with enhanced RASMC proliferation. Although these data do not prove that the stimulatory effects of IL-4 and IL-13 on RASMC proliferation are attributed to arginase induction, or are mediated by cAMP, they are consistent with these possibilities. IL-4 and other vasoactive and inflammatory cytokines have been shown to promote the growth of other cell types including porcine aorta smooth muscle cells (35), human adrenal capillary endothelial cells (53), and mouse skin fibroblasts (30). On the other hand, IL-4 and cAMP have been reported to inhibit proliferation of vascular smooth muscle cells from other sources (38, 48, 55). The reasons for these differences in results are not apparent but may be attributed to differences in cell types and/or cell culture conditions. Our findings that IFN-gamma and dexamethasone inhibit the induction of AI in RASMC are consistent with the well-documented cytostatic actions of glucocorticosteroids and IFN-gamma in vascular smooth muscle (16, 25).

Increased proliferation of vascular smooth cells contributes to intimal hyperplasia during atherosclerosis and restenosis (41, 42). The endogenous factors regulating vascular smooth muscle cell growth in response to arterial injury are not well understood. Both arginase and ornithine decarboxylase are intimately involved in cell proliferation. Arginase catalyzes the conversion of arginine to ornithine, and ornithine decarboxylase catalyzes the subsequent conversion of ornithine to putrescine. The production of polyamines plays an essential role in cell proliferation (19, 50). We hypothesize that at least one consequence of upregulating AI expression in RASMC may be the enhanced synthesis of polyamines, which may explain the enhancement of cell proliferation observed in this study. Accordingly, upregulation of AI expression caused by IL-4 and IL-13 might play an important role in the pathophysiology of atherosclerosis, angiogenesis, and restenosis, which result from a complex cascade of events involving abnormal proliferation of vascular smooth muscle cells.


    ACKNOWLEDGEMENTS

We thank Dr. Steven Gross for the kind gift of RASMC. We also thank Dr. Georgette M. Buga for providing expert technical support on the [3H]thymidine incorporation assay.


    FOOTNOTES

This research was supported by National Institutes of Health Grants HL-35014, HL-40922, and HL-58433 (to L. J. Ignarro) as well as GM-57384 (to S. M. Morris).

Address for reprint requests and other correspondence: L. J. Ignarro, Dept. of Molecular and Medical Pharmacology, UCLA School of Medicine, 23-120 CHS, Box 951735, 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. §1734 solely to indicate this fact.

Received 25 August 1999; accepted in final form 25 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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