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Regulation of the RON Receptor Tyrosine Kinase Expression in Macrophages: Blocking the RON Gene Transcription by Endotoxin-Induced Nitric Oxide^{1 ,2}

Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado School of Medicine and Denver Health Medical Center, Denver, CO 80204

	Abstract

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Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-, and TNF- regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF- plus IFN- abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF- plus IFN- induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (±)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-ß prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF- plus IFN- are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.

	Introduction

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Receptor-type protein tyrosine kinases are a group of transmembrane proteins that play a critical role in inflammation, wound healing, and tissue remodeling (1, 2, 3). The common architecture of these molecules includes an extracellular ligand binding domain, a transmembrane segment, and a large cytoplasmic portion with intrinsic tyrosine kinase activities (4). Many receptor tyrosine kinases such as those for CSF-1, hepatocyte growth factor (HGF),⁴ epidermal growth factor (EGF), and macrophage-stimulating protein (MSP) (5, 6, 7, 8) are expressed in macrophages. Stimulation of these receptors with their individual ligands induces tyrosine phosphorylation and increases protein kinase activity (9). The activated receptors transduce multiple signals that regulate macrophage inflammatory activities such as growth, survival, chemotactic migration, and phagocytosis (8, 10). Deregulation of growth factor receptor expression impairs development, differentiation, and inflammatory activities of macrophages (11, 12, 13). Thus, receptor tyrosine kinases, together with their specific ligands, constitute an important step in maintaining and regulating inflammatory reaction (1).

The RON receptor tyrosine kinase is a member of the MET protooncogene family (14), a distinct subfamily of receptor tyrosine kinases. The cDNA encoding human RON was originally cloned from a transformed foreskin keratinocyte cell line (14). The murine homologue of RON was cloned from hemopoietic stem cells and named stem cell-derived tyrosine kinase (STK) (15). For simplicity, we will refer to RON/STK as RON in this report. The ligand for RON was identified as MSP (16, 17, 18), also known as HGF-like protein (19). MSP is a serum protein originally identified by its stimulatory activities in mouse peritoneal resident macrophages (20). RON is expressed in certain types of tissue macrophages including peritoneal macrophages and osteoclasts (8, 21). In macrophages, RON activation results in cell shape change, migration, or phagocytosis (20). However, RON activation also leads to inhibition of inducible NO production by macrophages stimulated with LPS and inflammatory cytokines such as IFN- and TNF- (22, 23). Recent studies using mice with the disrupted RON gene (knockout) have demonstrated that inactivation of the RON gene increases the susceptibility of mice to LPS-induced inflammation and septic shock (13). These effects are due in part to unbalanced production of NO by RON^-/- macrophages (13). These data suggest that RON plays an important role in regulating inflammatory activities of macrophages during inflammation and septic shock (13, 22, 23).

The present studies were designed to study how RON is regulated in macrophages during inflammation. At present, information related to the regulation of RON expression in primary macrophages by inflammatory mediators is not available. Therefore, we sought to study RON expression in mouse peritoneal resident macrophages using various stimuli including LPS, cytokines, and NO donors. Our results demonstrate that the expression of RON in macrophages is regulated by LPS and TNF- plus IFN-. LPS- or inflammatory cytokine-induced NO is one of the major mediators that affects transcription of the RON gene in macrophages.

	Materials and Methods

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Mice and cells

C3H/HeN mice were purchased from Taconic (Germantown, NY). Mice were used at an age of 4–6 mo. Induction of acute inflammation in mice was conducted by i.p. injection of 10 µg LPS in 0.2 ml of PBS. Mice injected with PBS were used as controls. Peritoneal macrophages were collected by lavage of peritoneal cavity with 10 ml of RPMI 1640 containing 1% of FBS. Cells were lysed immediately or cultured in FBS-free RPMI 1640 at 37°C in a humidified incubator containing 5% CO₂ in air.

Reagents

Purified human mature plasma MSP was kindly provided by Dr. E. J. Leonard (National Cancer Institute, Frederick, MD). A DNA fragment containing the human RON gene promoter (24) was provided by Dr. R. Breathnach (Institut National de la Santé et de la Recherche Médicale, Paris, France). The fragment was subcloned into the luciferase report vector pGL3-basic (Promega, Madison, WI) and designated as pGL3-RONP. Mouse mAb to mouse RON was provided by Dr. T. Suda (Kumamoto University, Kumamoto, Japan). Rabbit anti-mouse RON IgG was prepared as previously described (17). Mouse mAb to phosphotyrosine (clone 4G10) was obtained from Upstate Biotechnology (Lake Placid, NY). Mouse recombinant IFN-, TNF-, IL-1ß, IL-6, and TGF-ß were obtained from Boehringer Mannheim (Indianapolis, IN). LPS from Escherichia coli serotype 055:B5 was obtained from Life Technologies (Gathersburg, MD). Chemicals including an inducible NO syntase (iNOS) inhibitor N^G-monomethyl-L-arginine (L-NMMA), a negative control N^G-monomethyl-D-arginine (D-NMMA), and NO donors (±)-S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) were obtained from Calbiochem (San Diego, CA). All other reagents were obtained from Sigma (St. Louis, MO).

Immunoprecipitation and Western blotting

The procedures were performed as described previously (16). Briefly, macrophages were lysed in 0.4 ml of lysis buffer (0.1 M Tris, pH 7.6, containing 0.15 M NaCl₂, 2 mM EDTA, 0.5% Nonidet P-40, 0.5% Triton X-100, 100 µM sodium vanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 20 µg/ml soybean trypsin inhibitor). Cellular proteins were immunoprecipitated with 1 µg mAb to RON bound to protein G Sepharose beads and separated in 8% acrylamide gel under reduced conditions. Western blotting was performed as described (16). Rabbit IgG to RON or mAb 4G10 was used as detecting Abs. Goat anti-mouse or rabbit IgG conjugated with peroxidase was used as secondary Ab. The reaction was developed with enhanced chemiluminescent reagents (Amersham, Arlington Heights, IL) and exposed to film. In some instances, the membrane was treated with SDS 2-ME erasure buffer (16) and reprobed with other Abs.

RNA isolation and Northern blot analysis

Briefly, total RNAs were extracted from cells using RNAzol B (Biotecx Laboratories, Houston, TX) and size-fractionated in a 1% agarose gel containing 2.2 M formaldehyde. RNA was transferred to a nylon membrane (Boehringer Mannheim) and UV cross-linked. The hybridization procedures were conducted as described previously (22). The mouse RON cDNA fragment labeled with [³²P]dCTP (Amersham) was used as a specific probe. The ß-actin cDNA fragment was used as control. Membranes were exposed to film at -80°C with intensifying screens.

Assay for NO^-₂ production

Macrophages at 2 x 10⁶ cells/ml were incubated in RPMI 1640 in 200 µl/well in a 96-well tissue culture plate. Cells were stimulated with LPS or cytokines. Culture fluids were collected 36 h after incubation. Synthesis of NO was determined by measuring NO^-₂, a stable reaction production of NO with molecular oxygen, using Griess reagents as described previously (22). The OD of each sample were measured by an ELISA reader at the wavelength of 570 nm. NO₂ concentrations were calculated by comparison with a standard curve prepared with NaNO₂.

Cell transfection and luciferase assay

Raw264.7 cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM with 10% FBS. For transfection, cells were plated in six-well plates (1 x 10⁶ cells/well) for 24 h and then transfected with 2 µg pGL3-RONP vector or pGL-3-Basic vector using transfection reagent Lipofectamine (Life Technologies). All cells were also transfected with pRL-SV40 control vector (Promega) for normalizing transfection efficiencies. The transfected cells were cultured for 16 h and then treated with 1 µg/ml LPS, 200 µM GSNO, or 50 µm SNAP for 24 h. Cells were harvested in lysis buffer and assayed for luciferase activity according to the manufacturer’s protocol. The amount of luciferase activity in each sample was quantified by a Turner Designs Luminometer TD-20/20 (Promega).

Macrophage chemotaxis assay

The assay was performed using a multiwell chemotactic chamber as described previously (20). Briefly, bottom wells were filled with different amounts of MSP and covered with a polycarbonate membrane. Top wells were filled with 50 µl macrophage suspension (2 x 10⁶ cells/ml) treated with or without LPS or NO donors. After a 3.5-h incubation, the membrane was air-dried and stained with Diff-Quik. The migrated cells were counted under the microscope in three randomly selected areas. The results were expressed as the percentage of input cells that migrated.

	Results

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Reduction of RON expression in peritoneal macrophages collected from mice injected with LPS

The loss of RON expression in acute exudate peritoneal macrophages has been reported in Con A-injected mice (8), although the reasons are unknown. To determine whether a similar effect occurs during bacterial product-induced inflammation, mice were injected i.p. with 10 µg LPS and peritoneal macrophages were collected to determine RON expression by Western blotting. Results are shown in Fig. 1. As a control, peritoneal macrophages from PBS-injected mice express RON. No significant changes in RON levels were observed. In contrast, macrophages from LPS-injected mice did not express RON, indicating that administration of LPS in vivo results in the loss of RON. The kinetics of LPS regulation of RON expression are shown in Fig. 2. The inhibitory effect could be seen as early as 8 h after LPS treatment. Also, the effect of LPS on RON lasted at least for 3 days, suggesting that LPS-induced RON inhibition occurs at a very short time and lasts for a long period.

View larger version (52K): [in this window] [in a new window]   FIGURE 1. Inhibition by LPS in vivo of RON expression in peritoneal macrophages. Peritoneal macrophages were collected 24 h after LPS (10 µg/mouse) administration. Cells from five mice were pooled in each group. Equal amounts of cellular proteins were used for immunoprecipitation with rabbit anti-RON IgG. After SDS-PAGE, RON was detected by the same rabbit anti-RON IgG in Western blotting.

View larger version (58K): [in this window] [in a new window]   FIGURE 2. Time course of LPS-induced RON inhibition in peritoneal macrophages. Peritoneal macrophages were collected from LPS-injected mice at different time intervals. Cells were pooled from five mice in each group. Immunoprecipitation and Western blotting were performed as described in Fig. 1.

To determine whether RON expression in macrophages could be regulated in vitro, peritoneal resident macrophages were cultured in serum-free RPMI 1640 and stimulated with LPS. RON expression was determined by Western blotting. Results are shown in Fig. 3. Consistent with the data from in vivo experiments, LPS stimulation results in inhibition of RON expression by cultured macrophages. As expected, macrophages stimulated with LPS produced high levels of NO (nitrite: 35 ± 7.4 µM). Unstimulated cells did not produce NO (nitrite: 0.87 ± 0.2 µM).

View larger version (58K): [in this window] [in a new window]   FIGURE 3. LPS-induced inhibition of RON expression in cultured peritoneal macrophages. Peritoneal resident macrophages (5 x 106 cell/dish) were stimulated with LPS (1 µg/ml) for 24 h in serum-free RPMI 1640. Unstimulated cells serve as control (CNL). Immunoprecipitation and Western blotting were performed as described in Fig. 1.

To test whether LPS inhibits RON mRNA expression, Northern blot analysis was performed using total RNAs isolated from peritoneal macrophages. These cells were collected 24 h after mice were injected with LPS. Results are shown in Fig. 4. In vivo injection of LPS significantly decreased the levels of RON mRNA expression by peritoneal macrophages (Fig. 4A, lanes 1 and 2), indicating that LPS inhibits RON mRNA expression. This effect was tested also in vitro using cultured macrophages. After LPS stimulation, the total RNAs were isolated. The results are shown in Fig. 4A, lanes 3 and 4. Again, LPS inhibited the RON mRNA expression, indicating that the effect of LPS acts on the mRNA levels. Results in Fig. 4B show the levels of ß-actin mRNA hybridized with radiolabeled ß-actin cDNA fragments in the same membrane, indicating the similar amounts of loaded RNA samples.

View larger version (51K): [in this window] [in a new window]   FIGURE 4. LPS-induced inhibition of RON mRNA expression by peritoneal macrophages. A, Peritoneal macrophages were harvested from mice 24 h after LPS injection. Collected cells were pooled and immediately processed for RNA isolation (in vivo experiments). For in vitro experiments, macrophages from PBS injected mice were cultured for 24 h in the presence of 1 µg/ml LPS. Unstimulated cells were used as control (CNL). Total RNAs were used for Northern blot analysis. B, The same membrane was treated with erasure buffer and reprobed with radiolabeled ß-actin cDNA fragments. Results indicate that the relative equal amounts of RNAs were loaded.

Because LPS induces cytokine production from macrophages, we wanted to determine whether other inflammatory mediators such as IFN- and TNF- were capable of regulating the RON expression. Peritoneal resident macrophages were stimulated with IFN-, TNF-, or their combinations, and the RON expression was determined by Western blotting. Results are shown in Fig. 5. IFN- or TNF- alone have no effect on RON expression. However, the combination of these two cytokines results in complete inhibition of RON expression. We also tested IL-1, IL-6 or their combination in inhibiting RON expression. No inhibition was observed (data not shown).

View larger version (55K): [in this window] [in a new window]   FIGURE 5. Inhibitory effect of TNF- and IFN- on RON expression by cultured peritoneal macrophages. Peritoneal resident macrophages (4 x 106 cells/dish) were incubated in serum-free RPMI 1640 and stimulated for 24 h with 1 µg/ml LPS, 10 ng/ml TNF-, 500 U/ml of IFN-, or the combination of two cytokines. Cells in medium were used as control (CNL). RON was immunoprecipitated with mAb to mouse RON. Expression levels of RON were determined in Western blotting using rabbit anti-RON IgG as described in Fig. 1.

Because LPS or TNF- plus IFN- induce NO production in macrophages (22) and NO is capable of regulating growth factor gene expression (25), we sought to determine whether NO plays a role in regulating RON expression. Two NO donors, GSNO and SNAP, were included in macrophage cultures to determine their effects on RON expression. Results are shown in Fig. 6A. Both NO donors strongly inhibited the RON expression by peritoneal resident macrophages. The inhibitory effects are comparable to those of LPS. The results also show that IFN- enhanced the inhibitory effect of LPS. Likewise, cytokines, such as TGF-ß and MSP, known to inhibit iNOS expression, also prevented LPS-induced inhibitory effects and sustained the RON expression. However, TGF-ß or MSP alone had no effect on the RON expression (data not shown). To demonstrate that NO is involved in LPS- or cytokine-induced RON inhibition, we tested the effect of the specific iNOS inhibitor L-NMMA on LPS-induced RON inhibition. The results are shown in Fig. 6B. L-NMMA prevented the inhibitory effect of LPS. In contrast, D-LNMMA, which is a negative control for L-NMMA, had no effect. The levels of nitrite accumulated in culture supernatants were also measured. The results are shown in Fig. 7. The addition of L-NMMA, TGF-ß, or MSP significantly inhibited LPS-induced NO production by macrophages. These results suggest that NO induced by LPS or cytokine is one of the principle molecules involved in regulating RON expression.

View larger version (70K): [in this window] [in a new window]   FIGURE 6. Effect of NO donors or iNOS inhibitors on macrophage RON expression. A, Peritoneal resident macrophages (4 x 106 cells/dish) were stimulated for 24 h with 200 µM GSNO or 50 µM SNAP. Cells stimulated with 1 µg/ml of LPS or in medium alone (CNL) were used as controls. In addition, macrophages were stimulated with LPS in combination with 500 U/ml IFN-, 10 ng/ml TGF-ß, or 5 nM MSP. B, Macrophages were stimulated for 24 h with 1 µg/ml of LPS in the presence of 2.5 µM L-NMMA or D-NMMA. Immunoprecipitation was performed using mouse mAb to RON. Rabbit anti-RON IgG was used as detecting Abs in Western blotting. The band intensity in A was analyzed by densitometric scanning. The value from the control band was set as 100.

View larger version (14K): [in this window] [in a new window]   FIGURE 7. Effect of L-NMMA, TGF-ß, and MSP on LPS-induced NO production by peritoneal resident macrophages. The conditions of cell stimulation were the same as those described in Fig. 6. Nitrite in culture fluids was determined by Griess reaction. Data are shown as mean ± SD of triplicate wells. Similar results were obtained from one additional experiment.

To study the inhibitory effects of NO in more detail, we used the mouse macrophage-like cell line Raw264.7 and tested the effects of LPS and NO donor on the RON gene promoter activities. Results are shown in Fig. 8. In Raw264.7 cells transiently transfected with a luciferase reporter vector containing the major promoter region of the RON gene, luciferase activities were increased 8.7-fold in comparison with cells transfected with the pGL3 basic vector. The increased promoter activities were reduced up to 85% when cells were treated with LPS. Similarly, the luciferase activities were inhibited by NO donors GSNO or SNAP. The luciferase activities were reduced by 74% in GSNO-treated cells and 69% in SNAP-treated cells, respectively.

View larger version (14K): [in this window] [in a new window]   FIGURE 8. Inhibition of the RON gene promoter activities by LPS and NO donors. Raw264.7 cells in six-well plates (1 x 106 cells/well) were transfected with 2 µg pGL3-RONP vector and stimulated with LPS, GSNO, or SNAP as detailed in Materials and Methods. The luciferase activities were quantified by a luminometer. Experiments were repeated three times. Results shown here are from one representative experiment.

To determine whether GSNO or SNAP not only inhibits RON expression but also affects MSP-induced RON activation and cellular functions, peritoneal resident macrophages were pretreated with GSNO, SNAP, or LPS for 1 h and then stimulated with MSP. RON phosphorylation was determined by Western blotting. Results are shown in Fig. 9A. Pretreatment of cells with LPS for 1 h did not affect the MSP-induced RON phosphorylation. However, pretreatment of cells with NO donors GSNO or SNAP significantly reduced the RON phosphorylation. The data presented in Fig. 9B show that the decreased levels of tyrosine phosphorylation of RON in GSNO- or SNAP-treated cells are not due to reduced RON protein expression. The levels of RON in both cases remained the same. MSP-induced macrophage migration was also tested in the presence of NO donors. Results are shown in Fig. 10. Control macrophages migrated toward MSP in a MSP concentration-dependent manner. The maximal migration was seen when 1 or 4 nM of MSP was used. In contrast, GSNO- or SNAP-treated macrophages did not respond to MSP. Only a marginal effect of MSP was observed. Similarly, LPS-treated macrophages also did not migrate well. These data indicate that NO not only inhibits macrophage RON expression but also blocks the RON activation leading to impaired macrophage migration.

View larger version (62K): [in this window] [in a new window]   FIGURE 9. Effect of GSNO or SNAP on MSP-induced tyrosine phosphorylation of RON. A, Peritoneal resident macrophages (4 x 106 cells/dish) were cultured in serum-free RPMI 1640 and pretreated with 1 µg/ml LPS, 200 µM GSNO, or 50 µM SNAP at 37°C for 1 h. After treatment, cells were washed twice and stimulated at 37°C with 5 nM MSP for 10 min. Cells cultured in medium only (first left lane) were used as control. RON was immunoprecipitated with mAb to RON. Phosphotyrosine of RON was detected using mAb 4G10 in Western blotting. B, The same membrane was treated with erasure buffer and reprobed with rabbit anti-RON IgG to determine the amounts of RON loaded on the membrane. One of two experiments with similar results is shown.

View larger version (16K): [in this window] [in a new window]   FIGURE 10. Effect of LPS, GSNO, or SNAP on MSP-induced macrophage migration. Peritoneal resident macrophages were pretreated at 37°C with 1 µg/ml LPS, 200 µM GSNO, or 50 µM SNAP for 1 h in serum-free RPMI 1640. Cells were then used in migration assay as described in Materials and Methods. , MSP alone; •, MSP with LPS; , MSP with GSNO; and , MSP with SNAP. The data are from one of three experiments with similar results. SD was <10%.

	Discussion

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The effect of inflammatory stimuli in vivo on RON expression was first observed in peritoneal macrophages collected from Con A-injected mice (8). In these mice, the expression levels of RON in acute exudate macrophages were dramatically reduced 1 day after Con A administration (8, 23). However, RON expression reappeared on macrophages collected at the later stages of inflammation (8, 23). These studies provide some clues indicating that the RON expression is regulated during the inflammation and occurs in the acute phase (early stages) of inflammation. It also suggests that Con-A or inflammatory mediators produced during Con A-induced peritonitis may act on macrophages and subsequently block RON expression. The data provided in this report support this conclusion. We show that the administration of the nonlethal dose of LPS in vivo inhibits RON expression by peritoneal macrophages at both protein and mRNA levels. These effects can be reproduced in vitro using cultured macrophages with LPS and the combination of inflammatory cytokines such as TNF- plus IFN-.

The findings that cytokines such as IFN- and TNF-, when used in combination, inhibit RON expression by macrophages are of particular interest to us. We show that the inhibitory effects of TNF- plus IFN- are comparable to those of LPS. Their inhibitory effects can be antagonized by TGF-ß. In contrast, other cytokines such as IL-1 and IL-6 have no effects on RON expression. These data suggest that although these cytokines all have inflammatory activities, their roles in regulating RON expression are different. TNF- and IFN- are two unique inflammatory mediators produced by macrophages during bacteria-induced inflammation (30, 31, 32). Their effects on macrophages usually result in increased inflammatory reactions (30, 31, 32). They activate macrophages by regulating cellular activities such as cytotoxicity and inflammatory mediator production (30, 31, 32). We reason that the inhibition of RON expression by the combination of TNF- and IFN- may account for their abilities to increase macrophage inflammatory activities. In searching the literature about the effects of cytokines on RON expression, we found a report published by Chen et al. (33). They show that a human monocytic cell line THP-1 could be induced to express RON mRNA during cell differentiation into macrophages. IFN- and TNF- facilitate the RON mRNA expression (33). Why the same cytokine exerts the opposite effect on RON expression is unclear and needs further investigation. However, it is possible that the inhibitory or enhancing effects of these two cytokines may depend on the maturation status of monocytes/macrophages. During inflammation, LPS-induced IFN- and TNF- inhibit RON expression by mature macrophages. In contrast, these two cytokines may promote RON expression during the process of monocyte differentiation. Because THP-1 is a tumor cell line and significantly different from primary macrophages, other mechanisms may also apply.

It is established that LPS, TNF-, or IFN- act on macrophages through different receptors and signaling pathways (30, 31, 34). LPS activates macrophage by interacting with CD14 and transduces signals through Toll-like proteins (34, 35). TNF- or IFN- bind to their individual receptors and stimulate JAK/STAT activities or TNF receptor-associated proteins (36, 37). The question we asked is how the combination of TNF- and IFN- exerts similar effects on RON expression as LPS. A reasonable hypothesis is that the inhibition is achieved by a common mechanism. It has been shown that like LPS, TNF- plus IFN- has the ability to induce iNOS expression in macrophages (22). Moreover, NO is a well-known immunoregulator (38). These results prompted us to seek whether NO is involved in LPS- or cytokine-induced RON inhibition. Our data suggest that this is the case. First, the inhibition of RON expression is accompanied by increased NO production. The levels of accumulated NO in culture supernatants from LPS-stimulated macrophages are comparable to those from cytokine-stimulated cells (22). Second, blocking NO production prevents the LPS-induced RON inhibition. In this case, both TGF-ß and MSP, known to inhibit NO production (22, 39), sustained the RON expression. Third, NO donors, GSNO and SNAP, directly inhibit the expression of the RON protein by macrophages. They also inhibit the promoter activities of the RON gene. Considering these facts, we concluded that NO is a major factor involved in LPS or inflammatory cytokine-induced RON inhibition. We speculate that NO may impair the functions of the transcription factors essential for RON gene transcription. In this regard, it has been shown that NO inhibits the binding of transcription factors to the promoter region of CSF-1 gene (40). It is also possible that NO may increase the activities of transcription repressors that block RON gene expression. Several transcription factor-binding sites such as SP1 and AP-2 have been identified in the promoter sequence of the RON gene. Therefore, it will be of great interest in the future to determine which transcription factor(s) is affected by NO in controlling RON gene expression.

	Acknowledgments

 
We thank Drs. E. J. Leonard (National Cancer Institute, Frederick, MD) for providing pure MSP; R. Breathnach (Institut National de la Santé et de la Recherche Médicale, Paris, France) for the DNA fragment of the human RON gene promoter; and T. Suda (University of Kumamoto, Kumamoto, Japan) for the mAb to mouse RON. We also thank Dr. D. Brown for critically reading the manuscript. The assistance of A. L. Kurtz in manuscript preparation is also appreciated.

	Footnotes

 
¹ This work is supported by National Institutes of Health Grant RO1 AI43516 (to M.H.W.).

² This paper is dedicated to Prof. Dr. Hans-Dieter Flad, Chairman of the Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany, on the occasion of his retirement.

³ Address correspondence and reprint request to Dr. Ming-Hai Wang, Department of Medicine, UCHSC/Denver Health Medical Center, Mail Box 4000, 777 Bannock Street, Denver, CO 80204. E-mail address:

⁴ Abbreviations used in this paper: HGF, hepatocyte growth factor; D-NMMA, N^G-monomethyl-D-arginine; EGF, epidermal growth factor; GSNO, S-nitrosoglutathione; L-NMMA, N^G-monomethyl-L-arginine; MSP, macrophage stimulating protein; SNAP, (±)-S-nitroso-N-acetylpenicillamine; STK, stem cell-derived tyrosine kinase; iNOS, inducible NO synthase.

Received for publication September 16, 1999. Accepted for publication January 20, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Beck, P. L, D. K. Podolsky. 1999. Growth factors in inflammatory bowel disease. Inflamm. Bowel. Dis. 5:44.[Medline]
2. Werner, S.. 1998. Keratinocyte growth factor: a unique player in epithelial repair processes. Cytokine Growth Factor Rev. 9:153.[Medline]
3. Kim, W. J., G. K. Gittes, M. T. Longaker. 1988. Signal transduction in wound pharmacology. Arch. Pharm. Res. 21:487.
4. Hanks, S. K., A. M. Quinn, T. Hunter. 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42.[Abstract/Free Full Text]
5. Yue, X., P. Favot, T. L. Dunn, A. I. Cassady, D. A. Hume. 1993. Expression of mRNA encoding the macrophage colony-stimulating factor receptor (c-fms) is controlled by a constitutive promoter and tissue-specific transcription elongation. Mol. Cell. Biol. 13:3191.[Abstract/Free Full Text]
6. Camussi, G, G. Montrucchio, E. Lupia, R. Soldi, P. M. Comoglio, F. Bussolino. 1997. Angiogenesis induced in vivo by hepatocyte growth factor is mediated by platelet-activating factor synthesis from macrophages. J. Immunol. 158:1302.[Abstract]
7. Nolte, C., F. Kirchhoff, H. Kettenmann. 1997. Epidermal growth factor is a motility factor for microglial cells in vitro: evidence for EGF receptor expression. Eur. J. Neurosci. 9:1690.[Medline]
8. Iwama, A., M. H. Wang, N. Yamaguchi, N. Ohno, K. Okano, T. Sudo, M. Takeya, F. Gervais, C. Morissette, E. J. Leonard, T. Suda. 1995. Terminal differentiation of murine resident peritoneal macrophages is characterized by expression of the STK protein tyrosine kinase, a receptor for macrophage-stimulating protein. Blood 86:3394.[Abstract/Free Full Text]
9. Ullrich, A., J. Schlessinger. 1990. Signal transduction by receptor with tyrosine kinase activity. Cell 61:203.[Medline]
10. Tushinski, R. J., I. T. Oliver, L. J. Guilbert, P. W. Tynan, J. R. Warner, E. R. Stanley. 1982. Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cells selectively destroy. Cell 28:71.[Medline]
11. Stanley, E. R., K. L. Berg, D. B. Einstein, P. S. Lee, Y. G. Yeung. 1994. The biology and action of colony stimulating factor-1. Stem Cells 12:(Suppl. 1):15.
12. Muraoka, R. S., W. Y. Sun, M. C. Colbert, S. E. Waltz, D. P. Witte, J. L. Degen, S. J. Friezner Degen. 1999. The Ron/STK receptor tyrosine kinase is essential for peri-implantation development in the mouse. J. Clin. Invest. 103:1277.[Medline]
13. Correll, P. H., A. Iwama, S. Tondat, G. Mayrhofer, T. Suda, A. Bernstein. 1997. Deregulated inflammatory response in mice lacking the STK/RON receptor tyrosine kinase. Genes Funct. 1:69.[Medline]
14. Ronsin, C, F. Muscatelli, M. G. Mattei, R. Breathnach. 1993. A novel putative receptor protein tyrosine kinase of the met family. Oncogene 8:1195.[Medline]
15. Iwama, A, K. Okano, T. Sudo, Y. Matsuda, T. Suda. 1994. Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells. Blood 83:3160.[Abstract/Free Full Text]
16. Wang, M. H., C. Ronsin, M. C. Gesnel, L. Coupey, A. Skeel, E. J. Leonard, R. Breathnach. 1994. Identification of the ron gene product as the receptor for the human macrophage stimulating protein. Science 266:117.[Abstract/Free Full Text]
17. Wang, M. H., A. Iwama, A. Skeel, T. Suda, E. J. Leonard. 1995. The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein. Proc. Natl. Acad. Sci. USA 92:3933.[Abstract/Free Full Text]
18. Gaudino, G, A. Follenzi, L. Naldini, C. Collesi, M. Santoro, K. A. Gallo, P. J. Godowski, P. M. Comoglio. 1994. RON is a heterodimeric tyrosine kinase receptor activated by the HGF homologue MSP. EMBO J. 13:3524.[Medline]
19. Han, S., L. A. Stuart, S. J. Degen. 1991. Characterization of the DNF15S2 locus on human chromosome 3: identification of a gene coding for four kringle domains with homology to hepatocyte growth factor. Biochemistry 30:9768.[Medline]
20. Skeel, A, T. Yoshimura, S. D. Showalter, S. Tanaka, E. Appella, E. J. Leonard. 1991. Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity. J. Exp. Med. 173:1227.[Abstract/Free Full Text]
21. Kurihara, N, A. Iwama, J. Tatsumi, K. Ikeda, T. Suda. 1996. Macrophage-stimulating protein activates STK receptor tyrosine kinase on osteoclasts and facilitates bone resorption by osteoclast-like cells. Blood 87:3704.[Abstract/Free Full Text]
22. Wang, M. H., G. W. Cox, T. Yoshimura, L. A. Sheffler, A. Skeel, E. J. Leonard. 1994. Macrophage-stimulating protein inhibits induction of nitric oxide production by endotoxin- or cytokine-stimulated mouse macrophages. J. Biol. Chem. 269:14027.[Abstract/Free Full Text]
23. Chen, Y.-Q., J. H. Fisher, M. H. Wang. 1998. Activation of the RON receptor tyrosine kinase inhibits inducible nitric oxide synthase (iNOS) expression by murine peritoneal exudate macrophages: phosphatidylinositol-3 kinase is required for RON-mediated inhibition of iNOS expression. J. Immunol. 161:4950.[Abstract/Free Full Text]
24. Del Gatto, F, E. Gilbert, C. Ronsin, R. Breathnach. 1995. Structure of the promoter for the human macrophage stimulating protein receptor gene. Biochim. Biophys. Acta 1263:93.[Medline]
25. Tuder, R. M., B. E. Flook, N. F. Voelkel. 1995. Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia: modulation of gene expression by nitric oxide. J. Clin. Invest. 95:1798.
26. Lee, P. S., Y. Wang, M. G. Dominguez, Y. G. Yeung, M. A. Murphy, D. D. Bowtell, E. R. Stanley. 1999. The cbl protooncoprotein stimulates CSF-1 receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation. EMBO J. 18:3616.[Medline]
27. Elkabes, S., L. Peng, I. B. Black. 1998. Lipopolysaccharide differentially regulates microglial trK receptor and neurotrophin expression. J. Neurosci. Res. 54:117.[Medline]
28. Baccarini, M., P. Dello Sbarba, D. Buscher, A. Bartocci, E. R. Stanley. 1992. IFN-/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor. J. Immunol. 149:2656.[Abstract]
29. Messmer, U. K., U. K. Reed, B. Brune. 1996. Bcl-2 protects macrophages from nitric oxide-induced apoptosis. J. Biol. Chem. 271:20192.[Abstract/Free Full Text]
30. Rosenblum, M. G., N. J. Donato. 1989. Tumor necrosis factor : a multifaceted peptide hormone. Crit. Rev. Immunol. 9:21.[Medline]
31. Billiau, A, H. Heremans, K. Vermeire, P. Matthys. 1998. Immunomodulatory properties of interferon-: an update. Ann. NY Acad. Sci. 856:22.[Medline]
32. Gessani, S., F. Belardelli. 1998. IFN- expression in macrophages and its possible biological significance. Cytokine Growth Factor Rev. 9:117.[Medline]
33. Chen, Q., M. C. DeFrances, R. Zarnegar. 1996. Induction of met protooncogene (hepatocyte growth factor receptor) expression during human monocyte-macrophages differentiation. Cell Growth Differ. 7:821.[Abstract]
34. Ingalls, R. R, H. Heine, E. Lien, A. Yoshimura, D. Golenbock. 1999. Lipopolysaccharide recognition, CD14, and lipopolysaccharide receptors. Infect. Dis. Clin. North. Am. 13:341.[Medline]
35. Kopp, E. B., R. Medzhitov. 1999. The Toll-receptor family and control of innate immunity. Curr. Opin. Immunol. 11:13.[Medline]
36. Lehtonen, A, S. Matikainen, I. Julkunen. 1997. Interferons up-regulate STAT1, STAT2, and IRF family transcription factor gene expression in human peripheral blood mononuclear cells and macrophages. J. Immunol. 159:794.[Abstract]
37. Baker, S. J., E. P. Reddy. 1998. Modulation of life and death by the TNF receptor superfamily. Oncogene 17:3261.[Medline]
38. Nussler, A. K., T. R. Biliar. 1993. Inflammation, imunoregulation, and inducible nitric oxide synthase. J. Leukocyte Biol. 54:171.[Abstract]
39. Vodovotz, Y, C. Bogdan, J. Paik, Q. W. Xie, C. Nathan. 1993. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta. J. Exp. Med. 178:605.[Abstract/Free Full Text]
40. Peng, H. B., T. B. Rajavashisth, P. Libby, J. K. Liao. 1995. Nitric oxide inhibits macrophage-colony stimulating factor gene transcription in vascular endothelial cells. J. Biol. Chem. 270:17050.[Abstract/Free Full Text]

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