Identification of CXCR4 as a New Nitric Oxide-Regulated Gene in Human CD34 Cell - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 01:00 |只看该作者 |倒序浏览 |打印

作者：Yanyan Zhang, Monika Wittner, Hakim Bouamar, Peggy Jarrier, William Vainchenker, Fawzia Louache作者单位：INSERM U, Institut Gustave Roussy, PR, Villejuif, France
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      【摘要】  u: H$ L$ h- g" p9 Z! r
   As an intracellular second messenger, nitric oxide (NO) is increasingly implicated in the control of transcriptional machinery and gene expression. Here, we show that cell surface expression of CXCR4 on CD34  cells was increased in a dose- and time-dependent manner in response to NO donors. Augmented surface expression was correlated with an increase in CXCR4 mRNA level. A specific NO scavenger prevented the elevation in CXCR4 mRNA caused by NO donors, suggesting a direct signaling action mediated by NO on CXCR4 transcription. NO treatment had no significant effect on CXCR4 mRNA stability. However, induction of CXCR4 mRNA by NO was still observed in conditions in which initiation of translation was inhibited, suggesting that the NO effect must be mediated by a pre-existing protein. CXCR4 mRNA induction did not involve cGMP (guanosine 3', 5'-cyclic monophosphate) generation but was most likely mediated via oxidation of intracellular protein thiols. Finally, CD34  cells pretreated with NO donors exhibited an increased chemotactic response. This study demonstrates that the NO pathway can modulate CXCR4 expression in human CD34  cells and suggests that NO may play a critical role in the trafficking of hematopoietic progenitors.
      【关键词】 CD  cells Nitric oxide Stromal cell-derived factor- Gene expression Chemokine receptor CXCR Cell migration
   INTRODUCTION& z  n( h$ ^' |7 ]  O! }% e

The chemokine receptor CXCR4 is a member of a large family of seven-transmembrane domain receptors coupled to heterotrimeric Gi proteins. CXCR4 is the primary physiologic receptor for the CXC chemokine ligand 12/stromal cell-derived factor-1 (SDF-1)/pre-B cell growth stimulating factor and functions as an entry coreceptor for strains of HIV-1 .
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In the hematopoietic system, SDF-1 and CXCR4 play critical roles in the trafficking, transendothelial migration, proliferation, and differentiation of hematopoietic cells. The importance of CXCR4 and SDF-1 as regulators of hematopoietic stem cell (HSC) biology has been established with observations that mutant mice with targeted gene disruption for SDF-1 or CXCR4 displayed deficient colonization of their bone marrow (BM) by hematopoietic progenitor cells (HPCs) ." z' {+ v1 h( G  H# t
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In many types of cells, SDF-1-dependent functions are modulated via the levels of CXCR4 expressed at the cell surface. Previous studies have provided evidence that post-transcriptional regulations control CXCR4 membrane expression. Indeed, an important pool of CXCR4 can be detected inside the endosome compartment in many types of cells, including primary lymphocyte and CD34  cells, as well as in several transformed cell lines .
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Nitric oxide (NO) is a short-lived free radical that serves as a signaling molecule in a wide spectrum of pathophysiological and physiological processes, including inflammation, apoptosis, regulation of enzyme activity, and gene expression. NO is produced by NO synthase (NOS) using L-arginine and molecular oxygen as substrates, yielding NO and L-citrulline as products .
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NO donors exhibit a variety of effects upon HPCs. They inhibit burst-forming unit-erythroid (BFU-E) formation while displaying inhibitory or stimulatory effects on colony-forming unit-granulocyte and macrophage (CFU-GM) growth . Although NO plays an important role in the regulation of hematopoiesis, its role in the trafficking of HPCs has not been clearly defined.

In this study, we present a novel observation that NO donors induced CXCR4 expression in human CD34  cells. Increased CXCR4 expression in response to NO donors occurred at the transcriptional level and was mediated by a pre-existing protein through a cGMP-independent pathway. Using in vitro chemotactic assays, we show that CD34  cells pretreated with NO donors exhibited increased chemotactic response to SDF-1. This study further emphasizes the importance of NO in the regulation of hematopoietic progenitor homeostasis.
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MATERIALS AND METHODS2 w7 z& `' e. l8 e) H4 _( H6 Y
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R-phycoerythrin (PE)-conjugated HPCA2 (anti-CD34) monoclonal antibodies (mAbs), allophycocyanin (APC) or PE-conjugated 12G5 (anti-CXCR4) mAbs were obtained from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen). PE-conjugated immunoglobulin G1 (IgG1) and IgG2a mAb controls were obtained from BD Biosciences (Le Pont de Claix, France, http://www.bdbiosciences.com). APC-conjugated IgG2a mAbs were obtained from Immunotech (Luminy, France, http://www.immunotech.com).
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Reagents and Human Cytokines# F% \4 b9 s/ f: p( C5 _

DETA NONOate (NOC18) and Spermine NONOate (SPER/NO) were obtained from Alexis Biochemical (Lausen, Switzerland, http://www.alexis-corp.com). 3-Morpholinosydnonimine (SIN-1) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, Na (carboxy-PTIO) were obtained from Calbiochem (San Diego, http://www.emdbiosciences.com). Puromycin, actinomycin D (ActD), sodium nitroprusside (SNP), 8-bromo-cGMP (8-Br-cGMP), guanosine 3', 5'-cyclic monophosphorothioate, Rp-Isomer (Rp-cGMPS), dithiothreitol (DTT), propidium iodide, and AMD3100 were obtained from Sigma-Aldrich (St. Quentin Fallavier, France, http://www.sigmaaldrich.com). Recombinant human SDF-1 was obtained from R&D Systems, Inc. (Minneapolis, http://www.rndsystems.com).
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Cord Blood, BM, and Mobilized Peripheral Blood CD34  Cells

Cord blood (CB) samples from normal full-term newborn infants were obtained from a CB bank (Dr. Van Nifderick, Hôpital St. Vincent de Paul, Paris). Aliquots of cytapheresis products from patients with nonhematologic disease after mobilization by chemotherapy and granulocyte colony-stimulating factor (G-CSF) and BM of healthy patients undergoing hip surgery were obtained after informed consent. CD34  cells were separated using a magnetic cell sorting system (miniMACS; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) in accordance with manufacturer's recommendations. The purity of recovered cells was determined by flow cytometry, after staining with the PE-HPCA2 anti-CD34 mAb, to be more than 95%.! k1 x9 s3 T+ \+ u, X

Cell Culture and Treatments
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Freshly isolated CD34  cells were cultured in serum-free medium containing Iscove's modified Dulbecco's medium (Invitrogen, Cergy-Pontoise, France, http://www.invitrogen.com), penicillin, streptomycin, glutamine, 11.5 µmol/l -thioglycerol (Sigma-Aldrich), 1.5% bovine serum albumin (BSA) (Cohn's fraction V; Sigma-Aldrich), sonicated lipids, and iron-saturated human transferrin, as previously described .

CD34  cells (1 x 106 per milliliter) were exposed to NO donors at concentrations and times indicated in the Results section. In the studies using puromycin and ActD, cells were treated with 20 µg/ml puromycin or 5 µg/ml ActD for 20 minutes before the addition of 100 µM NOC18.

Cell Viability Assays

CD34  cells (1 x 106 per milliliter) were exposed to NOC18 at concentrations and times indicated. Treated and untreated cells were then stained with propidium iodide (1 µg/ml) at 4¡ãC for 10 minutes followed with flow cytometry analysis.
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Treated and untreated cells (1,000 cells per milliliter) were plated in methylcellulose medium (0.8% methylcellulose in Iscove's medium, 30% fetal calf serum, 1% BSA, and 10¨C4 mol/l 2-ß mercaptoethanol) supplemented with 10 ng/ml recombinant human IL-6, 100 U/ml recombinant human IL-3, 50 ng/ml recombinant human steel factor, 10 ng/ml recombinant human G-CSF (rhuG-CSF), and 2 U/ml recombinant human erythropoietin (rhuEpo). (rhuG-CSF and rhuEpo were a kind gift from Amgen, Thousand Oaks, CA, http://www.amgen.com.) Cultures were incubated at 37¡ãC in air supplemented with 5% CO2 and saturated with humidity. Colonies were scored on day 14 of culture.
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NO Assay8 V9 g  K; p; v: W: }
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NO production was measured by estimating the stable NO metabolite, nitrite, in conditioned medium using a spectrophotometric method based on the Griess reaction . Mobilized peripheral blood (MPB) CD34  cells (1 x 106/ml) were exposed to NOC18 at concentrations and times indicated. Culture supernatants (100 µl) were mixed with 100 µl of Griess reagent (1% sulfanilamide in 5% phosphoric acid, 0.1% naphthyl ethylenediamine dihydrochloride) and incubated for 10 minutes at room temperature. Nitrite concentrations were determined by measuring the absorbance at 550 nm in an enzyme-linked immunosorbent assay reader.

Flow Cytometry Analysis

CD34  cells were stained with an appropriate dilution of the antibody or the isotype-matched control. After washing, cells were suspended in phosphate-buffered saline, kept at 4¡ãC, and analyzed on a FACsort (Becton Dickinson, Erembodegen, Belgium, http://www.bdeurope.com) with the Cell Quest software package (Becton Dickinson).6 g. i7 w" ~# G# d3 q8 ]2 P  L0 b1 G" {. k: g
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Chemotactic Assay

Migration assays were performed with 5-µm-pore filters chambers (Transwell, 24-well cell clusters; Corning Life Sciences, Acton, MA, http://www.corning.com/lifesciences). Briefly, freshly isolated MPB CD34  cells were cultured for 4 hours in serum-free medium with or without the presence of NOC18 (100 µM). Cells were washed and suspended at a concentration of 2.5 x 106 cells per milliliter in serum-free medium. The cell suspension (100 µl) was placed in the upper chamber, whereas 600 µl of medium with or without SDF-1 at different concentrations was introduced in the lower chamber. The chambers were incubated for 4 hours at 37¡ãC in 5% CO2 and 95% air. Cells in the lower chamber were carefully recovered for counting on a FACsort. In the studies using AMD3100, cells were treated with NOC18 for 4 hours, washed, and incubated with 5 µM AMD3100 for 30 minutes before chemotactic assays. All assays were done in triplicate. Data are presented as the percentage of migration calculated by the following ratio: number of migrated cells in response to SDF-1 or medium alone per number of cells in the input.* L( \+ i3 v" P( \9 V+ P- ~

Real-Time Quantitative Polymerase Chain Reaction
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RNA isolation was performed using the SV total RNA isolation system (Promega Corporation, Madison, WI, http://www.promega.com). Reverse transcription (RT) was performed with Superscript II RNase H reverse transcriptase (Invitrogen). Primers for CXCR4 mRNA were as follows: CXCR4 mRNA sense: 5'-CGTGCCCTCCTGCTGACTATT-3', antisense: 5'-GCCAACCATGATGTGCTGAA-3', and probe: 5'-TTCATCTTTGCCAACG TCAGTGAGGCA-3'. Polymerase chain reaction (PCR) reactions were carried out in the ABI Prism GeneAmp 5700 Sequence Detection System (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com) using TaqMan Universal PCR Master Mix (Applied Biosystems, Courtaboeuf, France, http://www.appliedbiosystems.com). For calculation of fold augmentation, RNA amounts were normalized to ß-actin mRNA.

Statistics
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Results of experimental points obtained from multiple experiments were reported as the mean ¡À standard deviation. Statistical analysis was performed using the one-tailed Student t test for unpaired data.4 j' E& K0 X0 e5 L) g
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RESULTS; B: {3 j2 v+ D' ?8 q; Z

NO Donors Induce CXCR4 Expression on CD34  Cells5 i/ i) Q- m* I

To analyze the effects of NO on CXCR4 expression in HPCs, MPB CD34  cells were exposed to NOC18, an NO donor, at increasing concentrations for 4 hours. At concentrations up to 1 mM, NOC18 was not cytotoxic as assessed by propidium iodide (supplemental online Fig. 1A) and colony formation in methylcellulose culture (supplemental online Fig. 1B). After NOC18 treatment, real-time quantitative RT-PCR was performed on total mRNA, and CXCR4 mRNA level was normalized to ß-actin mRNA. In medium alone, CXCR4 mRNA was detected at basal levels (Fig. 1A). Treatment with NOC18 increased CXCR4 mRNA levels in a dose-dependent manner, with a maximal induction of 10-fold observed at 100 µM. In parallel experiments, cells were stained with an anti-CXCR4 antibody, and surface expression of the receptor was analyzed by flow cytometry. As shown in a representative histogram (Fig. 1B), the expression of CXCR4 on the cell surface was increased after NOC18 treatment in a dose-dependent manner. The mean fluorescence intensity acquired from three independent experiments for each NOC18 concentration revealed a highly significant peak at 100 µM (Fig. 1C), showing that the maximum increase in CXCR4 mRNA was correlated with the augmentation in CXCR4 membrane expression. Higher concentrations of NOC18 (500 µM and 1 mM) also induced a significant increase of CXCR4 mRNA levels. However, this increment occurred at a lesser extent than 100 µM. This may be due to an accumulation of by-products or cell toxicity induced at high NOC18 concentrations.
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Figure 1. NOC18 induces CXCR4 mRNA and membrane expression on mobilized peripheral blood (MPB) CD34  cells in a dose- and time-dependent manner. (A¨CD): MPB CD34  cells were exposed to increasing concentrations of NOC18 for 4 hours. (E, F): MPB CD34  cells were exposed to 100 µM NOC18 for the times indicated. (A, E): Total RNA was extracted and CXCR4 mRNA expression level was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. CXCR4 mRNA amounts were normalized to ß-actin mRNA. (B, F): Cells were labeled with PE-CXCR4 and analyzed by flow cytometry. Representative histograms show the membrane expression of CXCR4. (C, G): MFI values of CXCR4 expression shown in (B) and (F) were quantified. The results show the mean ¡À SD for three independent experiments (*, p 6 G4 O5 H2 ~4 a, m4 C* w* L
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NO release by NOC18 could be measured as nitrite, a stable oxidized product of NO that accumulates in the cell culture supernatant after NO donor addition . As expected, the accumulation of nitrite increased with the increasing concentrations of NOC18 (Fig. 1D), and therefore NO generation upon NOC18 addition was dose-dependent in our experimental conditions. Interestingly, the increased expression of CXCR4 mRNA was directly correlated with the accumulation of nitrite after NOC18 addition (Fig. 1D).5 o6 Z/ `8 @0 r1 r: m5 Z# T
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Next, NOC18 was used at a concentration of 100 µM to determine the time course of CXCR4 induction. The expression level of CXCR4 mRNA was rapidly increased up to twofold within 30 minutes after NOC18 addition, reached a maximum of approximately 10-fold within 2 hours, and thereafter slightly decreased (Fig. 1E). When the CXCR4 membrane expression was studied, a slight but significant increase was detected after 2 hours followed by a marked increase at 4 hours (Fig. 1F, 1G). Importantly, the accumulation of nitrite upon NOC18 addition was evident as early as 30 minutes (Fig. 1H), again indicating that the induction of CXCR4 mRNA was directly correlated with NO release in the cell culture supernatant.

To determine whether the effects of NOC18 on CXCR4 expression could be observed on CD34  cells from others sources, we exposed CD34  cells isolated either from CB or adult BM to 100 µM NOC18 for 4 hours and examined the expression of CXCR4 on the membrane. CXCR4 was increased in an order of magnitude similar to that observed with MPB CD34  cells (supplemental online Fig. 2A¨C2F). Only MPB CD34  cells were used to perform the experiments described hereafter.! d/ ^8 r* ^2 p0 s

To study whether the effects of NOC18 on CXCR4 expression could be reproduced with other NO donors, MPB CD34  cells were treated with SNP, SPER/NO, and SIN-1. Like NOC18, SPER/NO is a stabilized NO-amine complex. In neutral aqueous solution, the complex decomposes and immediately releases NO . Exposure of MPB CD34  cells to these different compounds resulted in a highly enhanced expression of membrane CXCR4, with a highest induction observed at 10 µM SNP, 50 µM SPER/NO, and 100 µM SIN-1 (Fig. 2A¨C2F). Thus, different structural families of NO donors induce CXCR4 expression in MPB CD34  cells.
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Figure 2. Effects of nitric oxide donors on the membrane expression of CXCR4. Mobilized peripheral blood CD34  cells were exposed to different concentrations of SNP (A, B) or SPER/NO (C, D) or SIN-1 (E, F) for 4 hours. Cells were then labeled with PE-CXCR4 and analyzed by flow cytometry. (A, C, E): Representative histograms showing the membrane expression of CXCR4. (B, D, F): MFI of CXCR4 expression. Results are the mean ¡À SD for three independent experiments (**, p

Requirement of Pure NO to Induce CXCR4 mRNA and Membrane Expression
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It has been shown that NO donors can generate by-products other than NO. To test the involvement of NO itself in the induction of CXCR4 expression, experiments were performed in the presence of carboxy-PTIO, an NO scavenger. MPB CD34  cells were treated with NOC18 (100 µM, 4 hours) in the absence or presence of carboxy-PTIO (150 µM), then analyzed for CXCR4 mRNA and membrane expression. Under our experimental conditions, carboxy-PTIO either alone or in combination with NOC18 did not affect the amount of total RNA recovered or the amount of ß-actin present (data not shown). As shown in Figure 3, expression of CXCR4 mRNA (Fig. 3A) and membrane protein (Fig. 3B, 3C) was not altered by carboxy-PTIO, whereas carboxy-PTIO completely abolished the effects of NOC18. These data point toward a direct role of NO itself in the modulation of CXCR4 expression.

Figure 3. Effect of the nitric oxide scavenger carboxy-PTIO on CXCR4 mRNA and membrane expression induced by NOC18. Mobilized peripheral blood CD34  cells were incubated at 37¡ãC without or with 150 µM carboxy-PTIO, 100 µM NOC18 alone, or both agents in combination for 4 hours. (A): Total RNA was extracted and CXCR4 mRNA expression level was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. CXCR4 mRNA amounts were normalized to ß-actin mRNA. (B): Cells were labeled with PE-CXCR4 and analyzed by flow cytometry. Representative histograms show the membrane expression of CXCR4. (C): MFI values of CXCR4 expression were quantified. The results show the mean ¡À SD for three independent experiments (**, p 2 T1 K; ?4 n, H1 |- C
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NOC18-Induced Expression of CXCR4 mRNA is Regulated at the Transcription Level

The increase in CXCR4 mRNA level upon NOC18 treatment could be due to an increased transcription, an enhanced stabilization, or both. To examine whether a de novo mRNA synthesis participated in the process of NOC18-induced CXCR4 mRNA expression, MPB CD34  cells were incubated with NOC18 (100 µM) in the presence or absence of the transcriptional inhibitor ActD or with ActD alone (5 µg/ml) for 1, 2, or 4 hours. As shown in Figure 4A, the increase in CXCR4 mRNA induced by NOC18 was completely abolished in the presence of ActD.8 g0 y0 ^8 I- ]% P7 f

Figure 4. Upregulation of CXCR4 expression by NOC18 is dependent on de novo mRNA synthesis. (A): Mobilized peripheral blood (MPB) CD34  cells were incubated at 37¡ãC without or with 100 µM NOC18 alone, 5 µg/ml ActD, or both agents in combination for different times. Total RNA was extracted and CXCR4 mRNA expression level was evaluated by real-time quantitative reverse transcription-polymerase chain reaction (PCR). CXCR4 mRNA amounts were normalized to ß-actin mRNA. (B): MPB CD34  cells were pretreated with or without 100 µM NOC18 for 2 hours. The medium was removed, and fresh medium containing 5 µg/ml ActD was added to the cells. The incubation was then continued for different times. Total RNA was extracted and CXCR4 mRNA expression was analyzed by quantitative real-time PCR. CXCR4 mRNA amounts were normalized to ß-actin mRNA. (C): Estimated half-life of CXCR4 mRNA. A representative result is shown with the arrow lines showing the estimated half-life values for CXCR4 mRNA. The experiment was repeated three times and gave comparable results. Abbreviations: ActD, actinomycin D; hr(s), hour(s).! n1 f" ]5 k4 V8 O1 `
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Next, the NOC18 effect on CXCR4 mRNA turnover was explored. To this end, MPB CD34  cells were first incubated with NOC18 for 2 hours, then ActD was added to the culture medium, and incubation was continued for an additional 2 hours. In control CD34  cells (untreated) exposed for 2 hours to ActD, the half-life of CXCR4 mRNA was approximately 1 hour. When cells were first treated to NOC18 during 2 hours and then exposed to ActD, the level of CXCR4 mRNA was increased, but the CXCR4 mRNA half-life was not significantly modified (t1/2 = 0.9 hour) (Fig. 4B, 4C). These results suggest that the upregulation of CXCR4 by NOC18 was likely mediated via a de novo mRNA synthesis rather than an mRNA stabilization.& @5 F) ?' i; e7 G  v# s. ~

NOC18-Induced CXCR4 mRNA Expression is Independent of De Novo Protein Synthesis4 ]# v' U" b' C
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To determine whether a de novo protein synthesis was required for NO-mediated induction of CXCR4 mRNA transcription, MPB CD34  cells were treated with NOC18 in the presence of puromycin, an inhibitor of translation initiation. MPB CD34  cells were incubated for 4 hours with NOC18 (100 µM) with or without the presence of puromycin at 20 µg/ml, a concentration that inhibits protein synthesis. The presence of puromycin did not alter the ability of NOC18 to induce CXCR4 mRNA expression (Fig. 5A). In contrast, as expected, the increase in surface CXCR4 protein expression was completely inhibited (Fig. 5B, 5C).

Figure 5. Upregulation of CXCR4 expression by NOC18 is mediated by a pre-existing protein. Mobilized peripheral blood CD34  cells were incubated at 37¡ãC without or with 20 µg/ml puromycin, 100 µM NOC18 alone, or both agents in combination for 4 hours. (A): Total RNA was extracted and CXCR4 mRNA expression level was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. CXCR4 mRNA amounts were normalized to ß-actin mRNA. (B): Cells were labeled with phycoerythrin (PE)-CXCR4 and analyzed by flow cytometry. Representative histograms show the membrane expression of CXCR4. (C): The mean fluorescence intensity (MFI) of CXCR4 expression was quantified. The results show the mean ¡À SD for three independent experiments. (**, p # P" N) l8 I( ]2 z, s

Taken together, these observations demonstrate that upregulation of CXCR4 mRNA transcription upon NOC18 treatment is not dependent on de novo protein synthesis, implying that a pre-existing protein is involved in this process." d# B9 D6 W  F' T6 ?

Inhibition of NO-Induced CXCR4 Expression4 Q& S; E- \( q, P! E! s

Many effects of NO are mediated through a direct activation of sGC which leads to an increase in the intracellular levels of cGMP . To determine whether the NOC18-induced increase in CXCR4 expression was secondary to an activation of sGC and hence cGMP, we first tested the effect of the membrane-permeable cGMP analog 8-Br-cGMP on CXCR4 expression. After 4 hours of exposure of MPB CD34  cells to 100 µM 8-Br-cGMP alone, CXCR4 surface expression remained unaltered (Fig. 6A, 6B). Next, we checked a possible involvement of cGMP by using Rp-cGMPS, a specific inhibitor of cGMP-dependent protein kinase. MPB CD34  cells were preincubated with 100 µM Rp-cGMPS for 30 minutes followed by the addition of 100 µM NOC18 for 4 hours. In this experimental setting, preincubation with Rp-cGMPS did not alter NO-induced CXCR4 expression (Fig. 6C, 6D), indicating that induction of CXCR4 expression by NO is likely independent of the formation of intracellular cGMP. Finally, NO can alter a wide variety of cellular functions via thiol modification by either nitrosylation or oxidation. To determine whether induction of CXCR4 expression by NO involves such a mechanism, MPB CD34  cells were treated with 200 µM of the thiol agent DTT 30 minutes before the addition of NOC18. As shown in Figure 6E and 6F, DTT alone had no effect on CXCR4 expression. However, in the presence of DTT, the effect of NOC18 on CXCR4 expression was significantly reduced, suggesting that NO may exert its effect through a redox-based mechanism.5 B& E/ T. U  l: [5 R

Figure 6. Upregulation of cell surface CXCR4 expression by nitric oxide is independent of activation of cGMP signal pathway, but dependent on redox regulation. (A): Mobilized peripheral blood (MPB) CD34  cells were incubated at 37¡ãC without or with 100 µM 8-Br-cGMP or 100 µM NOC18, for 4 hours. Cells were then labeled with phycoerythrin (PE)-CXCR4 and analyzed by flow cytometry. Representative histograms show the membrane expression of CXCR4. (C): MPB CD34  cells were incubated at 37¡ãC without or with 100 µM NOC18 or 100 µM NOC18 in combination with 100 µM cGMP inhibitor Rp-cGMPS for 4 hours. Representative histograms show the membrane expression of CXCR4. (E): MPB CD34  cells were incubated at 37¡ãC without or with 100 µM NOC18 for 4 hours. For the DTT treatment teams, cells were exposed to 200 µM DTT 30 minutes before the addition of 100 µM NOC18. Representative histograms show the membrane expression of CXCR4. (B, D, F): The mean fluorescence intensity (MFI) values of CXCR4 expressions shown in (A, C, E) were quantified. The results are shown as the mean ¡À SD for three independent experiments (**p

NO Donors Increase the Migration Property of CD34  Cells to SDF-1 In Vitro3 |2 Z6 l% C4 o0 T  M
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Because NO increases the membrane expression of CXCR4 on CD34  cells, we evaluated the functional effect of NOC18 on chemotaxis of CD34  cells to SDF-1. Freshly isolated MPB CD34  cells were cultured in the absence or presence of NOC18 (100 µM) for 4 hours, and then chemotaxis assays were performed in response to SDF-1. As shown in Figure 7A, NOC18 did not modify the motility of MPB CD34  in the absence of SDF-1. In contrast, the observed NO donor-dependent increase of CXCR4 surface expression was paralleled by an increase in the number of CD34  cells that migrate in response to SDF-1 for each dose tested (from 10 ng/ml to 1,000 ng/ml) as compared with corresponding untreated cells (Fig. 7A). To determine whether the SDF-1/CXCR4 interactions are involved in the increased SDF-1-mediated chemotaxis under NO influence, MPB CD34  cells were pretreated with AMD3100, a small molecule antagonist of CXCR4 , before chemotaxis assays. As shown in Figure 7A, pretreatment with AMD3100 inhibited the chemotactic response of MPB CD34  cells elicited by SDF-1 regardless of whether cells were exposed to NOC18.
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Figure 7. Chemotaxis assay to SDF-1. (A): Mobilized peripheral blood (MPB) CD34  cells were first cultured in the absence or presence of 100 µM NOC18 for 4 hours. Then, half of the cells were treated with 5 µM AMD3100 for half an hour. Cells were then subjected to in vitro transwell migration assays to different doses of SDF-1 (from 10 to 1,000 ng/ml) or medium alone. (B): MPB CD34  cells were cultured in the absence or presence of 100 µM NOC18, 10 µM SNP, 50 µM SPER/NO, or 100 µM SIN-1 for 4 hours. Then, cells were subjected to in vitro transwell migration assays to 300 ng/ml SDF-1 or medium alone. (C): MPB CD34  cells were incubated at 37¡ãC without or with 100 µM NOC18, 5 µg/ml ActD alone, or both agents in combination for 4 hours. Then, cells were subjected to in vitro transwell migration assays to 300 ng/ml SDF-1 or medium alone. All results represent the mean ¡À SD for one representative experiment done in triplicate. Similar results were obtained from four independent experiments. Abbreviations: ActD, actinomycin D; AMD, AMD3100; SDF-1, stromal cell-derived factor-1; SIN-1, 3-morpholinosydnonimine; SNP, sodium nitroprusside; SPER/NO, Spermine NONOate.
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Other types of NO donors also induced higher chemotaxis of CD34  cells to 300 ng/ml SDF-1 (Fig. 7B). This induction was completely inhibited by ActD (Fig. 7C), suggesting that the effect of NO on SDF-1-mediated migration requires new synthesis of CXCR4. Together, these data indicate that NO may modulate SDF-1-induced chemotactic activity through upregulation of CXCR4 expression.  }/ Z  G, N1 ]% W

DISCUSSION5 X3 s  Z7 @6 j

NO is recognized as an important regulator of gene expression because a broad number of genes are regulated by NO both in vitro and in vivo. The results reported herein add CXCR4 to the growing list of NO-regulated genes, as they demonstrate a robust induction of CXCR4 mRNA paralleled by surface protein expression in human CD34  cells after exposure to NO-generating agents. Previous studies have shown that NO was involved in the regulation of proliferation and differentiation of hematopoietic progenitors . This report shows that NO modulates SDF-1 chemotaxis of human CD34  cells and, thus, further strengthens the link between NO signaling and stem cell homeostasis., V8 t3 W# |% V3 A" t5 e, W7 \) r
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The effect of NO on CXCR4 gene expression was studied by using NOC18, an NO donor that releases pure NO spontaneously and shows burst release . In our study, carboxy-PTIO completely blocked the action of NOC18, suggesting that a requirement of pure NO must be necessary to induce CXCR4 mRNA and membrane expression. Although we cannot definitively rule out some minor effects of the potential by-products of NOC18, our data suggest a direct role of NO in the modulation of CXCR4 expression.
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The upregulation of CXCR4 mRNA expression appears to be essentially related to an increased level of transcription. This assumption is supported by the data showing that CXCR4 mRNA compared with ß-actin mRNA was markedly increased by NOC18 and that this increase was totally inhibited by ActD. In addition, the CXCR4 mRNA half-life was comparable in control untreated and NOC18-treated CD34  cells, indicating that the NOC18 action did not occur through an RNA stabilization mechanism. Although increased CXCR4 mRNA expression is a major mechanism by which NO alters CXCR4 membrane expression, it is unclear from the present results whether NO may also regulate CXCR4 protein stability or function. Further characterization of NO effects using pulse-chase experiments will be helpful in further understanding the regulation of CXCR4.

The results reported here document an important role for NO on CXCR4 mRNA transcription and suggest that the signaling pathways mediated by NO must activate or suppress some transcription or repressor factors involved in the regulation of CXCR4 expression. The transcription factors that regulate CXCR4 expression are not completely known. It has been shown that c-Myc enhances, whereas Yin-Yang 1 suppresses, its promoter activity . However, HIF proteins were not detected by Western blot analyses in NOC18-treated CD34  cells nor were HIFs translocated to the nucleus after NOC18 treatment (our unpublished data). To gain insight into the mechanisms involved in CXCR4 mRNA induction by NOC18, we tested whether de novo protein synthesis was required to induce CXCR4 mRNA upregulation. The increase in CXCR4 mRNA by NOC18 still occurred in puromycin-treated cells, indicating that NO-induced CXCR4 mRNA expression did not require de novo protein synthesis. These data suggest that a stable protein already present in CD34  cells may modulate CXCR4 transcription.

To determine whether NO treatment of CD34  cells might influence CXCR4 expression by modulating autocrine SDF-1 production by CD34 CD38  cells, the mRNA level of SDF-1 after NO treatment was analyzed by real-time quantitative RT-PCR. No significant difference was found between NO-treated cells and untreated cells, suggesting that the expression of SDF-1 may not represent a mechanism of induction of CXCR4 by NO (our unpublished data).

NO regulates gene expression by multiple pathways, including cGMP-dependent and -independent mechanisms (reviewed in . Whether one of these factors represents the molecular target of NO-mediated effect on CXCR4 remains to be determined.3 `/ E+ R  p' y1 @0 `: Q7 C* R

Independently of the mechanism(s) used by NO in the induction of CXCR4 expression, our results have implications for other areas of cell biology. Indeed, it was found that NO can increase the membrane expression of CXCR4 on CD34  cells and simultaneously increase their migration properties to SDF-1, which suggests that NO may regulate the trafficking of progenitor cells. There are several studies suggesting that NO regulates cell migration in other systems. For example, NO was shown to modulate leukocyte recruitment .2 B% a, _# w/ ^8 N  ~  o# |7 Y

This study shows for the first time that NO modulates the transcription activity of the CXCR4 gene in HPCs. Induction of CXCR4 mRNA expression by NO does not depend on de novo protein synthesis, suggesting that NO modification of a pre-existing protein is involved. Because there was evidence that NO could also impact proliferation and differentiation of HSCs (probably through induction of p21WAF1 ), it is clear that NO effects on the hematopoietic system are likely to be multifaceted. Further studies are needed to determine the numerous effects of NO on hematopoiesis and their precise mechanisms.+ s$ P2 Q* B; k% U

DISCLOSURES
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The authors indicate no potential conflicts of interest.; v+ S0 q5 j' O
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ACKNOWLEDGMENTS

This work was funded by the Institut National de la Sant¨¦ et de la Recherche M¨¦dicale, Institut Gustave Roussy (CRI-SPS-2003-02 to F.L.), and the Association pour la Recherche contre le Cancer (Grant 4309 to F.L.). Y.Z. and H.B. received a fellowship from the French Minist¨¨re de la Recherche, the Association pour la Recherche contre le Cancer, and la Soci¨¦t¨¦ Française d'h¨¦matologie. We thank Dr. Françoise Wendling for discussion and critical reading of the manuscript.( f+ \- T5 e* t- l9 u. u7 _
      【参考文献】! h& B& ?- I! ]- M! W$ b: x, A

& k" y: p( i% }- y- i* K; B
Bleul CC, Farzan M, Choe H et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 1996;382:829¨C832.
, A' {( K4 N0 J) @3 T3 Y
Oberlin E, Amara A, Bachelerie F et al. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1. Nature 1996;382:833¨C835.0 |1 ~5 G4 c9 P. o: {  i# G- r7 `1 K
  k. z3 J7 x& ^1 X9 F* U$ `: K
Zou YR, Kottmann AH, Kuroda M et al. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 1998;393:595¨C599.

Ma Q, Jones D, Borghesani PR et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci U S A 1998;95:9448¨C9453.8 z& |5 k9 }1 V9 ^, q

Ma Q, Jones D, Springer TA. The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity 1999;10:463¨C471.5 B8 x% ]6 x) K4 y9 d

Kawabata K, Ujikawa M, Egawa T et al. A cell-autonomous requirement for CXCR4 in long-term lymphoid and myeloid reconstitution. Proc Natl Acad Sci U S A 1999;96:5663¨C5667.
& A0 T9 h- f7 K" e- a
Petit I, Szyper-Kravitz M, Nagler A et al. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 2002;3:687¨C694.
5 C" d6 w4 g5 k
Levesque JP, Hendy J, Takamatsu Y et al. Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide. J Clin Invest 2003;111:187¨C196.. s' O1 U+ ^* E+ t

Christopherson KW 2nd, Hangoc G, Broxmeyer HE. Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34  progenitor cells. J Immunol 2002;169:7000¨C7008.

Forster R, Kremmer E, Schubel A et al. Intracellular and surface expression of the HIV-1 coreceptor CXCR-4/Fusin on various leukocyte subsets: Rapid internalization and recycling upon activation. J Immunol 1998;160:1522¨C1531.8 [, ]. t* p: T% Q5 B' Y

Zhang Y, Foudi A, Geay JF et al. Intracellular localization and constitutive endocytosis of CXCR4 in human CD34  hematopoietic progenitor cells. STEM CELLS 2004;22:1015¨C1029.
% y! X7 ]6 X& r
Tarasova NI, Stauber RH, Choi JK et al. Visualization of G protein-coupled receptor trafficking with the aid of the green fluorescent protein. Endocytosis and recycling of cholecystokinin receptor type A. J Biol Chem 1997;272:14817¨C14824.
3 A* R, E5 p6 W$ x0 r, x/ a2 L% ?
Cole SW, Jamieson BD, Zack JA. cAMP up-regulates cell surface expression of lymphocyte CXCR4: Implications for chemotaxis and HIV-1 infection. J Immunol 1999;162:1392¨C1400.
# i1 o3 ]. \% ]1 L9 A  s9 A+ |9 ~
Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845¨C848.

Jinquan T, Quan S, Jacobi HH et al. CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4  T lymphocytes are regulated by interleukin-4 and interleukin-10. Immunology 2000;99:402¨C410." t/ _( i$ E' C3 v

Bruhl H, Cohen CD, Linder S et al. Post-translational and cell type-specific regulation of CXCR4 expression by cytokines. Eur J Immunol 2003;33:3028¨C3037.' ~) W" }; ^# X( R
8 j  m+ [& [; a1 g
Juffermans NP, Paxton WA, Dekkers PE et al. Up-regulation of HIV coreceptors CXCR4 and CCR5 on CD4( ) T cells during human endotoxemia and after stimulation with (myco)bacterial antigens: The role of cytokines. Blood 2000;96:2649¨C2654.
+ i' V4 O/ b# h; w. \5 l1 |% y
Saccani A, Saccani S, Orlando S et al. Redox regulation of chemokine receptor expression. Proc Natl Acad Sci U S A 2000;97:2761¨C2766.( Q' ^' x$ Q6 k/ m- [% b  l0 n
' Y. c" n6 G0 F! ]3 K& k
Schioppa T, Uranchimeg B, Saccani A et al. Regulation of the chemokine receptor CXCR4 by hypoxia. J Exp Med 2003;198:1391¨C1402.

Staller P, Sulitkova J, Lisztwan J et al. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature 2003;425:307¨C311.) Y. E1 T/ \5 q0 H( t9 g( \2 u

Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J 1994;298:249¨C258.

Bogdan C. Nitric oxide and the regulation of gene expression. Trends Cell Biol 2001;11:66¨C75.

Denninger JW, Marletta MA. Guanylate cyclase and the NO/cGMP signaling pathway. Biochim Biophys Acta 1999;1411:334¨C350.

Reykdal S, Abboud C, Liesveld J. Effect of nitric oxide production and oxygen tension on progenitor preservation in ex vivo culture. Exp Hematol 1999;27:441¨C450.
  J( r2 ~: u5 j! P; K, h
Shami PJ, Weinberg JB. Differential effects of nitric oxide on erythroid and myeloid colony growth from CD34  human bone marrow cells. Blood 1996;87:977¨C982.

Michurina T, Krasnov P, Balazs A et al. Nitric oxide is a regulator of hematopoietic stem cell activity. Mol Ther 2004;10:241¨C248.# O) L: ~' L+ B8 Y
+ Q8 d6 V, Z. Q' f' t) W3 T5 o/ Z- |+ \+ k
Aicher A, Heeschen C, Mildner-Rihm C et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med 2003;9:1370¨C1376., u! [: |9 J) p/ Y3 O3 r1 k

Aicher A, Heeschen C, Dimmeler S. The role of NOS3 in stem cell mobilization. Trends Mol Med 2004;10:421¨C425.6 T" b  L6 o+ w/ K, ?% A1 O

Debili N, Wendling F, Katz A et al. The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors. Blood 1995;86:2516¨C2525.

Green LC, Wagner DA, Glogowski J et al. Analysis of nitrate, nitrite, and
; V1 Y' U' A- O/ X& c  Z
Kimura H, Ogura T, Kurashima Y et al. Effects of nitric oxide donors on vascular endothelial growth factor gene induction. Biochem Biophys Res Commun 2002;296:976¨C982., j$ O$ P! H2 q4 L

Fitzhugh AL, Keefer LK. Diazeniumdiolates: Pro- and antioxidant applications of the "NONOates". Free Radic Biol Med 2000;28:1463¨C1469.3 S+ `+ p* t9 h, d

Feelisch M, Ostrowski J, Noack E. On the mechanism of NO release from sydnonimines. J Cardiovasc Pharmacol 1989;14 (suppl 11):S13¨CS22.  Z4 \( I  ~$ l, ]7 m3 g! k2 `* s

Roos CM, Rich GF, Uncles DR et al. Sites of vasodilation by inhaled nitric oxide vs. sodium nitroprusside in endothelin-constricted isolated rat lungs. J Appl Physiol 1994;77:51¨C57.
1 E- `- g! I- H
Zhao Y, Brandish PE, Ballou DP et al. A molecular basis for nitric oxide sensing by soluble guanylate cyclase. Proc Natl Acad Sci U S A 1999;96:14753¨C14758.
& b" M% ]2 F! v3 Z! J; b7 w7 @
Bellamy TC, Wood J, Goodwin DA et al. Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses. Proc Natl Acad Sci U S A 2000;97:2928¨C2933.9 ?, B# ~: d( ]2 c

Hatse S, Princen K, Bridger G et al. Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4. FEBS Lett 2002;527:255¨C262.

Rubin JB, Kung AL, Klein RS et al. A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors. Proc Natl Acad Sci U S A 2003;100:13513¨C13518.( C( S9 m% Y6 b$ s$ g
  s0 s3 S$ ~% t
Keefer LK, Nims RW, Davies KM et al. "NONOates" (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: Convenient nitric oxide dosage forms. Methods Enzymol 1996;268:281¨C293.
/ B4 j0 H5 |  T& |( K
Yoshida K, Akaike T, Doi T et al. Pronounced enhancement of NO-dependent antimicrobial action by an NO-oxidizing agent, imidazolineoxyl N-oxide. Infect Immun 1993;61:3552¨C3555.
  W: n7 B) W- n( C
Bouton C, Demple B. Nitric oxide-inducible expression of heme oxygenase-1 in human cells. Translation-independent stabilization of the mRNA and evidence for direct action of nitric oxide. J Biol Chem 2000;275:32688¨C32693.! d* I9 L4 w" e3 b/ `7 r8 O' Q

Hasegawa A, Yasukawa M, Sakai I et al. Transcriptional down-regulation of CXC chemokine receptor 4 induced by impaired association of transcription regulator YY1 with c-Myc in human herpesvirus 6-infected cells. J Immunol 2001;166:1125¨C1131.$ u) W/ o' p" E2 q% C( G2 ?( `

Moriuchi M, Moriuchi H, Margolis DM et al. USF/c-Myc enhances, while Yin-Yang 1 suppresses, the promoter activity of CXCR4, a coreceptor for HIV-1 entry. J Immunol 1999;162:5986¨C5992.1 t/ }5 k3 u5 ^8 j0 V- D: |
9 k" ^" F( i! r1 h7 D
Palmer LA, Gaston B, Johns RA. Normoxic stabilization of hypoxia-inducible factor-1 expression and activity: Redox-dependent effect of nitrogen oxides. Mol Pharmacol 2000;58:1197¨C1203.6 w2 I$ i9 g' }. M7 K: t' U# U

Sandau KB, Faus HG, Brune B. Induction of hypoxia-inducible-factor 1 by nitric oxide is mediated via the PI 3K pathway. Biochem Biophys Res Commun 2000;278:263¨C267.( M* q7 K: R5 K- d! i
- L8 @5 H' }+ z; B/ m) `
Sandau KB, Fandrey J, Brune B. Accumulation of HIF-1alpha under the influence of nitric oxide. Blood 2001;97:1009¨C1015.

Pilz RB, Casteel DE. Regulation of gene expression by cyclic GMP. Circ Res 2003;93:1034¨C1046.

Gaston BM, Carver J, Doctor A et al. S-Nitrosylation signaling in cell biology. Mol Interv 2003;3:253¨C263., j% c! V( t) g6 A, n

Marshall HE, Merchant K, Stamler JS. Nitrosation and oxidation in the regulation of gene expression. Faseb J 2000;14:1889¨C1900., M8 \& o2 n, Y: S7 |5 k
) k) G, ?2 d* ~. h
Hickey MJ. Role of inducible nitric oxide synthase in the regulation of leucocyte recruitment. Clin Sci (Lond) 2001;100:1¨C12.: G8 E/ [+ m/ x
$ K* P) M, y6 p, i& c$ D
Kawasaki K, Smith RS Jr, Hsieh CM et al. Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis. Mol Cell Biol 2003;23:5726¨C5737.8 m% T  m) l5 b& v4 s+ _* m

Dubey RK, Jackson EK, Luscher TF. Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cell. Role of cyclic-nucleotides and angiotensin1 receptors. J Clin Invest 1995;96:141¨C149.

Cherla RP, Ganju RK. Stromal cell-derived factor 1 alpha-induced chemotaxis in T cells is mediated by nitric oxide signaling pathways. J Immunol 2001;166:3067¨C3074.% d/ B# M& [: s6 W, p
$ ?% _, {% h" h$ V! G9 Z% |. @
Gu Z, Kaul M, Yan B et al. S-Nitrosylation of matrix metalloproteinases: Signaling pathway to neuronal cell death. Science 2002;297:1186¨C1190.
9 X- X  Z' {9 z+ }8 [
Bauer PM, Buga GM, Ignarro LJ. Role of p42/p44 mitogen-activated-protein kinase and p21waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide. Proc Natl Acad Sci U S A 2001;98:12802¨C12807.

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