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Endocrinology Vol. 142, No. 1 81-88
Copyright © 2001 by The Endocrine Society

ARTICLES

Fibroblast Growth Factor-2 Stimulates Endothelial Nitric Oxide Synthase Expression and Inhibits Apoptosis by a Nitric Oxide-Dependent Pathway in Nb2 Lymphoma Cells1

Paul R. Murphy, Mireille Limoges, Faith Dodd2, Robert T. M. Boudreau3 and Catherine K. L. Too4

Departments of Physiology and Biophysics (P.R.M., M.L.), Biochemistry and Molecular Biology (F.D., R.T.M.B., C.K.L.T.), and Obstetrics and Gynecology (P.R.M., C.K.L.T.), Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Address all correspondence and requests for reprints to: Catherine L. K. Too, Ph.D., Department of Biochemistry and Molecular Biology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. E-mail: ctoo{at}is.dal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently reported that the rat Nb2 T lymphoma cells expressed messenger RNAs (mRNAs) encoding both fibroblast growth factor-2 (FGF-2) and the FGF receptor, suggesting possible paracrine and/or autocrine roles for FGF-2 in lymphoma cell function. We have also shown that the Nb2 cells expressed endothelial nitric oxide synthase (eNOS) and produced low levels of nitric oxide (NO) that inhibited apoptosis of PRL-deprived cells via a PRL-independent, bcl-2-mediated pathway. In this study the effects of PRL and FGF-2 on Nb2 cell survival and NO production were further investigated. The percentages of nonapoptotic cells in PRL-treated vs. PRL-deprived cultures after 6 days were 95% and 53%, respectively. Addition of FGF-2 to PRL-deprived Nb2 cells did not stimulate cell proliferation, but the onset of apoptosis was significantly inhibited, such that more than 85% of the cells remained nonapoptotic after 6 days. The steady state levels of bcl-2 and bag-1 mRNAs were low in PRL-deprived Nb2 cells, but were markedly increased by PRL or FGF-2. bcl-2 expression was induced within 1 h of PRL or FGF-2 addition and continued to increase to a level 20- to 25-fold above the control level within 24 h. bag-1 expression also increased within 1 h after the addition of PRL or FGF-2, was maximal within 8 h, and declined slowly thereafter. The levels of eNOS mRNAs were low but detectable in growth-arrested Nb2 cells, and PRL further down-regulated eNOS mRNA levels over the next 24 h. In contrast, FGF-2 significantly increased eNOS mRNA levels within 2 h to reach a peak 10-fold induction by 12 h. FGF-2 stimulation of eNOS mRNA was accompanied by a 2- to 3.5-fold increase in cellular levels of the eNOS protein and a 2.5-fold increase in serine-phosphorylated eNOS. However, the ratio of serine-phosphorylated eNOS vs. total cellular eNOS was unchanged, indicating that FGF-2 did not affect the serine phosphorylation status of eNOS. Nb2 cells produced low basal levels of NO, which increased with increasing L-arginine concentrations. PRL did not further increase NO release in the presence of L-arginine (0.1 or 1 mM), but FGF-2 significantly (P <= 0.05) increased NO release in the presence of 0.1 and 1 mM L-arginine. Furthermore, coincubation of aminoguanidine (NOS inhibitor) with FGF-2 completely abrogated the protective effect of FGF-2 on bcl-2 and bag-1 mRNA levels in PRL-deprived Nb2 cells. In summary, FGF-2 inhibited apoptosis of PRL-deprived Nb2 cells. This antiapoptotic action of FGF-2 appears to be mediated by stimulation of eNOS expression, increased levels of cellular NO, and stimulation of expression of the antiapoptotic genes bcl-2 and bag-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BASIC FIBROBLAST growth factor-2 (FGF-2) is the prototypic member of a family of at least 17 related genes encoding heparin-binding proteins with growth-, antiapoptotic-, and differentiation- promoting activities (1). FGF-2 stimulates a variety of physiological processes, including cell proliferation, differentiation, cell migration, and angiogenesis, and the unregulated expression of FGF-2 is associated with solid tumor growth and neovascularization. More recently, FGF-2 has been recognized as a hemopoietic cytokine. Receptors for FGF are expressed on the surface of a variety of leukemic cell lines and peripheral B and T cells (2). FGF-2 stimulates the proliferation and delays the senescence of bone marrow stromal cells in culture. T lymphocytes have been reported to express FGF-2 messenger RNA (mRNA) and produce heparin-binding FGF-like bioactivity (3, 4), raising the possibility of an autocrine or paracrine role for FGF in hemopoietic cell function.

It has been suggested that tissue-infiltrating T cells may contribute to the progression of disease such as atherosclerosis and cancer by local release of FGF-2 at the disease site (3). Alternatively, FGF-2 may stimulate the progression of lymphocytic tumors in an autocrine fashion. Increased levels of FGF-2 immunoreactivity have been detected in urine from patients with a wide variety of neoplastic diseases, including acute lymphoblastic leukemia (5) and chronic B cell lymphocytic leukemia (6). Furthermore, elevated intracellular levels of FGF-2-like immunoreactivity in lymphocytes of patients with chronic lymphocytic leukemia have been shown to correlate with the high risk stage of the disease (6). FGF-2 has antiapoptotic activity in vitro and has been reported to protect against fludarabine-induced apoptosis in human CLL lymphocytes and leukemic cell lines (6, 7). Inhibition of apoptosis plays an important role in the clonal expansion and progression of leukemic tumors, and several cytokines have been shown to protect B cell lymphocytic leukemia cells from undergoing apoptosis (8, 9, 10). However, the role(s) of FGF-2 in the pathogenesis of leukemias is not yet fully understood.

The rat Nb2-11 C lymphoma is a T cell-derived line that expresses high affinity PRL receptors (PRLr) and is dependent on PRL for survival and proliferation. Using differential display to identify PRL target genes whose mRNAs are expressed in Nb2–11 C cells in response to activation of the PRLr signaling cascade, we recently demonstrated rapid activation of expression of FGF-2 and of FGF-2-responsive mRNAs after PRL stimulation of quiescent Nb2–11 C cells (11). Furthermore, we also reported that low levels of nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) inhibited apoptosis and promoted the survival of Nb2–11 C cells deprived of PRL via a PRL-independent, bcl-2-mediated pathway (12). In the present study we compared the effects of PRL and FGF-2 on Nb2–11 C cell survival and proliferation. We demonstrate that FGF-2 stimulates bcl-2 and bag-1 expression and inhibits the onset of apoptosis in PRL-deprived, growth-arrested Nb2–11 C cells. We also demonstrate that the antiapoptotic effect of FGF-2 is exerted via the stimulation of eNOS expression, resulting in the release of low levels of NO in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rabbit anti-eNOS antibodies and monoclonal antiphosphoserine antibody (clone PSR-45) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Sigma, (St. Louis, MO), respectively. Terminal deoxynucleotidyl transferase was purchased from Promega Corp. (Madison, WI). Biotinylated deoxy-ATP (biotin-14-dATP), streptavidin-horseradish peroxidase, FBS, and lactogen-free horse serum (HS) were purchased from Life Technologies, Inc. (Burlington, Canada). Aminoguanidine hemisulfate was obtained from Research Biochemicals International (Natick, MA).

Cell culture
Suspension cultures of Nb2–11 C cells (henceforth referred to as Nb2) were maintained in Fischer’s medium for leukemic cells supplemented with ß-mercaptoethanol (10-4 M), 10% FBS as a source of lactogens, and 10% lactogen-free HS as previously described (13). The L-arginine content of the Fischer’s medium purchased from Sigma (15 mg/liter) was adjusted to a final concentration of 18.6 mg/liter to maintain optimal cell growth as previously described (12). Confluent Nb2 lymphoma cells (1.2 x 106 cells/ml) were growth arrested in the above medium but without 10% FBS (arrest medium) and used within 18–24 h. Nb2 cells used for in situ end labeling (ISEL) were arrested and maintained in Fischer’s medium from Life Technologies, Inc. (containing 18.6 mg/liter arginine) supplemented with ß-mercaptoethanol (10-4 M) and 10% HS. For some experiments, FGF-2 (0.3–0.6 ng/ml) or aminoguanidine (25 mM) was also added to the Fischer’s arrest medium.

RNA extraction and comparative RT-PCR
Total RNA was extracted from Nb2 cells using RNeasy mini kits (QIAGEN (Mississauga, Canada). DNA-free RNA samples were obtained by digesting extracts with deoxyribonuclease I (1 U/sample) for 30 min at 37 C followed by a phenol/chloroform extraction. Total RNA (2 µg) was used for RT and amplification by PCR as previously described (12), but with some modifications as indicated below. PCR reactions were performed with 2.5 U Taq DNA polymerase (Life Technologies, Inc.) using specific primers for eNOS, bcl-2, or bag-1 (see Table 1Go). The PCR buffer was optimized for each set of primers using the Opti-Prime PCR Optimization Kit (Stratagene, La Jolla, CA). Optimal amplification of eNOS and bcl-2 mRNAs was carried out in Opti-Prime 1X PCR buffer 5 containing 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, and 25 mM KCl, whereas bag-1 amplification was carried out in Opti-Prime 1 x PCR buffer 2 containing 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 75 mM KCl. PCR amplification was performed as follows: 94 C for 60 sec and 20–35 cycles of 94 C for 45 sec, 62 C for 45 sec, and 72 C for 60 s. For each RT-PCR product analyzed, appropriate conditions were established to ensure that the amplification reaction did not proceed past the exponential phase (14). The amplified products were analyzed on 1.6% agarose gels. Where applicable, primer pairs for PCR were chosen to span at least one intron-exon splice boundary to eliminate the possibility of amplification of contaminating genomic DNA. Levels of eNOS, bcl-2, and bag-1 product were standardized against the QuantumRNA 18S ribosomal RT-PCR product (Ambion, Inc., Austin, TX) amplified from the same RT reaction.


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  		Table 1. PCR primer sequences and their predicted RT-PCR product sizes

 
Coimmunoprecipitation and Western analysis
Coimmunoprecipitation of eNOS was performed as previously described (12). Briefly, Nb2 cells (~20 x 106 cells) were washed in PBS and resuspended in 1 ml cold RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 50 mM Na4P2O7·10 H2O] containing 1 mM Na3VO4, 1 mM NaF, 5 mM EDTA, 5 mM EGTA, 0.1% IGEPAL, 0.25% sodium deoxycholate, and fresh protease inhibitors (2 mM PMSF and 3 µg/ml each of antipain, leupeptin, and pepstatin). After 30 min at 4 C, cell lysates were passaged through 21-gauge needles, and another 10 µl 200 mM PMSF stock were added. After 30 min on ice, the samples were microcentrifuged at 15,000 x g for 20 min at 4 C. Anti-eNOS antibody was added to the 15,000 x g supernatant (total cell lysate) and incubated for 1 h at 4 C, and 20 µl Protein A/G Plus-Agarose (Santa Cruz Biotechnology, Inc.) were added for an overnight incubation with gentle mixing. Immunoprecipitates were collected by centrifugation at 3,000 rpm for 5 min at 4 C. The pellet was washed four times with 1.0 ml PBS containing 1 M NaCl and finally resuspended in 40 µl sample buffer. Equal volumes of each sample (~20 µl each) were loaded onto two separate 4–20% SDS-PAGE gels for Western analysis of eNOS or phosphoserine as previously described (11). Rabbit anti-eNOS (primary antibody) and donkey antirabbit Ig-horseradish peroxidase conjugate (secondary antibody) were used at 0.2 µg/ml and 1:5000, respectively. Monoclonal antiphosphoserine (primary) and goat antimouse Ig-horseradish peroxidase conjugate (secondary) were used at 1:500 and 1:1500, respectively. Immunoreactive signals were detected with SuperSignal ULTRA (Pierce Chemical Co., Rockford, IL).

NO assay
Total nitric oxide released into the culture medium by Nb2 lymphoma cells was determined using the Total Nitric Oxide Assay Kit (Assay Designs, Inc., Ann Arbor, MI) following the manufacturer’s instructions. Briefly, growth-arrested Nb2 cells were washed, resuspended in 10 mM HEPES buffer (pH 7.4), containing 5 mM glucose, 0.145 mM NaCl, 5 mM KCl, 1 mM CaCl2·2H2O and 0.5% (wt/vol) BSA. Cells (0.5 x 106 cells/0.5 ml) were aliquoted into 12 x 75-mm culture tubes in the presence of increasing concentrations of L-arginine (0–1.0 mM) and FGF-2 (0–0.6 ng/ml) and incubated for 210 min at 37 C in 95% air-5% CO2. The cells were pelleted at 2000 rpm (5 min), and the supernatants were collected. Samples were aliquoted into microtiter plates for the assay. Total nitrates were reduced to nitrites by nitrate reductase in the presence of NADH. Colorimetric detection of total nitrites in the samples and nitrite standards was performed with Griess reagents I and II and measured by absorption at 540–570 nm. The nitrite concentration was determined from a calibration curve of sodium nitrite standards.

ISEL assay
Apoptotic DNA fragmentation was quantified in situ as previously described (15), but with modifications for the Nb2 system (12). Briefly, Nb2 cells were cytospun (Shandon Cytospin 2, Fisher Scientific, Nepean, Canada) onto glass slides silinated in 2% 3-aminopropyltriethoxysilane in acetone and fixed in 4% paraformaldehyde. Cells were permeabilized with pepsin (0.5%; pH 2.0) for 30 min at 37 C and incubated in 2% H2O2 for 10 min at room temperature to inactivate endogenous peroxidases. Cells were equilibrated in terminal deoxynucleotidyl transferase (TdT) buffer [30 mM Tris (pH 7.2), 140 mM sodium cocadylate, and 1 mM cobalt chloride], and DNA fragments were then elongated with 12.5 pmol/µl biotin-14-dATP and 0.4 U/µl TdT enzyme in TdT buffer for 60 min at 37 C in a humidified chamber. The reaction was stopped by immersing slides in terminating buffer (300 mM NaCl and 30 mM sodium citrate, pH 8.0) for 15 min followed by incubation in 5% horse serum for 10 min at room temperature. The incorporated biotin label was detected by incubation with streptavidin-horseradish peroxidase for 45 min at 37 C. Finally, the cells were stained in 3-amino-9-carbazol (1 mg/ml) for 30–45 min at 37 C and counterstained with hematoxylin. Apoptotic cells (showing red nuclei) were identified microscopically and quantified by counting the number of labeled nuclei in a field of at least 300 cells.

Statistical procedures
ANOVA and Scheffé’s F test were performed using StatView 4.5, (Abacus Concepts, Inc., Berkeley, CA). In each case, the results shown represent at least three separate experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Divergent effects of PRL and FGF-2 on Nb2 lymphoma cell survival and proliferation
We recently reported that Nb2 cells express mRNAs encoding both FGF-2 and the FGF receptor, suggesting a possible autocrine role for this factor in lymphoma cell function (11). We therefore compared the effects of PRL and FGF-2 on Nb2 cell survival and proliferation. As shown in Fig. 1AGo, Nb2 cells cultured in Fischer’s medium containing 10% lactogen-free HS failed to proliferate in the absence of added PRL (control). Addition of PRL to the growth-arrested cultures resulted in a 3-fold increase in cell number over the 6-day assay period. In contrast, there was a 50% decrease in the number of viable cells in control cultures over the same time period. Addition of recombinant FGF-2 to growth-arrested cells did not stimulate Nb2 cell proliferation, but prevented the decline in viable cells observed in the untreated controls. ISEL demonstrated that the cell loss following PRL withdrawal was attributable to increased apoptosis, and that this process was inhibited by FGF-2. As shown in Fig. 1BGo, PRL-treated cultures consisted of 95% nonapoptotic cells, whereas the nonapoptotic fraction declined to 53% after 6 days of PRL deprivation. Addition of FGF-2 significantly inhibited the onset of apoptosis in these growth-arrested cultures, such that more than 85% of cells remained nonapoptotic after 6 days without PRL.



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  		Figure 1. Exogenous FGF-2 promotes cell survival and inhibits apoptotic cell death in Nb2 lymphoma cells. Confluent Nb2 cells (~1 x 106 cells/ml) were growth arrested in Fischer’s medium containing 10% HS. After 20 h of arrest (day 0), the cell density was reduced to approximately 0.2 x 106 cells/ml for the 6-day assay. PRL (10 ng/ml) or FGF-2 (0.6 ng/ml) was added, whereas control cells were left untreated. Cells were collected daily for measurement of cell number in a Coulter counter (A; n = 3) and ISEL assay (B) as described in Materials and Methods. B, The percentage of apoptotic cells was determined visually by counting the number of labeled nuclei in a minimum of 300 cells per field. In A and B, the SEs were consistently less than 10% of the means and are not shown. The data presented are from one of three independent experiments, each with similar results.

 
Regulation of bcl-2 and bag-1 expression by PRL and FGF-2
PRL has previously been reported to stimulate the expression of bcl-2 mRNA and Bag-1 protein, both of which have been implicated in the prevention of apoptosis (16, 17, 18). As shown in Fig. 2Go, the steady state levels of bcl-2 and bag-1 mRNAs were low in PRL-deprived Nb2 cultures, but were markedly increased by treatment with either PRL or FGF-2. bcl-2 expression was significantly induced within 1 h after the addition of PRL or FGF-2 and continued to increase to a level 20- to 25-fold above controls within 24 h. bag-1 expression also increased within 1 h after the addition of PRL or FGF-2, was maximal within 8 h, and declined slowly thereafter.



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  		Figure 2. FGF-2 and PRL both stimulate the expression of antiapoptotic genes. Confluent Nb2 cells were growth arrested for 20 h and then reduced to a cell density of 0.6 x 106 cells/ml. The cells were treated with PRL (10 ng/ml) or FGF-2 (0.6 ng/ml), whereas controls (0 h) were left untreated. The cells were harvested at the indicated times. Total RNA (2 µg/sample) extracted from these cells was used for RT-PCR to amplify bcl-2 and bag-1 mRNAs and 18S ribosomal RNA as described in Materials and Methods. PCR products were resolved in 1.6% agarose gels. The 18S ribosomal RNA was used to standardize relative expression. •, bcl-2; {square}, bag-1. The quantitative data in the lower panels are the mean ± SEM of three or four independent experiments.

 
FGF stimulates eNOS expression and NO synthesis in PRL-deprived Nb2 cells
We recently reported that Nb2 cells express exclusively the eNOS isoform, which plays an antiapoptotic role in these cells. Low levels of NO, produced by eNOS, stimulated bcl-2 expression and inhibited apoptosis in PRL-deprived Nb2 cells (12). Therefore, in this study we compared the effects of PRL and FGF-2 on eNOS expression and NO synthesis in these cells. As shown in Fig. 3AGo, eNOS mRNA levels were low, but detectable, in growth-arrested cells. Addition of PRL (10 ng/ml) resulted in a further decline in eNOS mRNA levels over the next 24 h (Fig. 3AGo, upper panel, and Fig. 3BGo). In contrast, FGF-2 (0.6 ng/ml) caused a significant increase in eNOS mRNA levels within 2 h, reaching a peak 10-fold induction by 12 h after FGF-2 addition (Fig. 3AGo, lower panel, and 3B). The activity of eNOS has been reported to be regulated posttranslationally by serine phosphorylation (19). To determine whether FGF-2 altered the phosphorylation state of the eNOS protein, Nb2 cell lysates were immunoprecipitated with the anti-eNOS antibody and probed with anti-eNOS or antiphosphoserine antibodies. Western analysis indicated that FGF-2 treatment induced a 2- to 3.5-fold increase in the cellular levels of the eNOS protein, which was maintained for up to 24 h (data not shown). FGF-2 treatment also induced a modest (2- to 2.5-fold) increase in the cellular content of serine-phosphorylated eNOS (data not shown), consistent with the increase in total cellular eNOS. However, the ratio of serine-phosphorylated eNOS as a fraction of total cellular eNOS was unchanged, indicating that FGF-2 does not affect the phosphorylation state of eNOS.



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  		Figure 3. FGF-2, but not PRL, stimulates eNOS mRNA expression. A, PRL-deprived, growth-arrested Nb2 cells were treated with PRL (10 ng/ml) or FGF-2 (0.6 ng/ml) for 0–24 h. Total RNA was extracted and used for RT-PCR of eNOS mRNA and 18S ribosomal RNA as described in Materials and Methods. PCR products were resolved in 1.6% agarose gels. B, Relative expression was determined against 18S ribosomal RNA. Results are the mean ± SEM of three independent observations.

 
Total NO released into the culture medium by Nb2 cells was evaluated in the presence of varying concentrations of arginine (the NO precursor), with or without addition of PRL or FGF-2 for 4 h (Fig. 4Go). Basal NO release was at the limit of detection and was not significantly increased by the addition of PRL in the presence of arginine at a concentration of 0.1 or 1 mM (Fig. 4AGo). In contrast, FGF-2 had no effect on NO release in the absence of arginine, but significantly (P <= 0.05) increased NO production in the presence of 0.1 and 1.0 mM arginine (Fig. 4BGo).



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  		Figure 4. The total NO concentration increases after FGF-2 treatment. Growth-arrested Nb2 cells were resuspended in 10 mM HEPES buffer containing 5 mM glucose and 0.5% BSA (wt/vol) and incubated for 210 min with PRL (A) 100 ng/ml) or (B) FGF-2 (0.6 ng/ml), and increasing concentrations of arginine (0–1 mM). The cells were then pelleted and the supernatants were assayed for total nitrite content (see Materials and Methods). Quantitative data from analysis of three separate experiments are expressed as the mean ± SEM. Asterisks indicate a significant difference from the matched control value (P <= 0.05).

 
Inhibition of NO synthesis prevents FGF-2 stimulation of antiapoptotic gene expression
To determine the role of NO in the regulation of antiapoptotic gene expression by FGF-2, we next examined the effect of the NO synthase inhibitor aminoguanidine (AG) on basal and FGF-2-stimulated expression of bcl-2 and bag-1 (Fig. 5Go). Removal of PRL from the culture medium resulted in a greater than 95% decline in bcl-2 expression over the next 24 h, whereas bag-1 levels decreased modestly (35%) over the same time period. As expected, addition of FGF-2 to PRL-deprived cultures prevented the decline in expression of both bcl-2 and bag-1. Incubation with aminoguanidine alone resulted in a modest suppression of both bcl-2 and bag-1 expression compared with time-matched control cultures, possibly due to inhibition of basal eNOS activity. However, coincubation of aminoguanidine with FGF-2 completely abrogated the protective effect of FGF-2 on both bcl-2 and bag-1 mRNA levels.



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  		Figure 5. Aminoguanidine (AG), a NOS inhibitor, blocks the antiapoptotic effect of FGF-2. Nb2 cells were growth arrested in Fischer’s medium supplemented only with 10% lactogen-free HS and with or without the addition aminoguanidine (25 mM) for 20 h. Growth-arrested Nb2 cells were then treated with FGF-2 (0.6 ng/ml) for 0, 8, and 24 h. Total RNA (2 µg/sample) was used for RT-PCR analysis of bcl-2 and bag-1 mRNAs and of 18S ribosomal RNA as described in Materials and Methods. PCR products were resolved in 1.6% agarose gels. Quantitative data shown in B are the mean ± SEM of three independent observations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NO has been reported to exert both proapoptotic and antiapoptotic effects in a variety of cell types, including cells of the immune system (reviewed in Refs. 20 and 21). Induction of high levels of NO by cytokine stimulation of inducible NOS (iNOS) expression in activated macrophages plays a role in the immunological defense against bacteria and tumor cells and has also been shown to induce apoptotic cell death in the macrophages that produce the NO. In contrast, there is mounting evidence for a physiological role for low levels of NO as a survival factor for cells of the immune system. Endogenous NO and NO produced from exogenously added NO donors have been shown to inhibit apoptosis of both B and T cells as well as eosinophils (22, 23, 24). Although the mechanism of this antiapoptotic effect of NO is not completely understood, NO has been reported to protect B cells by stimulation of sustained expression of the protooncogene bcl-2 at both the mRNA and protein levels (25). More recently, NO has been shown to fully protect against UVA-induced apoptosis by up-regulation of bcl-2 in keratinocytes (26).

PRL qualifies as an autocrine/paracrine growth factor in immune cells (reviewed in Ref. 27). It is synthesized by normal rat and human immune cells (28, 29, 30), which also express receptors for PRL (30, 31, 32). The mitogenic action of PRL in lymphoid cells is studied extensively in the rat Nb2 T lymphoma cell line, which expresses high affinity cell surface PRLr and is critically dependent on PRL for growth (33). In addition to its stimulatory effects on cell proliferation, PRL has been shown to promote the survival of Nb2 cells by inhibition of glucocorticoid-induced apoptosis (34, 35). The antiapoptotic actions of PRL in Nb2 cells are associated with rapid induction of bcl-2 and pim-1 mRNAs (16, 17). Overexpression of Bag-1, a Bcl-2- and Raf-binding protein, has also been shown to promote Nb2 cell viability and survival (18).

Recently, we reported that the Nb2 T lymphoma cells expressed exclusively the eNOS isoform, whereas inducible nitric oxide synthase (iNOS) and neural nitric oxide synthase (nNOS) were not detectable in these cells (12). We have also shown that in the absence of PRL, NO releasers alone promoted cell survival and inhibited apoptosis in Nb2 cells deprived of PRL for 5 days. Furthermore, we have confirmed work by others demonstrating stimulation of bcl-2 expression within 1 h of PRL treatment in these cells (12, 16, 17). However, we also showed that in the absence of PRL, expression of bcl-2 was up-regulated by L-arginine (NO substrate) or the NO releaser, diethylamine/nitric oxide complex (DEA/NO), at 2 and 8 h, respectively (12). Therefore, we concluded that the antiapoptotic action of low levels of NO produced by eNOS in the Nb2 cells acted through a PRL-independent, bcl-2-mediated pathway.

PRL rapidly stimulates FGF-2 gene expression and cellular (but not secreted) FGF-2 in Nb2 cell cultures (11). As FGF-2 is known to have antiapoptotic activity in hemopoietic cells, it was therefore of interest to compare the effects of PRL and FGF-2 on NO synthesis, Bcl-2 gene expression, apoptosis, and proliferation. Somewhat surprisingly, PRL was unable to mimic the effects of exogenous FGF-2 on NOS expression and NO production. This apparent discrepancy between the actions of PRL (which induces FGF-2 expression in these cells) and exogenous FGF-2 may result from differential trafficking of the endogenous form(s) of FGF-2 produced in these cells. FGF-1 and FGF-2 are unusual among the FGF family in their lack of a signal sequence for secretion. Although a number of studies have indicated that FGF is exported from some cells by a nonclassical mechanism (36), it is increasingly recognized that the action of FGF-2 in many cells involves a nuclear-targeted intracrine signaling pathway (37). We were unable to detect FGF-2 in Nb2-conditioned medium after PRL treatment, suggesting that FGF-2 may not be efficiently secreted by these cells under normal circumstances (11).

We speculate that endogenous FGF-2 induced by PRL treatment remains largely intracellular and mediates as yet unidentified intracrine effects, which may or may not be related to the survival-promoting effects of PRL. In contrast, extracellular (exogenously added) FGF-2 activates the cell surface FGF receptor, initiating a signaling pathway leading to activation of eNOS and induction of the antiapoptotic machinery. The antiapoptotic effects of exogenous FGF-2 in Nb2 cells appear to be mediated by a pathway distinct from the PRL-signaling pathway in these cells. The existence of such a pathway is supported by recent independent observations that indicate that exogenous FGF-2 stimulates expression of eNOS in other cell types (38) and up-regulates the expression of bcl-2 in B lymphocytic leukemia cell lines, resulting in delayed apoptosis (7). The role of NO in the antiapoptotic response is supported by recent reports that endogenously produced NO protects against apoptosis in correlation with Bcl-2 up-regulation in a variety of cell types, including endothelial cells (26), T lymphoma cells (12), and normal human B lymphocytes (39).

The physiological significance of this pathway is not yet clear. However, extracellular FGF-2 acting through the cell surface FGF receptor may play a role in some stages of normal T cell development. It has been suggested that FGF-2 stored in the extracellular matrix of the stromal cells may interact with the FGF receptor on immature T cells (40). Interaction between stromal cells and hemopoietic cells is critical for hemopoietic cell survival and differentiation. The potential pathological significance is supported by recent observations that exogenous FGF-2 up-regulates Bcl-2 in human B lymphomas (7), and that serum FGF-2 levels are a direct indicator of poor prognosis in non-Hodgkin’s lymphoma (41).

We have shown previously that Nb2 cells produce low basal levels of NO. NO production in the Nb2 cells increased with increasing concentrations of arginine (NOS substrate), but the addition of PRL did not further increase NO release (12). In contrast, the present study showed that arginine stimulation of NO production in Nb2 cells was significantly enhanced by the addition of FGF-2. We examined the expression of the various NOS mRNA species by RT-PCR amplification, using target-specific primers, and were unable to detect either iNOS or nNOS in Nb2 cells after 35 cycles, although these RNAs were readily detectable in the appropriate control tissues, LPS-treated rat colon and rat brain, respectively (Fig. 1Go in Ref. 12). Furthermore, iNOS protein was not detectable in Nb2 cell lysates by Western blotting regardless of treatment. We conclude that NO production in these cells appears to result solely from the action of eNOS. This is in agreement with previous studies indicating that FGF-2 regulates local vasomotor tone via a NO-signaling pathway through activation of eNOS in aortic, coronary, and uterine vascular endothelial cells (42, 43, 44). FGF-2 stimulation of the expression of eNOS mRNA and/or protein has also been demonstrated in bovine adrenal capillary endothelial cells (38) and ovine fetoplacental artery endothelial cells (45).

Although the NOS inhibitor AG is a selective inhibitor of iNOS, it is able to inhibit the other NOS isoforms with the relative potency iNOS > nNOS > eNOS (46) and has previously been used to inhibit eNOS activity in rat tissues (47). Coincubation of AG with FGF-2 completely abrogated the effect of FGF-2 on both bcl-2 and bag-1 mRNA levels in PRL-deprived Nb2 cells. AG and other purine analogs have previously been shown to rapidly (within 8 h) suppress LPS-stimulated NO release and Bcl-2 induction in cultured B lymphocytes, resulting in increased apoptosis, an effect that is reversible with NO donors (39). However, it must be noted that AG, like other purine analogs, has NO-independent antiproliferative effects by virtue of its structural analogy with physiological purines. Indeed, the structurally related analog 6-mercaptopurine is an important drug in the maintenance therapy of acute lymphoblastic leukemia. Although less potent than 6-mercaptopurine, AG has also been shown to inhibit the proliferation of normal B cells (39). We have previously demonstrated that AG inhibits Nb2 cell proliferation in a standard 6-day bioassay (12). Although NO donors (arginine, S-nitrose-N-acetylpenicillamine, 3-morpholinosydnonimine HCl, and DEA) rapidly induce Bcl-2 expression in Nb2 cells, they do not reverse the antiproliferative effects of AG in PRL-treated cells, indicating that the latter effect is not mediated by the NO signaling pathway, respectively (12).

In summary, the present study shows that FGF-2 promotes Nb2 cells survival and inhibits apoptosis of PRL-deprived Nb2 cells, in association with increased expression of eNOS and production of NO. Both endogenous NO (induced by FGF-2) and exogenous NO (produced from NO donors such as arginine and DEA) (12) stimulate the expression of bcl-2 and bag-1 with a similar time course. Although circumstantial, this temporal association supports the possibility that FGF-2 exerts its effects via a NOS-Bcl-2 pathway in these cells.


   Acknowledgments
 
We thank Dr. Geoffrey Rowden (Department of Pathology) for use of the Shandon Cytospin 2.


   Footnotes
 
1 This work was supported by Medical Research Council Grants MOP12597 (to P.R.M.) and MOP12895 (to C.K.L.T.). Back

2 Supported by a Medical Research Council of Canada-Burroughs Wellcome Studentship and Murray MacNeil Summer Studentship. Back

3 Supported by a Cancer Research and Education-Nova Scotia trainee award, with funding from the Faculty of Medicine, Dalhousie University. Back

4 Scholar of the Medical Research Council. Back

Received June 16, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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