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The Journal of Immunology, 1999, 162: 5853-5859.
Copyright © 1999 by The American Association of Immunologists

Fibroblast Growth Factor-1 (FGF-1) Enhances IL-2 Production and Nuclear Translocation of NF-{kappa}B in FGF Receptor-Bearing Jurkat T Cells1

Victor M. Byrd2, Dean W. Ballard, Geraldine G. Miller and James W. Thomas

Departments of Medicine and Microbiology/Immunology and Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fibroblast growth factors (FGFs) are heparin-binding proteins crucial to embryogenesis, angiogenesis, and wound healing. FGF-1 is abundantly expressed in the synovium in rheumatoid arthritis and in rejecting allografts, sites of chronic immune-mediated inflammation. The frequency of FGF-1-responsive T cells is increased in the peripheral blood of these disorders, and a high percentage of infiltrating T cells in rheumatoid arthritis synovium express receptors for FGF-1. To understand the action of FGF-1 in T cells, studies were initiated in Jurkat T cells that express the signaling isoform of FGF receptor-1. These experiments show that FGF-1 stimulation of Jurkat T cells provides a second signal that augments TCR-mediated IL-2 production. Analogous to costimulation via CD28, this activity is mediated through activation of Rel/{kappa}B, a family of transcription factors known to regulate IL-2 and other activation-inducible proteins. FGF-1 alone induces modest nuclear translocation of {kappa}B-binding proteins, and this translocation is enhanced by the combination of anti-CD3 and FGF-1. This NF-{kappa}B binding complex is composed of transcriptionally active p65(RelA)/p50 heterodimers and results primarily from the targeted degradation of I{kappa}B-{alpha}, an inhibitor that sequesters Rel/{kappa}B in the cytoplasm. These data are the first to show a connection between FGF-1 signaling and NF-{kappa}B activation outside of embryonic development. The signaling events that link FGF receptor-1 engagement and NF-{kappa}B activation in Jurkat are probably distinct from the CD28 costimulation pathway, since FGF-1-induced Rel/{kappa}B binding proteins do not contain significant levels of c-Rel and are not identical with the CD28 response complex.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acidic and basic fibroblast growth factors (FGF-1 and FGF-2)3 are prototypes of heparin binding growth factors that stimulate proliferation, migration, and differentiation of cells of neuroectodermal and mesodermal origin reviewed in Refs. (1, 2, 3, 4). They are expressed widely during normal embryonic development and often in adult tissues at sites of injury, where their angiogenic activity contributes to wound healing. FGF-1 expression is markedly increased in some sites of chronic immunologic injury, in particular the synovium in rheumatoid arthritis (RA) (5, 6, 7, 8) and in allografts undergoing chronic rejection (9, 10) where T cell infiltration is common. Early evidence of a direct connection between T cells and nonhemopoietic growth factors including FGF was first reported by Johnson and Torres (11). Their experiments demonstrated that CD8+ T cells produced IFN-{gamma} when stimulated by the combination of FGF and TCR engagement by superantigen, while neither stimulus alone induced cytokine production. Their data thus suggested that FGF and other peptide growth hormones provided a second or costimulatory signal required for cytokine production. More recent experiments show that a subset of CD4+ T cells expresses FGF receptor-1 (FGFR-1) (12, 13), a high affinity receptor tyrosine kinase that mediates FGF signaling in many other cell types (reviewed in 14). For CD4+ T cells that express FGFR-1, FGF-1 can provide the costimulatory signal required for IL-2 production in conjunction with anti-CD3 (12, 13). FGF-1 alone, however, does not induce IL-2 production, and in contrast to other cell types that express FGFR-1, FGF-1 alone is unable to induce T cell proliferation. Whether this reflects the dependence of T cells upon IL-2 for proliferation or differences in FGFR-1 signaling pathways in T cells vs other cell types is unknown.

FGFR signaling in other cell types induces receptor oligomerization and autophosphorylation with resultant tyrosine phosphorylation of phospholipase C{gamma} and activation of Ras/MAPK/extracellular signal-related kinase (ERK) (14, 15, 16), which are also activated by the TCR pathway. IL-2 production resulting from the combination of TCR and FGF-1 signaling, therefore, presumably reflects an integration of signals from these two pathways and their resultant transcription factor activation. Among the transcription factors that are activated by signaling through FGFR-1 in nonlymphoid cells are AP-1 and cAMP response element binding protein/activating transcription factor-2 (ATF-2) (17, 18), and most recently members of the FGF family were shown, for the first time, to induce NF-{kappa}B/Rel transcription factors in the developing limb bud (19, 20).

Engagement of CD28 by its receptor ligands on APCs is to date the predominant interaction between T cell and APC surface molecules that provide costimulation for IL-2 production, thus inducing T cell expansion and preventing anergy or apoptosis. Activation of NF-{kappa}B/Rel factors is a major target of the costimulatory activity of the CD28 pathway (21, 22, 23, 24). Indirect evidence suggests that FGF-1 may also have costimulatory function in vivo, especially at sights such as the synovium in active RA and the parenchyma of solid organ allografts, where FGF-1 production is excessive (5, 7, 8, 9, 10). Patients with RA and cardiac transplant recipients have 5- to 10-fold increases in the precursor frequency of FGF-responsive T cells in peripheral blood compared with normal subjects, and changes in the frequency of these cells over time are correlated with disease activity in RA (5, 12, 13). Enrichment of FGFR-1+ CD4+ T cells in RA synovium compared with peripheral blood suggests that local FGF-1 may contribute to T cell activation, differentiation, or survival within inflammatory sites.

To improve our understanding of the actions on T cells independent of these complex inflammatory events, we initiated studies using a Jurkat T cell line that expresses the signaling isoform of FGFR-1. Evidence for activation of NF-{kappa}B/Rel transcription factors by FGFs expressed in the developing limb and the parallels with CD28 costimulation in T cells prompted us to investigate whether FGFR-1 signaling activates NF-{kappa}B/Rel in Jurkat T cells. The results show that FGF-1 induces enhanced degradation of cytoplasmic I{kappa}B-{alpha} and prolonged nuclear translocation of transcriptionally active p50/RelA heterodimers. In contrast to costimulation via CD28, FGF-induced Rel/{kappa}B complexes contained low levels of c-Rel, and no binding to the CD28 response element (CD28RE) was observed in nuclear extracts from cells stimulated with FGF-1. The data demonstrate that some functions of FGF-1 outside embryonic development are mediated by members of the Rel/NF-{kappa}B family and that signals from a nonhemopoietic growth factor and the TCR pathways may intersect. The implications for these findings in the activation of nontransformed T cells is discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Jurkat T cells expressing FGFR-1ß have been described previously (25). Briefly, they were produced by stable transfection of a plasmid containing FGFR-1ß cDNA driven by the CD2 promoter and locus control region. These cells (clone C2-14) express approximately 30,000 high affinity FGFR and were used for all the experiments described here. The cells were routinely maintained in RPMI 1640 containing 10% FCS, 10 mM HEPES, 5 mM L-glutamine, and 1 mg/ml G418. For T cell stimulations, cells were cultured in six-well flat-bottom plates coated with anti-CD3 (1 µg/ml mAb JE6, provided by Dr. Stanford Stewart, Vanderbilt University, Nashville, TN). Recombinant human FGF-1 or FGF-2 (R&D Systems, Minneapolis, MN) or anti-CD28 (mAb 9.3, 1/2,500, provided by Dr. Jeffrey Ledbetter, Bristol Myers Squibb, Seattle, WA) were included as indicated. All stimulations with FGF-1 or FGF-2 included heparin (20 U/ml). For determinations of IL-2 protein production, supernatants were harvested at 24 h and were assayed by ELISA (BioSource, Camarillo, CA) according to the manufacturer’s specifications.

Nuclear and cytoplasmic extract preparation

Nuclear and cytoplasmic protein extracts were prepared as previously described (26) from 107 T cells harvested at each time point. Cells were washed in 1.0 ml of ice-cold PBS, and cell pellets were lysed in 100 µl of lysis buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.4% Nonidet P-40, 1 mM DTT, and 0.5 mM PMSF) in the presence of protease inhibitors (antipain, aprotinin, leupeptin, bestatin, phosphoramidon, and soybean trypsin inhibitor, all 5 µg/ml, and pepstatin 0.5 µg/ml). Cells were incubated on ice for 5 min. The cell lysates were subsequently centrifuged for 60 s at 14,000 x g, and supernatants were collected for cytoplasmic protein analysis. The nuclear pellet from lysis was washed briefly in 100 µl of lysis buffer and then subjected to high salt extraction in 50 µl of nuclear buffer (0.4 M NaCl, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF, in the presence of protease inhibitors) at 4°C with extensive shaking for 15 min. Cellular debris from both cytoplasmic and nuclear extracts was removed by centrifugation for 10 min at 14,000 x g, and fractions were stored at -70°C until analyzed. Protein concentrations were determined by spectrophotometric assay (Bio-Rad, Hercules, CA) according to the manufacturer’s specifications.

EMSA/supershift assays

For EMSA, 10 µg of nuclear protein was incubated with 32P-labeled duplex probe including 1) a palindromic NF-{kappa}B site derived from the IL-2R-{alpha} promoter (5'-CAACGGCAGGGGAATTCCCCTCTCCTT-3') (27), or 2) the CD28 response element (CD28RE; 5'-GATCGTTTAAAGAAATTCCAAA-3') from the IL-2 promoter. Incubations were performed in binding buffer (125 mM NaCl, 2.5 mM EDNA, 0.25 mM DTT, 0.25 mM PMSF, 0.5 mg/ml BSA, 1% Nonidet P-40, and 0.1 mg/ml double-stranded poly(dI-dC)) for 15 min at room temperature. Approximately 100,000 cpm of radiolabeled probe was used per sample. Resultant nucleoprotein complexes were separated on a 5% nondenaturing polyacrylamide gel in TBE buffer (90 mM Tris, 90 mM borate, 2.5 mM EDTA) and visualized by autoradiography. For both probes, the specificity of protein-oligonucleotide interactions was verified by competition of radiolabeled probe with an excess of unlabeled oligonucleotide. For supershift assays, DNA/protein complexes were incubated with rabbit polyclonal antisera to members of the Rel/{kappa}B family, including p65(RelA) (antiserum R567) (26), p50 (antiserum R393) (28), or preimmune sera.

Western blot analysis

Nuclear protein extracts were prepared from stimulated Jurkat T cells as detailed above. Nuclear proteins (60 µg) were separated by 10% SDS-PAGE, transferred to polyvinylidene membranes, and blocked with 5% nonfat milk in Tris-buffered saline/Tween. Proteins were subsequently immunoblotted with polyclonal antiserum specific for c-Rel (antiserum R453, 1/2000) (29). For Western blot analysis of cytosolic I{kappa}B, cytoplasmic protein extracts were prepared from stimulated Jurkat T cells as detailed above, except that T cells were stimulated in the presence of cycloheximide (50 µg/ml; Sigma, St. Louis, MO) to block the NF-{kappa}B-induced resynthesis of I{kappa}B inhibitors (30, 31). Cytoplasmic proteins (100 µg) were separated by 10% SDS-PAGE, transferred to polyvinylidene membranes, and blocked as detailed above. Proteins were immunoblotted with polyclonal antiserum specific for either I{kappa}B-{alpha} (antiserum R663, 1/2500) (26) or I{kappa}B-ß (antiserum C-20, 1/200; Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive proteins were detected with peroxidase-conjugated anti-rabbit IgG using an enhanced chemiluminescence assay (Amersham, Arlington Heights, IL).

Jurkat transient transfections and chloramphenicol acetyltransferase (CAT) assay

Jurkat C2-14 was transiently transfected by electroporation with a vector containing a CAT reporter gene driven by the NF-{kappa}B sequence from the HIV-1 promoter as previously described (32). Electroporation conditions (250 V, 960 µF, time constant 34) were optimized to a viable cell recovery of 50%. Cells were rested for 24 h in RPMI/10% FCS, followed by stimulation for 24 h and assay for CAT activity. CAT assays were performed on whole cell lysates by a diffusion-based liquid scintillation method (33).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FGF-1 and FGF-2 act as costimulatory signals for IL-2 production in FGFR-1-expressing T cells

Optimal production of IL-2 by T cells is dependent upon signals generated by TCR engagement and costimulatory signals provided by additional cell surface receptors. To examine the costimulatory effects of FGF-1, Jurkat T cells that express FGFR-1 were stimulated with anti-CD3 alone, FGF-1 alone, or anti-CD3 plus FGF-1 (Fig. 1Go). Unstimulated T cells and those stimulated with anti-CD3 alone (zero FGF point on the anti-CD3 plus FGF curve) or FGF-1 alone produced little IL-2. However, the combination of anti-CD3 with FGF-1 increased IL-2 production approximately 7-fold over that with anti-CD3 alone. Similar results were obtained with FGF-2 (Fig. 1Go), consistent with the fact that FGFR-1 binds both these FGF family members with high affinity. Positive control stimulations with anti-CD3 plus anti-CD28 resulted in significant IL-2 production (mean, 1423 pg/ml; data not shown). These data confirm previous findings that engagement of the TCR together with stimulation by FGF-1 induces both IL-2 production and proliferation, whereas FGF-1 alone is ineffective (12, 13).



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  		FIGURE 1. FGF-1 and FGF-2 provide costimulatory signals for IL-2 production in FGFR-1+ T cells. FGFR-1-transfected Jurkat cells (subline C2-14) were plated at 1 x 106/ml and stimulated with immobilized anti-CD3, FGF-1 (50 ng/ml), or increasing concentrations of FGF-1 or FGF-2 (0–100 ng/ml) in the presence of anti-CD3 as described in Materials and Methods. Cells were harvested after 24-h incubation at 37°C, and supernatants were assayed for IL-2 by ELISA. Results are expressed as the mean IL-2 production from three separate experiments. Positive control stimulations with anti-CD3 plus anti-CD28 resulted in IL-2 production with a mean of 1423 pg/ml (data not shown).

 
FGF-1 stimulation induces nuclear translocation of {kappa}B binding proteins

Activation of T cells via the TCR together with costimulation via CD28 result in nuclear translocation of Rel/NF-{kappa}B transcription factors (23, 34). We therefore examined whether FGF-1 alone or in combination with TCR engagement similarly activates NF-{kappa}B. Gel shifts were performed using a palindromic {kappa}B sequence derived from the IL-2R {alpha}-chain enhancer as a probe and nuclear extracts from cells that were either unstimulated or stimulated with FGF-1 alone (Fig. 2GoA). Modest induction of NF-{kappa}B binding activity was present by 30 min after addition of FGF-1 and remained detectable with relatively little change during 6 h of culture in the presence of FGF-1. In contrast, stimulation with both anti-CD3 and FGF-1 led to enhanced binding compared with either stimulus alone, and binding activity increased substantially over time up to 12 h in culture (Fig. 2GoB). As a positive control for NF-{kappa}B/Rel activation, we performed EMSA on T cells stimulated with anti-CD3 and anti-CD28 and detected a similar {kappa}B binding complex (Fig. 2GoB). Competition experiments with unlabeled competitor probe confirmed the specificity of binding (data not shown). To confirm binding by NF-{kappa}B family members, supershifts were performed with antisera to p50 and p65 and with control nonimmune serum on nuclear extracts from cells stimulated with anti-CD3 alone, anti-CD3 plus FGF-1, and positive controls stimulated with anti-CD3 plus anti-CD28 (Fig. 2GoC). The results demonstrate the presence of p50 and p65 in all complexes binding to the {kappa}B sequence. The data thus indicate that stimulation by FGF-1 results in nuclear translocation of NF-{kappa}B that is enhanced by the combination of TCR and FGFR signaling.



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  		FIGURE 2. FGF-1 signaling in T cells results in the nuclear translocation of Rel/{kappa}B proteins. A, The Jurkat line C2-14 was stimulated with FGF-1 (25 ng/ml). Nuclear extracts were prepared as detailed in Materials and Methods. Ten micrograms of protein was incubated with a 32P-labeled probe from the IL-2R{alpha} NF-{kappa}B enhancer sequence. The inducible NF-{kappa}B band is marked by an arrow. A lower m.w. band, present in all samples, represented nonspecific binding. B, Stimulation with the combination of anti-CD3 plus FGF-1. Jurkat cells were stimulated with FGF-1 alone for 1 h, anti-CD3 alone, or anti-CD3 plus FGF-1 (25 ng/ml) over a 12-h time course; with anti-CD3 plus anti-CD28 for 1 h; or with anti-CD28 alone for 1 h. Gel shifts with 10 µg of nuclear protein and the labeled NF-{kappa}B oligonucleotide were conducted as described in A above. Binding of inducible NF-{kappa}B proteins is indicated by an arrow. Shown is a representative gel from three experiments. C, Supershift assays for p50 and p65. Nuclear protein extracts from the 4 h points were incubated with labeled NF-{kappa}B probe for 10 min at room temperature, incubated with 1 µl of preimmune serum (lane 2), anti-p50 (lane 3), or anti-p65 (lane 4) for an additional 10 min, followed by electrophoretic separation and autoradiography. The {kappa}B binding complexes, indicated by the arrow, were supershifted by both anti-p50 and anti-p65.

 
FGF-1 costimulation does not result in increased nuclear translocation of c-Rel

As supershift assays using polyclonal antiserum directed against c-Rel did not produce convincing mobility shifts (data not shown), we performed Western blotting for c-Rel using nuclear extracts from Jurkat T cells stimulated with anti-CD3 plus either FGF-1 or anti-CD28 (Fig. 3Go). The results demonstrate no appreciable increase in c-Rel nuclear translocation following stimulation with FGF-1 alone or anti-CD3 alone and a very modest increase in c-Rel following stimulation with anti-CD3 plus FGF-1. In contrast, marked nuclear translocation of c-Rel was noted following stimulation with anti-CD3 in combination with anti-CD28. These data show that FGF-induced Rel/{kappa}B complexes differ from those activated by anti-CD28 pathways, as FGF-induced complexes contain little c-Rel.



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  		FIGURE 3. FGF-1 costimulation does not induce robust nuclear translocation of c-Rel proteins. Jurkat T cells were stimulated with FGF-1 alone, anti-CD3 alone, or anti-CD3 plus either FGF-1 or anti-CD28. Cells were harvested at the indicated time points. Sixty micrograms of nuclear protein per sample were separated by SDS-PAGE and immunoblotted with polyclonal anti-c-Rel. Immunoreactivity was detected following incubation with a peroxidase-labeled anti-rabbit IgG and enhanced chemiluminescence. Immunoreactive c-Rel is indicated by the arrow.

 
FGF-1 costimulation does not induce nuclear translocation of proteins that bind to the CD28 response element

Activation through CD28, but not other costimulators (23), results in the formation of a nuclear CD28 response complex (CD28RC) that binds to a sequence designated the CD28 response element (CD28RE) present in several genes including the IL-2 promoter (23, 35, 36). Since FGF-1 costimulation enhances nuclear translocation of p50 and p65, which are components of the CD28RC (23), we examined whether it also results in a complex that binds the CD28RE. EMSA was performed with a probe for the CD28RE sequence and nuclear extracts from cells stimulated with FGF-1 alone in combination with anti-CD3 or from controls stimulated with anti-CD3 plus anti-CD28 (Fig. 4Go). Nuclear extracts from controls stimulated with anti-CD3 plus anti-CD28 showed the expected binding to the CD28RE probe, while cells stimulated with FGF-1, anti-CD28 alone, anti-CD3 alone, or anti-CD3 plus FGF-1 did not. These results indicate that costimulation by FGF-1 does not activate formation of the protein complex that binds this element of the IL-2 promoter and that signaling pathways from CD28 and FGFR-1 are not identical.



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  		FIGURE 4. FGF-1 costimulation does not result in the nuclear translocation of proteins that bind the CD28RE. Jurkat T cells were stimulated for 4 h with FGF-1, with anti-CD28, or with immobilized anti-CD3 in the presence or the absence of FGF-1 or anti-CD28. Ten microgams of nuclear extract were incubated with 32P-labeled oligonucleotide probe from the CD28RE in the IL-2 enhancer. Anti-CD28-inducible binding by the CD28RC is indicated by the arrow.

 
NF-{kappa}B proteins activated by FGF-1 costimulation are transcriptionally active

To confirm that the {kappa}B binding complex induced by FGF-1 costimulation was capable of activating transcription, we performed transient transfections with an NF-{kappa}B-CAT reporter construct. As shown in Fig. 5Go, stimulation with either anti-CD3 or FGF-1 alone resulted in only a 2-fold increase in CAT activity above that in unstimulated cells. Costimulation with FGF-1 and anti-CD3 resulted in an average 5-fold increase in CAT activity, similar to the 7.4-fold increase seen with anti-CD3 plus anti-CD28. The results demonstrate that both FGF-1 and CD28 costimulatory pathways induce Rel/{kappa}B protein complexes that are transcriptionally active.



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  		FIGURE 5. NF-{kappa}B proteins activated by FGF-1 are transcriptionally active. Jurkat T cells were transiently transfected with an NF-{kappa}B-CAT reporter construct derived from the {kappa}B enhancer sequence from the HIV-1 virus as detailed in Materials and Methods. Results are expressed as the relative fold induction compared with that in resting T cells and represent the mean ± SEM from five different experiments.

 
Costimulation with FGF-1 targets I{kappa}B-{alpha} for degradation

Rel/NF-{kappa}B proteins are retained in an inactive state in the cytoplasm by inhibitors (I{kappa}B) that prevent their nuclear translocation (30, 31, 37). Activating signals result in phosphorylation of these inhibitors and their subsequent degradation (30, 31). Costimulation via CD28 has been reported to induce degradation of both I{kappa}B-{alpha} (38, 39) and I{kappa}B-ß (34). To determine whether FGF-1 stimulation similarly results in degradation of I{kappa}B, Western blots were performed for I{kappa}B-{alpha} and I{kappa}B-ß on cytoplasmic extracts of cells stimulated with anti-CD3 alone or anti-CD3 plus FGF-1. Because active NF-{kappa}B induces synthesis of I{kappa}B-{alpha} protein (30), cycloheximide was added to cultures to prevent new protein synthesis and therefore the resynthesis of I{kappa}B-{alpha} induced by active NF-{kappa}B. The results are shown in Fig. 6Go. Compared with stimulation with anti-CD3 alone, costimulation with FGF-1 resulted in enhanced degradation of I{kappa}B-{alpha} with earlier kinetics. Decreased levels of I{kappa}B-{alpha} were apparent by 30 min of stimulation, and degradation was virtually complete by 4 h (Fig. 6Go). In contrast, no degradation of I{kappa}B-ß was seen. Similar results were observed for degradation of I{kappa}B-{alpha} following costimulation with anti-CD28. There was a modest decrease in I{kappa}B-ß. These data on I{kappa}B-ß degradation induced by CD28 stimulation are similar to those reported by Lai and Tan (38) and recently by Kalli et al. (39), but differ from the findings of Harhaj et al. (34). The differences may lie in the use of PMA (34) rather than anti-CD3 in conjunction with anti-CD28.



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  		FIGURE 6. FGF-1 costimulation targets the degradation of I{kappa}B-{alpha}. Jurkat cells were stimulated with anti-CD3 in the presence or the absence of either FGF-1 or anti-CD28. Cytoplasmic protein extracts were prepared as described in Materials and Methods. One hundred micrograms of cytoplasmic protein extract was separated by SDS-PAGE and immunoblotted with rabbit antiserum specific for either I{kappa}B-{alpha} or I{kappa}B-ß. Bands were visualized following incubation with peroxidase-conjugated anti-rabbit IgG and enhanced chemiluminescence.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Signals delivered through FGFRs provide costimulatory pathways in a subset of T lymphocytes and may contribute to T cell activation at inflammatory lesions characterized by abundant FGF-1 expression (5, 12, 13). While the activation pathways in T cells following engagement of FGFR tyrosine kinases have yet to be fully elucidated, recent data have clarified FGF signaling pathways in nonhemopoietic cells. In NIH-3T3 cells, binding of FGF-1 to FGFR-1 results in receptor autophosphorylation on six tyrosine residues, including residues that are crucial for functional kinase activity in the catalytic domain (Y653, Y654), and additional residues (Y766) that are required for the recruitment of various signaling intermediates (14, 16, 40). These autophosphorylation events trigger the recruitment of SH2 domain proteins, including phospholipase C{gamma}, Grb2, and Sos (16). Recruitment of such SH2 domain proteins then triggers the activation of Ras-dependent signaling events and the induction of MAPK pathways (16, 41). It is not known whether autophosphorylation of tyrosine residues outside the catalytic domain is required for FGFR-1 signaling, as site-directed mutagenesis of these noncatalytic tyrosine residues does not inhibit activation of the Ras/MAPK pathway or cell proliferation (40). In addition, the data suggest that FGF downstream signaling events are shared with cAMP pathways, as FGF/Ras-mediated gene activation in neuroblastoma cells has been shown to induce transcription of the ATF/AP-1 factors c-Jun and ATF-3, which, in turn, bind to cAMP response element-2 (18). These data demonstrating induction of Fos and Jun proteins are consistent with the observation that FGF-2 induces the expression of c-Jun and c-Fos in NIH-3T3 cells (17), and that the FGF-inducible response element from the syndecan gene contains novel AP-1 binding motifs (41).

The data presented here are, to the best of our knowledge, the first direct demonstration of Rel/{kappa}B activation following interaction of FGF-1 with FGFR-1. Recent reports by two laboratories examining expression of NF-{kappa}B in the mesoderm of developing chick limb bud have demonstrated a relationship between other FGFs and activation of NF-{kappa}B proteins. In both studies either exogenous FGF-8 (19) or FGF-4 (20) was capable of reconstituting c-Rel mesodermal expression and normal limb formation following excision of the apical ectodermal ridge, a known source of FGFs that regulate limb development (19, 20). The induction of other Rel/{kappa}B proteins, such as p50 or p65, was not addressed in these reports, and activation of an intermediary protein remains a possibility. Nonetheless, taken together, the data suggest that FGFRs in both Jurkat T cells and mesoderm directly activate Rel/{kappa}B. It is not evident from the studies in chick limb which FGFRs mediate c-Rel activation; however, previous investigations have shown that functional FGFR-1 and FGFR-2 are required for normal limb development in vertebrates (42, 43). In Jurkat cells, we were unable to demonstrate substantial c-Rel activation by FGF-1 and FGFR-1. The results may suggest that FGF family members and distinct FGFRs differentially regulate NF-{kappa}B/Rel proteins. In addition, nontransformed T cells that express FGFR-1, such as those in RA synovium (5), may be regulated differently.

While the function of FGF-1 as a secondary signal for T cell activation is analogous to costimulation through CD28 pathways, our data indicate that these two pathways are distinct. The failure of FGF-1-induced nuclear proteins to bind with high affinity to the CD28RE coupled with the minimal c-Rel in these complexes indicate that the Rel/{kappa}B complexes are not identical in FGF-1 and CD28 pathways. Recent data show that while RelA (p65), NF-{kappa}B1 (p50), and c-Rel are all components of the CD28RC, only RelA and c-Rel directly bind to and trans-activate the CD28RE (22, 24). In addition, the induction of c-Rel by CD28 appears to be a critical determinant of CD28RE transcriptional activity, as overexpression of c-rel augments the activity of a reporter gene under the control of the CD28RE (23). In Jurkat T cells we have not been able to demonstrate the presence of substantial c-Rel in FGF-1-induced complexes, and this lack of c-Rel activation may provide one explanation for the relatively weak transcriptional activity of FGF-1-induced complexes and their inability to bind to the CD28RE. Finally, recent evidence suggests that CD28 costimulation and subsequent activation of the CD28RE requires binding of factors unrelated to Rel/{kappa}B proteins, including the high mobility group protein HMG I(Y) (44), and NF-MATp35 (45). Such proteins may not be recruited in response to FGF-1 signaling in T cells. These events may differ in other types of T cells, such that FGF-1 costimulation in nontransformed T cells in vitro or its effects within inflammatory sites may include c-Rel activation. Experiments are currently in progress to determine these possibilities. Similarly, we have observed that the combination of FGF-1 and anti-CD28 increases NF-{kappa}B translocation in Jurkat without increasing IL-2 production (data not shown). The outcome of these signals may also differ in nontransformed T cells, and experiments to characterize these events are in progress.

Differential degradation of the NF-{kappa}B cytoplasmic inhibitors I{kappa}B-{alpha} and I{kappa}B-ß was examined to determine how FGF-1 regulates the induction of Rel/{kappa}B complexes. Emerging data suggest that control of differential Rel/{kappa}B complex induction by various stimuli occurs at the level of targeted degradation of either I{kappa}B-{alpha} or I{kappa}B-ß. Degradation of I{kappa}B-{alpha} has been linked to the nuclear induction of p50/p65 heterodimers, the classic NF-{kappa}B complexes induced by a broad variety of stimuli, including mitogens (PMA) and various cytokines (GM-CSF, TNF-{alpha}) (34). In contrast, targeted degradation of I{kappa}B-ß is restricted to only a few stimulators, including LPS and the human T cell leukemia virus-1 Tax protein (34). CD28 signaling targets both {alpha} and ß isoforms, as this pathway has been recently shown to prolong both PMA- and anti-CD3-induced degradation of I{kappa}B-{alpha} and to induce the degradation of I{kappa}B-ß, an event that is not targeted by PMA activation alone (34, 38, 39). Our results show that FGF-1 targets the degradation of I{kappa}B-{alpha}, as FGF-1 plus anti-CD3 enhances and substantially prolongs the modest I{kappa}B-{alpha} degradation induced by anti-CD3 alone. Little effect on I{kappa}B- ß degradation was observed in our studies. This FGF-1 targeting of I{kappa}B-{alpha} is consistent with the FGF-1 induction of Rel/{kappa}B complexes that contain mostly p50 and p65, the heterodimer complex that is typically induced with I{kappa}B-{alpha} degradation.

While we demonstrate activation of Rel/{kappa}B proteins following FGF-1/FGFR-1 interaction, this could be a response unique to specific combinations of FGFs/FGFRs rather than the FGF family as a whole. Recent studies in NIH-3T3 cells suggest that an FGF-2-inducible response element in the syndecan-1 promoter does not bind NF-{kappa}B (41). In these studies in vivo footprinting revealed three motifs that bind nuclear factors in response to FGF-2 activation. While two of these motifs contained AP-1-like consensus sites, the remaining motif contained no known consensus sequences. Binding competition experiments with an array of unlabeled oligonucleotides in gel-shift experiments did not reveal inhibition of any protein complexes by NF-{kappa}B oligonucleotides, suggesting that Rel/{kappa}B proteins are not crucial for the FGF-2-responsive expression of syndecan-1 (41). Thus, while NF-{kappa}B is activated by FGF-1 in Jurkat T cells, it is likely that other proteins play critical roles in the transcriptional regulation of FGF-1-responsive elements in other types of cells. In addition, the complex array of cytokines and growth factors found in inflammatory sites where FGFR-1+ T cells accumulate may further alter the transcriptional regulation of FGF-1 responses.


   Acknowledgments
 
We thank Dr. Zhi-Liang Chu for assistance with the gel shift assays.


   Footnotes
 
1 This work supported by National Institutes of Health Grants (to V.M.B.) K08AR02017, R01AI33839 (to D.W.B.), R01HL53771 (to G.G.M.), and R01AR43653 (to J.W.T.); the Howard Hughes Medical Institute (D.W.B.), and American Heart Association Grant-in-Aid 94006080. Back

2 Address correspondence and reprint requests to Dr. Victor M. Byrd, Department of Medicine/Division of Rheumatology, Vanderbilt University Medical Center, T3219 MCN 21st Ave. and Garland, Nashville, TN 37232. E-mail address: Back

3 Abbreviations used in this paper: FGF, fibroblast growth factor; RA, rheumatoid arthritis; FGFR-1, FGF receptor-1; MAPK, mitogen-activated protein kinase; CD28RE, CD28 response element; ATF-2, activating transcription factor-2; CAT, chloramphenicol acetyltransferase; CD28RC, CD28 response complex. Back

Received for publication November 20, 1998. Accepted for publication March 1, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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