Abstract
To evaluate possible functional differences between basic fibroblast growth factor (FGF) 2 isoforms we analyzed the effects of the 18-kDa FGF-2 which mainly localizes in the cytosol and that of the nuclear-targeted 22.5-kDa form on FGF receptors (FGFR) expression. These peptides were expressed at low amounts through a retroviral-infection system. Point mutated FGF-2 cDNAs under the control of the beta-actin promoter were used to infect a pancreatic cell line (AR4 2J) which does not produce FGF-2. Saturation and competition binding studies with 125I-FGF-2 revealed a 3-fold increase in both high and low affinity receptors in cells expressing the 22.5-kDa form and a 2-fold increase only in the high affinity receptors in cells producing the 18-kDa form. Kd values and molecular weights of the high affinity receptors were unaffected. Increasing cell densities or cell treatment with exogenous FGF-2 resulted in FGFR down-regulation as in control cells. Neutralizing anti-FGF-2 antibodies and suramin did not affect receptor density in control and in cells producing the 22.5-kDa form but further increased by 60 and 80%, respectively, the receptor level in cells synthesizing the 18-kDa form. These data suggest the involvement of the intracellular stored FGF-2 in FGFR up-regulation. Although all cells expressed FGFR-1, -2, and -3 mRNA only the FGFR-1 transcript was found increased, 6-fold in 22.5-kDa expressing cells and 3-fold in cell producing the shortest secreted isoform. The increase in FGFR-1 mRNA levels in the 22.5-kDa expressing cells was due to enhanced stability of the transcript. Confocal microscopy detected the presence of FGFR-1 at the cell surface whereas secretory isoforms of the receptor were not observed. Reverse transcriptase-polymerase chain reaction did not reveal significant differences in the expression of FGFR-1 variants. In the 22.5-kDa expressing cells exogenous FGF-2 evoked a stronger translocation of the calcium-phospholipid-dependent PKC. These results indicate that the transfected FGF-2 isoforms up-regulated FGFR-1 mRNA and protein. The 22.5-kDa form acted by increasing FGFR-1 mRNA stability enhancing cell responses to exogenous FGF-2.
Highlights
To evaluate possible functional differences between basic fibroblast growth factor (FGF) 2 isoforms we analyzed the effects of the 18-kDa FGF-2 which mainly localizes in the cytosol and that of the nuclear-targeted 22.5-kDa form on FGF receptors (FGFR) expression
AR4-2J cells do not produce FGF-2 [21] and possess low levels of FGFR [22]. These cells were induced to stably express the two molecular forms of FGF-2 under the control of the -actin promoter. We report that both FGF-2 molecular forms up-regulate the high affinity receptors and the levels of the FGFR-1 mRNA
Up-regulation of FGFR in FGF-2-expressing Cells—The time course of 125I-FGF-2 binding at 4 °C was analyzed first
Summary
Cell Culture—Parental and transfected AR4-2J cells were maintained in Dulbecco’s modified Eagle’s medium containing 4.5 g/liter glucose (Life Technologies, Inc., Eragny, France) and supplemented with 10% fetal calf serum (Life Technologies, Inc.). A5 clones were transfected with the FGF-2 cDNA containing a stop codon upstream to the AUG initiation codon They synthesized only the 18-kDa form, at a concentration of about 0.5 ng/106 cells. After three new washes with PBS, cells were incubated 30 min at 37 °C in serum-free Dulbecco’s modified Eagle’s medium buffered at pH 7.4 with 25 mM Hepes, containing 0.2% gelatin. To assess the effect of exogenous FGF-2 on 125I-FGF-2 binding, cells were incubated for 3 h at 37 °C in medium containing 10% fetal calf serum in the presence of increasing concentrations of the recombinant 18-kDa FGF-2. The specificity of the PCR products was checked by using restriction enzymes, selected according to the sequence of the rat FGFR-1 cDNA using computer analysis (PC/GENE, IntelliGenetics, Mountain View, CA). PKC activity is determined by subtracting the activity measured in the absence of diacylglycerol and phosphatidylserine from that measured in the presence of both cofactors and expressed as picomoles of 32P incorporated into histone H1 per min and per mg of protein
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