ABSTRACT
The interaction of fibroblast growth factor 2 (FGF-2) with heparan sulfate proteoglycans (HSPG) has been demonstrated to enhance receptor binding and alter the intracellular distribution of internalized FGF-2. In the present study, the intracellular fate of FGF-2 was analyzed in vascular smooth muscle cells (VSMC) under native and HSPG-deficient conditions. HSPG-deficient cells were generated by treatment with sodium chlorate. Cells were incubated with FGF-2 at 37 degrees C for prolonged periods (0-48 h) to allow for FGF-2 uptake and processing. Processing of FGF-2 occurred in stages. Initially a family of low molecular weight (LMW) fragments (4-10 kDa) were detected that accumulated to much higher ( approximately 10-fold) levels in native compared to heparan sulfate-deficient cells. Pulse-chase experiments revealed that the half-life of these LMW intermediates was significantly greater in native ( approximately 18 h) compared to HSPG-deficient cells ( approximately 4 h). Rate constants for FGF-2 processing were derived by modeling the uptake and processing of FGF-2 as a set of first-order differential equations. The kinetic analysis indicated that the greatest differences between native and HSPG-deficient VSMC was in the formation of LMW and further suggested that these FGF-2 products appear to represent a stable subpool of internal FGF-2 that is favored in cells that contain HSPG. Thus, HSPG might function as a cellular switch between immediate and prolonged signal activation by heparin-binding growth factors such as FGF-2. In the absence of HSPG, FGF-2 can interact with and activate its receptor, yet in the presence of HSPG, FGF-2 might be able to mediate prolonged or unique biological responses through intracellular processes.
Subject(s)
Fibroblast Growth Factor 2/metabolism , Heparan Sulfate Proteoglycans/physiology , Intracellular Fluid/metabolism , Protein Processing, Post-Translational , Animals , Cells, Cultured , Cytoplasm/metabolism , Endocytosis , Guinea Pigs , Heparan Sulfate Proteoglycans/deficiency , Heparan Sulfate Proteoglycans/genetics , Humans , Intracellular Fluid/chemistry , Kinetics , Molecular Weight , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Peptide Fragments/metabolism , Protein Processing, Post-Translational/genetics , Time FactorsABSTRACT
Basic fibroblast growth factor (bFGF) is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated in a number of vascular disorders. bFGF interacts with high-affinity receptors and heparan sulfate proteoglycans (HSPG) at the cell surface. HSPG have been demonstrated to enhance bFGF binding to its receptors, yet no known role for HSPG in modulating postbinding events has been identified. In the present study, we analyzed bFGF internalization, intracellular distribution, degradation, and stimulation of DNA synthesis within native and HSPG-deficient VSMC. HSPG-deficient VSMC were generated by treating cells with sodium chlorate to inhibit the sulfation of HSPG. We found that stimulation of DNA synthesis by bFGF in chlorate-treated VSMC was markedly reduced as compared with native cells, even at doses of bFGF where receptor binding was similar in the two conditions. This was not a general lack of mitogenic potential, as the addition of calf serum, or epidermal growth factor, stimulated DNA synthesis to a similar extent in native and chlorate-treated cells. Analysis of the accumulation of internalized bFGF within cytoplasmic and nuclear fractions of native and HSPG-deficient VSMC showed striking differences. At early time points (0-2 h), nearly identical amounts of bFGF were observed in the cytoplasmic fractions under both conditions, yet significant amounts of bFGF were only found in the nuclear fractions of native cells. At later time points (2-48 h), the amount of cytoplasmic bFGF was significantly greater in the native compared to HSPG-deficient cells, and nuclear deposition of bFGF began to reach similar levels under both conditions. Furthermore, the intracellular half-life of bFGF was dramatically prolonged in native compared to HSPG-deficient cells, in part, due to decreased bFGF degradation in native cells. Thus, HSPG appears to accelerate nuclear localization, increase cytoplasmic capacity, and inhibit intracellular degradation of bFGF in VSMC. Modulation of intracellular processing of bFGF by HSPG might control the biological activity of bFGF in VSMC.
