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Serum-starvation induces the extracellular appearance of FGF-1
J T Shin1, S R Opalenik, J N Wehby
1Department of Surgery, School of Medicine, University of Alabama at Birmingham 35294, USA.
Abstract:
Autocrine/paracrine stimulation of cell growth by members of the fibroblast growth factor (FGF) family of polypeptides is dependent upon extracellular interactions with specific high affinity receptors at the cell surface. Acidic FGF (FGF-1) lacks a classical signal sequence for secretion, suggesting that intrinsic levels of this mitogen may not stimulate cell growth and utilizes a non-classical pathway to gain access to the extracellular compartment. To evaluate the biological potential of intracellular FGF-1 more rigorously, human cDNA sequences for the growth factor were introduced into primary murine embryonic fibroblasts using retrovirally mediated gene transfer. Heparin affinity, Western analysis, mitogenic assays, in situ immunohistochemical techniques, induction of tyrosine phosphorylation and antibody inhibition studies were used to demonstrate functionality of the FGF-1 transgene in this experimental model. Under normal culture conditions, cells constitutively expressing intracellular FGF-1 exhibited a slight growth advantage. In contrast, when maintained in reduced serum, these cells adopted a transformed phenotype and demonstrated an enhanced growth potential, induction of FGF-specific phosphotyrosyl proteins and the nuclear association of the growth factor. Analysis of the conditioned media from these stressed cells indicated that serum starvation induces the secretion of FGF-1 as latent high molecular mass complexes requiring reducing agents to activate its full biological potential.
Insights
Intracellular acidic fibroblast growth factor (FGF-1) can stimulate cell growth, especially under reduced serum conditions. Serum starvation induces FGF-1 secretion as inactive complexes that require reduction for activation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Fibroblast growth factors (FGFs) regulate cell growth via extracellular receptors.
- Acidic FGF (FGF-1) lacks a secretion signal, suggesting intracellular roles or non-classical secretion pathways.
Purpose of the Study:
- To investigate the biological potential of intracellular FGF-1.
- To determine the secretion mechanism and activation of FGF-1 under stress conditions.
Main Methods:
- Retroviral gene transfer to express FGF-1 in murine fibroblasts.
- Heparin affinity, Western analysis, and mitogenic assays.
- Immunohistochemistry, tyrosine phosphorylation, and antibody inhibition studies.
Main Results:
- Intracellular FGF-1 expression conferred a slight growth advantage under normal conditions.
- Reduced serum conditions induced a transformed phenotype, enhanced growth, and nuclear localization of FGF-1.
- Serum starvation triggered FGF-1 secretion as latent high molecular mass complexes activated by reducing agents.
Conclusions:
- Intracellular FGF-1 possesses biological activity and can be secreted via a non-classical pathway.
- Stress conditions activate FGF-1 secretion and nuclear translocation, enhancing cell growth.
- FGF-1 requires reduction for full biological potential, indicating complex regulation of its extracellular activity.