Cell Cycle-Dependent Nuclear Localization of Exogenously Added Fibroblast Growth Factor-1 in BALB/c 3T3 and Human Vascular Endothelial Cells

doi:10.1006/excr.1994.1353

Experimental Cell Research

Volume 215, Issue 2, December 1994, Pages 363-372

https://doi.org/10.1006/excr.1994.1353 Get rights and content

Abstract

Previous studies have shown that the presence of a functional nuclear targeting sequence in the primary structure of fibroblast growth factor (FGF)-1 correlates with its activity as a mitogen, but not with its potential for inducing receptor tyrosine phosphorylation, suggesting the presence of a yet undefined function of FGF-1 as a nuclear protein. In the present study we have investigated the cytosolic and nuclear localization of exogenously added FGF-1. FGF-1-specific monoclonal antibodies were raised. By an extensive screening, highly specific antibody clones were isolated. For both BALB/c 3T3 and human umbilical vein endothelial (HUVE) cells, immunofluorescence studies performed with those clones delineated that during G1 stage of cell cycle, FGF-1 transits from cytosol to nucleus. This was followed by a shift to the perinuclear and juxtanuclear region just prior to the onset of S-phase in BALB/c 3T3 cells. Confocal microscopical examinations confirmed that the nuclear staining resides throughout the nuclear matrix with some enrichment at the envelope boundary and in the nucleoli. Immunoblot analysis of the fractionated BALB/c 3T3 cells that had been induced to proliferate by serum and pulsed with exogenous FGF-1 at various timings revealed that the incorporation of exogenous FGF-1 into cytosol took place constantly, whereas the nuclear translocation significantly increased after 5 h following stimulation of the quiescent cells. The cytosolic form of FGF-1 is indicated to be present in soluble cytosolic fraction rather than membrane-enveloped compartments, endosomes, by the microinjection of anti FGF-1 antibody to HUVE cells cultured in the presence of FGF-1. The data demonstrate that the exogenously added FGF-1 is constantly endocytosed and fractioned into the cytosol soluble compartment, whereas its nuclear localization is regulated at the nuclear translocation level and takes place preferably at late G1 phase of the cell cycle.

References (0)

Cited by (74)

Intracellular partners of fibroblast growth factors 1 and 2 - implications for functions
2021, Cytokine and Growth Factor Reviews
Citation Excerpt :
Upon binding and activation of their specific receptors (FGFRs) they stimulate intracellular signaling pathways such as PKC/PLCγ, PI3K/Akt, and Ras/MAPK [2,3]. In addition to the signaling through the tyrosine kinase receptors, FGF1 and FGF2 have been shown to translocate across cellular membranes and reach the cytosol and nucleus, an unusual feature for growth factors [4–6]. Even though this characteristic is shared by these two FGFs, most of the studies on the intracellular fate have focused on FGF1, the best-studied member of canonical FGFs.
Fibroblast growth factors 1 and 2 (FGF1 and FGF2) are mainly considered as ligands of surface receptors through which they regulate a broad spectrum of biological processes. They are secreted in non-canonical way and, unlike other growth factors, they are able to translocate from the endosome to the cell interior. These unique features, as well as the role of the intracellular pool of FGF1 and FGF2, are far from being fully understood. An increasing number of reports address this problem, focusing on the intracellular interactions of FGF1 and 2. Here, we summarize the current state of knowledge of the FGF1 and FGF2 binding partners inside the cell and the possible role of these interactions. The partner proteins are grouped according to their function, including proteins involved in secretion, cell signaling, nucleocytoplasmic transport, binding and processing of nucleic acids, ATP binding, and cytoskeleton assembly. An in-depth analysis of the network of these binding partners could indicate novel, non-classical functions of FGF1 and FGF2 and uncover an additional level of a fine control of the well-known FGF-regulated cellular processes.
Translocation of Exogenous FGF1 and FGF2 Protects the Cell against Apoptosis Independently of Receptor Activation
2018, Journal of Molecular Biology
Citation Excerpt :
This discrepancy may arise from the fact that the conditions of experiment are very harsh for the cells, that is, starvation and the presence of FGFR inhibitor and two other compounds (Suppl. Fig. 5c). Despite the fact that the translocation of FGF1 and FGF2 is a well-known phenomenon [7,9,18,37], we performed a control experiment to confirm that in the conditions used throughout the experiments FGF1 and FGF2 were indeed translocated into the cell upon serum-starvation (Fig. 4c). Using cell fractionation [22], we showed that in starved NIH 3T3 cells exogenously added FGF1 and FGF2 were found in the cytosolic (C) and nuclear (N) fractions in the absence or presence of an FGFR inhibitor (PD173074) (Fig. 4c).
FGF1 and FGF2 bind to specific cell-surface tyrosine kinase receptors (FGFRs) and activate intracellular signaling that leads to proliferation, migration or differentiation of many cell types. Besides this classical mode of action, under stress conditions, FGF1 and FGF2 are translocated in a receptor-dependent manner via the endosomal membrane into the cytosol and nucleus of the cell. However, despite many years of research, the role of translocated FGF1 and FGF2 inside the cell remains unclear. Here, we reveal an anti-apoptotic activity of intracellular FGF1 and FGF2, which is independent of FGFR activation and downstream signaling. We observed an inhibition of cell apoptosis induced by serum starvation or staurosporine upon treatment with exogenous FGF1 or FGF2, despite the presence of highly potent FGFR inhibitors. Similar results were found when the tyrosine kinase of FGFR1 was completely blocked by a specific mutation. Moreover, the anti-apoptotic effect of the growth factors was abolished by known inhibitors of the translocation of FGF1 and FGF2 from the endosomes to the interior of the cell. Interestingly, FGF2 showed higher anti-apoptotic activity than FGF1. Since FGF2 is not phosphorylated by PKCδ and is present inside the nucleus longer than is FGF1, we speculated that the different activities could reflect their diverse nuclear export kinetics. Indeed, we observed that FGF1 mutations preventing binding to nucleolin and therefore phosphorylation in the nucleus affect the anti-apoptotic activity of FGF1. Taken together, our data indicate that the translocation of FGF1 and FGF2 protects cells against apoptosis and promotes cell survival.
Fibroblast growth factor-12 (FGF12) translocation into intestinal epithelial cells is dependent on a novel cell-penetrating peptide domain: Involvement of internalization in the in vivo role of exogenous FGF12
2011, Journal of Biological Chemistry
The extracellular effect of fibroblast growth factor-12 (FGF12) remains unknown because FGF12 cannot activate any fibroblast growth factor receptors (FGFRs), and FGF12 is not currently thought to be released from cells. We reported previously that FGF12 plays an intracellular role in the inhibition of radiation-induced apoptosis. In this study, we demonstrated that recombinant FGF12 was able to be internalized into the cytoplasm of a rat intestinal epithelial cell line, IEC6, and this process was dependent on two novel cell-penetrating peptide (CPP) domains (CPP-M and CPP-C). In particular, CPP-C, composed of ∼10 amino acids, was identified as a specific domain of FGF12 and its subfamily in the C-terminal region (residues 140–149), although CPP-M was a common domain in the internal region of the FGF family. The absence of CPP-C from FGF12 or a mutation (E142L) in the CPP-C domain drastically reduced the internalization of FGF12 into cells. Therefore, CPP-C played an essential role in the internalization of FGF12. In addition, CPP-C was able to deliver other polypeptides into cells as a CPP because an FGF1/CPP-C chimeric protein was internalized into IEC6 cells more efficiently than wild-type FGF1. Finally, intraperitoneally added FGF12 inhibited radiation-induced apoptosis in the intestinal epithelial cells of BALB/c mice, and deletion of the CPP-C domain decreased the inhibition of the apoptosis. These findings suggest that exogenous FGF12 can play a role in tissues by translocating into cells through the plasma membrane, and the availability of this novel CPP provides a new tool for the intracellular delivery of bioactive molecules.
Translocation of exogenous FGF1 into cytosol and nucleus is a periodic event independent of receptor kinase activity
2011, Experimental Cell Research
Citation Excerpt :
It has previously been described that serum deprivation is required for efficient translocation into the cells, and that the translocation of FGF1 into cytosol and nucleus is delayed by 2–4 h compared to its endocytic uptake [15,20]. Also, a correlation between FGF1/FGF2 translocation and the G1 phase of the cell cycle has been observed, and it was therefore suggested that the translocation is somehow linked or restricted to G1 [20–23]. In this paper we have investigated the timing of the FGF1 translocation and the role of FGFR1 signalling in this process.
Fibroblast growth factor 1 (FGF1) has the property to become translocated from the extracellular space into the cell cytosol and nucleus. Membrane translocation of FGF1 occurs subsequent to endocytic uptake and is strictly FGF-receptor (FGFR) dependent. Here we have investigated the timing of FGF1 translocation in relation to FGFR1 signalling. We found that the translocation of FGF1 is a periodic event that occurs with 24 h intervals. Serum-starved cells translocated the growth factor with peak occurrences ~ 6 h, ~ 30 h, and ~ 54 h after the addition of FGF1. The periodic FGF1 translocation was totally independent of the FGFR1 tyrosine kinase activity as it proceeded unchanged when the kinase activity was chemically inhibited or the kinase domain was deleted. Furthermore, FGF1 translocation was not restricted to a particular phase of the cell cycle or dependent on cell cycle progression. The results demonstrate that the FGF1/FGFR1 complex constitutes a signalling module that independently of the receptor tyrosine kinase can convey a signal that initiates a strictly timed and periodic release of endocytosed FGF1 into the cytosol/nucleus.
Post treatment with an FGF chimeric growth factor enhances epithelial cell proliferation to improve recovery from radiation-induced intestinal damage
2010, International Journal of Radiation Oncology Biology Physics
A fibroblast growth factor (FGF) 1–FGF2 chimera (FGFC) was created previously and showed greater structural stability than FGF1. This chimera was capable of stimulating epithelial cell proliferation much more strongly than FGF1 or FGF2 even without heparin. Therefore FGFC was expected to have greater biologic activity in vivo. This study evaluated and compared the protective activity of FGFC and FGF1 against radiation-induced intestinal injuries.
We administered FGFC and FGF1 intraperitoneally to BALB/c mice 24 h before or after total-body irradiation (TBI). The numbers of surviving crypts were determined 3.5 days after TBI with gamma rays at doses ranging from 8 to 12 Gy.
The effect of FGFC was equal to or slightly superior to FGF1 with heparin. However, FGFC was significantly more effective in promoting crypt survival than FGF1 (p < 0.01) when 10 μg of each FGF was administered without heparin before irradiation. In addition, FGFC was significantly more effective at promoting crypt survival (p < 0.05) than FGF1 even when administered without heparin at 24 h after TBI at 10, 11, or 12 Gy. We found that FGFC post treatment significantly promoted 5-bromo-2′-deoxyuridine incorporation into crypts and increased crypt depth, resulting in more epithelial differentiation. However, the number of apoptotic cells in FGFC-treated mice decreased to almost the same level as that in FGF1-treated mice.
These findings suggest that FGFC strongly enhanced radioprotection with the induction of epithelial proliferation without exogenous heparin after irradiation and is useful in clinical applications for both the prevention and post treatment of radiation injuries.
Increased protein stability of FGF1 can compensate for its reduced affinity for heparin
2009, Journal of Biological Chemistry
Human FGF1 (fibroblast growth factor 1) is a powerful signaling molecule with a short half-life in vivo and a denaturation temperature close to physiological. Binding to heparin increases the stability of FGF1 and is believed to be important in the formation of FGF1·fibroblast growth factor receptor (FGFR) active complex. In order to reveal the function of heparin in FGF1·FGFR complex formation and signaling, we constructed several FGF1 variants with reduced affinity for heparin and with diverse stability. We determined their biophysical properties and biological activities as well as their ability to translocate across cellular membranes. Our study showed that increased thermodynamic stability of FGF1 nicely compensates for decreased binding of heparin in FGFR activation, induction of DNA synthesis, and cell proliferation. By stepwise introduction of stabilizing mutations into the K118E (K132E) FGF1 variant that shows reduced affinity for heparin and is inactive in stimulation of DNA synthesis, we were able to restore the full mitogenic activity of this mutant. Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat and/or proteolytic degradation and that heparin is not essential for a direct FGF1-FGFR interaction and receptor activation.

View all citing articles on Scopus

View full text

Regular ArticleCell Cycle-Dependent Nuclear Localization of Exogenously Added Fibroblast Growth Factor-1 in BALB/c 3T3 and Human Vascular Endothelial Cells

Abstract

Regular Article
Cell Cycle-Dependent Nuclear Localization of Exogenously Added Fibroblast Growth Factor-1 in BALB/c 3T3 and Human Vascular Endothelial Cells