Antoni Wiedlocha

Dual mode of signal transduction by externally added acidic fibroblast growth factor

Acidic fibroblast growth factor (aFGF), fused to diphtheria toxin and translocated into cells, stimulated DNA synthesis in toxin-resistant cells lacking functional aFGF receptors while having a high number of diphtheria toxin receptors. In NIH 3T3 cells that lack diphtheria toxin receptors, but have receptors for aFGF, both aFGF and the fusion protein induced tyrosine phosphorylation, but only aFGF as such entered the nuclei and stimulated DNA synthesis. The results indicate that signaling occurs partly through cell surface receptors and partly by transport of the growth factor into the cell.

Roles of Fibroblast Growth Factor Receptors in Carcinogenesis

The fibroblast growth factor receptors (FGFR) play essential roles both during development and in the adult. Upon ligand binding, FGFRs induce intracellular signaling networks that tightly regulate key biological processes, such as cell proliferation, survival, migration, and differentiation. Deregulation of FGFR signaling can thus alter tissue homeostasis and has been associated with several developmental syndromes as well as with many types of cancer. In human cancer, FGFRs have been found to be deregulated by multiple mechanisms, including aberrant expression, mutations, chromosomal rearrangements, and amplifications. In this review, we will give an overview of the main FGFR alterations described in human cancer to date and discuss their contribution to cancer progression. Mol Cancer Res; 8(11); 1439–52. ©2010 AACR.

Stimulation of Proliferation of a Human Osteosarcoma Cell Line by Exogenous Acidic Fibroblast Growth Factor Requires both Activation of Receptor Tyrosine Kinase and Growth Factor Internalization

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.

Tight folding of acidic fibroblast growth factor prevents its translocation to the cytosol with diphtheria toxin as vector.

A fusion protein of acidic fibroblast growth factor and diphtheria toxin A‐fragment was disulfide‐linked to the toxin B‐fragment. The complex bound specifically to diphtheria toxin receptors, and subsequent exposure to low pH induced the fusion protein to translocate to the cytosol. Heparin, inositol hexaphosphate and inorganic sulfate strongly increased the trypsin resistance of the growth factor part of the fusion protein, indicating tight folding, and they prevented translocation of the fusion protein to the cytosol. The data indicate that only a more disordered form of the growth factor is translocation competent.

Vesicle transmembrane potential is required for translocation to the cytosol of externally added FGF-1

Externally added fibroblast growth factor‐1 (FGF‐1) is capable of crossing cellular membranes to reach the cytosol and the nucleus in a number of cell types. We have monitored the translocation of the growth factor by two methods: phosphorylation of FGF‐1, and prenylation of an FGF‐1 mutant that contains a C‐terminal prenylation signal. Inhibition of endosomal acidification by ammonium chloride or monensin did not block the translocation of FGF‐1, whereas bafilomycin A1, a specific inhibitor of vacuolar proton pumps, blocked translocation completely. A combination of ionophores expected to dissipate the vesicular membrane potential (valinomycin plus monensin) also fully inhibited the translocation. The inhibition of translocation by bafilomycin A1 was overcome in the presence of monensin or nigericin, while ouabain blocked translocation under these conditions. The data indicate that translocation of FGF‐1 to cytosol occurs from the lumen of intracellular vesicles possessing vacuolar proton pumps, and that a vesicular membrane potential is required. Apparently, activation of vesicular Na+/K+‐ATPase by monensin or nigericin generates a membrane potential that can support translocation when the proton pump is blocked.

FGF-1: From Biology Through Engineering to Potential Medical Applications

Human fibroblast growth factor 1 (FGF-1) is one of the best characterized members of the FGF superfamily. FGF-1 is a powerful mitogen exhibiting strong action on numerous different cell types. It plays a role in various stages of development and morphogenesis, as well as in angiogenesis and wound healing processes. Engineering of FGFs can bring many advantages. Design and construction of different mutants can contribute to a better understanding of the mechanism of action of protein growth factors. Moreover, application of FGFs as recombinant polypeptides in the treatment of wound and fracture healing, cardiovascular diseases and neurodegenerative diseases seems to be a rational medical approach. However, low thermal stability and high sensitivity to proteases limit the potential pharmaceutical use of wild-type FGFs. Thus, advanced protein design techniques and recombinant protein production can help to obtain new variants of FGFs with radically increased thermodynamic stability, prolonged half-life and improved proteolytic resistance. Such studies can provide a good starting point to convert short-lived and/or sensitive growth factors to effective therapeutic proteins.

Indirubin-3'-monoxime inhibits autophosphorylation of FGFR1 and stimulates ERK1/2 activity via p38 MAPK.

Indirubin-3′-monoxime is a derivative of the bis-indole alkaloid indirubin, an active ingredient of a traditional Chinese medical preparation that exhibits anti-inflammatory and anti-leukemic activities. Indirubin-3′-monoxime is mainly recognized as an inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3. It inhibits proliferation of cultured cells, mainly through arresting the cells in the G1/S or G2/M phase of the cell cycle. Here, we report that indirubin-3′-monoxime is able to inhibit proliferation of NIH/3T3 cells by specifically inhibiting autophosphorylation of fibroblast growth factor receptor 1 (FGFR1), blocking in this way the receptor-mediated cell signaling. Indirubin-3′-monoxime inhibits the activity of FGFR1 at a concentration lower than that required for inhibition of phosphorylation of CDK2 and retinoblastoma protein and cell proliferation stimulated by fetal calf serum. The ability of indirubin-3′-monoxime to inhibit FGFR1 signaling was similar to that of the FGFR1 inhibitor SU5402. In addition, we found that indirubin-3′-monoxime activates long-term p38 mitogen-activated protein kinase activity, which stimulates extracellular signal-regulated kinase 1/2 in a way unrelated to the activity of FGFR1. Furthermore, we show that indirubin-3′-monoxime can inhibit proliferation of the myeloid leukemia cell line KG-1a through inhibition of the activity of the FGFR1 tyrosine kinase. The data presented here demonstrate previously unknown activities of indirubin-3′-monoxime that may have clinical implications.

Increased protein stability of FGF1 can compensate for its reduced affinity for heparin

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.

Inability of the Acidic Fibroblast Growth Factor Mutant K132E to Stimulate DNA Synthesis after Translocation into Cells

Acidic fibroblast growth factor (aFGF) is a potent mitogen. It acts through activation of specific cell surface receptors leading to intracellular tyrosine phosphorylation cascades, but several reports also indicate that aFGF enters cells and that it has an intracellular function as well. The aFGF(K132E) mutant binds to and activates fibroblast growth factor receptors equally strongly as the wild-type, but it is a poor mitogen. We demonstrate that aFGF(K132E) enters NIH 3T3 cells and is transported to the nuclear fraction like wild-type aFGF. A fusion protein of aFGF(K132E) and diphtheria toxin A-fragment (aFGF(K132E)-DT-A) and a similar fusion protein containing wild-type aFGF (aFGF-DT-A) were reconstituted with diphtheria toxin B-fragment. Both fusion proteins were translocated to the cytosol by the diphtheria toxin pathway and subsequently recovered from the nuclear fraction. Whereas translocation of aFGF-DT-A stimulated DNA synthesis in U2OSDR1 cells lacking functional fibroblast growth factor receptors, aFGF(K132E)-DT-A did not. The mutation disrupts a protein kinase C phosphorylation site in the growth factor making it unable to be phosphorylated. The data indicate that a defect in the intracellular action of aFGF(K132E) is the reason for its strongly reduced mitogenicity, possibly due to inability to be phosphorylated.

FGF-1 and FGF-2 Require the Cytosolic Chaperone Hsp90 for Translocation into the Cytosol and the Cell Nucleus

Similarly to many protein toxins, the growth factors fibroblast growth factor 1 (FGF-1) and FGF-2 translocate from endosomes into the cytosol. It was recently found that certain toxins are dependent on cytosolic Hsp90 for efficient translocation across the endosomal membrane. We therefore investigated the requirement for Hsp90 in FGF translocation. We found that low concentrations of the specific Hsp90 inhibitors, geldanamycin and radicicol, completely blocked the translocation of FGF-1 and FGF-2 to the cytosol and the nucleus. The drugs did not interfere with the initial binding of FGF-1 to the growth factor receptors at the cell-surface or with the subsequent internalization of the growth factors into endosomes. The activation of known signaling cascades downstream of the growth factor receptors was also not affected by the drugs. The data indicate that the drugs block translocation from endosomes to the cytosol implying that Hsp90 is required for translocation of FGF-1 and FGF-2 across the endosomal membrane.