Faculty Bibliography

Fibroblast growth factor 1 (FGF1) and FGF2, the prototypic members of the FGF family of growth factors, have been implicated in a variety of physiological and pathological processes. Unlike most other FGFs, FGF1 and FGF2 are ubiquitously expressed and are not efficiently secreted. Gene knockouts in mice have previously demonstrated a role for FGF2 in brain development, blood pressure regulation, and wound healing. The relatively mild phenotypic defects associated with FGF2 deletion led to the hypothesis that the continued expression of other FGFs partially compensated for the absence of FGF2 in these mice. We now report our generation of mice lacking FGF1 and their use, in combination with our previously described FGF2 null mice, to produce mice lacking both FGF1 and FGF2. FGF1-FGF2 double-knockout mice are viable and fertile and do not display any gross phenotypic defects. In the double-knockout mice we observed defects that were similar in extent to those previously described for the FGF2 null mice. Differences in the organization of neurons of the frontal motor cortex and in the rates of wound healing were observed. We also observed in FGF2(-/-) mice and in FGF1-FGF2 double-knockout mice novel impairments in hematopoiesis that were similar in severity. Essentially no abnormalities were found in mice lacking only FGF1. Our results suggest that the relatively mild defects in FGF2 knockout animals are not a consequence of compensation by FGF1 and suggest highly restricted roles for both factors under normal developmental and physiological conditions

The MET proto-oncogene, encoding the tyrosine kinase receptor for HGF, controls genetic programs leading to cell growth, invasiveness, and protection from apoptosis. Recently, MET mutations have been identified in hereditary and sporadic forms of papillary renal carcinoma (PRC). Introduction of different naturally occurring mutations into the MET cDNA results in the acquisition of distinct biochemical and biological properties of transfected cells. Some mutations result in a high increase in tyrosine kinase activity and confer transforming ability in focus forming assays. These mutants hyperactivate the Ras signaling pathway. Other mutations are devoid of transforming potential but are effective in inducing protection from apoptosis and sustaining anchorage-independent growth. These Met(PRC) receptors interact more efficiently with the intracellular transducer Pi3Kinase. The reported results show that MET(PRC) mutations can be responsible for malignant transformation through different mechanisms, either by increasing the growth ability of cells or by protecting cells from apoptosis and allowing accumulation of other genetic lesions.-Giordano, S., Maffe, A., Williams, T. A., Artigiani, S., Gual, P., Bardelli, A., Basilico, C., Michieli, P., Comoglio, P. M. Different point mutations in the met oncogene elicit distinct biological properties

Mutations in the genes encoding for Met, Ret and Kit receptor tyrosine kinases invariably result in increased kinase activity and in the acquisition of transforming potential. However, the requirement of receptor ligands for the transformation process is still unclear. We have investigated the role of hepatocyte growth factor (HGF), the high-affinity ligand for Met, in mutant Met-mediated cell transformation. We provide evidence that the transforming potential displayed by mutant forms of Met found in human cancer is not only sensitive but entirely dependent on the presence of HGF, by showing that mutant Met transforms NIH3T3 fibroblasts, which produce endogenous HGF, but is not able to transform epithelial cells, unless exogenous HGF is supplied. Accordingly, mutant Met-induced transformation of NIH3T3 cells can be inhibited by HGF antagonists and increased by HGF stimulation. We also show that an engineered Met receptor which contains an oncogenic mutation but is impaired in its ability to bind HGF completely loses its transforming activity, which can be rescued by causing receptor dimerization using a monoclonal antibody. These results indicate that point mutations resulting in Met kinase activation are necessary but not sufficient to cause cell transformation, the latter being dependent on ligand-induced receptor dimerization. They also suggest that mutant Met-driven tumour growth depends on the availability and tissue distribution of active HGF, and provide proof-of-concept for the treatment of mutant-Met related pathologies by HGF-antagonizing drugs

Several genetic forms of human dwarfism have been linked to activating mutations in FGF receptor 3, indicating that FGF signaling has a critical role in chondrocyte maturation and skeletal development. However, the mechanisms through which FGFs affect chondrocyte proliferation and differentiation remain poorly understood. We show here that activation of FGF signaling inhibits chondrocyte proliferation both in a rat chondrosarcoma (RCS) cell line and in primary murine chondrocytes. FGF treatment of RCS cells induces phosphorylation of STAT-1, its translocation to the nucleus, and an increase in the expression of the cell-cycle inhibitor p21WAF1/CIP1. We have used primary chondrocytes from STAT-1 knock-out mice to provide genetic evidence that STAT-1 function is required for the FGF mediated growth inhibition. Furthermore, FGF treatment of metatarsal rudiments from wild-type and STAT-1(-/-) murine embryos produces a drastic impairment of chondrocyte proliferation and bone development in wild-type, but not in STAT-1(-/-) rudiments. We propose that STAT-1 mediated down regulation of chondrocyte proliferation by FGF signaling is an homeostatic mechanism which ensures harmonious bone development and morphogenesis

We identified Ark, the mouse homolog of the receptor tyrosine kinase Axl (Ufo, Tyro7), in a screen for novel factors involved in GnRH neuronal migration by using differential-display PCR on cell lines derived at two windows during GnRH neuronal development. Ark is expressed in Gn10 GnRH cells, developed from a tumor in the olfactory area when GnRH neurons are migrating, but not in GT1-7 cells, derived from a tumor in the forebrain when GnRH neurons are postmigratory. Since Ark (Ax1) signaling protects from programmed cell death in fibroblasts, we hypothesized that it may play an antiapoptotic role in GnRH neurons. Gn10 (Ark positive) GnRH cells were more resistant to serum withdrawal-induced apoptosis than GT1-7 (Ark negative) cells, and this effect was augmented with the addition of Gas6, the Ark (Ax1) ligand. Gas6/Ark stimulated the extracellular signal-regulated kinase, ERK, and the serine-threonine kinase, Akt, a downstream component of the phosphoinositide 3-kinase (PI3-K) pathway. To determine whether ERK or Akt activation is required for the antiapoptotic effects of Gas6/Ark in GnRH neurons, cells were serum starved in the absence or presence of Gas6, with or without inhibitors of ERK and PI3-K signaling cascades. Gas6 rescued Gn10 cells from apoptosis, and this effect was blocked by coincubation of the cells with the mitogen-activated protein/ERK kinase (MEK) inhibitor, PD98059, or wortmannin (but not rapamycin). These data support an important role for Gas6/Ark signaling via the ERK and PI3-K (via Akt) pathways in the protection of GnRH neurons from programmed cell death across neuronal migration

Embryonic development requires a complex program of events which are directed by a number of signaling molecules whose expression must be rigorously regulated. We previously showed that expression of Fgf4, which plays an important role in postimplantation development and growth and patterning of the limb, is regulated in EC cells by the synergistic interaction of Sox2 and Oct-3 with the Fgf4 EC cell-specific enhancer. To verify whether this mechanism was also operating in vivo, and to identify new elements controlling Fgf4 gene expression in distinct developmental stages, we have analyzed the expression of LacZ reporter plasmids containing different fragments of the Fgf4 gene in transgenic mouse embryos. Utilizing these transgenic constructs we have been able to recapitulate, for the most part, Fgf4 gene expression during embryonic development. We show here that most of the cis-acting regulatory elements determining Fgf4 embryonic expression are located in conserved regions within the 3' UTR of the gene. The EC cell-specific enhancer is required to drive gene expression in the ICM of the blastocyst, and its activity requires the Sox and Oct-proteins binding sites. We were also able to identify specific and distinct enhancer elements that govern postimplantation expression in the somitic myotomes and the limb bud AER. The myotome-specific elements contain binding sites for bHLH myogenic regulatory factors, which appear to be essential for myotome expression. Finally, we present evidence that the very restricted pattern of expression of Fgf4 transcripts in the AER results from the combined action of positive and negative regulatory elements located 3' of the Fgf4 coding sequences. Thus the Fgf4 gene relies on multiple and distinct regulatory elements to achieve stage- and tissue-specific embryonic expression.

The assumption that genes encoding tyrosine kinase receptors could play a role in human cancers has been confirmed by the identification of oncogenic mutations in the kinase domain of RET and KIT. Recently, homologous residues were found mutated in MET, in papillary renal carcinomas (PRCs). The link coupling these genetic lesions to cellular transformation is still unclear. METPRC mutations result in increased kinase activity and-in some instances, i.e., M1250T substitution-in changes in substrate specificity. A direct correlation occurs between the transforming potential of METPRC mutants and their ability to constitutively associate with signal transducers through two phosphorylated tyrosines (Y1349VHVNATY1356VNV) located in the receptor tail. Substitution of these 'docking tyrosines' with phenylalanines leaves unaffected the altered properties of the kinase but abrogates transformation and invasiveness in vitro. Uncoupling the receptor from signal transducers with a tyrosine-phosphorylated peptide derivative (YpVNV) inhibits invasive growth induced by METPRC mutants. These data indicate that constitutive receptor coupling to downstream signal transducers is a key mechanism in neoplastic transformation driven by mutated MET and suggest a therapeutic strategy to target neoplastic diseases associated with this oncogene

Choroidal neovascularization (CNV) is the major cause of severe visual loss in patients with age-related macular degeneration. Laser treatment is helpful for a minority of patients with CNV, and development of new treatments is hampered by a poor understanding of the molecular signals involved. Several lines of evidence have suggested that basic fibroblast growth factor (FGF2) plays a role in stimulating CNV. In this study, we tested this hypothesis using mice with targeted disruption of the FGF2 gene in a newly developed murine model of laser-induced CNV. One week after krypton laser photocoagulation in C57BL/6J mice, 34 of 60 burns (57%) showed fluorescein leakage and 13 of 16 (81%) showed histopathological evidence of CNV. At 2 weeks, CNV was detected in 9 of 10 burns (90%) in which a bubble had been observed at the time of the laser treatment. Electron microscopy showed fenestrated vessels with large lumens within choroidal neovascular lesions. Two weeks after laser-induced rupture of Bruch's membrane, 27 of 36 burns (75%) contained CNV in FGF2-deficient mice compared with 26 of 30 (87%) in wild-type control mice, a difference that is not statistically significant. This study demonstrates that FGF2 is not required for the development of CNV after laser-induced rupture of Bruch's membrane and provides a new model to investigate molecular mechanisms and anti-angiogenic therapy in CNV

Basic fibroblast growth factor (FGF2) is constitutively expressed in the retina and its expression is increased by a number of insults, but its role in the retina is still uncertain. This study was designed to test the hypothesis that altered expression of FGF2 in the retina affects the development of retinal neovascularization. Mice with targeted disruption of the Fgf2 gene had no detectable expression of FGF2 in the retina by Western blot, but retinal vessels were not different in appearance or total area from wild-type mice. When FGF2-deficient mice were compared with wild-type mice in a murine model of oxygen-induced ischemic retinopathy, they developed the same amount of retinal neovascularization. Transgenic mice with a rhodopsin promoter/Fgf2 gene fusion expressed high levels of FGF2 in retinal photoreceptors but developed no retinal neovascularization or other abnormalities of retinal vessels; in the ischemic retinopathy model, they showed no significant difference in the amount of retinal neovascularization compared with wild-type mice. These data indicate that FGF2 expression is not necessary nor sufficient for the development of retinal neovascularization. This suggests that agents that specifically antagonize FGF2 are not likely to be useful adjuncts in the treatment of retinal neovascularization and therapies designed to increase FGF2 expression are not likely to be complicated by retinal neovascularization

Basic fibroblast growth factor (FGF2) is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiological and pathological processes, including limb development, angiogenesis, wound healing, and tumor growth, but its physiological role is still unclear. To determine the function of FGF2 in vivo, we have generated FGF2 knockout mice, lacking all three FGF2 isoforms, by homologous recombination in embryonic stem cells. FGF2(-/-) mice are viable, fertile and phenotypically indistinguishable from FGF2(+/+) littermates by gross examination. However, abnormalities in the cytoarchitecture of the neocortex, most pronounced in the frontal motor-sensory area, can be detected by histological and immunohistochemical methods. A significant reduction in neuronal density is observed in most layers of the motor cortex in the FGF2(-/-) mice, with layer V being the most affected. Cell density is normal in other regions of the brain such as the striatum and the hippocampus. In addition, the healing of excisional skin wounds is delayed in mice lacking FGF2. These results indicate that FGF2, although not essential for embryonic development, plays a specific role in cortical neurogenesis and skin wound healing in mice, which, in spite of the apparent redundancy of FGF signaling, cannot be carried out by other FGF family members