1. k-FGF protoncogene expression is associated with murine testicular teratogenesis, but is not involved during mouse testicular development
J M de Anta, M Monzó, B Peris, D Ruano Histol Histopathol. 1997 Jan;12(1):33-41.
The k-FGF gene, which belongs to the family of the fibroblast growth factor genes, is implicated in tumoral and developmental processes. It is expressed in embryonal carcinoma cells, in embryonic stem cells, during limb and tooth formation and in some germ cell tumors. However, the expression of this protooncogene during testicular development as well its relationship to spontaneous teratogenesis have not been determined. Here we investigate k-FGF expression during testicular development in mice, as well as in a spontaneous testicular teratoma (STT) and in the OTT6050 teratocarcinoma (TC) by Northern blotting, RT-PCR and it situ hybridization. Several data indicate that k-FGF gene contains downstream regulatory sequences which bind octamer factors. One of these transcription factors which binds to k-FGF enhancer is Oct-4. Although the k-FGF gene is activated by Oct-4 in embryonal carcinoma and embryonic stem cells and Oct-4 is expressed in the germ cells of the embryo, our results indicate that there is no detectable k-FGF expression in mouse testicular germ cells at any stage of development. This indicates that Oct-4 does not activate transcription of the k-FGF gene in mouse germ cells, and that k-FGF is not implicated during testicular development. We also show that there is a high k-FGF expression in the experimental OTT6050 TC, but only very low levels in a murine differentiated STT, suggesting that k-FGF activation may be responsible for the genesis and development of STT, behaving as a marker of malignancy in these neoplasms.
2. Transcriptional regulation of the murine k-fgf gene
A Rizzino, E Rosfjord Mol Reprod Dev. 1994 Sep;39(1):106-11. doi: 10.1002/mrd.1080390116.
Embryonal carcinoma (EC) cells provide a useful model system for studying the roles of growth factors during early mammalian development. In 1988, we determined that EC cells express a member of the fibroblast growth factor (FGF) family that cannot be detected after EC cells undergo differentiation. Attempts to understand how differentiation regulates the production of FGFs led to the finding that EC cells express the fibroblast growth factor k-FGF (FGF-4), whereas there is a large decrease in the steady state levels of k-FGF mRNA when EC cells differentiate. This suggested that transcription of the k-fgf gene is repressed when EC cells differentiate. To investigate this possibility, we prepared a series of reporter gene constructs containing various regions of the murine k-fgf gene. These constructs were transfected into two mouse EC cell lines and one mouse embryonic stem (ES) cell line. We determined that the mouse 5' flanking region cannot support expression of the reporter gene. In both EC and ES cell lines, expression of the reporter gene is elevated greatly by the addition of a 316 bp region from the third exon of the murine k-fgf gene. Sequence analysis of the 316 bp region identified one and possibly two conserved octamer binding motifs. These sequences are likely to be involved in regulation of the k-fgf gene, because differentiation of EC cells is known to reduce the expression of octamer binding proteins, including Oct-3. To test the possible role of octamer binding proteins, we examined the expression of our reporter gene constructs in F9-differentiated cells and in PYS-2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
3. The K-fgf/hst oncogene induces transformation through an autocrine mechanism that requires extracellular stimulation of the mitogenic pathway
D Talarico, C Basilico Mol Cell Biol. 1991 Feb;11(2):1138-45. doi: 10.1128/mcb.11.2.1138-1145.1991.
The K-fgf/hst oncogene encodes a secreted growth factor of the fibroblast growth factor (FGF) family. The ability of K-fgf-transformed cells to grow in soft agar and in serum-free medium is inhibited by anti-K-FGF neutralizing antibodies, consistent with an autocrine mechanism of transformation. The transformed properties of clones that express high levels of K-FGF are, however, only partially affected. To better define the autocrine mechanism of transformation by K-fgf and to determine whether receptor activation could occur intracellularly, we constructed two mutants of the K-fgf cDNA. Deletion of the sequences encoding the signal peptide suppressed K-fgf ability to induce foci in NIH 3T3 cells. A few morphologically transformed colonies were observed in cotransfection experiments, and they were found to express high levels of cytoplasmic K-FGF. However, their ability to grow in serum-free medium and in soft agar was inhibited by anti-K-FGF antibodies. Addition of a sequence encoding the KDEL endoplasmic reticulum and Golgi retention signal to the K-fgf cDNA led to accumulation of the growth factor in intracellular compartments. The ability of the KDEL mutant to induce foci in NIH 3T3 cells was much lower than that of the wild-type cDNA, and also in this case the transformed phenotype was reverted by anti-K-FGF antibodies. These and other findings indicate that the transformed phenotype of cells expressing a nonsecretory K-FGF is due to the extracellular activation of the receptor by the small amounts of growth factor that these cells still release. Thus, transformation by K-fgf appears to be due to an autocrine growth mechanisms that requires activation of the mitogenic pathway at the cell surface.
