The K-fgf/hst oncogene induces transformation through an autocrine mechanism that requires extracellular stimulation of the mitogenic pathway.

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.     

Transformation of NIH 3T3 cells with basic fibroblast growth factor or the hst/K-fgf oncogene causes downregulation of the fibroblast growth factor receptor: reversal of morphological transformation and restoration of receptor number by suramin

When NIH 3T3 cells were transfected with the cDNA for basic fibroblast growth factor (bFGF), most cells displayed a transformed phenotype. Acquisition of a transformed phenotype was correlated with the expression of high levels of bFGF (Quarto et al., 1989). Cells that had been transformed as a result of transfection with bFGF cDNA had a decreased capacity to bind 125I-bFGF to high affinity receptors. NIH 3T3 cells transfected with bFGF cDNA that expressed lower levels of bFGF were not transformed and had a normal number of bFGF receptors. NIH 3T3 cells transfected with the hst/Kfgf oncogene, which encodes a secreted molecule with 45% homology to bFGF, also displayed a transformed phenotype and decreased numbers of bFGF receptors. However, NIH 3T3 cells transfected with the H-ras oncogene were transformed but had a normal number of bFGF receptors. Thus, transformation by bFGF-like molecules resulted in downregulation of bFGF receptors. Receptor number was not affected by cell density for both parental NIH 3T3 cells and transformed cells. In the cells transfected with bFGF cDNA that were not transformed, the receptors could be downregulated in response to exogenous bFGF. Conditioned medium from transformed transfected cells contained sufficient quantities of bFGF to downregulate bFGF receptors on parental NIH 3T3 cells. Thus, the downregulation of bFGF receptors seemed related to the presence of bFGF in an extracytoplasmic compartment. Treatment of the transformed transfected NIH 3T3 cells with suramin, which blocks the interaction of bFGF with its receptor, reversed the morphological transformation and restored receptors almost to normal numbers. These results demonstrate that in these cells bFGF transforms cells by interacting with its receptor and that bFGF and hst/K-fgf may use the same receptor.     

Autocrine growth stimulation by secreted Kaposi fibroblast growth factor but not by endogenous basic fibroblast growth factor

We studied the different potentials of a secreted and a nonsecreted member of the fibroblast growth factor (FGF) family to induce autocrine growth stimulation in human adrenal cortex carcinoma cells (SW-13). These epithelial cells express basic FGF (bFGF) cell surface receptors, and picomolar concentrations of bFGF suffice to induce anchorage-independent growth. The requirement for exogenously added bFGF contrasts with the intracellular storage of biologically active bFGF in SW-13 cells greater than 10,000-fold in excess of the concentration needed to stimulate anchorage independent growth. To study whether the expression of a secreted FGF would alter the growth phenotype of these cells, we transfected them with an expression vector coding for the Kaposi-fgf (K-fgf) oncogene. In contrast to controls, K-fgf-transfected cells secrete significant amounts of biologically active K-fgf protein into the growth media, show up to 50-fold increased colony formation in soft agar, and grow into rapidly progressing, highly vascularized tumors in athymic nude mice. A reversible inhibition of the autocrine growth stimulation in vitro is brought about by the polyanionic compound suramin. We conclude that FGF has to be released from SW-13 cells to function fully as a growth stimulator in vitro and in vivo.     

Cleavage of K-FGF produces a truncated molecule with increased biological activity and receptor binding affinity

The K-FGF/HST (FGF-4) growth factor is a member of the FGF family which is efficiently secreted and contains a single N-linked glycosylation signal. To study the role of glycosylation in the secretion of K-FGF, we mutated the human K-fgf cDNA to eliminate the glycosylation signal and the mutated cDNA was cloned into a mammalian expression vector. Studies of immunoprecipitation from the conditioned medium of cells expressing this plasmid revealed that the lack of glycosylation did not impair secretion, however the unglycosylated protein was immediately cleaved into two NH2-terminally truncated peptides of 13 and 15 kD, which appeared to be more biologically active than the wild-type protein. These two proteins also showed higher heparin binding affinity than that of wt K-FGF. We have expressed in bacteria the larger of these two proteins (K140), in which the NH2-terminal 36 amino acids present in the mature form of K-FGF have been deleted. Mitogenicity assays on several cell lines showed that purified recombinant K140 had approximately five times higher biological activity than wild-type recombinant K-FGF. Studies of receptor binding showed that K140 had higher affinity than wt K-FGF for two of the four members of FGF receptor's family, specifically for FGFR-1 (flg) and FGFR-2 (bek). K140 also had increased heparin binding ability, but this property does not appear to be responsible for the increased affinity for FGF receptors. Thus removal of the NH2-terminal 36 amino acids from the mature K-FGF produces growth factor molecules with an altered conformation, resulting in higher heparin affinity, and more efficient binding to FGF receptors. Although it is not clear whether cleavage of K-FGF to generate K140 occurs in vivo, this could represent a novel mechanism of modulation of growth factor activity.     

An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family

We recently reported the cloning of a rearranged human oncogene following transfection of DNA from Kaposi's sarcoma into NIH 3T3 cells. To identify the protein(s) encoded in two novel mRNAs of 3.5 and 1.2 kb expressed in NIH 3T3 transformants, we constructed a cDNA library. One of the cDNA clones isolated (KS3) corresponded to the 1.2 kb mRNA and transformed NIH 3T3 cell when inserted into a mammalian expression vector. The 1152 nucleotide KS3 cDNA encodes a protein of 206 amino acids with significant homology to the growth factors basic FGF and acidic FGF. Expression of the KS3 product as a bacterial fusion protein or in COS cells allowed us to determine that both proteins had significant growth-promoting activity and that the COS cell protein was glycosylated. Thus one of the mRNAs transcribed from the KS oncogene encodes a growth factor that could transform cells by an autocrine mechanism and appears to represent a new member of the FGF family.     

Retention of fibroblast growth factor 3 in the Golgi complex may regulate its export from cells.

The fibroblast growth factors (FGFs) fall into two distinct groups with respect to their mode of release from cells. Whereas FGF1 and FGF2 lack conventional signal peptides, the remaining members have typical features of secreted proteins. However, the behavior of mouse FGF3 is anomalous, since, despite entering the secretory pathway and undergoing primary glycosylation, its release from transfected COS-1 cells is very inefficient compared with that of FGF4 and FGF5. To investigate the unusual properties of FGF3, we analyzed the processing, secretion, and intracellular localization of a series of site-directed mutants as well as chimeras produced by fusing parts of FGF3, FGF4, and FGF5. Wild-type FGF3 was shown to accumulate in an immature form in the Golgi complex, from where it is slowly released into the extracellular matrix. Removing or relocating the Asn-linked glycosylation site further impaired its release, and exchanging the signal peptide or carboxy terminus had little effect. In contrast, a chimeric protein with an amino terminus from FGF5 was efficiently secreted and biologically active in cell transformation assays. The data suggest that a structural feature of FGF3 involving the amino-terminal region and glycosylation site has a significant bearing on its passage through the Golgi complex and may regulate the secretion of the ligand.     

A retrovirus carrying the K-fgf oncogene induces diffuse meningeal tumors and soft-tissue fibrosarcomas.

The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained all of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF.     

The sensitivity to growth factors of NIH 3T3 cells transformed by the v-myc oncogene

Transformation of NIH 3T3 cells, induced by v-myc oncogene, activates a proliferative potential of the cells cultivated in the serum-free medium, and reduces the ratio of 3H-Tdr incorporation into the cells grown in the presence of 10% fetal serum in comparison to those grown in the serum-free medium. The v-myc transformed cells (NIH 3T3-v-myc) as well as the untransformed ones are very responsive to insulin. On the other hand, the epidermal growth factor, able to stimulate proliferation of NIH 3T3 cells, exert no effects on the NIH 3T3-v-myc cells. The NIH 3T3-v-myc cells cultivated in the medium, containing 2.5% human plasma enriched with thrombocytes, have the same proliferative characteristics as cells grown in the thrombocyte-free plasma. It is concluded that transformation of NIH 3T3 cells induced by v-myc oncogene may reduce a requirement for thrombocyte-released growth factors and EGF but not for insulin.     

Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF.     

Growth of myoblasts in lipoprotein-supplemented,serum-free medium: Regulation of proliferation by acidic and basic fibroblast growth factor

Summary BC₃H1 myoblast cells seeded at low density on gelatin-coated dishes and exposed to a 1∶1 (vol/vol) mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, insulin, and basic or acidic fibroblast growth factor (FGF). This serum-free medium combination supported cell multiplication at a rate equal to that of serum-supplemented medium, and at low cell input (10³ cells/35-mm dish). It also allowed serial transfer of the cultures under serum-free conditions. HDL seems to promote cell survival and to act as progression factor allowing cells to divide when exposed to either basic or acidic FGF. When the potency of basic and acidic FGF were compared, acidic FGF was 20-fold less potent than basic FGF.     

Functional phylogeny relates LET-756 to fibroblast growth factor 9

Fibroblast growth factors (FGFs) are secreted regulatory proteins involved in various developmental processes. In vertebrates, the FGF superfamily comprises 22 members. In non-vertebrates, six FGF genes have been identified in Ciona intestinalis, three in Drosophila melanogaster, and two (let-756 and egl-17) in Caenorhabditis elegans. The core of LET-756 shares a 30-50% sequence identity with the various members of the superfamily. The relationships between vertebrate and non-vertebrate FGFs are not clear. We made chimeric FGFs by replacing the core region of LET-756 by the cores of various mammalian, fly, and worm FGFs. LET-756 deleted in its core region was no longer able to rescue the lethal phenotype of a let-756 null mutant, and only chimeras containing the cores of FGFs 9, 16, and 20 showed rescue capacity. This core contains an internal motif of six amino acid residues (EFISIA) whose deletion or mutation abolished both the rescue activity and FGF secretion in the supernatant of transfected COS-1 cells. Chimera containing the core of C. intestinalis FGF9/16/20, a potential ortholog of FGF9 lacking the complete EFISIA motif, was not able to rescue the lethal phenotype or be secreted. However, the introduction of the EFISIA motif restored both activities. The data show that the EFISIA motif in the core of LET-756 is essential for its biological activity and that FGFs 9, 16, and 20, which contain that motif, are functionally close to LET-756 and may be evolutionary related. This non-classical mode of secretion using an internal motif is conserved throughout evolution.     

Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction.

Fibroblast growth factor (FGF) receptor (FGFR) gene family consists of at least four receptor tyrosine kinases that transduce signals important in a variety of developmental and physiological processes related to cell growth and differentiation. Here we have characterized the binding of different FGFs to FGFR-4. Our results establish an FGF binding profile for FGFR-4 with aFGF having the highest affinity, followed by K-FGF/hst-1 and bFGF. In addition, FGF-6 was found to bind to FGFR-4 in ligand competition experiments. Interestingly, the FGFR-4 gene was found to encode only the prototype receptor in a region where both FGFR-1 and FGFR-2 show alternative splicing leading to differences in their ligand binding specificities and to secreted forms of these receptors. Ligands binding to FGFR-4 induced receptor autophosphorylation and phosphorylation of a set of cellular polypeptides, which differed from those phosphorylated in FGFR-1-expressing cells. Specifically, the FGFR-1-expressing cells showed a considerably more extensive tyrosine phosphorylation of PLC-gamma than the FGFR-4-expressing cells. Structural and functional specificity within the FGFR family exemplified by FGFR-4 may help to explain how FGFs perform their diverse functions.     

Transformed phenotype conferred to NIH/3T3 cells by ectopic expression of heparin-binding growth factor 1/acidic fibroblast growth factor

Summary Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF-1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels. This work was supported by grants from the National Cancer Institute (RO1 CA45611), The March of Dimes Birth Defects Foundation (No. 6-549) and The Ohio Cancer Research Associates, Inc. I.-M.C. is a recipient of The Research Career Development Award (KO4 CA01369) from the National Institutes of Health.     

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10⁶ cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin. This revised version was published online in July 2006 with corrections to the Cover Date.     

FGF3 from Xenopus laevis.

Fibroblast growth factor 3 (FGF3) was first identified as the product of a cellular oncogene activated by mouse mammary tumour virus but its normal role appears to be in the developing embryo. To gain further insights into its function, we have isolated sequences encoding the FGF3 homologue in Xenopus laevis, XFGF3. COS-1 cells transfected with XFGF3 cDNA express a 31 kDa product, p31, generated by signal peptide cleavage and Asn-linked glycosylation at the single consensus site. This product is secreted and becomes associated with the cell surface and extracellular matrix. Proteolytic cleavage of p31 in the extracellular compartment results in an amino-terminally truncated product, p27, that is also glycosylated. Both p31 and p27 bind quantitatively to heparin-Sepharose and can be displaced from the cell surface and extracellular matrix by soluble heparin. Conditioned medium containing these two proteins is capable of inducing transient morphological transformation of NIH3T3 cells and of stimulating DNA synthesis in quiescent C57MG and BALB/MK cells which express different isoforms of FGF receptors 1 and 2. Since XFGF3 behaves very differently from its mouse counterpart, we constructed chimeras in which amino-terminal sequences from XFGF3 were fused with carboxy-terminal sequences from mouse FGF3. Increasing the contribution from mouse FGF3 led to a more restricted host range for the chimeric ligand.     

Vitamin D enhances mitogenesis mediated by keratinocyte growth factor receptor in keratinocytes

The hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and keratinocyte growth factor (KGF) belong to the network of autocrine and paracrine mediators in the skin. Both were shown to modulate keratinocyte proliferation, to reverse epidermal atrophy, to increase wound healing, and to reduce chemotherapy-induced alopecia. The overlap between their activities may suggest that vitamin D exerts some of its actions by modulation of KGF activities in the skin. This notion was examined by using HaCaT keratinocytes cultured in serum-free medium in the absence of exogenous growth factors and in the presence of the EGF receptor tyrosine kinase inhibitor AG 1478 that blocks their autonomous proliferation. These cells could be stimulated to proliferate by different fibroblast growth factors (FGFs). The relative mitogenic efficacy of basic FGF, acidic FGF, or KGF was in correlation with their affinities for the KGF receptor (KGFR). Forty-eight hour co-treatment with 1,25(OH)(2)D(3) enhanced KGFR-mediated cell proliferation in a dose dependent manner. Both ERK1/2 and c-Jun N-terminal kinase (JNK) were activated by the FGFs. Treatment with 1,25(OH)(2)D(3) increased the activation of ERK but reduced the activation of JNK. Treatment with 1,25(OH)(2)D(3) increased the levels of KGFR in the presence but not in the absence of KGF, probably due to inhibition of ligand-induced receptor degradation. Inhibition of protein kinase C with bisindolylmaleimide did not interfere with the effect of 1,25(OH)(2)D(3) on KGFR-mediated ERK activation. Our results support the notion that the paracrine KGF-KGFR system in the skin can act in concert with the autocrine vitamin D system in keratinocytes to promote keratinocyte proliferation and survival under situations of stress and injury.     

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.