Expression and localization of members of the fibroblast growth factor family in the bovine mammary gland.

The goal of the study was to examine the expression and localization of members of the fibroblast growth factor family in the bovine mammary gland during different developmental and functional stages. Mammary tissue was obtained from German Brown Swiss cows (n = 23) during defined stages of mammogenesis (before and during pregnancy), lactogenesis, peak and late lactation, and involution. Extracted mRNA was analyzed by reverse transcription polymerase chain reaction and RNase protection assay. The tissue content of fibroblast growth factor-1 and fibroblast growth factor-2 was determined by radioimmunoassay, and the localization of fibroblast growth factor-2 was determined by immunohistochemistry. The highest mRNA concentration for fibroblast growth factor-1, -2, and -7 and their receptors was detected in the glands of virgin heifers or primigravid heifers during involution; less abundant fibroblast growth factor mRNA was detected during lactogenesis and galactopoiesis. Tissue protein concentrations of fibroblast growth factor-1 and fibroblast growth factor-2 showed similar tendencies. Immunoreactive fibroblast growth factor-2 was observed during mammogenesis and involution in endothelial cells, ductal epithelial cells, myoepithelial cells, and some alveolar cells. After positive staining, fibroblast growth factor-2 could only be observed in endothelial and myoepithelial cells during lactogenesis and could only be observed in myoepithelial cells during galactopoiesis. Expression, tissue concentration, and distinct localization suggest that fibroblast growth factors may be important in the local regulation of the bovine mammary gland.

VEGF receptor subtypes KDR and FLT1 show different sensitivities to heparin and placenta growth factor.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor which binds to two structurally related tyrosine kinase receptors denoted KDR and FLT1. To compare the interaction of VEGF with each receptor, cell lines which express individual receptor subtypes were identified using Northern blot hybridization. Bovine aortic endothelial (ABAE) cells and WM35 melanoma cells were found to express KDR, while FLT1 was primarily expressed on SK-MEL-37. Both receptor subtypes were detected on another melanoma cell line (WM9). Heparin augmented VEGF binding to KDR-expressing cells (ABAE and WM35), but inhibited VEGF binding to FLT1-expressing cells (SK-MEL-37 and WM9). The concentration of heparin required for half maximal stimulation of VEGF binding to KDR-expressing cells (500 ng/ml) was 25 times greater than that required for half maximal inhibition of binding to FLT1-expressing cells (20 ng/ml). In WM9 cells, the effect of heparin was bimodal; low concentration inhibited, while higher concentrations stimulated binding of 125I-VEGF. Placenta growth factor (PIGF-1) is a recently described growth factor structurally similar to VEGF. PIGF-1 had a negligible or no effect on 125I-VEGF binding to KDR-expressing cells (ABAE, WM35), but did complete for binding to FLT1-expressing cells (SK-MEL-37 and WM9). Addition of heparin had no effect on its ability to compete for binding with 125I-VEGF. The data indicate differential regulation of the two VEGF receptors by heparin and extended specificity of FLT1 receptor, but not KDR, for binding PIGF-1 growth factor.

Basic fibroblast growth factor and its receptor messenger ribonucleic acids are expressed in human ovarian epithelial neoplasms.

OBJECTIVE: Our purpose was to determine whether basic fibroblast growth factor is present in, and synthesized by, human ovarian epithelial neoplasms and to evaluate the expression of gene for the basic fibroblast growth factor receptor. STUDY DESIGN: The synthesis of basic fibroblast growth factor and its receptor was investigated in seven primary human ovarian epithelial neoplasms. Neoplastic tissues were homogenized and the cytoplasmic extracts purified by heparin-sepharose chromatography with a linear salt gradient of 0.6 to 3 mol/L sodium chloride in Tris-hydrochloric acid. The in situ synthesis of basic fibroblast growth factor and its receptor was demonstrated by polymerase chain reaction. Total ribonucleic acid was reverse transcribed and then amplified with two oligonucleotide primers specific for the bovine and human basic fibroblast growth factor gene and its human receptor gene. RESULTS: As assessed by both bioassay and radioimmunoassay a peak of basic fibroblast growth factor-like activity was present in all tumors in the chromatographic fractions eluted with 3 mol/L sodium chloride. The mitogenic effect on bovine adrenocortical endothelial cell proliferation varied from 35% to 153% above control cultures. Levels of basic fibroblast growth factor-like immunoreactivity were between 4 and 33 ng/ml. Qualitatively similar results were obtained after purifying the cytoplasmic extract of dispersed human ovarian tumor cells. The mitogenic effect was completely abolished by a specific neutralizing anti-basic fibroblast growth factor antibody. Single major deoxyribonucleic acid bands of the expected size (354 and 661 bp) were detected in all tumors studied. The identify of this material with the human basic fibroblast growth factor sequence was confirmed by restriction enzyme analysis. CONCLUSION: These data demonstrate that both basic fibroblast growth factor and its receptor are present in and synthesized by human ovarian tumor cells. Thus basic fibroblast growth factor might stimulate their abnormal proliferation through an autocrine mechanism.

Fos expression induces growth arrest in tumorigenic neuroretina cells.

E26-infected chicken neuroretina (CNR) cells expressing P135gag-myb-ets are non-transformed and growth can be stimulated by basic fibroblast growth factor (bFGF). In contrast, MHE226-infected CNR cells, which express p61/63myc in addition to the P135gag-myb-ets of E26, are transformed, tumorigenic cells and are growth inhibited by bFGF. We looked for differences in both cells types which could help to understand the molecular basis of the bFGF response. We found marked differences in c-fos expression. In MHE226-CNR cells c-fos mRNA was induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) and bFGF, both inhibitors of MHE226-infected cell growth. In contrast, serum, a strong growth inducer, did not stimulate c-fos expression. In E26-infected cells all of these factors induced cell growth and c-fos expression. MHE226-CNR cells superinfected with v-fosFBJ-expressing retrovirus were inhibited in their growth and unresponsive to bFGF. Introduction of v-fosFBJ in E26 CNR cells transformed them but they remained sensitive to bFGF. These results suggest that fos is involved in the induced growth arrest of MHE226-CNR cells.

v-erbA cooperates with bFGF in neuroretina cell transformation.

We have studied the transforming ability of the oncogenes erbA and/or erbB in chicken neuroretina (CNR) cells and the effect of basic fibroblast growth factor (bFGF) on the transformed phenotype. The erbA oncogene alone was only transforming in the presence of bFGF. In contrast, cells expressing erbB as well as erbA + erbB were transformed in a bFGF-independent manner and were unresponsive to the growth factor. We studied whether other oncogenes could also block the cooperation between erbA and bFGF. Cytoplasmic or membrane-bound oncogenes (src, ras, or mill raf) increased the transforming potential of erbA but rendered the cells unresponsive to bFGF. Conversely, the nuclear oncogenes tested (fos and myb-ets + myc) also cooperated with erbA in CNR cell transformation but the cells remained responsive to the growth factor. A likely explanation is that CNR cells carrying the cytoplasmic but not the nuclear oncogenes have already activated the bFGF signal transduction pathway.

Transcriptional regulation of vascular endothelial growth factor gene expression in ovarian bovine granulosa cells.

The expression of vascular endothelial growth factor (VEGF) in cultured bovine granulosa cells has been studied. As shown by northern blot analysis, granulosa cells express the VEGF gene. Analysis of the VEGF transcripts by the polymerase chain reaction technique shows that granulosa cells express predominantly the smallest VEGF coding forms (VEGF121 and VEGF164). Since in the promoter region of the VEGF gene there are four potential AP-1 sites and two potential AP-2 sites we have studied if TPA and forskolin could regulate VEGF gene expression. TPA induces VEGF transcription in a time- and dose-dependent fashion. Maximal VEGF mRNA levels are detected 6 h after TPA treatment. Induction apparently requires de novo protein synthesis since it does not occur when translation is inhibited by cycloheximide. Forskolin, a naturally occurring diterpene that activates adenylylcyclase, also increases VEGF mRNA content in a time-dependent manner. Induction does not require de novo protein synthesis and, in contrast to TPA, induction is strongly potentiated by cycloheximide. Luteotrophic hormone, a known activator of adenylylcyclase, also induces VEGF transcription. These results imply that granulosa cells may be a source of VEGF which could play a role in the angiogenic process associated with ovulation and corpus luteum formation.

Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.

Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.

Expression and purification of biologically active human OSF-1 in Escherichia coli.

OSF-1 (also known as pleiotrophin, HB-GAM, HBGF-8 or HBNF) is a heparin-binding, neurotrophic protein. Its tissue-specific expression in rats is developmentally regulated and the protein is highly conserved between species. The protein is believed to be involved in neuronal development. Previous experiments in our laboratory showed that OSF-1 is primarily expressed in brain and bone. The biological function of OSF-1 in bone is unknown. In order to overcome the limited availability of the native protein, we now report on the high-level expression of human OSF-1 in Escherichia coli. The protein is present in the form of inclusion bodies, which were isolated and solubilized. The partially purified protein was refolded and further purified employing heparin sepharose chromatography. N-terminal sequence determination revealed the same amino acid sequence as the natural mature protein. The isolated backfolded recombinant human OSF-1 did promote neurites outgrowth in primary cultures of cortical neurons.

Glycosylation of vascular endothelial growth factor is not required for its mitogenic activity.

We have stably expressed the cDNA encoding the 165 amino-acid long form of human vascular endothelial growth factor (VEGF) in BHK-21 cells. VEGF was partially purified from the conditioned medium of transfected cells using heparin-sepharose affinity chromatography. The partially purified VEGF was mitogenic for various types of endothelial cells and inhibited the binding of pure [125I]VEGF to its receptors. Western blot analysis, using anti-VEGF antibodies, revealed a 47 kDa VEGF homodimer in the partially purified VEGF fraction. Preincubation of the transfected cells with the N-glycosylation inhibitor tunicamycin resulted in the conversion of the 47 kDa VEGF homodimer into a smaller, deglycosylated form of 42 kDa. Partially purified preparations of the deglycosylated VEGF displayed a mitogenic activity that was similar to that of the glycosylated form and efficiently inhibited the binding of native [125I]VEGF to the VEGF receptors of bovine aortic arch derived endothelial cells.

Definition of functional domains in P135gag-myb-ets and p48v-myb proteins required to maintain the response of neuroretina cells to basic fibroblast growth factor.

The v-myb- and v-ets-containing E26 retrovirus induces the proliferation of chicken neuroretina (CNR) cells in minimal medium. Proliferation of E26 CNR cells is strongly stimulated by basic fibroblast growth factor (bFGF). The v-myb-containing avian myeloblastosis virus also induces the proliferation of infected CNR cells stimulated by bFGF. Both E26 CNR and avian myeloblastosis virus CNR cells are able to form colonies in soft agar in the presence of bFGF. This suggests that the v-myb product, a nuclear sequence-specific DNA-binding protein which activates gene expression in transient transfection assays, plays a role in the proliferative response of the infected CNR cells. To determine the structure-function relationships of P135gag-myb-ets and p48v-myb, we have used deletion mutants expressed in retroviral vectors and have analyzed their effect on CNR cell proliferation as well as their effect on the CNR cell response to bFGF. We show that v-ets is not required for bFGF stimulation but increases the proliferation of CNR cells in minimal medium. In the v-myb mutants, the gag sequences derived from the helper virus increase the potency of the myb gene. The carboxyl-terminal domain required for the growth and transformation of myeloid cells and needed for maximal trans-activation in transient DNA transfection assays in fibroblasts was not required for the growth and bFGF response of CNR cells. In contrast, the domain encompassing amino acids 240 to 301 (containing part of the transcriptional activation domain of v-myb) was absolutely required for the response of CNR cells to bFGF and could be functionally replaced by the carboxyl-terminal transcriptional activation domain of the VP16 protein of herpes simplex virus.

The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing.

Vascular endothelial growth factor (VEGF) is an apparently endothelial cell-specific mitogen that is structurally related to platelet-derived growth factor. By Northern blot and protein analyses, we show that VEGF is produced by cultured vascular smooth muscle cells. Analysis of VEGF transcripts in these cells by polymerase chain reaction and cDNA cloning revealed three different forms of the VEGF coding region, as had been reported in HL60 cells. The three forms of the human VEGF protein chain predicted from these coding regions are 189, 165, and 121 amino acids in length. Comparison of cDNA nucleotide sequences with sequences derived from human VEGF genomic clones indicates that the VEGF gene is split among eight exons and that the various VEGF coding region forms arise from this gene by alternative splicing: the 165-amino-acid form of the protein is missing the residues encoded by exon 6, whereas the 121-amino-acid form is missing the residues encoded by exons 6 and 7. Analysis of the VEGF gene promoter region revealed a single major transcription start, which lies near a cluster of potential Sp1 factor binding sites. The promoter region also contains several potential binding sites for the transcription factors AP-1 and AP-2; consistent with the presence of these sites, Northern blot analysis demonstrated that the level of VEGF transcripts is elevated in cultured vascular smooth muscle cells after treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate.

Basic fibroblast growth factor expression is regulated by corticotropin in the human fetal adrenal: a model for adrenal growth regulation.

Human fetal adrenal growth after midgestation is very rapid and appears to be dependent upon pituitary adrenocorticotropin (ACTH) in vivo. We hypothesized that the regulation of fetal adrenal growth by ACTH is mediated by ACTH-stimulated local growth factor production. As we have found basic fibroblast growth factor (bFGF) to be a potent mitogen for human fetal adrenal cells in culture, we conducted studies to determine whether bFGF is synthesized by the human fetal adrenal gland and whether bFGF gene expression in primary cultures of human fetal adrenal cells is regulated by ACTH. Bioassayable bFGF-like activity was detected in extracts of whole human fetal adrenal glands and primary cultures of human fetal adrenal cells. Northern blot analysis of total RNA from whole human fetal adrenal glands revealed a characteristic 7-kilobase bFGF mRNA, indicating that the fetal adrenal bFGF bioactivity was most likely due to local synthesis. Slot blot and ribonuclease protection analysis showed that bFGF mRNA was present in very low amounts in total RNA from primary cultures of unstimulated human fetal adrenal cells but was increased 2- to 3-fold in cells exposed to 10 nM ACTH-(1-24) or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate for 24 hr. bFGF mRNA was localized to adrenocortical cells and not fibroblasts by in situ hybridization. bFGF mRNA was barely detectable in unstimulated cells, whereas it was markedly increased in cells exposed to either ACTH or 8-bromoadenosine 3',5'-cyclic monophosphate. These data support our hypothesis that the regulation of human fetal adrenal growth by ACTH at midgestation may be mediated by the stimulation of local growth factor production, and we suggest that bFGF may play a major role in this process.

Characterization of the receptors for vascular endothelial growth factor.

Vascular endothelial growth factor (vEGF) is a recently discovered mitogen for endothelial cells. It is also a potent angiogenic factor. We have characterized the vEGF receptors of endothelial cells using both binding and cross-linking techniques. Scatchard analysis of equilibrium binding experiments revealed two types of high-affinity binding sites on the cell surfaces of bovine endothelial cells. One of the sites has a dissociation constant of 10(-12) M and is present at a density of 3 x 10(3) receptors/cell. The other has a dissociation constant of 10(-11) M, with 4 x 10(4) receptors/cell. A high molecular weight complex containing 125I-vEGF is formed when 125I-vEGF is cross-linked to bovine endothelial cells. This complex has an apparent molecular mass of 225 kDa. Two other faintly labeled complexes with apparent molecular masses of 170 and 195 kDa also are detected. Reduction in the presence of dithiothreitol causes a substantial increase in the labeling intensity of the 170- and 195-kDa complexes, suggesting that these complexes are derived from the 225-kDa complex by reduction of disulfide bonds. The labeling of the vEGF receptors was inhibited by an excess of unlabeled vEGF but not by high concentrations of several other growth factors. Suramin and protamine, as well as several species of lectins, inhibited the binding. The expression of functional vEGF receptors was inhibited when the cells were preincubated with tunicamycin, indicating that glycosylation of the receptor is important for the expression of functional vEGF receptors. Pretreatment with swainsonine on the other hand, did not prevent formation of functional receptors. However, the mass of the 225-kDa complex is decreased by 20 kDa when 125I-vEGF is cross-linked to swainsonine-treated endothelial cells.

Iris-derived melanocytes contain a growth factor that resembles basic fibroblast growth factor.

A melanocyte growth stimulating factor has been purified from bovine iris melanocytes and identified as being closely related to the basic form of fibroblast growth factor (bFGF). This conclusion was based on the behavior of the melanocyte-derived growth factor when submitted to heparin-Sepharose affinity chromatography, as well as on its ability to cross-react with bFGF in radioimmuno- and radioreceptor assays. The ability of neutralizing bFGF antibodies to block cell proliferation in response to the melanocyte growth factor further confirms that it is closely related to bFGF. Since melanocytes express the 3.7-kb and 7.0-kb bFGF transcript, the possibility exists that uncontrolled expression of melanocyte-derived bFGF could be involved in the malignant transformation of melanocytes into melanoma cells.

Fibroblast growth factor. Chemical structure and biologic function.

Basic and acidic fibroblast growth factor (FGF) are two closely related growth factors that interact with the same receptor. Structurally related to FGF are five different oncogenes that have been involved in mammary, stomach, and bladder cancers but whose precise functions in physiologic processes are presently unknown. Both basic and acidic FGF exert similar biologic effects on a wide range of mesoderm- and neuroectoderm-derived cells, acting both as morphogens and mitogens. In addition, basic FGF has been shown to act as a ventrovegetalizing factor, inducing, at the midblastulae stage, mesoderm formation from animal pole cells destined to form ectodermal structures. This primordial role explains the wide variety of tissues, ranging from the central and peripheral nervous system to the vascular system, that are affected by FGF during the late embryonic, neonatal, and adult phases of development.

Comparison of the ability of basic and acidic fibroblast growth factor to stimulate the proliferation of an established keratinocyte cell line: modulation of their biological effects by heparin, transforming growth factor beta (TGF beta), and epidermal growth factor (EGF).

The bioactivity of both bFGF and aFGF in the BALB/MK-1 cell line has been compared to that of EGF. Our results indicate that, for that cell type, aFGF was far more potent than bFGF in inducing cell proliferation. In the presence of heparin, aFGF was as potent as EGF. In addition, excess bFGF has an inhibitory effect on the proliferation of MK cells exposed to a saturating concentration of aFGF, therefore acting as a partial agonist of aFGF. Surprisingly, bFGF, although it had low biological activity, was capable of synergizing the effect of EGF. In its presence, cultures exposed to saturating concentration of EGF have a final cell density 3- to 4-fold higher than that of counterpart cultures exposed to EGF alone. TGF beta, which in previous studies has been shown to inhibit the growth of keratinocytes, also inhibited the growth of BALB/MK-1 cells in response to either bFGF or aFGF. These studies suggest a role for FGF in regulating BALB/MK proliferation. aFGF provides positive growth signals which can be negatively modulated by excess bFGF or TGF beta, while bFGF, although a poor mitogen, could act by potentiating the effect of subsaturating concentrations of EGF.

Vascular endothelial growth factor: a new member of the platelet-derived growth factor gene family.

Using applications of the polymerase chain reaction (PCR) technique, cDNA clones have been isolated encoding bovine vascular endothelial growth factor (VEGF), a mitogen with specificity for vascular endothelial cells. Analysis of the clones indicates that VEGF can exist in two forms, probably due to alternative RNA splicing. The amino acid sequences predicted from the clones also show that VEGF shares homologies of about 21% and 24% respectively with the A and B chains of human platelet-derived growth factor (PDGF), and has complete conservation of the eight cysteine residues found in both mature PDGF chains. The homology is not reflected in function, however, since the cell types responsive to VEGF are distinct from those responsive to homo- and heterodimers of the PDGF chains.