Regulation of fibroblast growth factor 2 and fibroblast growth factor receptors by transforming growth factor beta in human osteoblastic MG-63 cells.

Fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) are important regulators of bone cell function. Although FGF-2 is a major modulator of bone cell function, its expression and regulation in human osteoblasts have not been investigated. We examined FGF-2 messenger RNA (mRNA) expression and regulation in the human osteosarcoma MG-63 cells. Northern analysis revealed that MG-63 cells expressed FGF-2 mRNA transcripts of 7, 4, 2.2, and 1.3 kilobases (kb). In the absence of serum, treatment with transforming growth factor beta (TGF-beta; 0.1-10 ng/ml) increased all FGF-2 mRNA transcripts. Maximal increase was seen with 1 ng/ml of TGF-beta. TGF-beta increased FGF-2 mRNA expression within 2 h and this was sustained for 24 h. Phorbal myristate acetate (PMA; 1 microM) also increased FGF-2 mRNA at 6 h. Time course studies showed that TGF-beta did not significantly alter FGFR1 or FGFR2 mRNA expression in MG-63 cells. Western blotting with anti-human FGF-2 revealed that MG-63 cells synthesize three isoforms of FGF-2 protein of approximately 18, 22/23, and 24 kDa, which were increased after either 6 h or 24 h of treatment with TGF-beta. Increased FGF-2 mRNA and protein expression in response to TGF-beta was markedly reduced by the protein kinase A (PKA) inhibitor H-89. Immunogold labeling of MG-63 cells treated with TGF-beta showed increased labeling for FGF-2 and FGFR2 in the nuclei. In contrast, TGF-beta treatment significantly decreased FGFR1 labeling in the nuclei. These data show that TGF-beta regulates FGF-2 gene expression in human osteosarcoma cells. Furthermore, TGF-beta modulates the cellular localization of FGF-2 and its receptors.

Interleukin-1 regulates FGF-2 mRNA and localization of FGF-2 protein in human osteoblasts.

Interleukin-1 (IL-1) and basic fibroblast growth factor (FGF-2) are potent stimulators of osteoclast formation. However, the role of FGF-2 in the responses to IL-1 in bone has not been reported. We examined the effect of IL-1 on FGF-2 mRNA and protein expression in human osteosarcoma MG-63 osteoblasts, normal human osteoblasts (NHOB), and osteoblasts from osteoarthritic patients (F2 and F13). IL-1 increased FGF-2 mRNA expression in osteoblasts within 1.5 to 3 h. Multiple FGF-2 protein isoforms were expressed in human osteoblasts. Twenty-four hours of treatment of MG-63 and NHOB cells with IL-1 increased the high-molecular-weight(HMW, 22/24 kDa) and low-molecular-weight (LMW, 18 kDa) FGF-2 proteins intracellularly. In contrast, IL-1 preferentially increased the LMW protein signal intracellularly as well as on the cell surface of F2 and F13 osteoblasts. We conclude that IL-1 is a major stimulator of FGF-2 expression in human osteoblasts. Furthermore, selective increases in the exportable LMW protein in osteoblasts from osteoarthritic patients may be of clinical relevance.

CUG-initiated FGF-2 induces chromatin compaction in cultured cardiac myocytes and in vitro.

Fibroblast growth factor-2 (FGF-2) is a mitogen found in CUG-initiated 21-25 kDa ("hi") or AUG-initiated 16-18 kDa ("lo") forms. Previously we demonstrated that "hi"-but not "lo"-FGF-2 caused a distinct nuclear phenotype characterized by apparently condensed chromatin present as separate clumps in the nucleus of cardiac myocytes. In this manuscript we investigated whether these effects were related to apoptosis or mitosis and whether they reflected a direct effect of "hi" FGF-2 on chromatin. Myocytes overexpressing "hi" FGF-2 and presenting the clumped chromatin phenotype: (i) were not labeled above background with antibodies to phosphorylated histones H1 and H3 used as indicators of mitotic chromatin condensation; (ii) did not stain positive for TUNEL; (iii) their nuclear lamina, visualized by anti-laminB immunofluorescence, appeared intact; (iv) neither caspase inhibitors, nor Bcl-2 or "lo" FGF-2 overexpression prevented the manifestation of the compacted nuclear phenotype. Purified recombinant "hi" FGF-2 was more potent than "lo" FGF-2 in promoting the condensation/aggregation of chick erythrocyte chromatin partially reconstituted with histone H1 in vitro. We conclude that the DNA phenotype induced by "hi" FGF-2 in cardiac myocytes likely reflects a direct effect on chromatin structure that does not require the engagement of mitosis or apoptosis. By affecting chromatin compaction "hi" FGF-2 may contribute to the regulation of gene expression.

Translocation of FGF2 to the cell surface without release into conditioned media.

Like most cells in culture, stably transfected COS-1 cells (CF18) that constitutively overexpress basic fibroblast growth factor (FGF2) do not release the growth factor into conditioned media. Yet, when cells were biotinylated, 30% of the total cell-associated immunoreactive FGF2 was detected on the cell surface. Under similar conditions, up to 70% of the total immunoreactive FGF2 in transfected endothelial cells (MAE ZIP) or untransfected rat (C6) and human (U87MG) glioblastoma cell lines was detected on their cell surface. When peripheral plasma membrane proteins were removed from the cell surface with 0.1 M sodium carbonate, the amount of exported FGF2 was significantly reduced, whereas cell viability was unaffected. FGF2 then reappeared on the cell surface in a time-dependent manner. Ouabain, a cardenolide previously shown to inhibit the export of FGF2 from transiently transfected COS-1 cells, blocked the appearance of FGF2 onto the surface of transfected CF18 cells and MAE ZIP cells but had no detectable effect on C6 and U87MG cells. The observation that the translocation of FGF2 onto the cell surface is dissociated from its release into conditioned medium is consistent with FGF2's being rarely found in biological fluids but always cell associated and in the extracellular matrix. The findings point to a role played by the protein export pathway in controlling FGF2 activity and the normal physiological function that this growth factor plays in cell growth and differentiation. The widely accepted presumption that the absence of FGF2 in conditioned media reflects its inability to exit the cell needs to be reevaluated.

Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals.

Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nuclear-targeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways.

Transcriptional regulation of fibroblast growth factor-2 expression in human astrocytes: implications for cell plasticity.

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs identified a unique -555/-513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na+,K+-ATPase.

Basic fibroblast growth factor (FGF-2) is one of a select group of proteins that can exit cells through an alternate, endoplasmic reticulum/Golgi apparatus independent exocytic pathway. This alternate pathway has been termed protein export. In an attempt to better understand this process, we have identified a family of related compounds, "cardenolides," that inhibit FGF-2 export. The cardenolides inhibit FGF-2 export in a time and concentration dependent fashion. Inhibition of FGF-2 export is specific in that the cardenolides have no effect on conventional protein secretion as measured by their inability to block release of the secreted protein human chorionic gonadotropin-alpha. Because cardenolides are known to inhibit ion transport activity mediated by Na+,K+-ATPase, we investigated whether there are functional interactions between FGF-2 and their only known molecular target: the alpha-subunit of Na+, K+-ATPase. Export of FGF-2 from COS-1 cells is selectively inhibited when co-transfected with expression vectors encoding the alpha-subunit and FGF-2. Moreover, antibodies to the alpha-subunit specifically co-immunoprecipitate FGF-2 along with the alpha-subunit while conversely, antibodies to FGF-2 specifically co-immunoprecipitate the alpha-subunit along with FGF-2. Finally, the ion transporting activities of the Na+,K+-ATPase can be uncoupled from protein export. Varying the external concentration of K+ has little effect on export of FGF-2. Taken together, these data: 1) identify a novel activity for cardenolides; 2) suggest a previously unknown role for the alpha-subunit of Na+, K+-ATPase in FGF-2 export; and 3) raise the possibility that the alpha-subunit itself may be an integral component of this alternate exocytic pathway mediating translocation of cytosolic FGF-2 to the cell surface.

Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cells.

FGF-2 has been implicated in the neoplastic transformation of glioma cells and in the transition of normal quiescent astrocytes to a proliferating, reactive state. In the present study we have observed that in human glial cells, levels and subcellular localization of FGF-2 are different in quiescent and proliferating cells. FGF-2 was detected in the cytoplasm of non-reactive astrocytes in human brain sections. In contrast FGF-2 was located within the cytoplasm and nuclei of reactive astrocytes in gliotic brain tissue and in neoplastic cells of glioma tumors. In vitro, FGF-2 was found predominantly in the nucleus of subconfluent proliferating astrocytes, but was detected only in the cytoplasm of density arrested quiescent astrocytes. Our results suggest that reduced cell contact stimulates nuclear accumulation of FGF-2, accompanying mitotic activation of reactive human astrocytes. FGF-2 was constitutively localized to the nucleus of continuously proliferating glioma cells independent of cell density. A role for intracellular FGF-2 was further suggested by the observation that glioma cells that are not stimulated to proliferate by extracellular FGF-2 proliferated faster when transfected with FGF-2 expressing vectors. This increased proliferation correlated with nuclear accumulation of FGF-2. Cell proliferation was attenuated by 5'-deoxy-5'-methylthioadenosine, a FGF-2 receptor tyrosine kinase inhibitor that acts within the cell, but was unaffected by myo-inositol hexakis [dihydrogen phosphate] that disrupts FGF-2 binding to plasma membrane receptors. Our results indicate that FGF-2 serves as a nuclear regulator of proliferation in astrocytic cells. In glioma cells, the constitutive presence of FGF-2 in the nucleus may promote proliferation that is insensitive to cell contact inhibition.

Promoter regions involved in density-dependent regulation of basic fibroblast growth factor gene expression in human astrocytic cells.

Expression of mitogenic basic fibroblast growth factor (bFGF) in the central nervous system is inhibited by direct cell contact and is implicated in reactive and neoplastic transformation of astrocytes. The molecular mechanisms controlling expression of bFGF were examined in cultures of human astrocytes. Cell-density-dependent depletion of bFGF mRNA levels parallels changes in bFGF gene protein. Regulation of transcription of a bFGF luciferase reporter gene containing an upstream region (bp -1800 to +314) of the bFGF gene promoter mimicks the density-dependent regulation of the endogenous bFGF gene in transfected astrocytes. Deletion analysis has identified a fragment (bp -650 to -513) and sequences further downstream (bp -274 to +314) as the regions required for the regulation of bFGF gene activity by cell density. Unlike in astrocytes, changing the cell density of glioma cell cultures does not affect the levels of bFGF protein and mRNA. bFGF luciferase constructs were expressed at the same level in high- or low-density cultures of glioma cells, indicating altered regulation of the bFGF gene promoter. Electrophoretic mobility shift assays showed binding of nuclear proteins to a fragment of bFGF gene promoter from bp -650 to -453. This binding was abolished by a deletion of the upstream cell-density-responsive region (bp -650 to -512). Binding was observed with nuclear extracts from subconfluent astrocytes but was reduced in extracts from confluent astrocytes. Our results indicate that induction of bFGF in astrocytes upon reduction of cell density is mediated transcriptionally by positive trans-acting factors interacting with bFGF promoter. In contrast, nuclear proteins from glioma cells bind to the promoter region from bp -650 to -453 independent of cell density. Thus, the constitutive binding of trans-acting factor(s) to the region of the bFGF promoter from bp -650 to -453 may be responsible for the continuous expression of bFGF that leads to the uncontrolled growth of glioma cells.

Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.

Regulation of growth factor mRNA levels in the eyes of diabetic rats.

The underlying etiology of diabetic microvascular disease remains unknown. To examine the potential contribution of basic fibroblast growth factor (bFGF), which is an angiogenic factor, and insulin-like growth factor-I (IGF-I) to the development of diabetic microvascular disease, bFGF and IGF-I mRNA levels were measured in tissues of control, diabetic, and insulin-treated diabetic rats. Diabetes was induced in rats by intravenous injection of streptozotocin (STZ) 65 mg/kg, and the rats were maintained for 21 days. bFGF mRNA levels increased threefold in the eyes of diabetic versus control rats, whereas a consistent change in bFGF mRNA levels was not observed in other tissues. In contrast, IGF-I mRNA levels decreased in the eyes and other tissues, including kidney, lung, and skeletal muscle, of diabetic as compared with control rats. Insulin treatment prevented the diabetes-induced increase in bFGF and decrease in IGF-I mRNA levels. Acidic FGF (aFGF) mRNA levels were unchanged in eyes from diabetic versus control rats. In partially purified retinas, diabetes increased bFGF mRNA levels twofold as compared with levels in control retinas, whereas IGF-I mRNA levels decreased to 58% of control levels in retinas from diabetic rats. Insulin treatment again prevented the diabetes-induced increase in IGF-I mRNA levels in the retina but had no effect on the diabetes-induced increase in bFGF mRNA levels. bFGF peptide levels were minimally increased in diabetic versus control retinas.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative export of FGF-2 occurs through an alternative, energy-dependent, non-ER/Golgi pathway.

Although basic fibroblast growth factor (bFGF/FGF-2) is found outside cells, it lacks a conventional signal peptide sequence; the mechanism underlying its export from cells is therefore unknown. Using a transient COS-1 cell expression system, we have identified a novel membrane-associated transport pathway that mediates export of FGF-2. This export pathway is specific for the 18-kD isoform of FGF-2, is resistant to the anti-Golgi effects of Brefeldin A, and is energy-dependent. In FGF-2-transfected COS-1 cells, this ER/Golgi-independent pathway appears to be constitutively active and functions to quantitatively export metabolically-labeled 18-kD FGF-2. Co-transfection and co-immunoprecipitation experiments, using a vector encoding the cytoplasmic protein neomycin phosphotransferase, further demonstrated the selectivity of this export pathway for FGF-2. When neomycin phosphotransferase was appended to the COOH-terminus of 18-kD FGF-2, the chimera was exported. However, the transmembrane anchor sequence of the integral membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) blocked export. The chimeric protein localized to the plasma membrane with its FGF-2 domain extracellular and remained cell-associated following alkaline carbonate extraction. Taken together, the data suggest that FGF-2 is "exported" from cells via a unique cellular pathway, which is clearly distinct from classical signal peptide-mediated secretion. This model system provides a basis for the development and testing of therapeutic agents which may block FGF-2 export. Such an intervention may be of considerable use for the treatment of angiogenesis-dependent diseases involving FGF-2.

Regulation of bFGF gene expression and subcellular distribution of bFGF protein in adrenal medullary cells.

Basic fibroblast growth factor (bFGF), a potent mitogenic/neurotrophic factor, controls the development and plasticity of many types of neural cells. In adrenal chromaffin cells, the appearance of bFGF protein coincided with the establishment of functional innervation, suggesting induction by trans-synaptic signals. In cultured bovine adrenal medullary cells Western blot analysis revealed 18-, 23-, and 24-kD bFGF isoforms in the cytosolic and nuclear fractions. Stimulation of acetylcholine nicotinic receptors or hormonal angiotensin II receptors or the direct stimulation of adenylate cyclase with forskolin or protein kinase C (PKC) with PMA increased the content of all bFGF isoforms. Increases in the levels of intracellular bFGF did not result in detectable presence of bFGF proteins in culture medium. Instead, bFGF proteins accumulated in the cytoplasm or the nucleus depending on whether PKC or cAMP pathways were activated. The long-term nuclear forskolin-induced accumulation of bFGF was prevented by cycloheximide or by antisense bFGF oligonucleotide and was also accompanied by an increase in bFGF mRNA. We used luciferase reporter plasmids containing the human bFGF promoter to show that the induction of bFGF resulted from transcriptional activation of the bFGF gene and was mediated by regulatory sequences located upstream from its transcription start site. Stimulation of bFGF gene expression by forskolin and PMA was synergistic and was mediated through different promoter regions. The results suggest that stimulation by cAMP and PKC is mediated through novel cis elements. The regulation of bFGF protein content also involves posttranscriptional mechanisms since changes in the levels of individual bFGF isoforms were different depending on whether cells were treated with carbachol or angiotensin II, forskolin, or PMA. The present study indicates that bFGF is an intracrine cytoplasmic-nuclear factor, whose expression is regulated by trans-synaptic and hormonal stimuli and which may act as a direct mediator of genomic responses to afferent stimulation.

Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells.

Mutations of the p53 gene are found in various human cancers. The frequency of its mutation is reported to increase during tumor progression in most tumors. In human gliomas, mutations of the p53 gene are found in about one-third of the malignant forms and in few of the benign ones, indicating their possible involvement in tumor progression. On the other hand, we have recently shown that basic fibroblast growth factor (basic FGF) plays a crucial role in tumor progression as an autocrine growth factor in tissues of human gliomas. Therefore, we hypothesized that p53 might regulate the promoter activity of the basic FGF gene, which has several GC boxes and no typical TATA box. In this study, cotransfection assays using human glioblastoma and hepatocellular carcinoma cells and establishment of stable cell lines expressing mutant-type p53 were performed. The basic FGF gene promoter was demonstrated to be regulated by p53 at the transcriptional level and its basal core promoter was found to be responsive to p53. Expression of endogenous basic FGF was also demonstrated to be activated by mutant type p53. Wild-type p53 repressed gene expression of the basic FGF and its mutant activated it in vitro, implying one of the possible pathways in tumor progression.

Basic fibroblast growth factor (bFGF) regulates tyrosine hydroxylase and proenkephalin mRNA levels in adrenal chromaffin cells.

bFGF is a neurotrophic protein expressed in various regions of the adult peripheral and central nervous system. The present study was undertaken to examine the role of bFGF in multihormonal, catecholaminergic and enkephalinergic cells of the adrenal medulla (AM). Western blot analysis revealed the presence of at least three bFGF isoforms (18, 22/23, and 24 kDa) in cultured bovine AM cells. Incubation of AM cells with the exogenous 18 kDa bFGF produced time-dependent increases in tyrosine hydroxylase (TH) and proenkephalin (PEK) mRNA, with maximal changes occurring at 12 h (TH) or 24 h (PEK) of bFGF exposure. Effects of bFGF on TH and PEK mRNA were non-additive with increases induced by exposure of AM cells to nicotine, the depolarizing agent veratridine, or the adenylate cyclase activator forskolin. These data indicate that bFGF effects may occur through intracellular pathways accessed during transsynaptic induction of TH and PEK genes. The increases in PEK mRNA induced by nicotine or bFGF were inhibited by the calcium antagonist TMB-8. TMB-8 also inhibited bFGF-induced increases in TH mRNA as well. However, treatment with TMB-8 increased basal levels of TH mRNA. The addition of bFGF increased endogenous levels of c-fos mRNA, c-Fos and c-Fos-related proteins, suggesting that bFGF may activate TH and PEK gene expression through a calcium-AP1 transcriptional regulatory pathway. Immunohistochemical analysis revealed the presence of bFGF-immunoreactivity (bFGF-IR) in the cytoplasm and in the nucleus of AM cells. Incubation of cells with exogenous bFGF produced time-dependent increases of nuclear bFGF-IR.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in basic fibroblast growth factor RNA and protein levels in human primary melanocytes and metastatic melanoma cells.

Cultivation of human melanocytes requires several growth factors for cell proliferation. For example, basic fibroblast growth factor (bFGF) is an essential growth agent for melanocyte proliferation in vitro and has been proposed to be an autocrine growth factor in human melanoma cells. Studies using either anti-bFGF antibodies or antisense oligonucleotides partially inhibited the proliferation of human melanoma cells. However, one group was unable to detect bFGF RNA transcripts in human melanoma cells using a human complementary DNA probe. These contradictory results prompted us to investigate the bFGF gene expression in human primary melanocytes and metastatic melanoma cells using Southern, Northern, and Western blot analyses. No gross rearrangements in the bFGF gene were detected in the genomic DNA. Although high levels of bFGF RNA transcripts were detected in melanocytes, no bFGF protein was detected using Western blot analysis. In contrast, melanoma cells expressed much lower levels of bFGF RNA transcripts, and cells from three of four cell strains synthesized the multiple isoforms of bFGF protein. In one of the melanoma cell strains, no bFGF protein was detected using Western blot analysis. Although three of four melanoma cell strains expressed bFGF protein, this molecule does not appear to function as an autocrine growth factor, and expression of the bFGF protein was not a consistent alteration in all melanoma cell strains.

Basic fibroblast growth factor in cells derived from Dupuytren's contracture: synthesis, presence, and implications for treatment of the disease.

Dupuytren's contracture (DC) is associated with fibroblast and endothelial cell proliferation. We have identified a fibroblast and endothelial cell mitogen, basic fibroblast growth factor (FGF), in cells derived from this tissue and characterized the effects of this growth factor on DC cells. Northern blot analysis of DC cells reveals the presence of basic FGF mRNA species, and the DC cells coexpress multiple forms of basic FGF. Radioreceptor assays establish that the DC cells have high-affinity binding sites for basic FGF and proliferate in response to exogenous recombinant basic FGF. Furthermore, a conjugate between saporin (a ribosome-inactivating protein) and basic FGF, which is cytotoxic to cells possessing the basic FGF receptor, is also cytotoxic to DC cells. The possibility that basic FGF-saporin could be a potential therapeutic agent for prevention of recurrence of the disease after surgery is discussed.

Selective expression of high molecular weight basic fibroblast growth factor confers a unique phenotype to NIH 3T3 cells.

The phenotypes of NIH 3T3 cells transfected with basic fibroblast growth factor (bFGF) cDNAs that express only the high molecular weight (HMW) forms of bFGF, the 18-kDa form, or all forms were examined. Cells producing the 18 kDa or all forms of bFGF were transformed at high levels of growth factor expression but were nontransformed at low levels. Cell producing low levels of HMW forms of bFGF were growth impaired when compared with the parental cells. These cells tended to form multinucleated giant cells, did not grow in soft agar, were nontumorigenic, had a normal bFGF receptor number, and had a nontransformed morphology. Cells expressing high levels of HMW bFGFs had a transformed morphology and were tumorigenic. These data suggest a specific functional role for HMWbFGF.

Multiple forms of bFGF: differential nuclear and cell surface localization.

The single copy gene for human basic fibroblast growth factor (bFGF) has been shown to encode not one but multiple proteins of 24, 23, 22 and 18 kD. Although bioactivities of the 18 kD protein are currently used to define bFGF gene function, it is not yet known if the three larger proteins have these same bioactivities or whether they will serve to define new bFGF gene functions. In this report we present a comparative study describing the de novo synthesis, transport, processing and intracellular location of individual bFGF isoforms. Data from cDNA mutagenesis and COS cell expression experiments show that individual isoforms are differentially localized to either the cell surface or to the nucleus. The 24, 23 and 22 kD proteins (CUG-mediated initiation) exclusively localize in the nucleus while the 18 kD protein (AUG-mediated initiation) is preferentially exported onto the cell surface, but is not released into the surrounding culture medium. Specific CUG or AUG translation initiation codons are necessary and sufficient for the synthesis of each isoform examined and thereby, indirectly, mediate differential localization. Since bFGF does not contain the characteristic signals predicted for cell surface or nuclear targeting, our continuing studies will either unmask its functionally equivalent domain(s) or will identify the requisite participation of yet unknown cellular components.