Member Of FGF Family Research Articles

Anabolic effect of aminoterminally truncated fibroblast growth factor 4 (FGF4) on bone

Fibroblast growth factor 4 (FGF4), a member of the FGF family, plays several important roles in bone development during embryogenesis. Systemic administration of FGF4 increases bone mass in rats, which suggests the potential therapeutic usefulness of this growth factor in treatment for osteopenia and in bone regeneration. We investigated the length of FGF4 required to exert its anabolic effects, because this information may be useful in developing new molecules to mimic the effects of FGF4. Because the active site of FGF family molecules is in the carboxylterminal region, we produced aminoterminally truncated recombinant human FGF4s (rhFGF4s) of different sizes. Human FGF4 cDNA containing almost the full length of the coding region (573 bp, 191 amino acid residues) was inserted into pUC18 vector and then deleted from the 5′ end using the ExoIII system. Each of the deleted FGF4 cDNAs was subcloned into a pET29(+) expression vector. Differently sized recombinant proteins were expressed in the BL21(DE3)pLysS Escherichia coli strain and then purified. The growth-stimulative effects on NIH3T3 cells of each recombinant protein were examined by means of MTT colorimetric assay. Full-length and the shortened recombinant proteins, which stimulated NIH3T3 cell growth, were then subcutaneously administered into male ddY mice (6 weeks old) every day for 2 weeks. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). The rhFGF4 of 134 amino acid residues, the region homologous to other members of the FGF family, exerted a growth-stimulative effect on NIH3T3 cells comparable to the full-length version of FGF4; however, the shortest version, with 111 amino acid residues, showed a limited growth-stimulative effect. Systemic administration of the rhFGF4 of 134 amino acid residues increased the bone mineral density (BMD) of femurs at a dose of 0.1 mg/kg, which was comparable to that of the full-length rhFGF4. DEXA analysis, pQCT analysis, soft X-ray photos, and contact microradiographs revealed an increase in femoral trabecular bone in FGF4-treated animals; an increase in bone formation was also evident upon histomorphometric analysis. These results indicate that the region of FGF4 that is homologous to other FGF family members provides a sufficient anabolic effect in bone and that this recombinant protein is potentially useful as a therapeutic agent in bone.

Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo.

Fgf8 and Fgf4 encode FGF family members that are coexpressed in the primitive streak of the gastrulating mouse embryo. We have analyzed the phenotype of Fgf8(-/-) embryos and discovered that they fail to express Fgf4 in the streak. In the absence of both FGF8 and FGF4, epiblast cells move into the streak and undergo an epithelial-to-mesenchymal transition, but most cells then fail to move away from the streak. As a consequence, no embryonic mesoderm- or endoderm-derived tissues develop, although extraembryonic tissues form. Patterning of the prospective neuroectoderm is greatly perturbed in the mutant embryos. Anterior neuroectoderm markers are widely expressed, at least in part because the anterior visceral endoderm, which provides signals that regulate their expression, is not displaced proximally in the absence of definitive endoderm. Posterior neuroectoderm markers are not expressed, presumably because there is neither mesendoderm underlying the prospective neuroectoderm nor a morphologically normal node to provide the inductive signals necessary for their expression. This study identifies Fgf8 as a gene essential for gastrulation and shows that signaling via FGF8 and/or FGF4 is required for cell migration away from the primitive streak.

Fibroblast growth factor-8 protects cultured rat hippocampal neurons from oxidative insult

Basic fibroblast growth factor (bFGF) has been reported to have neuroprotective properties following excitotoxic, metabolic, and oxidative insults. We report here that another FGF family member, FGF-8 is able to protect rat hippocampal cultures from oxidative stress. The b isoform of FGF-8 protected hippocampal cultures from hydrogen peroxide with an EC50 of approximately 25 ng/ml. In a time course study, using pre-, co-, post-treatment paradigms, we report that bFGF and FGF-8b were neuroprotective when added as a pre-treatment, co-treatment, and even at 2 h post-insult. Using neuronal enriched cultures, we demonstrate that bFGF and FGF-8b neuroprotection partially results from a direct action of the growth factors on neurons. The direct action on neurons may work in concert with normal and FGF-stimulated glial secretion products to give the full FGF protective effect. FGF-8b showed maximal protection at 50 ng/ml, whereas bFGF showed maximal protection at 10 ng/ml. Despite requiring higher concentrations to elicit protection, FGF-8b is able to attain levels of protection equivalent to that of bFGF (attenuation of 75–80% of hydrogen peroxide induced death). We also report that bFGF and FGF-8b are able to protect the human neuroblastoma cell line, SK-N-MC, from peroxide-induced LDH release by 50%. From these studies, we conclude that FGF-8b is another member of the FGF family which may show in vivo efficacy for the treatment of oxidative insults, such as stroke.

A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo.

Morphogenesis depends on the precise control of basic cellular processes such as cell proliferation and differentiation. Wnt5a may regulate these processes since it is expressed in a gradient at the caudal end of the growing embryo during gastrulation, and later in the distal-most aspect of several structures that extend from the body. A loss-of-function mutation of Wnt5a leads to an inability to extend the A-P axis due to a progressive reduction in the size of caudal structures. In the limbs, truncation of the proximal skeleton and absence of distal digits correlates with reduced proliferation of putative progenitor cells within the progress zone. However, expression of progress zone markers, and several genes implicated in distal outgrowth and patterning including Distalless, Hoxd and Fgf family members was not altered. Taken together with the outgrowth defects observed in the developing face, ears and genitals, our data indicates that Wnt5a regulates a pathway common to many structures whose development requires extension from the primary body axis. The reduced number of proliferating cells in both the progress zone and the primitive streak mesoderm suggests that one function of Wnt5a is to regulate the proliferation of progenitor cells.

Pretreatment with intravenous FGF-13 reduces infarct volume and ameliorates neurological deficits following focal cerebral ischemia in rats

Fibroblast growth factor-13 (FGF-13), novel member of FGF family has recently been molecularly cloned as a result of high throughput sequencing of a ovarian cancer cell, hippocampal, and kidney cDNA libraries. The human gene encodes for a protein with a molecular weight of 22 kDa that is most homologous to FGF-8 (70% similarity). In the current study, we tested the effects of intravenously administered FGF-13 in a model of permanent focal cerebral ischemia in Sprague–Dawley rats. FGF-13 or the vehicle was administered systematically via the tail vein 30 min prior, and 30 min and 24 h after the occlusion of the left middle cerebral artery (MCAo). Animals were weighed and evaluated behaviorally prior to and at 24 and 48 h after MCAo. The volume of cerebral infarct and swelling were determined using an image analysis system (BioQuant) and cresyl violet stained sequential sections from the forebrain region. Histopathology was evaluated to compare the therapeutic effects. We found a 63% reduction in infarct volume in FGF-13- vs. vehicle-treated animals (infarct volume was 21.9±3.8% in vehicle- and 8.1±1.6% in FGF-13-treated rats, p=0.0016) and a moderate inhibition of brain swelling by FGF-13. The reduction in infarct volume and brain swelling were associated with improvement of clinical deficits in FGF-13 treated animals ( p<0.001). Histopathological examination determined that nervous tissue was better preserved in FGF-13 treated rats than those of controls. These data show that pretreatment with intravenous FGF-13 reduces infarct size and ameliorates neurological deficits following permanent focal cerebral ischemia in rats.

Recombinant Rat Fibroblast Growth Factor-16: Structure and Biological Activity

Fibroblast growth factor-16 (FGF-16) is the most recent member of the FGF family to be cloned. Since the biologic activity of rat FGF-16 (rFGF-16) was unknown, and this protein has no apparent signal sequence, we transformed its entire cDNA intoEscherichia colifor high-level expression and further characterization of this novel protein. An attempt was made to purify the expressed protein from the supernatant of mechanically lysed cells using sequential cation-exchange chromatography. This resulted in a gradual loss of the protein as precipitate throughout the purification process. In addition to precipitation during purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the partially purified materials showed a cluster of protein bands around 20k to 29k. Sequence analysis of the major bands indicated that two N-terminal truncations had occurred, duringE. colifermentation, purification, or both. The largest truncation resulted in the removal of the 34 N-terminal amino acids, including the initiation codon methionine. We cloned d34 rFGF-16, expressed the gene inE. coli,and developed a purification process for this form. Even with this truncated form, precipitation was a problem. We were largely able to overcome this problem, however, by including EDTA throughout the purification process. We have characterized the structure of purified d34 rFGF-16 extensively using circular dichroism, Fourier transform infrared spectroscopy, and sedimentation velocity analysis. These studies revealed that the protein has a distinct tertiary structure, consists primarily of β-strands, has a weak tendency to self-associate, and is fairly extended. We then performed biologic assays which showed that d34 rFGF-16 induces oligodendrocyte proliferationin vitro,and induces hepatocellular proliferation and increased liver weightin vivo.In summary, FGF-16, a novel FGF family member, has both unique structural and biological properties.

Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2.

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.

Structure and Expression of a Novel Member, FGF-16, of the Fibroblast Growth Factor Family

We have isolated cDNA encoding a novel member (207 amino acids) of the FGF family from the rat heart by homology-based polymerase chain reaction. As this protein is the 16th documented member of the FGF family, we tentatively term it FGF-16. Among FGF family members, FGF-16 is most similar (73% amino acid identity) to FGF-9. We have also determined the structure of human FGF-16 with high amino acid sequence identity (98.6%) to rat FGF-16. Although the predicted FGF-16 amino acid sequence lacks a typical signal sequence, recombinant rat FGF-16 was efficiently secreted by Sf9 insect cells infected with recombinant baculovirus containing the cDNA.FGF-16mRNA was predominantly expressed in the rat heart among the adult major tissues examined. The expression profile ofFGF-16mRNA was quite different from those of other members of the FGF family. In rat embryos,FGF-16mRNA was predominantly expressed in the brown adipose tissue. However, the expression decreased greatly after birth. These results indicate that FGF-16 in embryos might play a role in development of the brown adipose tissue.

FGF-8Is Associated with Anteroposterior Patterning and Limb Regeneration inXenopus

FGF-8has attracted attention particularly because of its importance for limb development in the chick and mouse, although it also has a number of earlier expression domains in these species. We have now cloned anFGF-8homologue fromXenopusin which it is easier to do functional studies on early development. There is no maternal expression, while zygotic expression is highest in the gastrula and neurula stages.XFGF-8is expressed as a ring around the blastopore and subsequently in the tail bud. There are several domains in the head including the hatching gland, the branchial clefts, and the midbrain–hindbrain border. At later stages there is a prominent band of expression in the limb bud epidermis. Although there is no morphological apical ridge, this band of expression suggests that theXenopuslimb bud contains a cryptic region with a similar ability to stimulate mesenchymal outgrowth. The mesoderm-inducing activity of XFGF-8 is somewhat lower than that of other FGFs, while the posteriorizing activity is similar. These differences are probably due to the different receptor specificity. The posterior expression and high posteriorizing activity suggest that XFGF-8 contributes to the patterning of the anterior–posterior axis by FGF family members during gastrulation. In contrast to the amniotes,Xenopuslimb buds can regenerate following damage. We show that regeneration is correlated with the reexpression ofXFGF-8in the distal epidermis, suggesting that this ability is critical for successful limb regeneration.

Mouse fibroblast growth factor 10: cDNA cloning, protein characterization, and regulation of mRNA expression.

Fibroblast growth factor 7 (FGF-7) or keratinocyte growth factor (KGF), is a potent and specific mitogen for epithelial cells. We have recently identified a novel human FGF-7 homologue, named FGF-10. To study the expression of this new FGF family member and its regulation in wound repair, we cloned the mouse FGF-10 (mFGF-10) cDNA. The encoded protein is 92% identical to human FGF-10 and 91% identical to rat FGF-10. When expressed in mammalian 293 cells, the mFGF-10 protein was glycosylated but remained cell- or extracellular matrix-associated. Upon addition of heparin, mFGF-10 protein was released into the media. mRNA encoding mFGF-10 was relatively abundant in lung, skin, brain and heart. In the skin, both FGF-7 and mFGF-10 were expressed in the dermal, but not the epidermal compartment. In contrast to FGF-7, mFGF-10 expression was not induced during cutaneous wound repair. In cultured fibroblasts, expression of mFGF-10 was strongly repressed by transforming growth factor beta and tumor necrosis factor alpha, whereas epidermal growth factor and interleukin-1beta had no effect. These results demonstrate a differential regulation of mFGF-10 and FGF-7 expression in vitro and during the wound healing process.

A common RNA structural motif involved in the internal initiation of translation of cellular mRNAs.

The 5'-non-translated regions (5'NTR) of human immunoglobulin heavy chain binding protein (BiP), Antennapedia (Antp) ofDrosophilaand human fibroblast growth factor 2 (FGF-2) mRNAs are reported to mediate translation initiation by an internal ribosome binding mechanism. In this study, we investigate predicted features of the higher order structures folded in these 5'NTR sequences. Statistical analyses of RNA folding detected a 92 nt unusual folding region (UFR) from 129 to 220, close to the initiator AUG in the BiP mRNA. Details of the structural analyses show that the UFR forms a Y-type stem-loop structure with an additional stem-loop in the 3'-end resembling the common structure core found in the internal ribosome entry site (IRES) elements of picornavirus. The Y-type structural motif is also conserved among a number of divergent BiP mRNAs. We also find two RNA elements in the 5'-leader sequence of human FGF-2. The first RNA element (96 nt) is 2 nt upstream of the first CUG start codon located in the reported IRES element of human FGF-2. The second (107 nt) is immediately upstream of the authentic initiator AUG of the main open reading frame. Intriguingly, the folded RNA structural motif in the two RNA elements is conserved in other members of FGF family and shares the same structural features as that found in the 5'NTR of divergent BiP mRNAs. We suggest that the common RNA structural motif conserved in the diverse BiP and FGF-2 mRNAs has a general function in the internal ribosome binding mechanism of cellular mRNAs.

Construction of a sensitive enzyme immunoassay for human fibroblast growth factor 9.

Hybridomas secreting monoclonal antibodies (MAbs) against human fibroblast growth factor 9 (FGF-9) were established using recombinant human (rh) FGF-9 N33 as the immunogen. Among these, MAb 150-59 demonstrated a potent neutralizing activity against FGF-9. It arrested the FGF-9-induced growth of BALB/c 3T3 A31 cells at an equimolar dose of the factor. It also abrogated the in vivo thrombopoietic activity of FGF-9. Mitogenic activity of several other FGF family members such as FGF-1, FGF-2, and FGF-4 was not neutralized by this MAb. A sensitive sandwich enzyme immunoassay for FGF-9 was developed employing MAbs 150-59 and 13-3. The detection limit of this system was 3 pg/well. In this assay system, FGF-1 and FGF-2 were not cross-reactive up to 1 microgram/well. Using this system, the distribution of FGF-9 in rat organs was examined. FGF-9 could be detected only in the extract of rat cerebellum. Also, we detected a high amount of FGF-9 in the culture supernatant of certain cell lines originated from human tumor. These findings suggest that this enzyme immunoassay system may be used to clarify biological meaning of FGF-9.

Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms.

Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in either proliferating or differentiated skeletal muscle cells. FGF-I and FGF-2 proteins were present in proliferating skeletal muscle cells, but undetectable after terminal differentiation. We show that transfection of expression constructs encoding FGF-1 or FGF-2 mimics the effects of exogenously applied FGFs, inhibiting skeletal muscle cell differentiation and stimulating DNA synthesis. These effects require activation of an FGF tyrosine kinase receptor as they are blocked by transfection of a dominant negative mutant FGF receptor. Transient transfection of cells with FGF-1 or FGF-2 expression constructs exerted a global effect on myoblast DNA synthesis, as greater than 50% of the nontransfected cells responded by initiating DNA synthesis. The global effect of cultures transfected with FGF-2 expression vectors was blocked by an anti-FGF-2 monoclonal antibody, suggesting that FGF-2 was exported from the transfected cells. Despite the fact that both FGF-l and FGF-2 lack secretory signal sequences, when expressed intracellularly, they regulate skeletal muscle development. Thus, production of FGF-1 and FGF-2 by skeletal muscle cells may act as a paracrine and autocrine regulator of skeletal muscle development in vivo.

Conservation of Ligand Specificity between the Mammalian and Amphibian Fibroblast Growth Factor Receptors

We have previously cloned and sequenced a newt keratinocyte growth factor receptor (KGFR) cDNA which exhibited a unique spatial and temporal expression pattern in the regenerating newt limb. In this report, we further characterize the biochemical and functional properties of this newt KGFR. A stable Chinese hamster ovary transfectant overexpressing the newt KGFR was capable of binding both 125I-fibroblast growth factor-1 (FGF-1) and 125I-FGF-7 but not 125I-FGF-2, indistinguishable from the human KGFR. Scatchard analysis and cross-linking studies further support the conclusion that FGF-1 and FGF-7 are the ligands for the newt KGFR. In addition to their ability to bind to FGFs, both the human and the newt KGFR are also capable of repressing differentiation in mouse MM14 myoblasts. MM14 cells express FGFR1 and are repressed from differentiation by FGF-1, FGF-2, and FGF-4 but not FGF-7. Co-transfection of MM14 cells with either a human or newt KGFR expression construct conferred a response to FGF-7 as determined by a human alpha-cardiac actin/luciferase reporter construct. The response to FGF-7 was similar to the endogenous FGF response as FGF-7 prevented MM14 myoblasts from undergoing terminal differentiation. Thus, both the human and the newt KGFRs transduce signals similar to those transduced via the endogenous mouse FGFR1. Together these data indicate that this newly isolated newt KGFR is a functional receptor as it binds two FGF family members with high affinity and mediates signaling in skeletal muscle myoblasts. Because the binding pattern of the newt KGFR is similar to the pattern observed for its mammalian counterpart, it emphasizes the strict conservation that this ligand/receptor system has undergone through evolution.

Effects of Oxygen on Wound Responses to Growth Factors: Kaposi's FGF, but not Basic FGF Stimulates Repair in Ischemic Wounds

Kaposi's fibroblast growth factor (K-FGF, FGF-4) is a newer member of FGF family with uncharacterized wound healing properties. Basic fibroblast growth factor (bFGF, FGF-2) has been well studied and accelerates repair in normal and impaired wound healing models. K-FGF and bFGF are known to have similar biological effects in tissue culture, and both stimulate fibroblast and endothelial cell proliferation. The rabbit dermal ulcer model was used to examine the effects of bFGF and K-FGF under ischemic and nonischemic conditions. We found bFGF was ineffective in stimulating healing under ischemic conditions even at high doses (30 micrograms/wound). However, when the ischemic wounds were treated with bFGF (5 micrograms/wound) plus hyperbaric oxygen therapy, it was highly effective again as previously found under nonischemic conditions (P < 0.05). In contrast K-FGF stimulated repair in both nonischemic and ischemic wounds (P < 0.05). These results suggest that wound oxygen content differentially regulates responsiveness to bFGF and that K-FGF is biologically active in hypoxic wounds.

Differential temporal and spatial gene expression of fibroblast growth factor family members during mouse mammary gland development.

Mammary gland development is dependent upon local regulatory factors as well as systemic hormones to mediate gland morphogenesis and associated mesenchymal-epithelial interactions. FGF-3 (int-2) has been implicated as an oncogenic growth factor produced locally in mouse mammary tumor virus-induced mammary tumorigenesis. The observation that FGF-3 is not expressed during normal mammary development as well as the high degree of cellular proliferation and angiogenesis that accompany mammary gland growth suggest roles for other FGF family members in this process. In this study, we have examined expression of FGF family members at various stages of mouse mammary growth and tumorigenesis. FGF-1, FGF-2, FGF-4, and FGF-7 were expressed during the ductal stage of mammary development. The majority of FGF-1 gene expression was in the luminal epithelial cells, whereas FGF-2 expression was in the mammary stroma and possibly the myoepithelial cells. The presence of mammary epithelium induced the expression of both FGF-2 and FGF-7 in the stroma. FGF-1 and FGF-2 expression declined during pregnancy and dropped again during lactation, but quantitative analysis showed a much more dramatic decrease in FGF-2 expression. FGF-7 transcripts were also detected during pregnancy and lactation, but an alternate transcript size was observed at these stages. FGF-1, FGF-2, and FGF-7 transcripts were detected in mammary preneoplasias, tumors, and immortal cell lines, but at levels less than those seen during normal mammary growth. These results support the hypothesis that FGF family members play a role in local regulation of mammary development. The differential spatial and temporal pattern of FGF-1, FGF-2 and FGF-7 gene expression indicate that they each have unique functions in the gland.

Antisense Oligodeoxynucleotides Suppress Basic Fibroblast Growth Factor Expression in Glioma Cell Lines and Primary Cultures of Neural Crest Cells

Basic fibroblast growth factor (bFGF) is a heparin-binding protein implicated in the differentiation, proliferation, and maintenance of cells in the central and peripheral nervous systems. Methods for modulating bFGF expression would, therefore, be useful for examining the function(s) of bFGF in neural tissue. Here we evaluate the effects of bFGF-specific antisense oligonucleotides on bFGF expression in two distinct cell culture systems. The first culture system employed an established human glioma cell line expressing hl~h levels of bFGF protein. Inhibition of bFGF expression in this system by antisense oligonucleotides inhibited bFGFs mitogenic effects. The second system utilized primary cultures derived from peripheral nerve explants. in these cultures, neural crest-derived melanocyte/Schwann cell progenitors from peripheral nerve undergo a transformation into melanocytes in response to treatment with bFGF or phorbol esters. Antisense primers directed to two different sites of bFGF mRNA suppressed this phorbol este-rinduced melanogenesis in peripheral nerve explants in a saturable and specific manner. Sense strand primers or sequence-scrambled antisense primers were ineffective in this regard. Western blot and immunocytochemical analyses demonstrated that only antisense primers significantly reduced the specific activity of bFGF protein in glioma cell or phorbol ester-treated peripheral nerve culture extracts, confirming that these antisense oligonucleotides interfered with bFGF protein expression.The application of FGF-specific antisense primers should prove useful in elucidating the role of FGF family members in nervous system development and maintenance.

Effects of acid and basic fibroblast growth factor and heparin on resorption of cultured fetal rat long bones

We tested acid and basic fibroblast growth factor (aFGF and bFGF), members of the heparin binding FGF family, for their ability to stimulate bone resorption as measured by the release of previously incorporated 45Ca from cultured fetal rat long bones in the presence and absence of heparin. Purified low-molecular-weight heparin (LMW heparin) at 5-125 micrograms/ml had no direct stimulatory effect. There was little effect from aFGF (10(-11)-10(-8) M) alone, but increased resorption was observed in the presence of LMW heparin. With bFGF, increased bone resorption was observed at 10(-9) M but not at 10(-8) M. The stimulatory effects of aFGF and bFGF in the presence of LMW heparin were not blocked by the addition of indomethacin (10(-6) M), which blocks prostaglandin production, or hydroxyurea (10(-3) M), which blocks DNA synthesis. However, pretreatment with aphidicolin (3 x 10(-5) M), a potent inhibitor of DNA synthesis, blocked the effect of acid FGF and diminished the effect of bFGF. These results indicate that both aFGF and bFGF can stimulate bone resorption by a prostaglandin-independent mechanism, particularly in the presence of heparin. The activation of FGF-mediated bone resorption by heparin could play a role in producing the osteoporosis that has been described with heparin therapy and mastocytosis.