Form Of Fibroblast Growth Factor Research Articles

Targeting HER2 and FGFR-positive cancer cells with a bispecific cytotoxic conjugate combining anti-HER2 Affibody and FGF2

Breast cancer remains a significant global health challenge, necessitating the development of effective targeted therapies. This study aimed to create bispecific targeting molecules against HER2 and FGFR1, two receptors known to play crucial roles in breast cancer progression. By combining the high-affinity Affibody ZHER2:2891 and a modified, stable form of fibroblast growth factor 2 (FGF2), we successfully generated bispecific proteins capable of simultaneously recognizing HER2 and FGFR1. Two variants were designed: AfHER2-sFGF2 with a shorter linker and AfHER2-lFGF2 with a longer linker between the HER2 and FGFR1-recognizing proteins. Both proteins exhibited selective binding to HER2 and FGFR1, with AfHER2-lFGF2 demonstrating simultaneous binding to both receptors. AfHER2-lFGF2 exhibited superior internalization compared to FGF2 in FGFR-positive cells and significantly increased internalization compared to AfHER2 in HER2-positive cells. To enhance their therapeutic potential, highly potent cytotoxic agent MMAE was conjugated to the targeting proteins, resulting in protein-drug conjugates. The bispecific AfHER2-lFGF2-vcMMAE conjugate demonstrated exceptional cytotoxic activity against HER2-positive, FGFR-positive, and dual-positive cancer cell lines that was significantly higher compared to monospecific conjugates. These data indicate the beneficial effect of simultaneous targeting of HER2 and FGFR1 in precise anticancer medicine and contribute valuable insights into the design and potential of bispecific targeting molecules for breast cancer treatment.

Intra-articular sprifermin therapy reduces total knee replacements: accumulated evidence from translational and clinical data

Purpose: Osteoarthritis (OA) is a degenerative disease with high prevalence, creating an unmet medical need for drugs to protect and regenerate cartilage. Sprifermin, a truncated form of fibroblast growth factor 18 (rhFGF18), is being investigated as a potential disease-modifying OA drug (DMOAD). Sprifermin has been shown to increase cartilage volume in the knees of OA patients. Studies published about the mode of action clearly demonstrate anabolic effects. Accumulated analysis of data available from preclinical experiments and clinical data was performed to investigate the effects of sprifermin on late stage OA tissues as well as the rate of total knee replacements (TKR) in late stage OA patients.

Promising targets for therapy of osteoarthritis: a review on the Wnt and TGF-β signalling pathways.

Osteoarthritis (OA) is the most common chronic joint disorder worldwide, with a high personal burden for the patients and an important socio-economic impact. Current therapies are largely limited to pain management and rehabilitation and exercise strategies. For advanced cases, joint replacement surgery may be the only option. Hence, there is an enormous need for the development of effective and safe disease-modifying anti-OA drugs. A strong focus in OA research has been on the identification and role of molecular signalling pathways that contribute to the balance between anabolism and catabolism in the articular cartilage. In this context, most insights have been gained in understanding the roles of the transforming growth factor-beta (TGF-β) and the Wingless-type (Wnt) signalling cascades. The emerging picture demonstrates a high degree of complexity with context-dependent events. TGF-β appears to protect cartilage under healthy conditions, but shifts in its receptor use and subsequent downstream signalling may be deleterious in aged individuals or in damaged cartilage. Likewise, low levels of Wnt activity appear important to sustain chondrocyte viability but excessive activation is associated with progressive joint damage. Emerging clinical data suggest some potential for the use of sprifermin, a recombinant forms of fibroblast growth factor 18, a distant TGF-β superfamily member, and for lorecivivint, a Wnt pathway modulator.

Assessment of cartilage markers in synovial fluid from the forward study provide insight to the biphasic effect of sprifermin

Purpose: Sprifermin is a truncated form of fibroblast growth factor (FGF) 18 known to induce chondrocyte proliferation and type II collagen formation [1,2]. Data from preclinical investigations show that cartilage formation happens in different phases after therapy with sprifermin, starting with a phase of cartilage degradation during the induction of proliferation of chondrocytes followed by a phase of cartilage formation/production of extracellular matrix. The aim was to investigate the effect of intra-articular (IA) administrated sprifermin on cartilage turnover as compared to placebo by measurement of markers in longitudinal synovial fluid samples of patients participating in the FORWARD study. Methods: Synovial fluids (SF) from participants with baseline and at least one follow-up sample (baseline (BL) at three consecutive weeks in six month intervals through to week (wk) 80, fig.A available from the phase II clinical trial evaluating the efficacy and safety of intraarticularly delivered sprifermin [3] were selected for the investigations. Biochemical markers were measured in available SF samples of the placebo (saline IA, n=38) and the highest sprifermin dose group (100 mg/IAx4, n=59). Each included patient had baseline and at least one FU sample available. Samples were pretreated with ultrasound and centrifugation to decrease viscosity. Markers measured were PRO-C2 (type II collagen formation), huARGS (aggrecan degradation), and FBN-C (fibronectin). Markers were technically validated for synovial fluid measurement. Data were normalized to baseline to investigate the median change over time. Results: Baseline mean (SD) levels of the markers in SF at BL were: PRO-C2, 21.4 (13.6) ng/mL, huARGS, 1117 (516) pM and FBN-C, 2556 (1959) ng/mL. PRO-C2 was initially decreased (from BL to wk 2) after injection with sprifermin; however, the level was increased at the beginning of a new injection cycle followed by a decrease after injection of sprifermin (Fig.B). Overall PRO-C2 levels increased over time in therapy with sprifermin, while no change was observed for the placebo arm. huARGS showed a similar pattern as PRO-C2 - there was an overall increase in ARGS over time in the sprifermin group (fig.C). Interestingly ARGS continuously decreased over time in the placebo group. FBN-C is continuously increased after injection’s cycles, whereas no effect was seen in the placebo group (fig.D). Conclusions: Confirmatory of the preclinical investigations we saw a biphasic response on cartilage turnover after injection with sprifermin. Cartilage formation and chondrocyte proliferation was only modulated by sprifermin, and cartilage degradation (ARGS) was temporal induced and reduced by sprifermin and saline injections, respectively.1. Gigout A, Guehring H, Froemel D, Meurer A, Ladel C, Reker D, et al. Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix. Osteoarthr Cartil. 2017;25. 2. Reker D, Kjelgaard-Petersen CF, Siebuhr AS, Michaelis M, Gigout A, Karsdal MA, et al. Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo. J Transl Med. 2017;15. 3. Hochberg MC, Guermazi A, Guehring H, Aydemir A, Wax S, Fleuranceau-Morel P, et al. Effect of Intra-Articular Sprifermin vs Placebo on Femorotibial Joint Cartilage Thickness in Patients With Osteoarthritis. JAMA. 2019

Abstract 88: Comparative tumorigenesis of progranulin and fibroblast growth factor 4 and in adrenocortical carcinoma cells.

Abstract Adrenal carcinomas are almost always fatal, but the mechanisms that influence transition from low malignancy adenomas to metastatic carcinomas are ill defined. SW-13 adrenocortical carcinoma cells are not tumorigenic in nude mice and do not support anchorage-independent growth at low cell density unless stimulated by secreted forms of fibroblast growth factors (FGF) family, such as FGF4, or the growth factor-like proteins progranulin (PGRN) which is over produced by many cancers. This growth factor-dependent acquisition of tumorigenesis may model the transition from benign to malignant adrenocortical cancer. Both PGRN and FGF4 were shown to stimulate phosphorylation of MEK1/2 in SW-13 cells. The aim of this study is to investigate the tumorigenic mechanisms of PGRN and FGF, in particular to identify “core” malignancy responses shared by both tumorigenic pathways. We employed Affymetrix Human Genome U133A microarrays to examine the transcriptome profiles in SW-13 cells with elevated PGRN or FGF4. We employed Ingenuity Pathway Analysis (IPA®) to visualize differentially expressed genes in the context of biological pathways. In SW-13 cells with elevated PGRN, those transcripts that were significantly up-regulated were related to cell morphology, cellular assembly and organization, nervous system development and function, carbohydrate metabolism, molecular transport. In cells with elevated FGF4, significantly up-regulated molecules were associated with lipid metabolism, molecular transport, small molecule biochemistry, cell death. 80 transcripts that were up-regulated in common by FGF and PGRN were identified, and were significantly associated with gene expression, cell death, cellular development. 28 of those genes were related to cancer, including nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, v-rel reticuloendotheliosis viral oncogene homolog (avian), BCL2-associated X protein, early growth response 4, nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100), etc. 65 FGF and PGRN common down-regulated genes were identified, and were significantly associated with reproductive system development and function, cellular development, cellular growth and proliferation. We found, however, that the majority of genes were regulated differentially between PGRN and FGF. Our results suggest that PGRN and FGF4 share common signals which contribute to tumorgenesis, but also reveal that the common malignant phenotype attributed to both PGRN and FGF4 differs significantly at the transcriptional level suggesting unique pathways linking PGRN and FGF to tumor progression in these cells. Citation Format: Yonghua Zhang, Amin Ismail, Andrew Bateman. Comparative tumorigenesis of progranulin and fibroblast growth factor 4 and in adrenocortical carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 88. doi:10.1158/1538-7445.AM2013-88

Multilayered microcapsules for the sustained-release of angiogenic proteins from encapsulated cells

BackgroundMultilayered alginate microcapsules with a permselective poly-L-ornithine membrane can be used for the dual purpose of encapsulating cells in the inner core and sustained release of angiogenic proteins from the outer layer. The aim of this study was to examine the encapsulation and release of a novel chimeric form of fibroblast growth factor–1 (FGF-1) from the outer layer of alginate microcapsules. MethodsHeparin-binding growth-associated molecule bound to FGF-1 (HB-GAM/FGF-1) was encapsulated in the outer layer of multilayered alginate microbeads constructed using varying alginate conditions. The encapsulation and release of the chimera was quantified. ResultsThe outer layer was able to encapsulate and release HB-GAM/FGF-1 for up to 30 days. The outer layer made with 1% alginate of high mannuronic acid content provided the fastest release, while 1.25% high guluronic acid content alginate displayed the longest duration of release. ConclusionsThe outer layer of multilayered alginate microbeads can be used for the encapsulation and long-term release of HB-GAM/FGF-1.

Abstract 34: Oncogenic tyrosine kinases phosphorylate and inhibit PKM2 to provide a metabolic advantage to tumor growth

Abstract Cancer cells show increased aerobic glycolysis and enhanced lactate production compared to healthy cells, a phenomenon known as the Warburg effect. Cell surface growth factor receptors, which often carry tyrosine kinase activities in their cytoplasmic domains, are overexpressed in many human cancers and are believed to play a key role in determining cell metabolism. Thus, we explored the hypothesis that tyrosine kinase signaling, which is commonly increased in tumors, regulates the Warburg effect and contributes to tumorigenesis and maintenance of the tumor. We performed phospho-proteomics studies and found that oncogenic forms of fibroblast growth factor (FGF) receptor type 1 (FGFR1) inhibit the pyruvate kinase M2 isoform (PKM2) in cancer cells. Pyruvate kinase is a rate-limiting enzyme during glycolysis and catalyzes the production of pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. Recent studies demonstrated that the enzymatic activity of the pyruvate kinase M2 isoform (PKM2) is inhibited by phosphotyrosine binding; moreover, these researchers found that PKM2 is crucial for aerobic glycolysis and provides a growth advantage to tumors. However, it remains unclear which tyrosine kinase pathways are physiologically responsible for this inhibition of PKM2 activity and which protein factors undergo tyrosine phosphorylation, allowing them to bind to and thereby inhibit PKM2. We found that PKM2 is itself tyrosine phosphorylated in cancer cells and such a physiological modification of PKM2 promotes the switch to aerobic glycolysis from oxidative phosphorylation. FGFR1 directly phosphorylates PKM2 at tyrosine residue 105 (Y105). This inhibits the formation of active, tetrameric PKM2 by disrupting binding of the PKM2 cofactor fructose-1,6-bisphosphate (FBP). Moreover, we found that phosphorylation of PKM2 Y105 is common in human cancers. Immunoblotting revealed that PKM2 is phosphorylated at Y105 in diverse human breast cancer, lung cancer, prostate cancer and leukemia cell lines. Oncogenic tyrosien kianses including BCR-ABL, FLT3-ITD and JAK2 also directly phosphorylate PKM2 Y105 in in vitro kinase assays using purified proteins. Furthermore, the presence of a PKM2 mutant in which phenylalanine is substituted for Y105 (Y105F) in cancer cells leads to decreased cell proliferation under hypoxic conditions, increased oxidative phosphorylation with reduced lactate production, and reduced tumor growth in xenografts in nude mice. Our findings suggest that tyrosine phosphorylation regulates PKM2 to provide a metabolic advantage to tumor cells, thereby promoting tumor growth. This may represent a common, short-term molecular mechanism underlying the Warburg effect in both leukemias and solid tumors, in addition to the chronic changes believed to be regulated by transcription factors, including hypoxia inducible factor 1 and Myc. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 34.

Polypeptide growth factors in bone matrix.

The presence of many types of polypeptide growth factors in the mineralized extracellular matrix of bone is now well established. These factors are generally referred to as bone-derived growth factors (BDGFs), and are similar, or possibly identical, to the following species; platelet-derived growth factor (PDGF); acidic and basic forms of fibroblast growth factor (aFGF, bFGF); transforming growth factor beta (TGF-beta); and insulin-like growth factor 1 (IGF-1). Several osteoinductive factors, such as bone morphogenetic protein (BMP) and osteogenin, a skeletal growth factor (SGF), and osteoblast-derived BDGFs, have also been identified. Complete description of the biological functions of these BDGFs which are relevant to bone will ultimately require specific bioassays involving specific cell types in vitro, as well as in vivo animal implant models. Studies with primary rat osteoblast-like cells exposed either to mixed BDGFs, pure TGF-beta, or heparin-purified PDGF, aFGF, or bFGF from bovine bone have shown a general dose-dependent mitogenic effect. Phenotypic changes which accompany the BDGF-induced wave of proliferation include: decreased osteocalcin secretion and a reduction in 1,25-(OH)2 vitamin D3-stimulated osteocalcin synthesis; reduced alkaline phosphatase specific activity; decreased cyclic AMP responsiveness to parathyroid hormone (PTH); and increased collagen synthesis. Bone exhibits the most complex spectrum of growth factor activities of any tissue yet described. In bovine bone powder free of blood and cartilage contamination, the volume concentration of mitogens is up to 20 times greater than that in serum. Bone cells and other indigenous cell types must be considered as possible sources of the BDGFs, in addition to sequestration from blood. Mechanisms for the unmasking or release of BDGFs from the mineralized matrix that result in local action on osteoblasts, endothelial cells, and other target cells are undoubtedly important for the development and maintenance of bone tissue.

Transcription Factor Ets-1 Regulates Fibroblast Growth Factor-1-Mediated Angiogenesis in vivo: Role of Ets-1 in the Regulation of the PI3K/AKT/MMP-1 Pathway

We previously demonstrated that a modified secreted form of fibroblast growth factor 1 (FGF-1), a prototypic member of the FGF family, has the ability to stimulate angiogenesis in an in vivo model of angiogenesis, the so-called chick chorioallantoic membrane assay or CAM. We recently defined the importance of the phosphatidylinositol 3-kinase/AKT pathway in FGF-1-mediated angiogenesis in this model using specific pharmacological inhibitors. In our continuing efforts to define the molecular signaling pathway regulating FGF-1-induced angiogenesis in vivo, we utilized a transcription factor activity assay and identified transcription factor Ets-1 as a critical effector of FGF-1-induced angiogenesis. Both activity and mRNA expression levels of the Ets-1 molecule were increased in response to FGF-1 overexpression in CAMs, as documented by electrophoretic mobility shift assay (gel shift) and reverse transcription real-time PCR techniques, respectively. Furthermore, the delivery of Ets-1 antisense (AS) into CAM tissues effectively reduced angiogenesis in the CAM assay. In addition, both Ets-1 AS-treated chicken CAMs and cultured endothelial cells exhibited a reduction in matrix metalloproteinase 1 gene expression levels. The Ets-1 AS-treated endothelial cells also demonstrated a reduction in migration. These data suggest that Ets-1 activation is a requisite for FGF-1-mediated angiogenesis in vivo. Therefore, Ets-1 might be a potential target for the generation of inhibitor drugs for the treatment of FGF-dependent pathological angiogenesis such as metastatic tumors, rheumatoid arthritis and diabetic retinopathy.

Cancer Therapy Through Control of Cell Migration

Cell migration plays a pivotal role in a many biological process that are essential for development, repair, and pathogenesis. Thus, inhibition of migration has the potential of limiting or suppressing the development of various diseases. Much of the focus on the therapeutic treatment of cancer has involved compounds that target cell proliferation and subsequent cell death. However, targeting migration is another approach that has not been pursued but holds promise for alternative means of therapy. One such potential therapeutic is a small protein that inhibits the migration of a number of cell types. This protein is derived from the amino terminal end of the 24 kDa form of fibroblast growth factor, and suppresses migration in the presence of a variety of growth factors. Analysis of the protein in mouse models shows that it inhibits in vivo angiogenesis and tumor growth at low concentrations. Thus, inhibition of migration is a viable alternative to more traditional methods of therapeutically treating tumors. Further study of the mechanism of inhibition can lead to the development of novel drugs targeting a distinctive cell process.

NoBP, a nuclear fibroblast growth factor 3 binding protein, is cell cycle regulated and promotes cell growth.

Secreted and nuclear forms of fibroblast growth factor 3 (FGF3) have opposing effects on cells. The secreted form stimulates cell growth and transformation, while the nuclear form inhibits DNA synthesis and cell proliferation. By using the yeast two-hybrid system we have identified a nucleolar FGF3 binding protein (NoBP) which coimmunoprecipitated and colocalized with FGF3 in transfected COS-1 cells. Characterization of the NoBP binding domain of FGF3 exactly matched the sequence requirements of FGF3 for its translocation into the nucleoli, suggesting that NoBP might be the nucleolar binding partner of FGF3 essential for its nucleolus localization. Carboxyl-terminal domains of NoBP contain linear nuclear and nucleolar targeting motifs which are capable of directing a heterologous protein beta-galactosidase to the nucleus and the nucleoli. While NoBP expression was detected in all analyzed proliferating established cell lines, NoBP transcription was rapidly downregulated in the promyelocytic leukemia cell line HL60 when induced to differentiate. Analysis on the expression pattern of NoBP mRNA throughout the cell cycle in HeLa cells synchronized by lovastatin demonstrated a substantial upregulation during the late G(1)/early S phase. NoBP overexpression conferred a proliferating effect onto NIH 3T3 cells and can counteract the inhibitory effect of nuclear FGF3, suggesting a role of NoBP in controlling proliferation in cells. We propose that NoBP is the functional target of nuclear FGF3 action.

Subcellular and developmental expression of alternatively spliced forms of fibroblast growth factor 14

Fibroblast growth factors (FGFs) 11–14 comprise a subfamily of FGFs with poorly defined biological function. Here we characterize two isoforms of FGF14 (FGF14-1a and FGF14-1b) that result from the alternative usage of two different first exons. We demonstrate that these isoforms have differential subcellular localization and that they are differentially expressed in various adult tissues. Using in situ hybridization we show that Fgf14 is widely expressed in brain, spinal cord, major arteries and thymus between 12.5 and 14.5 days of mouse embryonic development. We also show that during cerebellar development, Fgf14 is first observed at postnatal day 1 in post mitotic granule cells, and later in development, in migrating and post migratory granule cells. The developmental expression pattern of Fgf14 in the cerebellum is complementary to that of Math1, a marker for proliferating granule cells in the external germinal layer.

Ocular Neovascularization: Clarifying Complex Interactions

Neovascularization within the eye contributes to visual loss in several ocular diseases, the most common of which are proliferative diabetic retinopathy, neovascular age-related macular degeneration, and retinopathy of prematurity. Together, these three diseases afflict persons in all stages of life from birth through late adulthood and account for most instances of legal blindness. Retinopathy of prematurity (ROP) occurs in premature neonates. Normally, the retina becomes completely vascularized at full term. In the premature baby, the retina remains incompletely vascularized at the time of birth. Rather than continuing in a normal fashion, vasculogenesis in the premature neonatal retina becomes disrupted. Abnormal new proliferating vessels develop at the juncture of vascularized and avascular retina. These abnormal new vessels grow from the retina into the vitreous, resulting in hemorrhage and tractional detachment of the retina. Although laser ablation of avascular peripheral retina may halt the neovascular process if delivered in a timely and sufficient manner, some premature babies nevertheless go on to develop retinal detachment. Surgical methods for treating ROP-related retinal detachments in neonates have limited success at this time because of unique problems associated with this surgery, such as the small size of the eyes and the extremely firm vitreoretinal attachments in neonates. Diabetic retinopathy is the leading cause of blindness in adults of working age. In persons with diabetes mellitus, retinal capillary occlusions develop, creating areas of ischemic retina. Retinal ischemia serves as a stimulus for neovascular proliferations that originate from pre-existing retinal venules at the optic disk or elsewhere in the retina posterior to the equator. Severe visual loss in proliferative diabetic retinopathy (PDR) results from vitreous hemorrhage and tractional retinal detachment. Again, laser treatment (panretinal photocoagulation to ischemic retina) may arrest the progression of neovascular proliferations in this disease but only if delivered in a timely and sufficiently intense manner. Some diabetic patients, either from lack of ophthalmic care or despite adequate laser treatment, go on to sustain severe visual loss secondary to PDR. Vitrectomy surgery can reduce but not eliminate severe visual loss in this disease. Age-related macular degeneration is the leading cause of severe visual loss in persons over 65 years old. In contrast to ROP and PDR, in which neovascularization emanates from the retinal vasculature and extends into the vitreous cavity, AMD is associated with neovascularization originating from the choroidal vasculature and extending into the subretinal space. Choroidal neovascularization causes severe visual loss in AMD patients because it occurs in the macula, the area of retina responsible for central vision. The stimuli which lead to choroidal neovascularization are not understood. Laser ablation of the choroidal neovascularization may stabilize vision in selected patients. However, only 10% to 15% of patients with neovascular AMD have lesions judged to be appropriate for laser photocoagulation according to current criteria. Retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular age-related macular degeneration are but three of the ocular diseases which can produce visual loss secondary to neovascularization. Others include sickle cell retinopathy, retinal vein occlusion, and certain inflammatory diseases of the eye. These, however, account for a much smaller proportion of visual loss caused by ocular neovascularization. Additional treatments beyond laser photocoagulation and vitrectomy surgery are needed to improve outcomes in these patients. Pharmacological antiangiogenic therapy can potentially assist in prevention of the onset or progression of ocular neovascularization and is a current goal of many research laboratories and pharmaceutical companies.

Protection of Rat Myocardium by Mitogenic and Non-Mitogenic Fibroblast Growth Factor During Post-Ischemic Reperfusion

The effects of acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) and a non mitogenic form of FGF1 on myocardial ischemia and reperfusion were assessed. Rats underwent 10 minutes of coronary artery occlusion followed by 24 hours of reperfusion. Creatinine kinase content of the affected myocardium showed that both fibroblast growth factors 1 and 2 effectively protected against ischemia reperfusion injury (p < 0.01), and that the vasoactive but nonmitogenic form of the FGF1 was equally protective (p < 0.01 versus control + vehicle). The results were confirmed by light and electron-microscopy histological studies. Histological evaluations after treatment with the non-mitogenic fibroblast growth factor 1 showed that it did not generate the severe hyperplasia and connective tissue disorganization observed with the native mitogenic proteins.The possibility of using a non-mitogenic form of fibroblast growth factor for cardio-protection circumvents many of the potentially undesirable effects that may derive from systemically introducing broad spectrum acting fibroblast growth factors in vivo. This myocardial protection observed 24 hours after the treatment with fibroblast growth factors, and the efficacy of the non-mitogenic form of the protein, also suggest that the protective effect of fibroblast growth factors may be due to the increased blood flow rather than to angiogenesis.

FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement.

A constitutively active form of fibroblast growth factor 2 (FGFR2) was identified in rat osteosarcoma (ROS) cells by an expression cloning strategy. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C terminus generated from chromosomal rearrangement with a novel gene, designated FGFR activating gene 1 (FRAG1). While the removal of the C terminus slightly activates FGFR2, the presence of the FRAG1 sequence drastically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAG1 is ubiquitously expressed and encodes a predicted protein of 28 kDa lacking significant structural similarity to known proteins. Epitope-tagged FRAG1 protein showed a perinuclear localization by immunofluorescence staining. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C terminus alteration through a chromosomal fusion with FRAG1.

Progression to steroid autonomy is accompanied by altered sensitivity to growth factors in S115 mouse mammary tumour cells

Progression to steroid autonomy is a major clinical problem in the treatment of steroid-sensitive tumours. Molecular mechanisms remain unknown but recent hypotheses imply a role for growth factors in this progression. Since S115 + A androgen-responsive mouse mammary tumour cells provide a model system to study this phenomenon in vitro, we have used this model to investigate growth factor gene expression and sensitivity during progression from a steroid sensitive to insensitive state. S115 + A androgen-responsive cells showed a positive proliferative response, morphological response and increased saturation density to various forms of fibroblast growth factor (FGF) and transforming growth factor beta (TGF beta) in both monolayer and suspension culture. A marked synergy was noted, however, between FGF and TGF beta in promoting growth in suspension culture. S115 + A cells possessed mRNA for both acidic FGF (aFGF) and TGF beta 1, both of which were increased by testosterone. Progression to androgen insensitivity was associated with a reversal of growth factor response such that all growth factor responses became generally inhibitory on growth of the unresponsive cells but with a particularly striking synergistic action between FGF and TGF beta 1 on inhibition of both monolayer and suspension growth. Levels of aFGF and TGF beta 1 mRNAs remained low in steroid-insensitive S115-A cells, indicating that loss of response was not associated with any constitutive upregulation of endogenous production of one of these growth factors. The scientific and clinical implications are discussed.

Over-expression of fibroblast growth factors in Xenopus embryos

A number of forms of fibroblast growth factor (FGF) were over-expressed within Xenopus embryos by injection of synthetic FGF mRNAs into fertilized eggs. Injected embryos showed abnormalities in development which were mainly secondary to a disruption of gastrulation movement. The effects observed after injection of bFGF mRNA, however, were much less severe than those observed after injection of an altered form of bFGF mRNA which differs only by the addition of a signal sequence for secretion, or of another member of the FGF family, kFGF, which is normally efficiently secreted. All forms of FGF caused the induction of mesoderm in animal cap explants isolated from blastulae, but the amount of bFGF mRNA required to induce the formation of significant levels of mesoderm was higher by a factor of over a hundred than that of the FGFs which contain a signal sequence for secretion. Over-expressed bFGF accumulated in the nuclei of bastulae but did not necessarily cause mesoderm formation. These results show that FGFs must be secreted from the cells in which they are synthesised in order to act efficiently as mesoderm inducing factors and suggest that bFGF itself, which does not contain a signal sequence for secretion, is unlikely to be directly involved in mesoderm induction during early embryonic development.

A naturally occurring secreted form of fibroblast growth factor (FGF) receptor 1 binds basic FGF in preference over acidic FGF.

Alternatively spliced variants of fibroblast growth factor receptor 1 mRNA are predicted to encode secreted forms and membrane-bound forms of receptors. The predicted amino acid sequences of these receptor variants differ in a portion of the extracellular region. In this study, we characterized the function of one of these splice variants which was predicted by its cDNA to be a secreted FGF receptor. We expressed this secreted form of the human FGFR1 (sFGFR1) in Chinese hamster ovary cells. The sFGFR1 protein oligomerized upon ligand binding. Surprisingly, the sFGFR1 preferentially bound basic FGF over acidic FGF. In cross-linking experiments, the sFGFR1 showed higher binding affinity for basic FGF (Kd approximately 30 nM) than for acidic FGF (Kd greater than 300 nM). These results suggest that this secreted form of FGF receptor has an unusual ligand binding specificity that may be important for its biological role in vivo.

Retinoic acid potentiates interleukin-1- and fibroblast growth factor-induced human synovial fibroblast proliferation

All trans-retinoic acid (ATRA) and related compounds, at concentrations ranging from 10(-8) to 10(-6) M, augmented the proliferation of human synovial fibroblasts (HSN) stimulated by human interleukin-1 alpha or -beta (IL-1 alpha, IL-beta) and both the acidic and basic forms of fibroblast growth factor (FGFa, FGFb). In contrast, ATRA had no effect on human tumor necrosis factor alpha (TNF alpha)-induced HSN proliferation. The potentiation of HSN proliferation was completely dependent on the presence of IL-1 or FGF since HSN were unresponsive to ATRA alone. The mechanism by which ATRA enhances IL-1-induced HSN proliferation does not appear mediated by changes in the affinity or number of IL-1 receptors expressed by HSN; however, treatment with dexamethasone (DEX, 10(-6)M) resulted in a twofold increase in IL-1 receptor number. ATRA inhibited both IL-1 beta- and TNF alpha-induced secretion of prostaglandin-E2 (PGE2), a potent feedback inhibitor of cytokine-stimulated HSN proliferation. However, the synergistic effect of ATRA on IL-1- or FGF-induced proliferation did not appear related to the secretion of cyclooxygenase products since ATRA had no effect on TNF alpha-induced HSN proliferation and indomethacin was included in all HSN proliferation experiments. The results of this study suggest that ATRA may contribute to the pathology of chronic arthritic disease by potentially causing increased growth of the joint-destroying pannus.

Ligand-induced transphosphorylation between different FGF receptors.

Recent evidence shows that different fibroblast growth factors (FGF) bind with similar high affinities to two FGF receptors (FGFR) called flg and bek. In order to explore the mechanism of FGFR tyrosine autophosphorylation, we have generated cell lines which co-express a kinase-negative mutant of FGFR and an active form of FGFR. The following transfected NIH 3T3 cells were generated: (i) cells which express a shorter truncated form of bek (two Ig domains) together with a kinase-negative mutant of full length bek (bek K517A), (ii) cells which express wild-type bek together with kinase-negative flg (flg K514A) and (iii) cells co-expressing wild-type flg together with bek K517A. Immunoprecipitations with either bek-or flg-specific antisera followed by immunoblotting indicated that the double transfectants express the desired receptor species. The addition of acidic FGF (aFGF) to the various cell lines followed by immunoprecipitation with anti-FGFR antibodies and immunoblotting with anti-phosphotyrosine specific antibodies indicated that aFGF induces tyrosine phosphorylation of the kinase-negative FGFR mutants. These results show that tyrosine autophosphorylation of the kinase-negative FGFR is mediated by a transphosphorylation mechanism and that both homologous (bek----bek) and heterologous (bek----flg and flg----bek) transphosphorylation occurs in living cells. Recent evidence shows that tyrosine autophosphorylation of receptors with tyrosine kinase activities is essential for mediating interactions with signaling molecules. Therefore, heterologous transphosphorylation could amplify the response of cells to various forms of FGFs and their cognate receptors.