FGFR1 Isoforms Research Articles

Abstract 1876: Preclinical efficacy of fibroblast growth factor ligand trap HGS1036 in lung carcinoma models with genomic amplification of FGFR1

Abstract Lung cancer is the most common cause of cancer-related death in industrialized countries. In 2010, it is estimated that cancer of the lung and bronchus will be responsible for 157,300 deaths in the US (Jermal et al. 2010). A number of recent publications have reported the genomic amplification of the fibroblast growth factor receptor 1 gene (FGFR1) in lung cancer (Weiss et al. 2010; Voortman et al. 2010). Subtype analysis indicates that approximately 22% of squamous non-small cell lung cell carcinomas (NSCLC) and 27-33% of small cell lung cancer (SCLC) have amplification of the FGFR1 gene. Amplification of the FGFR1 gene in lung cancer cell lines is associated with over-expression of the FGFR1 receptor protein and increased signaling through the FGFR1 pathway resulting in increased cell proliferation and tumorigenesis. HGS1036 (formerly FP-1039) is a soluble fusion protein consisting of the extracellular domain of human FGFR1 isoform α-IIIc linked to the Fc region of human immunoglobulin G1 (IgG1). HGS1036 acts as a ligand “trap” that sequesters multiple fibroblast growth factor (FGF) family ligands, blocking their ability to bind to and activate multiple FGF receptors. We investigated the preclinical efficacy of HGS1036 in preclinical lung cancer models with genomic FGFR1 amplification as a novel therapeutic strategy. A panel of lung cancer cell lines (n=5) were identified that displayed genomic FGFR1 amplification on chromosome 8. Two of the cell lines, DMS53 and DMS114, were derived from the tumors of patients with SCLC. The other three cell lines, NCI-H1581, NCI-H520 and NCI-H1703, were derived from the tumors of patients with NSCLC. Examination of in vitro drug sensitivity indicated that all 5 cell lines were sensitive to HGS1036. Treatment of NCI-H1581, NCI-H520, NCI-H1703, DMS53 and DMS114 cells with HGS1036 in vitro resulted in a 52%, 71%, 46%, 47% and 82% reduction in cell proliferation, respectively, as determined by CellTiterGlo. Sensitivity to HGS1036 in vitro generally correlated with FGFR1 copy number and total FGFR1 protein expression. HGS1036 treatment of mice bearing FGFR1 amplified lung cancer xenografts resulted in tumor growth inhibition in all five FGFR1-amplified models examined (p=<0.01). The degree of tumor growth inhibition, as assessed by area-under-the-curve analysis, varied from 31-84% depending on the FGFR1 amplified lung model examined. These experiments demonstrate that lung tumor cell lines with genomic FGFR1 amplification can be sensitive to FGF-ligand blockade by HGS1036 and provide preclinical rationale for the clinical study of HGS1036 in the treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1876. doi:1538-7445.AM2012-1876

Abstract 284: FGFR2 is required for growth and survival in a rare subset of FGFR2 amplified colorectal cancer

Abstract FGFR2 amplification has been reported in subsets of gastric cancer and breast cancer. We describe FGFR2 amplification in a cell line derived from a colorectal carcinoma arising in a young patient with ulcerative colitis. The cell line has features of endocrine differentiation, representing a rare (< 1%) subset of colorectal cancer. FGFR2 amplification in this cell line results in an overexpressed and highly activated FGFR2 kinase. The FGFR selective inhibitor PD173074 blocked FGFR2 phosphorylation and inhibited Erk, Akt, and S6 ribosomal protein phosphorylation. FGFR2 inhibition resulted in profound cell death as measured by robust accumulation of cells in subG1 by flow cytometry and PARP cleavage by western analysis. A structurally unrelated FGFR2 inhibitor, MK2461, also caused cell death and inhibited downstream signaling pathways. RTK arrays revealed coactivation of EGFR family kinases, and a functional role for these kinases is being investigated. Interestingly, an FGFR2 isoform in this cell line migrated at 160kD, in contrast to the 140kD isoform observed in SNU16 and other cell lines. However, C-terminal FGFR2 antibodies did not react with the 160kD FGFR2 isoform, suggesting that it has a C-terminal deletion similar to the FGFR2 isoform in OCUM2M and KatoIII cell lines. We are currently defining the frequency of FGFR2 overexpression and activation in colon cancer tissue microarrays. The strong growth inhibition and induction of cell death observed in vitro suggests that colorectal cancer tumor cells harboring FGFR2 amplification / activation may show similar response to FGFR inhibitor therapy. 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 284.

FGFR2 Abnormalities Underlie a Spectrum of Bone, Skin, and Cancer Pathologies

Fibroblast growth factor receptor (FGFR)2 is regulated on the basis of the balance of FGFs, heparan-sulfate proteoglycans, FGFR2 isoforms, endogenous inhibitors, and microRNAs. FGFR2 signals cross-talk with hedgehog, bone morphogenetic protein, and other regulatory networks. Some cases of congenital skeletal disorders with an FGFR2 mutation show skin phenotypes, including acne, cutis gyrata, and acanthosis nigricans. Gain-of-function mutations or variations of human FGFR2 occur in estrogen receptor-positive breast cancer, diffuse-type gastric cancer, and endometrial uterine cancer. Oral administration of AZD2171 or Ki23057 inhibits in vivo proliferation of cancer cells with aberrant FGFR2 activation in rodent therapeutic models. However, loss-of-function mutations of FGFR2 are reported in human melanoma. Conditional Fgfr2b knockout in the rodent epidermis leads to increased macrophage infiltration to the dermis and adipose tissue, epidermal thickening accompanied by basal-layer dysplasia and parakeratosis, and the promotion of chemically induced squamous-cell carcinoma. Dysregulation of FGFR2 results in a spectrum of bone and skin pathologies and several types of cancer.

Novel FGFR2 deletion in a patient with Beare–Stevenson‐like syndrome

Novel <i>FGFR2</i> deletion in a patient with Beare–Stevenson‐like syndrome

Histone-Acetylated Control of Fibroblast Growth Factor Receptor 2 Intron 2 Polymorphisms and Isoform Splicing in Breast Cancer

Recent genome-wide association studies have identified fibroblast growth factor receptor (FGFR)2 as one of a few candidate genes linked with breast cancer susceptibility. In particular, the disease-predisposing allele of FGFR2 is inherited as a 7.5-kb region within intron 2 that harbors eight single nucleotide polymorphisms. The relationship between these single nucleotide polymorphisms and FGFR2 gene expression remains unclear. Here we show the common occurrence of polymorphisms within the intron 2 region in a panel of 10 breast cancer cell lines. High FGFR2-expressing cell lines such as MCF-7 cells displayed polymorphic sequences with constitutive histone acetylation at multiple intron 2 sequences harboring putative transcription binding sites. Knockdown of Runx2 or CCAAT enhancer binding protein beta in these cells resulted in diminished endogenous FGFR2 gene expression. In contrast FGFR2-negative MDA-231 cells were wild type and showed evidence of histone 3/4 deacetylation at the rs2981578, rs10736303, and rs7895676 disease-associated alleles that harbor binding sites for Runx2, estrogen receptor, and CCAAT enhancer binding protein beta, respectively. Histone deacetylation inhibition with trichostatin A resulted in enhanced acetylation at these intron 2 sites, an effect associated with robust FGFR2 reexpression. Isoform analysis proved reexpression of the FGFR2-IIIc variant the splicing of which was positively influenced by trichostatin A-mediated recruitment of the Fas-activated serine/threonine phosphoprotein survival protein. Our findings highlight the potential role of histone acetylation in modulating access to selected polymorphic sites within intron 2 as well as downstream splicing sites in generating variable FGFR2 levels and isoforms in breast cancer.

Co-receptor Requirements for Fibroblast Growth Factor-19 Signaling

FGF19 is a unique member of the fibroblast growth factor (FGF) family of secreted proteins that regulates bile acid homeostasis and metabolic state in an endocrine fashion. Here we investigate the cell surface receptors required for signaling by FGF19. We show that betaKlotho, a single-pass transmembrane protein highly expressed in liver and fat, induced ERK1/2 phosphorylation in response to FGF19 treatment and significantly increased the interactions between FGF19 and FGFR4. Interestingly, our results show that alphaKlotho, another Klotho family protein related to betaKlotho, also induced ERK1/2 phosphorylation in response to FGF19 treatment and increased FGF19-FGFR4 interactions in vitro, similar to the effects of betaKlotho. In addition, heparin further enhanced the effects of both alphaKlotho and betaKlotho in FGF19 signaling and interaction experiments. These results suggest that a functional FGF19 receptor may consist of FGF receptor (FGFR) and heparan sulfate complexed with either alphaKlotho or betaKlotho.

Skeletal overgrowth is mediated by deficiency in a specific isoform of fibroblast growth factor receptor 3

Fibroblast growth factor receptor 3 (FGFR3) plays an important role in the control of chondrocyte proliferation and differentiation, a process critical for normal development of the skeleton. To reveal the contributions of the epithelial Fgfr3b isoform and the mesenchymal Fgfr3c isoform to skeletal overgrowth seen in mice, in which both isoforms have been inactivated (Fgf3c(-/-) mice), we have generated mice in which each of the two Fgfr3 isoforms has been selectively inactivated. Whereas no apparent phenotype was detected in Fgfr3b(-/-) mice, strong stimulation of chondrocyte proliferation in the growth plates of Fgf3c(-/-) mice caused dramatic skeletal overgrowth and other skeletal abnormalities resembling the phenotype of mice deficient in both Fgfr3 isoforms. In addition, Fgfr3c(-/-) mice exhibited decreased bone mineral density in the cortical and trabecular bone, whereas the bone mineral density of Fgfr3b(-/-) mice resembled that of WT mice. These experiments demonstrated that the mesenchymal Fgfr3c isoform is responsible for controlling chondrocyte proliferation and differentiation that mediate normal skeletal development, whereas the epithelial Fgfr3b isoform does not contribute toward this process.

Fibroblast growth factor receptor 2 tyrosine kinase is required for prostatic morphogenesis and the acquisition of strict androgen dependency for adult tissue homeostasis

The fibroblast growth factor (FGF) family consists of 22 members and regulates a broad spectrum of biological activities by activating diverse isotypes of FGF receptor tyrosine kinases (FGFRs). Among the FGFs, FGF7 and FGF10 have been implicated in the regulation of prostate development and prostate tissue homeostasis by signaling through the FGFR2 isoform. Using conditional gene ablation with the Cre-LoxP system in mice, we demonstrate a tissue-specific requirement for FGFR2 in urogenital epithelial cells--the precursors of prostatic epithelial cells--for prostatic branching morphogenesis and prostatic growth. Most Fgfr2 conditional null (Fgfr2(cn)) embryos developed only two dorsal prostatic (dp) and two lateral prostatic (lp) lobes. This contrasts to wild-type prostate, which has two anterior prostatic (ap), two dp, two lp and two ventral prostatic (vp) lobes. Unlike wild-type prostates, which are composed of well developed epithelial ductal networks, the Fgfr2(cn) prostates, despite retaining a compartmented tissue structure, exhibited a primitive epithelial architecture. Moreover, although Fgfr2(cn) prostates continued to produce secretory proteins in an androgen-dependent manner, they responded poorly to androgen with respect to tissue homeostasis. The results demonstrate that FGFR2 is important for prostate organogenesis and for the prostate to develop into a strictly androgen-dependent organ with respect to tissue homeostasis but not to the secretory function, implying that androgens may regulate tissue homeostasis and tissue function differently. Therefore, Fgfr2(cn) prostates provide a useful animal model for scrutinizing molecular mechanisms by which androgens regulate prostate growth, homeostasis and function, and may yield clues as to how advanced-tumor prostate cells escape strict androgen regulations.

The mouse seminal vesicle shape mutation is allelic withFgfr2

The mouse seminal vesicle shape (svs) mutation is a spontaneous recessive mutation that causes branching morphogenesis defects in the prostate gland and seminal vesicles. Unlike many other mutations that reduce prostatic and/or seminal vesicle branching, the svs mutation dramatically reduces branching without reducing organ growth. Using a positional cloning approach, we identified the svs mutant lesion as a 491 bp insertion in the tenth intron of Fgfr2 that results in changes in the pattern of Fgfr2 alternative splicing. An engineered null allele of Fgfr2 failed to complement the svs mutation proving that a partial loss of FGFR2(IIIb) isoforms causes svs phenotypes. Thus, the svs mutation represents a new type of adult viable Fgfr2 allele that can be used to elucidate receptor function during normal development and in the adult. In the developing seminal vesicles, sustained activation of ERK1/2 was associated with branching morphogenesis and this was absent in svs mutant seminal vesicles. This defect appears to be the immediate downstream effect of partial loss of FGFR2(IIIb) because activation of FGFR2(IIIb) by FGF10 rapidly induced ERK1/2 activation, and inhibition of ERK1/2 activation blocked seminal vesicle branching morphogenesis. Partial loss of FGFR2(IIIb) was also associated with down-regulation of several branching morphogenesis regulators including Shh, Ptch1, Gli1, Gli2, Bmp4, and Bmp7. Together with previous studies, these data suggest that peak levels of FGFR2(IIIb) signaling are required to induce branching and sustain ERK1/2 activation, whereas reduced levels support ductal outgrowth in the prostate gland and seminal vesicles.

379 POSTER The role of Src-family kinases in the activation of the EGFR following chemotherapy

Mounting experimental evidence has suggested that the trophic environment of cells in culture is an important determinant of their vulnerability to the cytotoxic effects of reactive oxidants such as peroxynitrite (ONOO−). However, acidic fibroblast growth factor (FGF-1)-induced signaling renders some cells more sensitive and others resistant to the cytotoxic effects of ONOO−. To determine whether alternatively spliced fibroblast growth factor receptor (FGFR-1) isoforms are responsible for this differential response, we have stably transfected FGFR-negative rat brain-derived resistant vessel endothelial cells (RVEC) with human cDNA sequences encoding either FGFR-1α or FGFR-1β. FGF-1 treatment of RVEC(R-1α) transfectants enhanced ONOO−-mediated cell death in a manner dependent upon FGFR-1 tyrosine kinase, MEK/Erk 1/2 kinase, and p38 MAP kinase activities and independent of Src-family kinase (SFK) activity. FGF-1 treatment of RVEC(R-1β) transfectants inhibited the cytotoxic effects of ONOO− in a manner dependent upon FGFR-1 tyrosine kinase, MEK/Erk 1/2 kinase, and SFK activities and independent of p38 MAP kinase activity. FGF-1-induced preactivation of both FGFR-1 tyrosine and Erk 1/2 kinases was detected in both RVEC(R-1α) and RVEC(R-1β) transfectants. FGF-1-induced preactivation of p38 MAPK was restricted to RVEC(R-1α) transfectants, whereas, ligand-induced preactivation of SFK was limited to RVEC(R-1β) transfectants. Collectively, these results both reemphasize the role of extracellular trophic factors and their receptor-mediated signaling pathways during cellular responses to oxidant stress and provide a first indication that the alternatively spliced FGFR-1 isoforms induce differential signal transduction pathways.

Gene Expression Profiling of Fibroblast Growth Factor Ligands and their Receptors in Rat Hepatic Stellate Cells (HSC) and In In Vitro Transdifferentiated Myofibroblasts (MFB)

Hepatic stellate cells (HSC) are pericytes of the sinusoidal compartment and represent the major site of vitamin A storage. After hepatic injury, HSC are activated and may play a key role in the regeneration of the liver. The liver has an amazing capacity to regenerate back to normal cell mass in a short time which requires a rise in cytokines and growth factors to promote proliferation and migration of various cell types. Activation of HSC is accompanied by morphological and metabolic changes including increased synthesis and secretion of cytokines and growth factors. FGFs and their receptors are important regulators of repair of various tissues and organs by stimulation of cell migration and proliferation. FGF signals are transduced via four related FGF receptor tyrosine kinases and their variants. In this study, we employed low density oligonucleotide arrays and reverse transcriptase-polymerase chain reaction (RT-PCR) to identify the expression profiles of different FGFs, their receptors and antagonists in hepatic stellate cells. The expression panel was compared with that obtained from in vitro transdifferentiated myofibroblasts (MFB). The expression analysis revealed that HSC and MFB express mRNA of four FGF ligands: FGF4, FGF5, FGF9 and FGF10. Freshly isolated HSC express FGF-R1(IIIc), FGF-R2(IIIc), FGF-R3(IIIc) and FGFR4–17 mRNA. Surprisingly, in vitro transdifferentiated MFB express the FGF-R1(IIIb) isoform in addition. Although HSC and MFB have the cognate receptors to the FGF ligands they express, both cell types are non-responsive to these FGF ligands, while the same FGF ligands induce proliferation and DNA synthesis in hepatocytes suggesting a paracrine function. The related Sprouty and Spred protein families are recently discovered negative regulators of FGF receptor signaling. Since HSC and MFB express mRNA of Sprouty 1, 2 and 4 and of Spred 1 and 2, our findings may suggest that members of both inhibitor families function in a negative feedback mechanism controlling the autocrine action of FGFs in HSC.

Alternative Splicing of Fibroblast Growth Factor Receptor 3 Produces a Secreted Isoform That Inhibits Fibroblast Growth Factor–Induced Proliferation and Is Repressed in Urothelial Carcinoma Cell Lines

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that play key roles in proliferation, differentiation, and tumorigenesis. FGFR3 was identified as the major family member expressed in both normal human urothelium and cultured normal human urothelial (NHU) cells and was expressed as the IIIb isoform. We also identified a splice variant, FGFR3 Delta8-10, lacking exons encoding the COOH-terminal half of immunoglobulin-like domain III and the transmembrane domain. Previous reports have assumed that this is a cancer-specific splice variant. We showed that FGFR3 Delta8-10 is a normal transcript in NHU cells and is translated, N-glycosylated, and secreted. Primary urothelium expressed high levels of FGFR3 transcripts. In culture, levels were reduced in actively proliferating cells but increased at confluence and as cells approached senescence. Cells overexpressing FGFR3 IIIb showed FGF1-induced proliferation, which was inhibited by the addition of FGFR3 Delta8-10. In bladder tumor cell lines derived from aggressive carcinomas, there were significant alterations in the relative expression of isoforms including an overall decrease in the proportion of FGFR3 Delta8-10 and predominant expression of FGFR3 IIIc in some cases. In summary, alternative splicing of FGFR3 IIIb in NHU cells represents a normal mechanism to generate a transcript that regulates proliferation and in bladder cancer, the ratio of FGFR3 isoforms is significantly altered.

Fibroblast Growth Factor (FGF)-1, FGF2, FGF7 and FGF Receptors are Uniformly Expressed in Trophoblast Giant Cells During Restricted Trophoblast Invasion in Cows

The bovine placenta is characterized by a limited invasion of trophoblast giant cells (TGC). In contrast to mononuclear trophoblast cells (MTC), TGC are non-polarized cells, which migrate and fuse with single uterine epithelial cells throughout gestation. Fibroblast growth factors (FGF) were shown to be associated with the migratory activity of cells, cell differentiation and angiogenesis, and due to its localization in trophoblast cells were proposed as important regulating factors in hemochorial placentae of rodents and humans, and the (syn)epitheliochorial placenta of pig and sheep. Since migrating bovine TGC are of epithelial origin, but exhibit similarities to mesenchymal cells we hypothesize that the restricted trophoblast invasion in cattle is characterized by a specific FGF expression pattern. Therefore, the spatiotemporal expression of specific FGF factor:receptor pairs, either acting on cells of mesenchymal origin or on epithelial cells was examined in bovine placental tissues throughout gestation and prepartum by immunohistochemistry, semiquantitative RT-PCR and in situ hybridization. FGF1 protein was found in trophoblast, caruncular epithelium (CE) and stroma (CS), stroma of chorionic villi (SCV), and in fetal and maternal blood vessels. FGF2 signals dominated in maternal vascular endothelia (VE), immature TGC, and MTC, whereas staining in other cell types was clearly weaker. FGF7 protein was detected in fetal and maternal blood vessel as well as in immature TGC and MTC predominantly at the chorionic plate. FGFR immunoreaction was localized in immature TGC, MTC, and to a clearly lesser extent in CS, CE and fetal and maternal blood vessels. Mature TGC stained negatively for all examined factors and FGFR. The corresponding mRNAs specific for FGF1, -2, -7, total FGFR, and FGFR2 isoforms IIIb and IIIc were colocalized in immature TGC, whereas hybridization was substantially lower in CE and absent in CS, SCV and mature TGC throughout gestation, but switched to CS and VE immediately prepartum. Semiquantitative RT-PCR revealed higher mRNA levels for FGF1, FGFR, and FGFR2IIIc in cotyledons compared to caruncles (p<0.05), whereas it was the opposite with FGF2 (p<0.001). FGF7 and FGFR2IIIb mRNA levels did not differ between caruncles and cotyledons. Significant changes (p<0.05) of mRNA levels related to gestational age were found for FGF1 and FGFR2IIIc, but not for FGF2, -7, total FGFR, and FGFR2IIIb. The specific localization of all examined FGF family members in TGC suggests that TGC, apart from their classical function as producers of hormonal products, play other important roles in the regulation of bovine placentomal growth, differentiation and angiogenesis.

Analysis of the Biochemical Mechanisms for the Endocrine Actions of Fibroblast Growth Factor-23

Fibroblast growth factor (FGF)-23 has emerged as an endocrine regulator of phosphate and of vitamin D metabolism. It is produced in bone and, unlike other FGFs, circulates in the bloodstream to ultimately regulate phosphate handling and vitamin D production in the kidney. Presently, it is unknown which of the seven principal FGF receptors (FGFRs) transmits FGF23 biological activity. Furthermore, the molecular basis for the endocrine mode of FGF23 action is unclear. Herein, we performed surface plasmon resonance and mitogenesis experiments to comprehensively characterize receptor binding specificity. Our data demonstrate that FGF23 binds and activates the c splice isoforms of FGFR1-3, as well as FGFR4, but not the b splice isoforms of FGFR1-3. Interestingly, highly sulfated and longer glycosaminoglycan (GAG) species were capable of promoting FGF23 mitogenic activity. We also show that FGF23 induces tyrosine phosphorylation and inhibits sodium-phosphate cotransporter Npt2a mRNA expression using opossum kidney cells, a model kidney proximal tubule cell line. Removal of cell surface GAGs abolishes the effects of FGF23, and exogenous highly sulfated GAG is capable of restoring FGF23 activity, suggesting that proximal tubule cells naturally express GAGs that are permissive for FGF23 action. We propose that FGF23 signals through multiple FGFRs and that the unique endocrine actions of FGF23 involve escape from FGF23-producing cells and circulation to the kidney, where highly sulfated GAGs most likely act as cofactors for FGF23 activity. Our biochemical findings provide important insights into the molecular mechanisms by which dysregulated FGF23 signaling leads to disorders of hyper- and hypophosphatemia.

Regulation of Fibroblast Growth Factor-2 Activity by Human Ovarian Cancer Tumor Endothelium

Fibroblast growth factor-2 (FGF-2) is a potent angiogenic cytokine that is dependent on heparan sulfate for its biological activity. We have investigated the relationship among heparan sulfate, FGF-2, and the signal-transducing receptors in human, advanced-stage, serous ovarian adenocarcinoma. Using a unique molecular probe, FR1c-Ap, which consisted of a soluble FGF receptor 1 isoform IIIc covalently linked to an alkaline phosphatase moiety, the distribution of heparan sulfate that had the ability to support the formation of a heparan sulfate/FGF-2/FGFR1 isoform IIIc alkaline phosphatase heparan sulfate construct complex was determined. This may be taken as a surrogate marker for the distribution of biologically active heparan sulfate and was distributed predominantly in endothelial cells and stroma but was absent from adenocarcinoma cells. In situ hybridization revealed the expression of FGFR1 mRNA in the endothelium and reverse transcription-PCR confirmed the presence of FGFR1 isoform IIIc but not isoform IIIb. The presence of FGF-2 around tumor endothelium was detected through immunohistochemistry. Double-staining techniques showed that heparan sulfate was found predominantly at the basal aspect of the endothelium and suggested that syndecan-3 might function as one of the proteoglycans involved in FGF-2 signaling in the endothelium. The data suggest that the entire extracellular signaling apparatus, consisting of FGF-2, biologically active heparan sulfate, and FGFRs capable of responding to FGF-2, is present in ovarian cancer endothelium, thereby highlighting the cytokine and its cognate receptor as potential targets for the antiangiogenic treatment of this disease.

Expression pattern of fibroblast growth factors (FGFs), their receptors and antagonists in primary endothelial cells and vascular smooth muscle cells

Fibroblast growth factors (FGFs) are important angiogenic growth factors. While basic FGF (FGF2) is well established as a potent inducer of angiogenesis much less is known about other FGFs possibly expressed by EC. We investigated the expression of all known FGFs, their main tyrosine kinase receptors and antagonists by RT-PCR analysis in human umbilical vascular endothelial cells (HUVECs) to obtain a complete expression profile of this important growth factor system in model endothelial cells (EC). In addition to FGFR1IIIc, which is considered as the major FGF receptor in EC, HUVECs express similar levels of FGFR3IIIc, detectable amounts of FGFR2IIIc and a new FGF receptor without an intracellular kinase domain (FGFR5). HUVECs express several secreted FGFs, including FGF5, 7, 8, 16 and 18 and two members of the fibroblast growth factor homologous factors (FHFs), not yet reported to be expressed in EC. The expression panel was compared with that obtained from human vascular smooth muscle cells (VSMCs) and human aortic tissue. Human umbilical artery smooth muscle cells (HUASMCs) and HUVECs express the identical FGF receptor and ligand panel implicating that both cell types act, according the FGF signals more as an entity than as individual cell types. Expression of Fgf1, 2, 7, 16 and 18 and the antagonists Sprouty 2,3 and 4 was demonstrated for all analysed cDNAs. The IIIc isoforms of FGFR1 and 2 and the novel FGFR5 were expressed in the aorta, but expression of the FGF receptor 3 was not detected in cDNAs derived from aortic tissue. In the VSMC of rat aortic tissue and in HUASM cultured cells we could demonstrate FGF18 immunoreactivity in the nucleus of the cells. The expression of several secreted FGFs by EC may focus the view more on their paracrine effects on neighbouring cells during tissue regeneration or tumor formation.

Proliferation of Neointimal Smooth Muscle Cells after Arterial Injury: DEPENDENCE ON INTERACTIONS BETWEEN FIBROBLAST GROWTH FACTOR RECEPTOR-2 AND FIBROBLAST GROWTH FACTOR-9

The growth factor signaling mechanisms responsible for neointimal smooth muscle cell (SMC) proliferation and accumulation, a characteristic feature of many vascular pathologies that can lead to restenosis after angioplasty, remain to be identified. Here, we examined the contribution of fibroblast growth factor receptors (FGFRs) 2 and 3 as well as novel fibroblast growth factors (FGFs) to such proliferation. Balloon catheter injury to the rat carotid artery stimulated the expression of two distinctly spliced FGFR-2 isoforms, differing only by the presence or absence of the acidic box, and two distinctly spliced FGFR-3 isoforms containing the acidic box and differing only by the presence of either the IIIb or IIIc exon. Post-injury arterial administration of recombinant adenoviruses expressing dominant negative mutant forms of these FGFRs were used to assess the roles of the endogenous FGFR isoforms in neointimal SMC proliferation. Dominant negative FGFR-2 containing the acidic box inhibited such proliferation by 40%, whereas the dominant negative FGFR-3 forms had little effect. Expression of FGF-9, known to be capable of binding to all four neointimal FGFR-2/-3 isoforms, was abundant within the neointima. FGF-9 markedly stimulated both the proliferation of neointimal SMCs and the activation of extracellular signal-related kinases 1/2, effects which were abrogated by the administration of antisense FGF-9 oligonucleotides to injured arteries and the expression of the dominant negative FGFR-2 adenovirus in cultured neointimal SMCs. These studies demonstrate that, although multiple FGFRs are induced in neointimal SMCs following arterial injury, specific interactions between distinctly spliced FGFR-2 isoforms and FGF-9 contribute to the proliferation of these SMCs.

Fgfr mRNA isoforms in craniofacial bone development

Mutations in genes encoding for fibroblast growth factor receptors (FGFRs) have been identified as causes of both chondrodysplasias and craniosynostoses, both of which cause abnormalities in the growth and development of the craniofacial region. FGFRs form mRNA splicing isoforms, each with distinct ligand binding specificity and tissue distribution. These confer specific biological functions on these isoforms. Although it is known that FGFRs are expressed at numerous locations during early mouse development, including the craniofacial area, relatively little is known about the expression of the splicing isoforms during craniofacial bone development. To address this, we have performed a detailed survey to detect these genes in the developing mouse craniofacial region. We have analyzed the developing mouse mandible, calvaria, and cranial base, in particular the spheno-occipital synchondrosis, a key centre of craniofacial growth. Fgfr1c was detected weakly in osteoblastic cells in both the developing calvarial and mandibular bones. Fgfr3b and Fgfr3c were found chiefly in proliferating chondrocytes of the cranial base synchondroses and the mandibular condyle. Fgfr2b transcripts were most notably detected in the perichondria of the mandibular condyle and the cranial base. Fgfr2c transcripts were detected with high intensity in differentiating osteoblasts at the sutural osteogenic fronts of the calvarial bones. In addition, Fgfr2c was also expressed in the perichondria of the mandibular condyle and the cranial base. These expression patterns suggest both differing and similar functions for -b and -c isoforms. The former is exemplified by Fgfr1 transcripts, which show distinct differences in their distribution, being mutually exclusive. Similar functions are suggested by the overlapping expression patterns of the -b and -c isoforms of both Fgfr2 and Fgfr3. Fgfr4 transcripts were found in developing muscles. These data help to explain the disturbances in craniofacial growth exhibited by both patients and the growing number of transgenic mice carrying mutations in genes encoding FGFRs/Fgfrs.

IL-15 and its role in rheumatoid arthritis

Background: IL-15 is involved in all phases of rheumatoid arthritis. Recently we have shown that rheumatoid arthritis synovial fibroblasts (RASF) express both IL-15 and functional IL-15 receptor [1]. Objective: The aim of present study was to identify pathways that are regulated by autocrine IL-15 (IL-15R) in RASF. Methods: RASF were transfected with plasmid encoding IL-15R antagonist (CRB-15, Cardion AG) or control constructs. RNA from transient transfectants were used for Microarray analysis. The differential expression of genes obtained by microarray analysis was verified by SYBR Green real-time PCR. The expression of IL-15Rα, cell proliferation and the expression of p16 and p21 were evaluated in stably transfected cells. Results: The IL-15R antagonist produced by transfected RASF blocked the endogenous IL-15/IL-15Rα interaction, which resulted in an inhibition of cell proliferation (45 ± 10%) via an increase of the expression of p16. In addition, we found that inhibition of IL-15Rα induced the expression of mRNA for FGFR-3. Since two isoforms of FGFR-3 have been identified (FGFR-3b and FGFR-3c) [2], we tested the effect of IL-15Rα inhibition on their expression. In contrast to FGFR-3b, the level of mRNA for FGFR-3c was strongly increased in cells transfected with the IL-15R antagonist (4.71 ± 2.5 in transient transfectants and 6.1 ± 1 fold in stable transfectants). FGFR-3c isoform binds specifically FGF-9, but also FGF-2 [2]. Besides FGFR-3, FGF-2 that is abundant in RA joints binds to FGFR-1. In vitro studies revealed that FGFR-1 transmits a potent mitogenic signal, whereas FGFR-3 usually has no stimulatory effect or inhibits cell proliferation. In contrast to FGFR-3c, blocking of IL-15Rα did not change the mRNA expression for FGFR-1 in RASF. Moreover, we checked whether FGF-2 affects the expression of IL-15Rα. Indeed, FGF-2 strongly decreased the spontaneous and tumor necrosis factor alpha-triggered expression of IL-15Rα at the mRNA and protein levels. Conclusion: Our findings raise the possibility of a negative loop between FGF-2/FGFR-3c and IL-15/IL-15R signaling in RASF. Moreover, the activation of RASF by FGFs could depend on the ratio of FGFR-1/FGFR-3 expression, which is controlled by the endogenous IL-15/IL-15R system.

Alternatively spliced FGFR-1 isoform signaling differentially modulates endothelial cell responses to peroxynitrite

Mounting experimental evidence has suggested that the trophic environment of cells in culture is an important determinant of their vulnerability to the cytotoxic effects of reactive oxidants such as peroxynitrite (ONOO −). However, acidic fibroblast growth factor (FGF-1)-induced signaling renders some cells more sensitive and others resistant to the cytotoxic effects of ONOO −. To determine whether alternatively spliced fibroblast growth factor receptor (FGFR-1) isoforms are responsible for this differential response, we have stably transfected FGFR-negative rat brain-derived resistant vessel endothelial cells (RVEC) with human cDNA sequences encoding either FGFR-1α or FGFR-1β. FGF-1 treatment of RVEC(R-1α) transfectants enhanced ONOO −-mediated cell death in a manner dependent upon FGFR-1 tyrosine kinase, MEK/Erk 1/2 kinase, and p38 MAP kinase activities and independent of Src-family kinase (SFK) activity. FGF-1 treatment of RVEC(R-1β) transfectants inhibited the cytotoxic effects of ONOO − in a manner dependent upon FGFR-1 tyrosine kinase, MEK/Erk 1/2 kinase, and SFK activities and independent of p38 MAP kinase activity. FGF-1-induced preactivation of both FGFR-1 tyrosine and Erk 1/2 kinases was detected in both RVEC(R-1α) and RVEC(R-1β) transfectants. FGF-1-induced preactivation of p38 MAPK was restricted to RVEC(R-1α) transfectants, whereas, ligand-induced preactivation of SFK was limited to RVEC(R-1β) transfectants. Collectively, these results both reemphasize the role of extracellular trophic factors and their receptor-mediated signaling pathways during cellular responses to oxidant stress and provide a first indication that the alternatively spliced FGFR-1 isoforms induce differential signal transduction pathways.