Influence of NOS3 rs2070744 genotypes on hepatocellular carcinoma patients treated with lenvatinib.

We investigated whether or not nitric oxide synthase 3 (NOS3) rs2070744 genotypes can affect the response for lenvatinib treatment in patients with hepatocellular carcinoma (HCC). We evaluated the relation of the NOS3 rs2070744 genotypes to the tumor response, progression-free survival (PFS), and overall survival (OS) as the response for lenvatinib. We also examined the association between fibroblast growth factor receptor (FGFR) gene polymorphisms, a potential feature of lenvatinib, and the response. There were no significant differences between the studies for either PFS or OS, even though patients with the TT genotype had a longer mean PFS (hazard ratio [HR] 0.60; p = 0.069) and mean OS (HR 0.46; p = 0.075) than those with the TC/CC genotypes. However, patients with a single-nucleotide polymorphism (SNP) combination pattern of the NOS3 rs2070744 TC/CC and FGFR4 rs351855 CT/TT genotypes had a significantly shorter mean PFS (HR 2.56; p = 0.006) and mean OS (HR 3.36; p = 0.013) than those with the other genotypes. The NOS3 rs2070744 genotypes did not influence the clinical response. However, the SNP combination pattern of the NOS3 rs2070744 and FGFR4 rs351855 genotypes may be helpful as treatment effect predictors and prognostic factors for HCC patients treated with lenvatinib.

Fibroblast growth factor receptor (FGFR) alterations in squamous differentiated bladder cancer: a putative therapeutic target for a small subgroup.

Although drugable fibroblast growth factor receptor (FGFR) alterations in squamous cell carcinomas (SCC) of various entities are well known, little is known about FGFR modifications in squamous differentiated bladder cancer. Therefore, our study evaluated FGFR1-3 alterations as a putative therapeutic target in this subgroup. We analyzed 73 squamous differentiated bladder cancers (n = 10 pT2, n = 55 pT3, n = 8 pT4) for FGFR1-3 protein expression, FGFR1-3 copy number variations, FGFR3 chromosomal rearrangements (fluorescence in situ hybridization (FISH)) and FGFR3 mutations (SNapShot analysis). Only single cases displayed enhanced protein expression, most frequently FGFR3 overexpression (9.4% (6/64)). FISH showed no amplifications of FGFR1, 2 or 3. Break apart events were only slightly above the cut off in 12.1% (8/66) of cases and no FGFR3-TACC3 rearrangements could be proven by qPCR. FGFR3 mutations (p.S249C) were found in 8.5% (6/71) of tumors and were significantly associated with FGFR3 protein overexpression (p < 0.001), and unfavourable clinical outcome (p = 0.001). Our findings are consistent with the results of the TCGA data set for the "squamous-like" subtype of bladder cancer (n = 85), which revealed reduced overall expression of FGFR1 and FGFR2 in tumors compared to normal tissue, while expression of FGFR3 remained high. In the TCGA "squamous-like" subtype FGFR3 mutations were found in 4.9% and correlated with high FGFR3 RNA expression. Mutations of FGFR1 and FGFR2 were less frequent (2.4% and 1.2%). Hence, our comprehensive study provides novel insights into a subgroup of squamous differentiated bladder tumors that hold clues for novel therapeutic regimens and may benefit from FGFR3-targeted therapies.

Novel Bioluminescent Binding Assays for Ligand-Receptor Interaction Studies of the Fibroblast Growth Factor Family.

We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand-receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand-receptor interaction studies.

Roles of FGFR in oral carcinogenesis.

Fibroblast growth factor receptors (FGFRs) play essential roles in organ development during the embryonic period, and regulate tissue repair in adults. Accumulating evidence suggests that alterations in FGFR signalling are involved in diverse types of cancer. In this review, we focus on aberrant regulation of FGFRs in pathogenesis of oral squamous cell carcinoma (OSCC), including altered expression and subcellular location, aberrant isoform splicing and mutations. We also provide an overview of oncogenic roles of each FGFR and its downstream signalling pathways in regulating OSCC cell proliferation and metastasis. Finally, we discuss potential application of FGFRs as anti-cancer targets in the preclinical environment and in clinical practice.

Systemic and placental α-klotho: Effects of preeclampsia in the last trimester of gestation.

INTRODUCTION: α-klotho is an anti-aging protein, potentially important in preeclampsia (PE). Produced by kidney, brain and placenta, and by mRNA splicing is both a full-length membrane-bound and a truncated soluble protein in the circulation. The membrane-bound protein is an obligate co-receptor for fibroblast growth factor 23 (FGF23) and its action on receptor (FGFR), but ADAM proteinases also cause its shedding. The aims of this study were to investigate levels of maternal plasma, placental, and fetal membrane α-Klotho and their association with placental accelerated villous maturation (AVM) in PE. In addition, placental and membrane levels of ADAM17 and FGFR were measured in the same patients. METHODS: Maternal blood, placenta and fetal membranes from 61 women (31 with PE and 30 controls) between 32 and 40 weeks gestation were collected. Plasma α-klotho was measured by ELISA, and quantitative immunohistochemistry used for α-klotho, ADAM17 and FGFR1 in tissues. Placental AVM was histologically assessed. RESULTS: Maternal plasma levels of α-Klotho were higher in PE compared to controls (p = 0.01) and patients with the highest levels had significantly less AVM (p = 0.03). α-Klotho, ADAM17, and FGFR were all present in syncytiotrophoblast and cytotrophoblast of membranes. Between 32 and 40 weeks gestation, all placental levels decreased in controls respectively (p = 0.04, p = 0.004, p = 0.05), but not in PE. Fetal membrane levels were unchanged. DISCUSSION: Maternal plasma α-Klotho was increased in PE and its levels associated with reduced placental AVM. Changes in placental α-Klotho, ADAM17, and FGFR suggest their involvement in the pathophysiology of PE.

Prognostic roles for fibroblast growth factor receptor family members in malignant peripheral nerve sheath tumor.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are rare, highly malignant, and poorly understood sarcomas. The often poor outcome of MPNST highlights the necessity of identifying prognostic predictors for this aggressive sarcoma. Here, we investigate the role of fibroblast growth factor receptor (FGFR) family members in human MPNSTs. RESULTS: aCGH and bioinformatics analysis identified frequent amplification of the FGFR1 gene. FISH analysis revealed that 26.9% MPNST samples had amplification of FGFR1, with both focal and polysomy patterns observed. IHC identified that FGFR1 protein expression was positively correlated with FGFR1 gene amplification. High expression of FGFR1 protein was associated with better overall survival (OS) and was an independent prognostic predictor for OS of MPNST patients. Additionally, combined expression of FGFR1 and FGFR2 protein characterized a subtype of MPNST with better OS. FGFR4 protein was expressed 82.3% of MPNST samples, and was associated with poor disease-free survival. MATERIALS AND METHODS: We performed microarray-based comparative genomic hybridization (aCGH) profiling of two cohorts of primary MPNST tissue samples including 25 patients treated at The University of Texas MD Anderson Cancer Center and 26 patients from Tianjin Medical University Cancer Institute and Hospital. Fluorescence in situ hybridization (FISH) was used to validate the gene amplification detected by aCGH analysis. Another cohort of 63 formalin-fixed paraffin-embedded MPNST samples (including 52 samples for FISH assay) was obtained to explore FGFR1, 2, 3, and 4 protein expression by immunohistochemical (IHC) analysis. CONCLUSIONS: Our integrated genomic and molecular studies provide evidence that FGFRs play different prognostic roles in MPNST.

A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma.

Herein, we have identified cross-talk between the Hippo and fibroblast growth factor receptor (FGFR) oncogenic signaling pathways in cholangiocarcinoma (CCA). Yes-associated protein (YAP) nuclear localization and up-regulation of canonical target genes was observed in CCA cell lines and a patient-derived xenograft (PDX). Expression of FGFR1, -2, and -4 was identified in human CCA cell lines, driven, in part, by YAP coactivation of TBX5. In turn, FGFR signaling in a cell line with minimal basal YAP expression induced its cellular protein expression and nuclear localization. Treatment of YAP-positive CCA cell lines with BGJ398, a pan-FGFR inhibitor, resulted in a decrease in YAP activation. FGFR activation of YAP appears to be driven largely by FGF5 activation of FGFR2, as siRNA silencing of this ligand or receptor, respectively, inhibited YAP nuclear localization. BGJ398 treatment of YAP-expressing cells induced cell death due to Mcl-1 depletion. In a YAP-associated mouse model of CCA, expression of FGFR 1, 2, and 4 was also significantly increased. Accordingly, BGJ398 treatment was tumor-suppressive in this model and in a YAP-positive PDX model. These preclinical data suggest not only that the YAP and Hippo signaling pathways culminate in an Mcl-1-regulated tumor survival pathway but also that nuclear YAP expression may be a biomarker to employ in FGFR-directed therapy.

The involvement of fibroblast growth factor receptor signaling pathways in dermatofibroma and dermatofibrosarcoma protuberans.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) control a wide range of biological functions; however, their involvement in the pathogenesis of dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) is currently unknown. In this study, we first confirmed the histological diagnosis by detecting fusion COL1A1-PDGFB transcripts in DFSP, and examined the expression of all FGFRs (FGFR1-4), some of their ligands (FGF1, 2, 9), and forkhead box N1 (FOXN1) as a downstream target of FGFR3 in DF and DFSP by immunohistochemical analysis. Although we failed to detect the expression of FGF1 and FGF9 as specific ligands for FGFR3 in DF, overexpression of FGFR3 and FOXN1 was observed in the epidermal regions of DF, suggesting that the epidermal regions of DF were similar to seborrhoeic keratosis both in terms of histological features and the activation of FGFR3/FOXN1. In addition, strong expression of FGF2 and FGFR4 was observed in the tumor lesions of DF. Expression patterns of FGFR3/FOXN1 and FGF2/FGFR4 in DF were in contrast with those of DFSP. The activation of FGFR signaling pathways may be not only relevant to the pathogenesis of DF, but also very useful in the differential diagnosis of DF and DFSP.

G protein-coupled receptor heterodimerization in the brain.

G protein-coupled receptors (GPCRs) play critical roles in cellular processes and signaling and have been shown to form heteromers with diverge biochemical and/or pharmacological activities that are different from those of the corresponding monomers or homomers. However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems, the existence of such receptor heterocomplexes in the brain remains largely unknown, mostly because of the lack of appropriate methodology. Herein, we describe the in situ proximity ligation assay procedure underlining its high selectivity and sensitivity to image GPCR heteromers with confocal microscopy in brain sections. We describe here how the assay is performed and discuss advantages and disadvantages of this method compared with other available techniques.

The function of FGF signaling in the lens placode.

Previous studies suggested that FGF signaling is important for lens formation. However, the times at which FGFs act to promote lens formation, the FGFs that are involved, the cells that secrete them and the mechanisms by which FGF signaling may promote lens formation are not known. We found that transcripts encoding several FGF ligands and the four classical FGF receptors are detectable in the lens-forming ectoderm at the time of lens induction. Conditional deletion of Fgfr1 and Fgfr2 from this tissue resulted in the formation of small lens rudiments that soon degenerated. Lens placodes lacking Fgfr1 and 2 were thinner than in wild-type embryos. Deletion of Fgfr2 increased cell death from the initiation of placode formation and concurrent deletion of Fgfr1 enhanced this phenotype. Fgfr1/2 conditional knockout placode cells expressed lower levels of proteins known to be regulated by FGF receptor signaling, but proteins known to be important for lens formation were present at normal levels in the remaining placode cells, including the transcription factors Pax6, Sox2 and FoxE3 and the lens-preferred protein αA-crystallin. Previous studies identified a genetic interaction between BMP and FGF signaling in lens formation and conditional deletion of Bmpr1a caused increased cell death in the lens placode, resulting in the formation of smaller lenses. In the present study, conditional deletion of both Bmpr1a and Fgfr2 increased cell death beyond that seen in Fgfr2(CKO) placodes and prevented lens formation. These results suggest that the primary role of autocrine or paracrine FGF signaling is to provide essential survival signals to lens placode cells. Because apoptosis was already increased at the onset of placode formation in Fgfr1/2 conditional knockout placode cells, FGF signaling was functionally absent during the period of lens induction by the optic vesicle. Since the expression of proteins required for lens formation was not altered in the knockout placode cells, we can conclude that FGF signaling from the optic vesicle is not required for lens induction.

Myeloproliferative disorder FOP-FGFR1 fusion kinase recruits phosphoinositide-3 kinase and phospholipase Cgamma at the centrosome.

BACKGROUND: The t(6;8) translocation found in rare and agressive myeloproliferative disorders results in a chimeric gene encoding the FOP-FGFR1 fusion protein. This protein comprises the N-terminal region of the centrosomal protein FOP and the tyrosine kinase of the FGFR1 receptor. FOP-FGFR1 is localized at the centrosome where it exerts a constitutive kinase activity. RESULTS: We show that FOP-FGFR1 interacts with the large centrosomal protein CAP350 and that CAP350 is necessary for FOP-FGFR1 localisation at centrosome. FOP-FGFR1 activates the phosphoinositide-3 kinase (PI3K) pathway. We show that p85 interacts with tyrosine 475 of FOP-FGFR1, which is located in a YXXM consensus binding sequence for an SH2 domain of p85. This interaction is in part responsible for PI3K activation. Ba/F3 cells that express FOP-FGFR1 mutated at tyrosine 475 have reduced proliferative ability. Treatment with PI3K pathway inhibitors induces death of FOP-FGFR1 expressing cells. FOP-FGFR1 also recruits phospholipase Cgamma1 (PLCgamma1) at the centrosome. We show that this enzyme is recruited by FOP-FGFR1 at the centrosome during interphase. CONCLUSION: These results delineate a particular type of oncogenic mechanism by which an ectopic kinase recruits its substrates at the centrosome whence unappropriate signaling induces continuous cell growth and MPD.

Skin-homing receptors on effector leukocytes are differentially sensitive to glyco-metabolic antagonism in allergic contact dermatitis.

T cell recruitment into inflamed skin is dependent on skin-homing receptor binding to endothelial (E)- and platelet (P)-selectin. These T cell receptors, or E- and P-selectin ligands, can be targeted by the metabolic fluorosugar inhibitor, 4-F-GlcNAc, to blunt cutaneous inflammation. Compelling new data indicate that, in addition to T cells, NK cells are also recruited to inflamed skin in allergic contact hypersensitivity (CHS) contingent on E- and P-selectin-binding. Using a model of allergic CHS, we evaluated the identity and impact of NK cell E-selectin ligand(s) on inflammatory responses and examined the oral efficacy of 4-F-GlcNAc. We demonstrated that the predominant E-selectin ligands on NK cells are P-selectin glycoprotein ligand-1 and protease-resistant glycolipids. We showed that, unlike the induced E-selectin ligand expression on activated T cells upon exposure to Ag, ligand expression on NK cells was constitutive. CHS responses were significantly lowered by orally administered 4-F-GlcNAc treatment. Although E-selectin ligand on activated T cells was suppressed, ligand expression on NK cells was insensitive to 4-F-GlcNAc treatment. These findings indicate that downregulating effector T cell E- and P-selectin ligand expression directly correlates with anti-inflammatory efficacy and provides new insight on metabolic discrepancies of E-selectin ligand biosynthesis in effector leukocytes in vivo.

Selective over-expression of fibroblast growth factor receptors 1 and 4 in clinical prostate cancer.

Fibroblast growth factor receptors (FGFRs) mediate the tumourigenic effects of FGFs in prostate cancer. These receptors are therefore potential therapeutic targets in the development of inhibitors to this pathway. To identify the most relevant targets, we simultaneously investigated FGFR1-4 expression using a prostate cancer tissue microarray (TMA) and in laser capture microdissected (LCM) prostate epithelial cells. In malignant prostates (n = 138) we observed significant FGFR1 and FGFR4 protein over-expression in comparison with benign prostates (n = 58; p < 0.0001). FGFR1 was expressed at high levels in the majority of tumours (69% of grade 3 or less, 74% of grade 4 and 70% of grade 5), while FGFR4 was strongly expressed in 83% of grade 5 cancers but in only 25% of grade 1-3 cancers (p < 0.0001). At the transcript level we observed a similar pattern, with FGFR1 and FGFR4 mRNA over-expressed in malignant epithelial cells compared to benign cells (p < 0.0005 and p < 0.05, respectively). While total FGFR2 was increased in some cancers, there was no association between expression and tumour grade or stage. Transcript analysis, however, revealed a switch in the predominant isoform expressed from FGFR2IIIb to FGFR2IIIc among malignant epithelial cells. In contrast, protein and transcript expression of FGFR3 was very similar between benign and cancer biopsies. The functional effect of targeting FGFR4 in prostate cancer cells has not previously been investigated. In in vitro experiments, suppression of FGFR4 by RNA interference effectively blocked prostate cancer cell proliferation (p < 0.0001) and invasion (p < 0.001) in response to exogenous stimulation. This effect was evident regardless of whether the cells expressed the FGFR4 Arg388 or Gly388 allele. In parallel experiments, FGFR3 suppression had no discernible effect on cancer cell behaviour. These results suggest evidence of selective over-expression of FGFR1 and FGFR4 in clinical prostate cancer and support the notion of targeted inhibition of these receptors to disrupt FGF signalling.

Whole genome oligonucleotide-based array comparative genomic hybridization analysis identified fibroblast growth factor 1 as a prognostic marker for advanced-stage serous ovarian adenocarcinomas.

PURPOSE: To identify markers that can predict overall survival in patients with high-grade advanced stage serous adenocarcinomas. PATIENTS AND METHODS: Oligonucleotide array comparative genomic hybridization (aCGH) was performed on 42 microdissected high-grade serous ovarian tumor samples. aCGH segments were obtained and a prediction Cox model was built and validated by the standard leave one out analysis. Both DNA and mRNA copy numbers of selected genes located on the candidate aCGH segments were determined by quantitative polymerase chain reaction (qPCR) and quantitative reverse transcriptase PCR (qRT-PCR) analyses. The gene that showed the highest correlation was further validated on an independent set of specimens and was selected for further functional studies. RESULTS: Two chromosomal regions, 4p16.3 and 5q31-5q35.3, exhibited the strongest correlation with overall survival (P < .01). From the 5q31 region, fibroblast growth factor 1 (FGF-1) was selected for further validation study. FGF-1 mRNA copy number was significantly correlated with DNA copy number and protein expression levels (P = .021 and < .001), and both FGF-1 mRNA and protein levels were significantly associated with overall survival (P = .018 and .042). This association was validated for protein expression on an independent set of 81 samples, significant to P = .006. Further studies showed significant correlation between FGF-1 protein expression and CD31+ staining in the tumor stroma (P = .024). Finally, both cancer cells and endothelial cells treated with exogenous FGF-1 showed a significant increase in cell motility and survival. CONCLUSION: Amplification of FGF-1 at 5q31 in ovarian cancer tissues leads to increased angiogenesis, and autocrine stimulation of cancer cells, which may result in poorer overall survival in patents with high-grade advanced stage serous ovarian cancer.

Complete identification of E-selectin ligands on neutrophils reveals distinct functions of PSGL-1, ESL-1, and CD44.

The selectins and their ligands are required for leukocyte extravasation during inflammation. Several glycoproteins have been suggested to bind to E-selectin in vitro, but the complete identification of its physiological ligands has remained elusive. Here, we showed that E-selectin ligand-1 (ESL-1), P-selectin glycoprotein ligand-1 (PSGL-1), and CD44 encompassed all endothelial-selectin ligand activity on neutrophils by using gene- and RNA-targeted loss of function. PSGL-1 played a major role in the initial leukocyte capture, whereas ESL-1 was critical for converting initial tethers into steady slow rolling. CD44 controlled rolling velocity and mediated E-selectin-dependent redistribution of PSGL-1 and L-selectin to a major pole on slowly rolling leukocytes through p38 signaling. These results suggest distinct and dynamic contributions of these three glycoproteins in selectin-mediated neutrophil adhesion and signaling.

CD43 deficiency has no impact in competitive in vivo assays of neutrophil or activated T cell recruitment efficiency.

Using noncompetitive methodologies comparing CD43(+/+) and CD43(-/-) mice, it has been reported that CD43(-/-) leukocytes exhibit reduced recruitment efficiency to sites of inflammation. More recent analyses demonstrate that CD43 on activated T cells can function as an E-selectin ligand (E-SelL) in vitro, suggesting that CD43 might promote rolling interactions during recruitment of leukocytes and account for the reported recruitment deficits in CD43(-/-) T cells and neutrophils in vivo. Internally controlled competitive in vivo methods using fluorescent tracking dyes were applied to compare recruitment efficiency of CD43(+/+) vs CD43(-/-) activated T cells to inflamed skin and of peripheral blood neutrophils to inflamed peritoneum. A simple CFSE perfusion method was developed to distinguish arterial/venous vasculature and confirm appropriate extravasation through venules in a Con A-induced cutaneous inflammation model. In vivo recruitment of peripheral blood neutrophils to inflamed peritoneum was core 2 GlcNAcT-I dependent, but recruitment efficiency was not influenced by absence of CD43. There were also no significant differences in core 2 GlcNAcT-I-dependent, selectin-dependent, cutaneous recruitment of activated T cells from CD43(+/+) and congenic CD43(-/-) mice in either B6 or P-selectin(-/-) recipients despite biochemical confirmation that a CD43-specific E-SelL was present on activated T cells. We conclude that recruitment of neutrophils and activated T cells in these in vivo models is not influenced by CD43 expression and that if CD43 on activated T cells performs an E-SelL function in vivo, it contributes in a limited physiological context.

An essential role for zebrafish Fgfrl1 during gill cartilage development.

The vertebrate craniofacial skeleton develops via a complex process involving signaling cascades in all three germ layers. Fibroblast growth factor (FGF) signaling is essential for several steps in pharyngeal arch development. In zebrafish, Fgf3 and Fgf8 in the mesoderm and hindbrain have an early role to pattern the pouch endoderm, influencing craniofacial integrity. Endodermal FGF signaling is required for the differentiation and survival of postmigratory neural crest cells that form the pharyngeal skeleton. We identify a novel role for zebrafish Fgf receptor-like 1a (Fgfrl1a) that is indispensable during gill cartilage development. We show that depletion of Fgfrl1a is sufficient to abolish cartilage derivatives of the ceratobranchials. Using an Fgfrl1a-deficient model, we analyzed expression of genes critical for chondrogenesis in the different compartments of the developing pharyngeal arch. Fgfrl1a-depleted animals demonstrate typical neural crest specification and migration to populate the arch primordia as well as normal pouch segmentation. However, in the absence of Fgfrl1a, larvae fail to express the transcription factor glial cells missing 2 (gcm2), a gene necessary for cartilage and gill filament formation, in the ectodermal lining of the branchial arches. In addition, two transcription factors essential for chondrogenesis, sox9a and runx2b, fail to express within the mesenchymal condensations of the branchial arches. A duplicate zebrafish gene, fgfrl1b, has now been identified. We show that Fgfrl1b is also required for proper formation of all ventral cartilage elements and acts cooperatively with Fgfrl1a during gill cartilage formation.

FGF-10 and its receptor exhibit bidirectional paracrine targeting to urothelial and smooth muscle cells in the lower urinary tract.

Control of the regenerative properties of urothelial tissue would greatly aid the clinician in the management of urinary tract disease and disorders. Fibroblast growth factor 10 (FGF-10) is a mitogen which is particularly promising as a protein therapy for urothelial injury. The spatial synthesis, transport, targeting, and mechanistic pathway of FGF-10 and its receptor were studied in a human urothelial cell culture model and in fixed sections of lower urinary tract tissue. Synthesis of FGF-10 was restricted to mesenchymal fibroblasts, and secreted FGF-10 exhibited paracrine transport to two proximal sites, transitional epithelium and smooth muscle cell bundles, both of which were also the exclusive sites of FGF-10 receptor synthesis. The addition of recombinant FGF-10 to quiescent urothelial cells in vitro was sufficient to stimulate DNA synthesis. This stimulation was through a pathway independent of the epidermal growth factor receptor pathway. Deconvolution, light and transmission electron microscopic studies captured FGF-10 and its receptor in association with the urothelial cell surface, in cytoplasm, and within nuclei, observations that describe the mechanism that transduces the mitogenic signal in these tissues. Localization of the FGF-10 receptor to the superficial urothelial layer is clinically significant because intravesical administration of FGF-10 may provide the clinician a means to control the turnover of transitional epithelium in bladder disorders such as interstitial cystitis.

N-cadherin mediated cell contact inhibits germinal vesicle breakdown in mouse oocytes maintained in vitro.

The effect of granulosa cell contact on the ability of zona-free oocytes to undergo germinal vesicle breakdown (GVBD) was assessed using a granulosa cell co-culture system. Oocytes contacted granulosa cells in a site-specific manner such that their GV was away from the granulosa cells. Also contact with granulosa cells reduced the percentage of oocytes undergoing GVBD from about 40% to 15%. GVBD was inhibited by contact with granulosa cells but not a granulosa cell-secreted product, since oocytes cultured in the same culture, that were adjacent to the granulosa cell monolayer underwent GVBD at the same rate as controls. Similarly, media collected from granulosa cell cultures did not attenuate the rate of GVBD. The ability of granulosa cell contact to inhibit GVBD was equal to that of db-cAMP. Moreover, the ability of granulosa cells to inhibit GVBD was not mimicked by spontaneously immortalized granulosa cells. This cell specificity appeared to be related to N-cadherin, since granulosa cells and oocytes express N-cadherin and a N-cadherin antibody attenuates the effect of granulosa cell contact. The mechanism through which N-cadherin mediated cell contact maintains meiotic arrest is unknown. It is possible that homophilic N-cadherin binding between the granulosa cells and oocyte acts through a junxtacrine mechanism, which in part may lead in the activation fibroblast growth factor (FGF) receptors that are expressed by the oocyte. The involvement of FGF receptors is supported by the observations that FGF and a N-cadherin peptide known to activate FGF receptors inhibit GVBD.

The expression of fibroblast growth factors and their receptors in Hodgkin's lymphoma.

The expression of fibroblast growth factors (FGF1 and FGF2) and their receptors has been reported in a variety of human neoplasms, including haematological neoplasia. Fibroblast growth factors can promote tumour growth directly, or indirectly through promoting the growth of vessels. In the present study, we evaluated the expression of FGF1 and FGF2 as well as FGF receptors 1-4 (FGFR1-FGFR4) in 39 cases of Hodgkin's lymphoma, including all subtypes, as well as Hodgkin's lymphoma cell lines. FGF1 and FGF2 and their receptors FGFR2-FGFR4, but not FGFR1, were found to be expressed by the malignant cells in the majority of cases. Interestingly, only FGFR3, but none of the FGFs or the other FGFRs, was expressed by the Hodgkin's lymphoma cell lines. This indicates that only FGFR3 is constitutively expressed by Hodgkin's lymphoma cells, whereas FGFs and the other FGFRs are induced in vivo. Culture of the Hodgkin's cell lines under conditions of hypoxic stress could induce vascular endothelial growth factor (VEGF) but not FGF expression. FGFs, in contrast to VEGF, might be expressed in response to paracrine stimuli. In situ hybridization did not reveal FGFR3 gene amplification or translocation as the cause of constitutive FGFR3 expression, as has been shown in a subset of multiple myeloma. FGFR3 might be expressed as part of the Hodgkin's cell phenotype. The demonstration of widespread expression of FGFs and some of their receptors in Hodgkin's lymphoma suggests that FGFs are important for sustaining growth of the lymphoma and suggests that anti-FGF antibodies could be used as an adjuvant treatment.