Activation Of Fibroblast Growth Factor Signaling Research Articles

Abstract 992: IL-6/STAT3-mediated inflammatory signaling results in dysregulated fibroblast growth factor receptor activation and tumor cell signaling in human pancreatic adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy characterized by delayed diagnosis and the lowest five-year survival amongst all cancer sites. PDAC development and progression are attributed to chronic inflammation and associated tissue repair mechanisms which contribute to high density tumor formation and chemoresistance. PDAC-associated inflammation is, in part, owed to high levels of interleukin 6 (IL-6) and its activated pathways including JAK/STAT3. In Addition, Fibroblast growth factor (FGF) signaling pathways are known to be dysregulated in cancer and have been implicated in various malignancies including PDAC. As such, we sought to explore the association between IL-6-induced inflammatory signaling and FGF signaling activation. To observe this effect as it pertains to carcinogenesis and cancer progression we used four human cell lines ranging from healthy pancreatic ductal epithelial (HPDE) cells to various grade (G) PDAC cells. Cells were treated with IL-6 at a concentration of 100 ng/mL for 24 hours. The results showed increased phosphorylation of STAT3 in BxPC-3 (G2) and PANC-1 (G3) cells with no change noted in HPDE and SW1990 (G2, metastatic) cells. IL-6 stimulation likewise resulted in markedly increased phosphorylation of the fibroblast growth factor receptor substrate 2-alpha (FRS2-α) and its downstream component Erk1/2 in BxPC3 cells, indicating activation of FGF signaling through the MAPK/Erk pathway in our low grade PDAC cell model. In conclusion, the results demonstrate a potential link between IL-6 induced inflammatory signaling and dysregulation of FGF signaling in human PDAC. Activated STAT3 signaling seems to be involved in this process. Existing cancer therapies aimed at controlling growth factor signaling broadly target vascular endothelial or epithelial growth factor receptors (VEGF/EGFR) with marginal efficacy and adverse side effects in PDAC. Identifying the pivotal link between inflammatory signaling and dysregulation of fibroblast growth factor signaling will allow for the development of more precisely targeted and effective cancer therapy in pancreatic cancer. This may minimize clinically observed exaggerated fibrotic events within the tumor and may hence result in higher vulnerability for established chemotoxic therapies in the tumor cells. Citation Format: Karol Nawalaniec, Yuxiang Wang, Romana Moench, Minghua Cao, Yueming Luo, Li-Li Hsiao, Ana Maria Waaga-Gasser. IL-6/STAT3-mediated inflammatory signaling results in dysregulated fibroblast growth factor receptor activation and tumor cell signaling in human pancreatic adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 992.

Novel culture system via wirelessly controllable optical stimulation of the FGF signaling pathway for human and pig pluripotency

Stem cell fate is largely determined by cellular signaling networks and is heavily dependent on the supplementation of exogenous recombinant proteins into culture media; however, uneven distribution and inconsistent stability of recombinant proteins are closely associated with the spontaneous differentiation of pluripotent stem cells (PSCs) and result in significant costs in large-scale manufacturing. Here, we report a novel PSC culture system via wirelessly controllable optical activation of the fibroblast growth factor (FGF) signaling pathway without the need for supplementation of recombinant FGF2 protein, a key molecule for maintaining pluripotency of PSCs. Using a fusion protein between the cytoplasmic region of the FGF receptor-1 and a light-oxygen-voltage domain, we achieved tunable, blue light-dependent activation of FGF signaling in human and porcine PSCs. Our data demonstrate that a highly controllable optical stimulation of the FGF signaling pathway is sufficient for long-term maintenance of PSCs, without the loss of differentiation potential into three germ layers. This culture system will be a cost-effective platform for a large-scale stem cell culture.

Fibroblast Growth Factor (FGF) Signaling Protects Against Acute Pancreatitis-Induced Damage by Modulating Inflammatory Responses.

BackgroundAcute pancreatitis (AP) is a symptom of sudden pancreas inflammation, which causes patients severe suffering. In general, fibroblast growth factor (FGF) levels are increased and amylase and lipase activities are elevated during AP pathogenesis, but protein concentration are low. However, the mechanism through which FGF signaling regulates AP pathogenesis remains elusive.Material/MethodsThe concentrations of PGE2, TNF-α, sCRP, FGF1, and FGF2 in the serum samples of the AP group and healthy control group were detected by enzyme-linked immunosorbent assay. In addition, IκBα and p-IκBα levels were analyzed in the serum samples. Subsequently, the AP rat model was established, and FGF1, FGF2, anti-FGF1, and anti-FGF2 antibodies and Bay11-7082 were injected into AP rats. TNF-α, PAI-1 JNK, p-JNK, IκBα, and p-IκBα levels were also examined.ResultsResults showed that levels of PGE2, TNF-α, sCRP, p-IκBα, FGF1, and FGF2, as well as amylase and lipase activity were increased in patients with AP compared with those in healthy people. In addition, protein concentrations were lower in patients with AP than in the healthy group. Activation of FGF signaling by injecting FGF1 or FGF2 also inhibited AP-induced inflammation response in the pancreas and increased amylase and lipase activities, as well as protein concentration. However, the injection of FGF1 and FGF2 antibodies accelerated AP-mediated inflammation responses in the serum. In addition, Bay11-7082 injection inhibited AP activation of inflammation response and amylase and lipase activities. Protein concentration were also increased in AP rats.ConclusionsFGF signaling protects against AP-mediated damage by inhibition of AP-activating inflammatory responses.

FGF Signaling Directs the Cell Fate Switch from Neurons to Astrocytes in the Developing Mouse Cerebral Cortex.

During mammalian neocortical development, neural precursor cells generate neurons first and astrocytes later. The cell fate switch from neurons to astrocytes is a key process generating proper numbers of neurons and astrocytes. Although the intracellular mechanisms regulating this cell fate switch have been well characterized, extracellular regulators are still largely unknown. Here, we uncovered that fibroblast growth factor (FGF) regulates the cell fate switch from neurons to astrocytes in the developing cerebral cortex using mice of both sexes. We found that the FGF signaling pathway is activated in radial glial cells of the ventricular zone at time points corresponding to the switch in cell fate. Our loss- and gain-of-function studies using in utero electroporation indicate that activation of FGF signaling is necessary and sufficient to change cell fates from neurons to astrocytes. We further found that the FGF-induced neuron-astrocyte cell fate switch is mediated by the MAPK pathway. These results indicate that FGF is a critical extracellular regulator of the cell fate switch from neurons to astrocytes in the mammalian cerebral cortex.SIGNIFICANCE STATEMENT Although the intracellular mechanisms regulating the neuron-astrocyte cell fate switch in the mammalian cerebral cortex during development have been well studied, their upstream extracellular regulators remain unknown. By using in utero electroporation, our study provides in vivo data showing that activation of FGF signaling is necessary and sufficient for changing cell fates from neurons to astrocytes. Manipulation of FGF signaling activity led to drastic changes in the numbers of neurons and astrocytes. These results indicate that FGF is a key extracellular regulator determining the numbers of neurons and astrocytes in the mammalian cerebral cortex, and is indispensable for the establishment of appropriate neural circuitry.

Abstract A026: Expression of fibroblast growth factor receptor 1 isoforms and activation of different pathways in prostate cancer progression

Abstract Bone metastases typically develop in patients with advanced prostate cancer (PCa). Androgen deprivation is commonly used as treatment, but responses are short, and eventually the disease progresses to a castration-resistant form (CRPC). The fibroblast growth factor (FGF) axis is commonly altered during PCa progression. Our group and others have implicated the FGF axis in the pathogenesis of PCa progression in bone, identified it as a candidate target for therapy, and suggested that FGF receptor (FGFR)-1 mediates a positive feedback loop between PCa cells and bone cells. RNA sequencing studies of 183 human PCa samples indicated that the mean expression of FGFR-1 is the highest of all the FGFR family genes studied. Analyses of these FGFR-1 transcripts identified eight different protein-coding transcripts to be the most abundantly expressed with diverse human PCa tissue samples expressing different FGFR-1 isoforms. The overall goal of this project is to investigate implications of FGFR1/FGFR1 isoforms expression in the pathogenesis of PCa bone metastases. When we assessed the expression of the two best-characterized FGFR-1 isoforms (alpha (NM_023110.2) and beta (NM_023105.2), which represent the most abundant protein coding transcripts found in PCa, we observed that all patient-derived xenografts (PDXs) studied express significantly higher levels of FGFR-1 alpha than beta while PCa cell lines mostly express the beta isoform. Mining The Cancer Genome Atlas (TCGA) PCa data, we identified distinct patterns of gene expression associated with each FGFR-1 isoform (alpha and beta). Briefly, FGFR-1 beta (but not alpha) is significantly associated with pathways including the MAP-kinase signaling cascade, signaling by FGFR in disease and pathways in cancer. In vitro studies of FGF signaling activation in PCa cells expressing FGFR-1 isoforms alpha, beta or empty vector, confirmed these results. Male SCID mice injected intracardially with PC3-FGFR-1 alpha exhibited higher death rates compared to animals injected with either PC3-FGFR-1 beta or PC3-empty vector. Furthermore, higher number of metastases per mice as well as higher number of mice with bone metastases were observed in animals injected with PC3-FGFR-1 alpha. The fact that different prostate tumors express different FGFR-1 isoforms suggests that FGFR1 alpha and beta isoforms activate different genes or pathways in PCa cells and this may underlie, at least in part, PCa heterogeneity, pattern of progression, and differences in response to FGFR1 inhibitors. Further studies will warrant the understanding of the implications of FGFR-1 isoforms expression in the pathogenesis of PCa. Citation Format: Estefania Labanca, Jun Yang, Peter Shepherd, Justin Roberts, Michael Starbuck, Bradley Broom, Matthew Iyer, Christopher Logothetis, Arul Chinnaiyan, Nora Navone. Expression of fibroblast growth factor receptor 1 isoforms and activation of different pathways in prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A026.

ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

Inner cell Mass (ICM) specification into epiblast (Epi) and primitive endoderm (PrE) is an asynchronous and progressive process taking place between E3.0 to E3.75 under the control of the Fibroblast Growth Factor (FGF)/Extracellular signal-Regulated Kinase (ERK) signaling pathway. Here, we have analyzed in details the kinetics of specification and found that ICM cell responsiveness to the up and down regulation of FGF signaling activity are temporally distinct. We also showed that PrE progenitors are generated later than Epi progenitors. We further demonstrated that, during this late phase of specification, a 4 hours period of FGF/ERK inhibition prior E3.75 is sufficient to convert ICM cells into Epi. Finally, we showed that ICM conversion into Epi in response to inhibition during this short time window requires both transcription and proteasome degradation. Collectively, our data give new insights into the timing and mechanisms involved in the process of ICM specification.

Chordin and dickkopf-1b are essential for the formation of head structures through activation of the FGF signaling pathway in zebrafish

The ability of the Spemann organizer to induce dorsal axis formation is dependent on downstream factors of the maternal Wnt/β-catenin signaling pathway. The fibroblast growth factor (FGF) signaling pathway has been identified as one of the downstream components of the maternal Wnt/β-catenin signaling pathway. The ability of the FGF signaling pathway to induce the formation of a dorsal axis with a complete head structure requires chordin (chd) expression; however, the molecular mechanisms involved in this developmental process, due to activation of FGF signaling, remain unclear. In this study, we showed that activation of the FGF signaling pathway induced the formation of complete head structures through the expression of chd and dickkopf-1b (dkk1b). Using the organizer-deficient maternal mutant, ichabod, we identified dkk1b as a novel downstream factor in the FGF signaling pathway. We also demonstrate that dkk1b expression is necessary, after activation of the FGF signaling pathway, to induce neuroectoderm patterning along the anteroposterior (AP) axis and for formation of complete head structures. Co-injection of chd and dkk1b mRNA resulted in the formation of a dorsal axis with a complete head structure in ichabod embryos, confirming the role of these factors in this developmental process. Unexpectedly, we found that chd induced dkk1b expression in ichabod embryos at the shield stage. However, chd failed to maintain dkk1b expression levels in cells of the shield and, subsequently, in the cells of the prechordal plate after mid-gastrula stage. In contrast, activation of the FGF signaling pathway maintained the dkk1b expression from the beginning of gastrulation to early somitogenesis. In conclusion, activation of the FGF signaling pathway induces the formation of a dorsal axis with a complete head structure through the expression of chd and subsequent maintenance of dkk1b expression levels.

Smooth muscle FGF/TGFβ cross talk regulates atherosclerosis progression.

The conversion of vascular smooth muscle cells (SMCs) from contractile to proliferative phenotype is thought to play an important role in atherosclerosis. However, the contribution of this process to plaque growth has never been fully defined. In this study, we show that activation of SMC TGFβ signaling, achieved by suppression of SMC fibroblast growth factor (FGF) signaling input, induces their conversion to a contractile phenotype and dramatically reduces atherosclerotic plaque size. The FGF/TGFβ signaling cross talk was observed in vitro and in vivo. In vitro, inhibition of FGF signaling increased TGFβ activity, thereby promoting smooth muscle differentiation and decreasing proliferation. In vivo, smooth muscle‐specific knockout of an FGF receptor adaptor Frs2α led to a profound inhibition of atherosclerotic plaque growth when these animals were crossed on Apoe −/− background and subjected to a high‐fat diet. In particular, there was a significant reduction in plaque cellularity, increase in fibrous cap area, and decrease in necrotic core size. In agreement with these findings, examination of human coronary arteries with various degrees of atherosclerosis revealed a strong correlation between the activation of FGF signaling, loss of TGFβ activity, and increased disease severity. These results identify SMC FGF/TGFβ signaling cross talk as an important regulator of SMC phenotype switch and document a major contribution of medial SMC proliferation to atherosclerotic plaque growth.

Heterogeneities in Nanog Expression Drive Stable Commitment to Pluripotency in the Mouse Blastocyst.

The pluripotent epiblast (EPI) is the founder tissue of almost all somatic cells. EPI and primitive endoderm (PrE) progenitors arise from the inner cell mass (ICM) of the blastocyst-stage embryo. The EPI lineage isdistinctly identified by its expression of pluripotency-associated factors. Many of these factors have been reported to exhibit dynamic fluctuations of expression in embryonic stem cell cultures. Whether these fluctuations correlating with ICM fate choice occur invivo remains an open question. Using single-cell resolution quantitative imaging of a Nanog transcriptional reporter, we noted an irreversible commitment to EPI/PrE lineages invivo. A period of apoptosis occurred concomitantly with ICM cell-fate choice, followed by a burst of EPI-specific cell proliferation. Transitions were occasionally observed from PrE-to-EPI, but not vice versa, suggesting that they might be regulated and not stochastic. We propose that the rapid timescale of early mammalian embryonic development prevents fluctuations in cell fate.

Quantification of shape and cell polarity reveals a novel mechanism underlying malformations resulting from related FGF mutations during facial morphogenesis

Fibroblast growth factor (FGF) signaling mutations are a frequent contributor to craniofacial malformations including midfacial anomalies and craniosynostosis. FGF signaling has been shown to control cellular mechanisms that contribute to facial morphogenesis and growth such as proliferation, survival, migration and differentiation. We hypothesized that FGF signaling not only controls the magnitude of growth during facial morphogenesis but also regulates the direction of growth via cell polarity. To test this idea, we infected migrating neural crest cells of chicken embryos with replication-competent avian sarcoma virus expressing either FgfR2(C278F), a receptor mutation found in Crouzon syndrome or the ligand Fgf8. Treated embryos exhibited craniofacial malformations resembling facial dysmorphologies in craniosynostosis syndrome. Consistent with our hypothesis, ectopic activation of FGF signaling resulted in decreased cell proliferation, increased expression of the Sprouty class of FGF signaling inhibitors, and repressed phosphorylation of ERK/MAPK. Furthermore, quantification of cell polarity in facial mesenchymal cells showed that while orientation of the Golgi body matches the direction of facial prominence outgrowth in normal cells, in FGF-treated embryos this direction is randomized, consistent with aberrant growth that we observed. Together, these data demonstrate that FGF signaling regulates cell proliferation and cell polarity and that these cell processes contribute to facial morphogenesis.

Fibroblast growth factor signaling promotes physiological bone remodeling and stem cell self-renewal

Fibroblast growth factor (FGF) signaling activates many bone marrow cell types, including various stem cells, osteoblasts, and osteoclasts. However, the role of FGF signaling in regulation of normal and leukemic stem cells is poorly understood. This review highlights the physiological roles of FGF signaling in regulating bone marrow mesenchymal and hematopoietic stem and progenitor cells (MSPCs and HSPCs) and their dynamic microenvironment. In addition, this review summarizes the recent studies which provide an overview of FGF-activated mechanisms regulating physiological stem cell maintenance, self-renewal, and motility. Current results indicate that partial deficiencies in FGF signaling lead to mild defects in hematopoiesis and bone remodeling. However, FGF signaling was shown to be crucial for stem cell self-renewal and for proper hematopoietic poststress recovery. FGF signaling activation was shown to be important also for rapid AMD3100 or post 5-fluorouracil-induced HSPC mobilization. In vivo, FGF-2 administration successfully expanded both MSPCs and HSPCs. FGF-induced expansion was characterized by enhanced HSPC cycling without further exhaustion of the stem cell pool. In addition, FGF signaling expands and remodels the supportive MSPC niche cells. Finally, FGF signaling is constitutively activated in many leukemias, suggesting that malignant HSPCs exploit this pathway for their constant expansion and for remodeling a malignant-supportive microenvironment. The summarized studies, concerning regulation of stem cells and their microenvironment, suggest that FGF signaling manipulation can serve to improve current clinical stem cell mobilization and transplantation protocols. In addition, it may help to develop therapies specifically targeting leukemic stem cells and their supportive microenvironment.

HtrA1 Is a Novel Antagonist Controlling Fibroblast Growth Factor (FGF) Signaling via Cleavage of FGF8

Accumulating evidence suggests that HtrA1 (high-temperature requirement A1) is involved in modulating crucial cellular processes and implicated in life-threatening diseases, such as cancer and neuropathological disorders; however, the exact functions of this protease in vivo remain unknown. Here, we show that loss of HtrA1 function increases fibroblast growth factor 8 (FGF8) mRNA levels and triggers activation of FGF signaling, resulting in dorsalization in zebrafish embryos. Notably, HtrA1 directly cleaves FGF8 in the extracellular region, and this cleavage results in decreased activation of FGF signaling, which is essential for many physiological processes. Therefore, HtrA1 is indispensable for dorsoventral patterning in early zebrafish embryogenesis and serves as a key upstream regulator of FGF signaling through the control of FGF levels. Furthermore, this study offers insight into new strategies to control human diseases associated with HtrA1 and FGF signaling.

Cell Surface Heparan Sulfate Chains Regulate Local Reception of FGF Signaling in the Mouse Embryo

Heparan sulfate (HS) proteoglycans modulate the activity of multiple growth factors on the cell surface and extracellular matrix. However, it remains unclear how the HS chains control the movement and reception of growth factors into targeted receiving cells during mammalian morphogenetic processes. Here, we found that HS-deficient Ext2 null mutant mouse embryos fail to respond to fibroblast growth factor (FGF) signaling. Marker expression analyses revealed that cell surface-tethered HS chains are crucial for local retention of FGF4 and FGF8 ligands in the extraembryonic ectoderm. Fine chimeric studies with single-cell resolution and expression studies with specific inhibitors for HS movement demonstrated that proteolytic cleavage of HS chains can spread FGF signaling to adjacent cells within a short distance. Together, the results show that spatiotemporal expression of cell surface-tethered HS chains regulate the local reception of FGF-signaling activity during mammalian embryogenesis.

Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling

Spinal muscular atrophy (SMA), a devastating neurodegenerative disorder characterized by motor neuron loss and muscle atrophy, has been linked to mutations in the Survival Motor Neuron (SMN) gene. Based on an SMA model we developed in Drosophila, which displays features that are analogous to the human pathology and vertebrate SMA models, we functionally linked the fibroblast growth factor (FGF) signaling pathway to the Drosophila homologue of SMN, Smn. Here, we characterize this relationship and demonstrate that Smn activity regulates the expression of FGF signaling components and thus FGF signaling. Furthermore, we show that alterations in FGF signaling activity are able to modify the neuromuscular junction defects caused by loss of Smn function and that muscle-specific activation of FGF is sufficient to rescue Smn-associated abnormalities.

Sulfatase modifying factor 1–mediated fibroblast growth factor signaling primes hematopoietic multilineage development

Self-renewal and differentiation of hematopoietic stem cells (HSCs) are balanced by the concerted activities of the fibroblast growth factor (FGF), Wnt, and Notch pathways, which are tuned by enzyme-mediated remodeling of heparan sulfate proteoglycans (HSPGs). Sulfatase modifying factor 1 (SUMF1) activates the Sulf1 and Sulf2 sulfatases that remodel the HSPGs, and is mutated in patients with multiple sulfatase deficiency. Here, we show that the FGF signaling pathway is constitutively activated in Sumf1−/− HSCs and hematopoietic stem progenitor cells (HSPCs). These cells show increased p-extracellular signal-regulated kinase levels, which in turn promote β-catenin accumulation. Constitutive activation of FGF signaling results in a block in erythroid differentiation at the chromatophilic erythroblast stage, and of B lymphocyte differentiation at the pro–B cell stage. A reduction in mature myeloid cells and an aberrant development of T lymphocytes are also seen. These defects are rescued in vivo by blocking the FGF pathway in Sumf1−/− mice. Transplantation of Sumf1−/− HSPCs into wild-type mice reconstituted the phenotype of the donors, suggesting a cell autonomous defect. These data indicate that Sumf1 controls HSPC differentiation and hematopoietic lineage development through FGF and Wnt signaling.

Mesoderm migration in Drosophila is a multi-step process requiring FGF signaling and integrin activity

Migration is a complex, dynamic process that has largely been studied using qualitative or static approaches. As technology has improved, we can now take quantitative approaches towards understanding cell migration using in vivo imaging and tracking analyses. In this manner, we have established a four-step model of mesoderm migration during Drosophila gastrulation: (I) mesodermal tube formation, (II) collapse of the mesoderm, (III) dorsal migration and spreading and (IV) monolayer formation. Our data provide evidence that these steps are temporally distinct and that each might require different chemical inputs. To support this, we analyzed the role of fibroblast growth factor (FGF) signaling, in particular the function of two Drosophila FGF ligands, Pyramus and Thisbe, during mesoderm migration. We determined that FGF signaling through both ligands controls movements in the radial direction. Thisbe is required for the initial collapse of the mesoderm onto the ectoderm, whereas both Pyramus and Thisbe are required for monolayer formation. In addition, we uncovered that the GTPase Rap1 regulates radial movement of cells and localization of the beta-integrin subunit, Myospheroid, which is also required for monolayer formation. Our analyses suggest that distinct signals influence particular movements, as we found that FGF signaling is involved in controlling collapse and monolayer formation but not dorsal movement, whereas integrins are required to support monolayer formation only and not earlier movements. Our work demonstrates that complex cell migration is not necessarily a fluid process, but suggests instead that different types of movements are directed by distinct inputs in a stepwise manner.

Rapid Purification and High Sensitivity Analysis of Heparan Sulfate from Cells and Tissues: TOWARD GLYCOMICS PROFILING

Studies on glycosaminoglycans and proteoglycans (PGs) have been hampered by difficulties in isolation and analysis by traditional methods that are laborious and lack sensitivity and throughput. Here we demonstrate a simple method for rapid isolation of proteoglycans (RIP) employing phenol/guanidine/chloroform reagent to purify heparan sulfate (HS) PGs quantitatively from various tissues and cells. We further show that this generic purification methodology, when applied in concert with a BODIPYTM fluorescent label, permits structural analyses on RIP-purified HS at ∼1,000-fold higher sensitivity than standard UV detection methods and ∼10–100-fold higher sensitivity than previous fluorescence detection methods. The utility of RIP-BODIPY methodology was demonstrated by rapid profiling of HS structural composition from small tissue samples, multiple mouse organs, and as little as a few thousand cultured cells. It was also used to generate novel insights into in vivo structural changes in HS from Sulf1 knock-out mice for the first time that differed significantly from previous observations limited to tissue culture experiments. RIP was also applied to purify HS for bioassay testing, exemplified by cell assays of fibroblast growth factor signaling activation; this generated data from 2-O-sulfotransferase knock-out mice and revealed an unexpected deficiency in fibroblast growth factor activation by HS from heterozygous mice. These data demonstrate that RIP will underpin emerging efforts to develop glycomics profiling strategies for HS and other glycosaminoglycans to explore their structure-function relationships in complex biological systems.

The investigation of synovial genomic targets of bucillamine with microarray technique

To identify the molecular mechanisms of bucillamine activity, global gene expression analysis and pathway analysis were conducted using IL-1 beta-stimulated human fibroblast-like synovial cells (FLS). Normal human FLS were treated with IL-1 beta in the presence or absence of 10 and 100 microM bucillamine for 6 h. Total RNA was extracted and global gene expression levels were detected using a 44 k human whole genome array. Data were analyzed using Ingenuity pathway analysis. Numerous pathways were activated by IL-1 beta stimulation. At both concentrations, bucillamine suppressed nine signal pathways stimulated by IL-1 beta. Bucillamine effectively inhibited fibroblast growth factor (FGF) signaling and tight junction signaling activated by IL-1 beta in FLS. Suppression of these signal pathways may correlate with the pharmacologic mechanisms of bucillamine. In particular, the suppression of FGF signaling by bucillamine is remarkable because the activation of FGF signaling may be involved in rheumatoid arthritis pathology.

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.

FGF signaling network in the gastrointestinal tract (Review)

Fibroblast growth factor (FGF) signals are transduced through FGF receptors (FGFRs) and FRS2/FRS3- SHP2 (PTPN11)-GRB2 docking protein complex to SOS-RAS-RAF-MAPKK-MAPK signaling cascade and GAB1/GAB2-PI3K-PDK-AKT/aPKC signaling cascade. The RAS approximately MAPK signaling cascade is implicated in cell growth and differentiation, the PI3K approximately AKT signaling cascade in cell survival and cell fate determination, and the PI3K approximately aPKC signaling cascade in cell polarity control. FGF18, FGF20 and SPRY4 are potent targets of the canonical WNT signaling pathway in the gastrointestinal tract. SPRY4 is the FGF signaling inhibitor functioning as negative feedback apparatus for the WNT/FGF-dependent epithelial proliferation. Recombinant FGF7 and FGF20 proteins are applicable for treatment of chemotherapy/radiation-induced mucosal injury, while recombinant FGF2 protein and FGF4 expression vector are applicable for therapeutic angiogenesis. Helicobacter pylori, a causative pathogen for peptic ulcer diseases, chronic atrophic gastritis and gastric cancer, injects bacterial proteins into gastric epithelial cells by using Type IV secretion system, which leads to FGF signaling activation through FGF2 upregulation as well as CagA-dependent SHP2 activation. FGFR2 gene is preferentially amplified and overexpressed in diffuse-type gastric cancer. PD173074 is a small-molecule inhibitor for FGFR, while RO4396686 and SU6668 are small-molecule inhibitors for FGFR and other tyrosine kinases. Cocktail therapy using multiple protein kinase inhibitors could enhance the therapeutic effects for gastrointestinal cancer through the reduction of recurrence associated with somatic mutations of drug-target genes. Single nucleotide polymorphism (SNP) and copy number polymorphism (CNP) of genes encoding FGF signaling molecules will be identified as novel risk factors of gastrointestinal cancer. Personalized prevention and personalized medicine based on the combination of genetic screening and novel therapeutic agents could dramatically improve the prognosis of cancer patients.