Fibroblast Growth Factor Stimulation Research Articles

Abstract 39: Fibroblast growth factor induces neoplastic cell transformation through a non-canonical signaling pathway

Abstract Induction of cell proliferation is closely related with the cellular signaling pathway activation by stimulation of diverse growth factors such as epidermal growth factor (EGF) and fibroblast growth factor. The stimulation of growth factors induces activation of extracellular signal-regulated kinases (ERKs)/p90 ribosomal S6 kinases (p90RSK), resulting in induction of cell proliferation and cell transformation. Although fibroblast growth factor (FGF) is a well-known growth factor and acts as a ligand of FGF receptor (FGFR), a receptor tyrosine kinase, in cytoplasmic membrane, the tumor promoter potential has not been clearly understood. Here, we provided the evidences that FGF acted as a tumor promoter. We found that FGF-induced cell proliferation and anchorage-independent cell transformation were correlated with the induction of G1/S cell cycle transition. Importantly, we found that kaempferol targeted and inhibited FGFR phosphorylation by in vitro and ex vivo. Interestingly, FGF stimulation utilized a non-canonical signaling pathway to activate RSK2 and ATF-1, which was not transduced by EGF stimulation. We confirmed that kaempferol inhibited tyrosine phosphorylation of FGFR, resulted in nuclear accumulation of phospho-ATF-1 at Ser63. Importantly, kaempferol, PKC412, PD98059 and U0126 inhibited EGF-induced anchorage-independent cell transformation in JB6 Cl41 cells. In contrast, FGF-induced cell transformation in soft agar was not inhibited by PD98059 and U0126. Taken together, these results demonstrate that FGF acts as a tumor promoter and dual inhibition of kaempferol on the kinase activities of FGFR and RSK2 suppresses the FGF-induced neoplastic cell transformation through a non-canonical signaling pathway which is not utilized by EGF stimulation. Citation Format: Sun-Mi Yoo, Cheol-Jung Lee, Mee-Hyun Lee, Yong-Yeon Cho. Fibroblast growth factor induces neoplastic cell transformation through a non-canonical signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 39. doi:10.1158/1538-7445.AM2015-39

Fibroblast and Epidermal Growth Factors Utilize Different Signaling Pathways to Induce Anchorage-independent Cell Transformation in JB6 Cl41 Mouse Skin Epidermal Cells.

Background:Extracellular stimulation of cells with growth factors such as epidermal growth factor (EGF) induces cell proliferation and cell transformation. Although fibroblast growth factor (FGF) is a well-known family member of growth factors and acts as a ligand of FGF receptor (FGFR), a receptor tyrosine kinase, in cytoplasmic membrane, the tumor promoter potential of FGF has not been clearly understood.Methods:The role of FGF as a tumor promoter was determined measuring its effects of cell proliferation and transformation by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and anchorage-independent cell transformation assays, respectively. The antibody specificity of phospho-RSK2 Tyr529 was determined by Western blotting using a purified FGFR kinase domain in vitro and the membrane fraction of JB6 Cl41 cells ex vivo. The signaling pathways mediated by FGF or EGF were determined by the comparisons of phosphorylation inhibitory efficacy using signaling inhibitors including kaempferol.Results:FGF acted as a tumor promoter. FGF induced cell proliferation by stimulation of G1/S cell cycle transition, and anchorage-independent cell transformation in JB6 Cl41 cells. FGF-induced FGFR phosphorylation was suppressed by kaempferol treatment in a dose dependent manner. Interestingly, FGF stimulation utilized a non-canonical signaling pathway to activate RSK2 and activating transcription factor (ATF)-1, which was not transduced by EGF stimulation. Importantly, kaempferol inhibited tyrosine phosphorylation of FGFR by FGF stimulation and nuclear accumulation of phospho-ATF-1 at Ser63. Moreover, although kaempferol, 4’-N-benzoyl staurosporine (PKC412), 2-(2’-amino-3’-methoxyphenyl)oxanaphthalen-4-one (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)buta-diene (U0126) inhibited EGF-induced anchorage-independent cell transformation in JB6 Cl41 cells, FGF-induced cell transformation in soft agar was only inhibited by PKC412 and kaempferol, but not by PD98059 and U0126.Conclusions:FGF acts as a tumor promoter and dual inhibition of kaempferol on the kinase activities of FGFR3 and RSK2 suppresses the FGF-induced neoplastic cell transformation through a non-canonical signaling pathway which is not utilized by EGF stimulation.

Modulation of Fibroblast Growth Factor Signaling Is Essential for Mammary Epithelial Morphogenesis

Fibroblast growth factor (FGF) signaling is essential for vertebrate organogenesis, including mammary gland development. The mechanism whereby FGF signaling is regulated in the mammary gland, however, has remained unknown. Using a combination of mouse genetics and 3D ex vivo models, we tested the hypothesis that Spry2 gene, which encodes an inhibitor of signaling via receptor tyrosine kinases (RTKs) in certain contexts, regulates FGF signaling during mammary branching. We found that Spry2 is expressed at various stages of the developing mammary gland. Targeted removal of Spry2 function from mammary epithelium leads to accelerated epithelial invasion. Spry2 is up-regulated by FGF signaling activities and its loss sensitizes mammary epithelium to FGF stimulation, as indicated by increased expression of FGF target genes and epithelia invasion. By contrast, Spry2 gain-of-function in the mammary epithelium results in reduced FGF signaling, epithelial invasion, and stunted branching. Furthermore, reduction of Spry2 expression is correlated with tumor progression in the MMTV-PyMT mouse model. Together, the data show that FGF signaling modulation by Spry2 is essential for epithelial morphogenesis in the mammary gland and it functions to protect the epithelium against tumorigenesis.

Abstract A006: FGF-2 stimulates breast cancer growth activating ER and PR

Abstract Estrogen and progesterone receptors (ER and PR respectively) are prognostic markers and dictate therapeutic decisions in breast cancer patients. Although most of the evidence suggests that estrogens are the major etiological factors in breast cancer, there is experimental and epidemiological data that also points to the involvement of PR in mammary gland carcinogenesis and breast cancer growth. Both ER and PR are activated by their natural ligands or by ligand-independent pathways. Carcinoma-associated fibroblasts produce growth factors that stimulate epithelial cell proliferation. We have recently demonstrated that Fibroblast growth factor 2 (FGF-2) is able to activate PR and induce progestin-dependent tumor growth. In addition, we have demonstrated an interaction between ER and PR at the promoters of Cyclin D1 (CCND1) and MYC, which is necessary to mediate progestin-induced gene transcription and cell proliferation. The aim of this study was to evaluate the role of ER in FGF-2-induced cell proliferation. We used C4-HD and C4-HI tumors from the medroxyprogesterone acetate (MPA)-induced murine breast cancer model and two human cell lines, T47D and MCF-7. The C4-HD tumors only grow with the exogenous administration of progestins or FGF-2. High levels of activated ER expression were observed in C4-HD tumors growing in NOD/SCID female mice treated with FGF-2 (5 ug/day) or MPA (20 mg/depot). The inhibitor of FGF receptors (PD173074, 25 mg/kg) which inhibits C4-HI tumor growth and PR phosphorylation also reduced the expression and phosphorylation of ER. FGF-2 (50 ng/ml) stimulated cell proliferation of primary cultures of C4-HD and C4-HI tumor cells, and of T47D and MCF-7 cells (p<0.001). The antiestrogen ICI 182.780 (ICI, 1-100 nM), the FGFR inhibitors (PD 0.1 uM and BGJ398 1 nM), and siRNAs against ER, all blocked the FGF-2 stimulation (p<0.001) in the aforementioned models. An increase in pSer118 and pSer167 ER and in the nuclear interaction between ER with the coactivator AIB1 and with FOXA1, were observed in FGF-2-treated MCF-7 and T47D cells. Moreover, FGF-2 induced an increase in the expression of the estrogen responsive elements (ERE)-Luc reporter (p<0.001), that was blocked by ICI and PD (p<0.001). FGF-2 also mediated the up-regulation of two ER-regulated genes, MYC and pS2/TFF1 (p<0.001), which was inhibited by ICI (p<0.001). Finally, ER and FOXA1 were recruited to ERE sites at pS2/TFF1 promoter after FGF-2 incubation in MCF-7 cells (p<0.01). Our results indicate that FGF-2 mediates PR and ER activation in breast cancer cells, suggesting that the combination of antiestrogens, antiprogestins and FGF-2-signaling inhibitors may be an option in selected breast cancer patients. Citation Format: Tomás Guillardoy, María A. Gorostiaga, Claudia Lanari, Sebastián Giulianelli. FGF-2 stimulates breast cancer growth activating ER and PR. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A006.

SOCS-1/3 participation in FGF-2 signaling to modulate RANK ligand expression in paget's disease of bone

Paget's disease of bone (PDB) is a chronic focal skeletal disorder characterized by excessive bone resorption followed by disorganized new bone formation. Measles virus nucleocapsid (MVNP) is implicated in pathogenesis of PDB. RANK ligand (RANKL), a critical osteoclastogenic factor expressed on bone marrow stromal/preosteoblast cells is upregulated in PDB. We recently demonstrated that fibroblast growth factor-2 (FGF-2) which induces RANKL expression is elevated in PDB. In this study, we hypothesized that FGF-2 modulates suppressors of cytokine signaling (SOCS) to induce RANKL expression in PDB. We identified increased levels of SOCS-1/3 mRNA expression in bone marrow mononuclear cells derived from patients with PDB compared to normal subjects. Interestingly, conditioned media obtained from MVNP transduced osteoclast progenitor cells significantly increased SOCS-1/3 mRNA expression in stromal/preosteoblast cells. We next examined if SOCS participates in FGF-2 signaling to modulate RANKL gene expression. We showed that FGF-2 stimulation significantly increased SOCS-1/3 expression in human bone marrow stromal/preosteoblast cells. In addition, co-expression of SOCS-1/3 with hRANKL gene promoter-luciferase reporter plasmid in marrow stromal cells demonstrated a significant increase in promoter activity without FGF-2 stimulation. Furthermore, siRNA inhibition of STAT-1 suppresses FGF-2 increased SOCS-1/3 expression in these cells. Thus, our results suggest that SOCS participates in FGF-2 modulation of RANKL expression in PDB.

Potent stimulation of fibroblast growth factor 19 expression in the human ileum by bile acids

Fibroblast growth factor 19 (FGF19) is proposed to be a negative feedback regulator of hepatic bile acid (BA) synthesis. We aimed to clarify the distribution of FGF19 expression in human intestine and to investigate induction in a novel explant system. Ileal and colonic mucosal biopsies were obtained at endoscopy and analyzed for FGF19 transcript expression. Primary explants were incubated with physiological concentrations of various BA for up to 6 h, and expression of FGF19 and other genes was determined. FGF19 transcripts were detected in ileum but were unquantifiable in colon. No loss of FGF19 mRNA occurred as a consequence of the explant system. Ileal FGF19 transcript expression was induced 350-fold by 50 μM chenodeoxycholate (CDCA, n = 24, P < 0.0001) and 161-fold by 50 μM glycochenodeoxycholate (GCDCA, n = 12, P = 0.0005). The responses of other genes to CDCA or GCDCA (50 μM) were smaller: median increases of ileal bile acid binding protein, organic solute transporter-α and -β, and short heterodimer partner were 2.4- to 4.0-fold; apical membrane sodium bile acid transporter and farnesoid X receptor (FXR) showed little change. The EC50 for FGF19 transcript induction by CDCA was 20 μM. FGF19 protein concentrations were significantly higher in the culture fluid from BA-stimulated explants. FGF19 induction with cholate was 81% of that found with CDCA, but deoxycholate (40%) and lithocholate (4%) were significantly less potent. The synthetic FXR agonist obeticholic acid was much more potent than CDCA with a 70-fold FGF19 stimulation at 1 μM. We concluded that FGF19 expression in human ileum is very highly responsive to BA. Changes in FGF19 induction are a potential mechanism involved in disorders of BA homeostasis.

RhoD activated by fibroblast growth factor induces cytoneme-like cellular protrusions through mDia3C

The small GTPase RhoD regulates actin cytoskeleton to collapse actin stress fibers and focal adhesions, resulting in suppression of cell migration and cytokinesis. It also induces alignment of early endosomes along actin filaments and reduces their motility. We show here that a constitutively activated RhoD generated two types of actin-containing thin peripheral cellular protrusions distinct from Cdc42-induced filopodia. One was longer, almost straight, immotile, and sensitive to fixation, whereas the other was shorter, undulating, motile, and resistant to fixation. Moreover, cells expressing wild-type RhoD extended protrusions toward fibroblast growth factor (FGF) 2/4/8-coated beads. Stimulation of wild-type RhoD-expressing cells with these FGFs also caused formation of cellular protrusions. Nodules moved through the RhoD-induced longer protrusions, mainly toward the cell body. Exogenously expressed FGF receptor was associated with these moving nodules containing endosome-like vesicles. These results suggest that the protrusions are responsible for intercellular communication mediated by FGF and its receptor. Accordingly, the protrusions are morphologically and functionally equivalent to cytonemes. RhoD was activated by FGF2/4/8. Knockdown of RhoD interfered with FGF-induced protrusion formation. Activated RhoD specifically bound to mDia3C and facilitated actin polymerization together with mDia3C. mDia3C was localized to the tips or stems of the protrusions. In addition, constitutively activated mDia3C formed protrusions without RhoD or FGF stimulation. Knockdown of mDia3 obstructed RhoD-induced protrusion formation. These results imply that RhoD activated by FGF signaling forms cytoneme-like protrusions through activation of mDia3C, which induces actin filament formation.

RETRACTED: Fibroblast growth factor‐2 promotes catabolism via FGFR1‐Ras‐Raf‐MEK1/2‐ERK1/2 axis that coordinates with the PKCδ pathway in human articular chondrocytes

Fibroblast growth factor 2 (FGF-2) has been found to play an anti-anabolic and/or a catabolic role in adult human articular cartilage via regulation of multiple signaling pathways. Upon FGF-2 stimulation, a molecular crosstalk between the mitogen activated protein kinase (MAPK) and protein kinase C δ (PKCδ) pathways are initiated, where PKCδ positively regulates downstream MAPK signaling. In this study, we explored the relationship between fibroblast growth factor receptor 1 (FGFR1), Ras, and PKCδ in FGF-2 signaling in human articular chondrocytes. Pathway-specific inhibition using both chemical inhibitors and siRNA targeting FGFR1 demonstrated that, upon FGF-2 stimulation, FGFR1 controlled both Ras and PKCδ activation, which converged on the Raf-MEK1/2-ERK1/2 axis. No crosstalk was observed between Ras and PKCδ. Quantitative PCR analyses revealed that both Ras and PKCδ contributed to FGF-2-mediated upregulation of MMP-13, ADAMTS5, and repression of aggrecan gene. Correspondingly, FGF-2-mediated proteoglycan loss was effectively reversed by individual pathway-specific inhibitor of Ras, PKCδ, and ERK1/2 in both 3-dimensional alginate bead culture and cartilage organ culture systems. Our findings suggest that FGFR1 interacts with FGF-2 and then activates Ras and PKCδ, which concertedly drive MAPK signaling to mediate biological effects of FGF-2. Such an integration of dual inputs constitutes a novel mechanism of FGF-2 signaling cascade in human articular chondrocytes.

FGFR1 cleavage and nuclear translocation regulates breast cancer cell behavior.

FGF-10 and its receptors, FGFR1 and FGFR2, have been implicated in breast cancer susceptibility and progression, suggesting that fibroblast growth factor (FGF) signaling may be co-opted by breast cancer cells. We identify a novel pathway downstream of FGFR1 activation, whereby the receptor is cleaved and traffics to the nucleus, where it can regulate specific target genes. We confirm Granzyme B (GrB) as the protease responsible for cleavage and show that blocking GrB activity stopped FGFR1 trafficking to the nucleus and abrogates the promigratory effect of FGF stimulation. We confirm the in vivo relevance of our findings, showing that FGFR1 localized to the nucleus specifically in invading cells in both clinical material and a three-dimensional model of breast cancer. We identify target genes for FGFR1, which exert significant effects on cell migration and may represent an invasive signature. Our experiments identify a novel mechanism by which FGF signaling can regulate cancer cell behavior and provide a novel therapeutic target for treatment of invasive breast cancer.

Sa1303 Dramatic Stimulation of Fibroblast Growth Factor 19 Expression in the Human Ileum by Bile Acids

Sa1303 Dramatic Stimulation of Fibroblast Growth Factor 19 Expression in the Human Ileum by Bile Acids

Data-driven modelling of receptor tyrosine kinase signalling networks quantifies receptor-specific potencies of PI3K- and Ras-dependent ERK activation

Signal transduction networks in mammalian cells, comprising a limited set of interacting biochemical pathways, are accessed by various growth factor and cytokine receptors to elicit distinct cell responses. This raises the question as to how specificity of the stimulus-response relationship is encoded at the molecular level. It has been proposed that specificity arises not only from the activation of unique signalling pathways, but also from quantitative differences in the activation and regulation of shared receptor-proximal signalling proteins. To address such hypotheses, data sets with greater precision and coverage of experimental conditions will need to be acquired, and rigorous frameworks that codify and parameterize the inherently non-linear relationships among signalling activities will need to be developed. In the present study we apply a systematic approach combining quantitative measurements and mathematical modelling to compare the signalling networks accessed by FGF (fibroblast growth factor) and PDGF (platelet-derived growth factor) receptors in mouse fibroblasts, in which the ERK (extracellular-signal-regulated kinase) cascade is activated by Ras- and PI3K (phosphoinositide 3-kinase)-dependent pathways. We show that, whereas the FGF stimulation of PI3K signalling is relatively weak, this deficiency is compensated for by a more potent Ras-dependent activation of ERK. Thus, as the modelling would predict, the ERK pathway is activated to a greater extent in cells co-stimulated with FGF and PDGF, relative to the saturated levels achieved with either ligand alone. It is envisaged that similar approaches will prove valuable in the elucidation of quantitative differences among other closely related receptor signalling networks.

Fibroblast Growth Factor 2 Stimulation of Osteoblast Differentiation and Bone Formation Is Mediated by Modulation of the Wnt Signaling Pathway

Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/β-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and β-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and β-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3β, a negative regulator of Wnt/β-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted β-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.

Investigating the role of FGF‐2 in stem cell maintenance by global phosphoproteomics profiling

Human embryonic stem cells (hESCs) are of immense interest for regenerative medicine as a source of tissue replacement. Expansion of hESCs as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. One of the key growth factors that maintains this balance is fibroblast growth factor-2 (FGF-2). However, the underlying molecular mechanisms are poorly understood. We recently profiled specifically tyrosine phosphorylation events that occur during FGF-2 stimulation of hESCs (Ding et al., J. Cell. Physiol. 2010, 225, 417-428). Here, we complement this phosphoproteome profiling by analyzing temporal dynamics of mostly serine and threonine protein phosphorylation events. Our multi-dimensional strategy combines strong cation exchange chromatography to reduce the sample complexity followed by titanium dioxide off-line for the enrichment of phosphopeptides and dimethylation-based stable isotope labeling for quantification. This approach allowed us to identify and quantify 3261 unique proteins from which 1064 proteins were found to be phosphorylated in one or more residues (representing 1653 unique phosphopeptides). Approximately 40% of the proteins (553 unique phosphopeptides) showed differential phosphorylation upon FGF-2 treatment. Among those are several members of the canonical pathways involved in pluripotency and self-renewal (e.g. Wnt and PI3K/AKT), hESC-associated proteins such as SOX2, RIF1, SALL4, DPPA4, DNMT3B and 53 proteins that are target genes of the pluripotency transcription factors SOX2, OCT4 and NANOG. These findings complement existing pluripotency analyses and provide new insights into how FGF-2 assists in maintaining the undifferentiated state of hESCs.

Fibroblast growth factor‐2 enhances NK sensitivity of hepatocellular carcinoma cells

The roles of fibroblast growth factor-2 (FGF-2) in the hepatocellular carcinoma (HCC) development are still controversial. In this study, we investigated the expression of FGF-2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF-2 in NK sensitivity of HCC cells. The FGF-2 expressions were detected in the liver tissues of patients, but not in normal liver. The serum FGF-2 levels of the patients with CH, liver cirrhosis (LC) or HCC were significantly higher than those of healthy volunteers. The serum FGF-2 levels of patients decreased with the progression of chronic liver disease. HCC occurrence of LC patients with high levels of serum FGF-2 was significantly lower than that with low levels of serum FGF-2. Proinflammatory cytokines, such as IL-1β and IL-6, induced FGF-2 expressions in HCC cells and normal hepatocytes. FGF-2 stimulation resulted in increasing the expression of the membrane-bound major histocompatibility complex class I-related chain A (MICA), an NK activating molecule, and decreasing that of human leukocyte antigen (HLA) class I, an NK inhibitory molecule, on HCC cells. This did not occur with normal hepatocytes. Adding anti-FGF receptor-2 neutralizing antibody resulted in inhibiting the change of MICA and HLA class I expressions on FGF-2 stimulated HCC cells. FGF-2 stimulation on HCC cells resulted in increasing NK sensitivity against HCC cells. These findings indicate that FGF-2 produced by HCC cells or normal hepatocytes of chronic liver disease may play critical roles in eliminating HCC cells by innate immunity.

Tyrosine Phosphorylation Profiling in FGF-2 Stimulated Human Embryonic Stem Cells

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.

Fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes.

IntroductionCartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA.MethodsPrimary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity.ResultsChondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human articular chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human articular chondrocytes.ConclusionsFGFR1 is the major mediator with the degenerative potential in the presence of FGF-2 in human adult articular chondrocytes. FGFR1 activation by FGF-2 promotes catabolism and impedes anabolism. Disruption of the balance between FGFR1 and FGFR3 signaling ratio may contribute to the pathophysiology of OA.

Fibroblast growth factor‐2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction

Fibroblast growth factor-2 (FGF-2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF-2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF-2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3-kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF-2-stimulated migration of MPDL22 cells. Moreover, in response to FGF-2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)-1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF-2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti-CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF-2. Furthermore, an anti-CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF-2-induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF-2-mediated cell motility of PDL cells, suggesting that FGF-2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production.

FGF2 promotes Msx2 stimulated PC‐1 expression via Frs2/MAPK signaling

PC-1 is an enzymatic generator of pyrophosphate and a critical regulator of tissue mineralization. We previously showed that fibroblast growth factor-2 (FGF2) specifically induces PC-1 expression in calvarial pre-osteoblasts and that this occurs via a transcriptional mechanism involving Runx2. Because aberrant FGF signaling and Msx2 activity result in similar craniofacial skeletal defects and because Msx2 is an established regulator of osteoblastic gene expression, here we investigate Msx2 as an additional mediator of PC-1 gene expression. mRNA analysis and experiments utilizing PC-1 gene promoter/luciferase reporter constructs demonstrate that Msx2 promotes transcription of the PC-1 gene downstream of FGF2. Results indicate that both Msx2 and Runx2 are recruited to a conserved core Msx2 binding site within the PC-1 gene promoter upon FGF2 stimulation, and that Msx2 and Runx2 function together to induce PC-1 gene expression in osteoblastic cells. Here we show that FGF signaling promotes Msx2 transcriptional activity on the PC-1 gene promoter via the Frs2/MAPK signaling pathway. To our knowledge, this is the first report of Msx2 functioning as a transcriptional enhancer downstream of FGF2 in calvarial pre-osteoblasts. As activating mutations in FGF receptors and Msx2 result in similar craniofacial skeletal disorders, our findings support the idea that FGF signaling and Msx2 activity influence cranial osteogenesis via the same molecular mechanism.

Activation of AP-1 Transcription Factors Differentiates FGF2 and Vascular Endothelial Growth Factor Regulation of Endothelial Nitric-oxide Synthase Expression in Placental Artery Endothelial Cells

FGF2 (fibroblast growth factor 2), but not vascular endothelial growth factor (VEGF), stimulates sustained activation of ERK2/1 for endothelial NOS3 (nitric-oxide synthase 3) protein expression in ovine fetoplacental artery endothelial cells (oFPAEC). We deciphered herein the downstream signaling of ERK2/1 responsible for NOS3 expression by FGF2 in oFPAEC. FGF2, but not VEGF, increased NOS3 mRNA levels without altering its degradation. FGF2, but not VEGF, trans-activated sheep NOS3 promoter, and this was dependent on ERK2/1 activation. FGF2 did not trans-activate NOS3 promoters with deletions upstream of the consensus AP-1 site (TGAGTC A, -678 to -685). Trans-activation of wild-type NOS3 promoter by FGF2 was significantly inhibited when either the AP-1 or the cAMP-response element (CRE)-like sequence (TGCGTCA, -752 to -758) was mutated and was completely blocked when both were mutated. EMSA analyses showed that FGF2, but not VEGF, stimulated AP-1 and CRE DNA-protein complexes primarily composed of JunB and Fra1. Chromatin immunoprecipitation assays confirmed JunB/Fra1 binding to NOS3 promoter AP-1 and CRE elements in intact cells. FGF2, but not VEGF, stimulated JunB and Fra1 expressions; all preceded NOS3 up-regulation and were inhibited by PD98059. Down-regulation of JunB or Fra-1, but not c-Jun, blocked FGF2 stimulation of NOS3 expression and NO production. AP-1 inhibition suppressed FGF2 stimulation of NOS3 expression in human umbilical vein EC and uterine artery endothelial cells. Thus, FGF2 induction of NOS3 expression is mainly mediated by AP-1-dependent transcription involving JunB and Fra1 up-regulation via sustained ERK2/1 activation in endothelial cells.

FGF‐2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3‐K/GSK3β signaling

Fibroblast growth factor-2 (FGF-2) is widely used to culture human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cells. Despite its importance in maintaining undifferentiated hESC phenotype, a lack of understanding in the role of FGF-2 still exists. Here, we investigate the signaling events in hESC following the addition of exogenous FGF-2. In this study, we show that hESC express all forms of fibroblast growth factor receptors (FGFRs) which co-localize on Oct3/4 positive cells. Furthermore, downregulation of Oct3/4 in hESC occurs following treatment with an FGFR inhibitor, suggesting that FGF signaling may regulate Oct3/4 expression. This is also observed in iPS cells. Also, downstream of FGF signaling, both mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase pathways (PI3-K) are activated following FGF-2 stimulation. Notably, inhibition of MAPK and PI3-K signaling using specific kinase inhibitors revealed that activated PI3-K, rather than MAPK, can mediate pluripotent marker expression. To understand the importance of PI3-K activation, activation of Wnt/beta-catenin by FGF-2 was investigated. Wnt signaling had been implicated to have a role in maintaining of pluripotent hESC. We found that upon FGF-2 stimulation, GSK3beta is phosphorylated following which nuclear translocation of beta-catenin and TCF/LEF activation occurs. Interestingly, inhibition of the Wnt pathway with Dikkopf-1 (DKK-1) resulted in only partial suppression of the FGF-2 induced TCF/LEF activity. Prolonged culture of hESC with DKK-1 did not affect pluripotent marker expression. These results suggest that FGF-2 mediated PI3-K signaling may have a direct role in modulating the downstream of Wnt pathway to maintain undifferentiated hESC.