FGF19 Signaling Research Articles

Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin, Fgf, and sonic hedgehog function.

Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol's effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function. Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was used to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons. Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8, or Shh mRNA overexpression rescues ethanol-induced decreases in GAD1 and Atonal1a gene expression. These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis.

Aberrantly elevated microRNA-34a in obesity attenuates hepatic responses to FGF19 by targeting a membrane coreceptor β-Klotho

MicroRNA-34a (miR-34a) is the most highly elevated hepatic miR in obese mice and is also substantially elevated in patients who have steatosis, but its role in obesity and metabolic dysfunction remains unclear. After a meal, FGF19 is secreted from the ileum; binds to a hepatic membrane receptor complex, FGF19 receptor 4 and coreceptor β-Klotho (βKL); and mediates postprandial responses under physiological conditions, but hepatic responses to FGF19 signaling were shown to be impaired in patients with steatosis. Here, we show an unexpected functional link between aberrantly elevated miR-34a and impaired βKL/FGF19 signaling in obesity. In vitro studies show that miR-34a down-regulates βKL by binding to the 3' UTR of βKL mRNA. Adenoviral-mediated overexpression of miR-34a in mice decreased hepatic βKL levels, impaired FGF19-activated ERK and glycogen synthase kinase signaling, and altered expression of FGF19 metabolic target genes. Consistent with these results, βKL levels were decreased and hepatic responses to FGF19 were severely impaired in dietary obese mice that have elevated miR-34a. Remarkably, in vivo antisense inhibition of miR-34a in obese mice partially restored βKL levels and improved FGF19 target gene expression and metabolic outcomes, including decreased liver fat. Further, anti-miR-34a treatment in primary hepatocytes of obese mice restored FGF19-activated ERK and glycogen synthase kinase signaling in a βKL-dependent manner. These results indicate that aberrantly elevated miR-34a in obesity attenuates hepatic FGF19 signaling by directly targeting βKL. The miR-34a/βKL/FGF19 axis may present unique therapeutic targets for FGF19-related human diseases, including metabolic disorders and cancer.

Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

Previous studies have shown that starvation or consumption of a high fat, low carbohydrate (HF-LC) ketogenic diet induces hepatic fibroblast growth factor 21 (FGF21) gene expression in part by activating the peroxisome proliferator-activated receptor-α (PPARα). Using primary hepatocyte cultures to screen for endogenous signals that mediate the nutritional regulation of FGF21 expression, we identified two sources of PPARα activators (i.e. nonesterified unsaturated fatty acids and chylomicron remnants) that induced FGF21 gene expression. In addition, we discovered that natural (i.e. bile acids) and synthetic (i.e. GW4064) activators of the farnesoid X receptor (FXR) increased FGF21 gene expression and secretion. The effects of bile acids were additive with the effects of nonesterified unsaturated fatty acids in regulating FGF21 expression. FXR activation of FGF21 gene transcription was mediated by an FXR/retinoid X receptor binding site in the 5'-flanking region of the FGF21 gene. FGF19, a gut hormone whose expression and secretion is induced by intestinal bile acids, also increased hepatic FGF21 secretion. Deletion of FXR in mice suppressed the ability of an HF-LC ketogenic diet to induce hepatic FGF21 gene expression. The results of this study identify FXR as a new signaling pathway activating FGF21 expression and provide evidence that FXR activators work in combination with PPARα activators to mediate the stimulatory effect of an HF-LC ketogenic diet on FGF21 expression. We propose that the enhanced enterohepatic flux of bile acids during HF-LC consumption leads to activation of hepatic FXR and FGF19 signaling activity and an increase in FGF21 gene expression and secretion.

Pregnane X receptor activation induces FGF19-dependent tumor aggressiveness in humans and mice

The nuclear receptor pregnane X receptor (PXR) is activated by a range of xenochemicals, including chemotherapeutic drugs, and has been suggested to play a role in the development of tumor cell resistance to anticancer drugs. PXR also has been implicated as a regulator of the growth and apoptosis of colon tumors. Here, we have used a xenograft model of colon cancer to define a molecular mechanism that might underlie PXR-driven colon tumor growth and malignancy. Activation of PXR was found to be sufficient to enhance the neoplastic characteristics, including cell growth, invasion, and metastasis, of both human colon tumor cell lines and primary human colon cancer tissue xenografted into immunodeficient mice. Furthermore, we were able to show that this PXR-mediated phenotype required FGF19 signaling. PXR bound to the FGF19 promoter in both human colon tumor cells and "normal" intestinal crypt cells. However, while both cell types proliferated in response to PXR ligands, the FGF19 promoter was activated by PXR only in cancer cells. Taken together, these data indicate that colon cancer growth in the presence of a specific PXR ligand results from tumor-specific induction of FGF19. These observations may lead to improved therapeutic regimens for colon carcinomas.

Abstract 1106: Fibroblast growth factor FGF19 signaling promotes prostate cancer progression

Abstract Aberrant fibroblast growth factor (FGF) signaling can promote tumor development by directly driving cancer cell proliferation and survival, and by supporting tumor angiogenesis. Multiple FGFs have been found upregulated in prostate cancers, including FGF1, FGF2, FGF6, FGF8 and FGF17, all of which can activate FGF receptor 4 (FGFR4). FGFR4 is overexpressed in prostate cancer (PCa) and positively associated with aggressive PCa. FGF19 is a distinct member of FGF family in that it predominantly binds to FGFR4 with high affinity. In this present study we aimed to study the role of FGF19 in human PCa progression, and to determine whether the targeted suppression of FGF19/FGFR4 signaling has potential therapeutic benefits in PCa. Our results demonstrated that FGF19 is upregulated in human PCa compared to normal prostate tissues. FGF19 is expressed in an autocrine manner by all tested PCa cell lines. Exogenous FGF19 stimulates PCa cell proliferation, anchorage-independent growth, adhesion and invasion in vitro. The mRNAs of FGF19 co-receptors αKlotho and ßKlotho are expressed in 97.5% and 27.5% of PCa clinical samples, respectively; but αKlotho is expressed in only 57% normal prostate tissues, ßKlotho is barely detected in normal prostate tissues. Suppression of FGF19 by short hairpin RNA (shRNA) targeting FGF19 gene inhibits PCa cell proliferation, adhesion and invasiveness in vitro. Immunoprecipitation and western blot assays showed that FGF19 stimulates phosphorylation of FGFR4, FRS2α, Erk1/2 and p-38 MAPK, as well as MEK1/2 in both PC3 and DU145. Our data also revealed that FGF19 induced the serine/threonine protein kinase Akt phosphorylation in PCa cells. Lentiviral shRNA delivery was used for stable FGFR4 gene silencing in PC3 and LNCaP cells. The targeted knockdown of FGFR4 in PC3 and LNCaP cells resulted in significantly inhibited cell proliferation, invasion and remarkably reduced Akt phosphorylation. The upregulated expression of activated Caspase 8 and activated Caspase 3 in FGFR4 knockdown cells indicates that FGFR4 signaling inhibits PCa cell apoptosis. To determine if FGFR4 suppression impacts prostate tumor growth and metastasis in vivo we generated a PCa orthotopic xenograft model in which PC3-sh-FGFR4 or PC3-shV cells are injected directly into the prostates of nude mice in each group. The results revealed that the FGFR4 suppression in PCa cells significantly inhibited tumorigenicity in this model (P=0.01). The primary tumor weight was decreased by 63% in sh-FRGR4 tumors (p<0.05) and the proportion of mice with lymph node metastasis was decreased from 93.3% to 50% (p<0.01). The Ki67 immunohistochemistry showed a significant decrease of Ki67 positive cell percentage in PC3-sh-FGFR4 xenograft tumors (19.85%) compared to control group (35.93%, p<0.001). Our data indicated that FGF19/FGFR4 signaling may be a promising target in PCa therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1106. doi:10.1158/1538-7445.AM2011-1106

841 CIRCULATING LEVELS OF TNF-LIKE CYTOKINE 1A (TL1A) AND ITS DECOY RECEPTOR 3 (DCR3) IN NASH: MARKERS OF ADVANCED DISEASE

Background: FGF-19, a novel intestinal hormone, is secreted in the terminal ileum postprandially and during bile salt absorption. Non-alcoholic fatty liver disease (NAFLD) is common in obesity but the molecular mechanisms of NAFLD development in non-obese patients remain yet to be defined. FGF-19 might play a protective role in NAFLD by improving insulin sensitivity, increasing hepatic beta-oxidation and inhibiting lipogenesis. The aim of our study was to investigate FGF-19 serum concentrations in non-obese (BMI 30kg/m) patients and healthy volunteers in a fasted state and after a defined oral fat load. Patients and Methods: In total, 14 NAFLD patients (mean BMI 27.7 kg/m), 21 obese individuals and 16 healthy controls were recruited. FGF-19 levels were determined by ELISA after 10 hours of overnight fasting. Subsequently, all individuals received 1 g fat (Calogen) per kg body weight orally. FGF-19 serum concentrations were measured at baseline and after 2, 4 and 6 hours. No other food intake was permitted during this time but water was allowed ad libitum. Results: Basal serum FGF-19 levels in the fasted state do not differ between overweight NAFLD patients, obese subjects and normal controls. The oral fat load increases FGF-19 levels in all three groups. FGF-19 serum levels peak after 4 hours in all three groups and were highest in controls (378.9±202.1 pg/ml). In comparison to men, women display significantly higher FGF-19 levels after 6 hours (283.4±189.6 vs. 172.1±82.1 pg/ml, p < 0.05). Of note, non-obese NAFLD patients display the lowest FGF-19 levels at all time points. In particular, FGF-19 levels 2 hours post oral fat load are significantly lower (p = 0.004) in overweight NAFLD patients (143.7±61.4 pg/ml) as compared to both healthy volunteers and obese patients (287.4±177.7 pg/ml and 281.4±235.0 pg/ml respectively). Conclusions: We established an oral fat load test (“OFLT”), which demonstrated impaired FGF-19 release in non-obese NAFLD patients. The hepatic mechanisms consequences of impaired FGF-19 signaling require further investigation.

The Klotho gene family as a regulator of endocrine fibroblast growth factors

The Klotho gene encodes a single-pass transmembrane protein and functions as an aging-suppressor gene, which extends lifespan when overexpressed and accelerates the development of aging-like phenotypes when disrupted in mice. Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that regulates phosphate and vitamin D homeostasis. It has been shown that Klotho-deficient mice and Fgf23 knockout mice exhibit identical phenotypes. This observation led to the identification of Klotho as a cofactor essential for interactions between FGF23 and FGF receptors. In addition to the Klotho-FGF23 axis, recent studies has shown that betaKlotho, a Klotho family protein, also functions as a cofactor required for FGF19 and FGF21 signaling and determines the tissue-specific metabolic activities of FGF19 and FGF21. This review summarizes recent progress in understanding of Klotho and betaKlotho function in the regulation of tissue-specific metabolic activity of the endocrine fibroblast growth factors (FGF19, FGF21, and FGF23).

The Klotho gene family and the endocrine fibroblast growth factors

This review summarizes recent progress in understanding of Klotho and betaKlotho function in the regulation of tissue-specific metabolic activity of the endocrine fibroblast growth factors (FGF19, FGF21, and FGF23). The Klotho gene encodes a single-pass transmembrane protein and functions as an aging-suppressor gene, which extends lifespan when overexpressed and accelerates the development of aging-like phenotypes when disrupted in mice. FGF23 is a bone-derived hormone that regulates phosphate and vitamin D metabolism. It has been shown that Klotho-deficient mice and FGF23 knockout mice exhibit identical phenotypes. This observation led to the identification of Klotho as a cofactor essential for the interaction between FGF23 and fibroblast growth factor receptors. In addition to the Klotho-FGF23 axis, recent studies have shown that betaKlotho, a Klotho family protein, also functions as a cofactor required for FGF19 and FGF21 signaling and determines tissue-specific metabolic activities of FGF19 and FGF21. Recent mouse genetic studies have broadened our understanding of molecular pathways involved in mineral and bile acid homeostasis regulated by FGF23 and FGF19, respectively. The FGF19 subfamily of ligands requires the Klotho gene family of transmembrane proteins for their tissue-specific bioactivity. Further investigations on endocrine axes mediated by the Klotho family and FGF19 subfamily members are expected to provide new insights into the molecular mechanisms by which the endocrine fibroblast growth factors regulate bile acid, energy, and phosphate/vitamin D metabolism.

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.

Liver-specific Activities of FGF19 Require Klotho beta

Hepatocyte function is regulated by members of the fibroblast growth factor (FGF) family of proteins, but little is known about the specific molecular mechanisms of this endocrine pathway. FGF19 regulates bile acid homeostasis and gall bladder filling; FGF19 binds only to FGF receptor 4 (FGFR4), but its liver-specific activity cannot be explained solely by the distribution of this receptor. Although it has been suggested that Klotho beta (KLB) may have a role in mediating FGF19 activity, we have provided for the first time definitive evidence that KLB is required for FGF19 binding to FGFR4, intracellular signaling, and downstream modulation of gene expression. We have shown that FGFR4 is widely distributed in mouse, whereas KLB distribution is more restricted. Liver was the only organ in which both genes were abundantly expressed. We show that in mice, FGF19 injection triggers liver-specific induction of c-Fos and repression of CYP7A1. The tissue-specific activity of FGF19 supports the unique intersection of KLB and FGFR4 distribution in liver. These studies define KLB as a novel FGFR4 coreceptor required for FGF19 liver specific functions.

Fgf19 regulated by Hh signaling is required for zebrafish forebrain development

Fibroblast growth factor (Fgf) signaling plays important roles in brain development. Fgf3 and Fgf8 are crucial for the formation of the forebrain and hindbrain. Fgf8 is also required for the midbrain to form. Here, we identified zebrafish Fgf19 and examined its roles in brain development by knocking down Fgf19 function. We found that Fgf19 expressed in the forebrain, midbrain and hindbrain was involved in cell proliferation and cell survival during embryonic brain development. Fgf19 was also essential for development of the ventral telencephalon and diencephalon. Regional specification is linked to cell type specification. Fgf19 was also essential for the specification of γ-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes generated in the ventral telencephalon and diencephalon. The cross talk between Fgf and Hh signaling is critical for brain development. In the forebrain, Fgf19 expression was down-regulated on inhibition of Hh but not of Fgf3/Fgf8, and overexpression of Fgf19 rescued partially the phenotype on inhibition of Hh. The present findings indicate that Fgf19 signaling is crucial for forebrain development by interacting with Hh and provide new insights into the roles of Fgf signaling in brain development.

FGF19‐FGFR4 signaling elaborates lens induction with the FGF8‐L‐Maf cascade in the chick embryo

The fibroblast growth factor (FGF) family is known to be involved in vertebrate eye development. However, distinct roles of individual FGF members during eye development remain largely elusive. Here, we show a detailed expression pattern of Fgf19 in chick lens development. Fgf19 expression initiated in the forebrain, and then became restricted to the distal portion of the optic vesicle abutting the future lens placode, where FGF receptor 4 (Fgfr4), a receptor for FGF19, was expressed. Fgf8, a positive regulator for L-Maf, was expressed in a portion of the optic vesicle. To examine the role of FGF19 signaling during early eye development, Fgf19 was misexpressed near the presumptive lens ectoderm; however, no alteration in the expression of lens marker genes was observed. Conversely, a secreted form of FGFR4 was misexpressed to inhibit an FGF19 signal, resulting in the induction of L-Maf expression. To further define the relationship between L-Maf and Fgf19, L-Maf misexpression was performed, resulting in ectopic induction of Fgf19 expression by Hamburger and Hamilton's stage 12/13. Furthermore, misexpression of Fgf8 induced Fgf19 expression in addition to L-Maf. These results suggest that FGF19-FGFR4 signaling plays a role in early lens development in collaboration with FGF8 signaling and L-Maf transcriptional system.

Mouse FGF15 is the ortholog of human and chick FGF19, but is not uniquely required for otic induction

The inner ear develops from an ectodermal placode that is specified by inductive signals from the adjacent neurectoderm and underlying mesoderm. In chick, fibroblast growth factor ( Fgf)- 19 is expressed in mesoderm underlying the presumptive otic placode, and human FGF19 induces expression of otic markers in a tissue explant containing neural plate and surface ectoderm. We show here that mouse Fgf15 is the sequence homolog of chick and human Fgf19/FGF19. In addition, we show that FGF15, like FGF19, is sufficient to induce expression of otic markers in a chick explant assay, suggesting that these FGFs are orthologs. Mouse embryos lacking Fgf15, however, do not have otic abnormalities at E9.5–E10.5, suggesting that Fgf15 is not uniquely required for otic induction or early patterning of the otocyst. To compare FGF15 and FGF19 signaling components and assess where signals potentially redundant with FGF15 might function, we determined the expression patterns of Fgf15 and Fgf19. Unlike Fgf19, Fgf15 is not expressed in mesoderm underlying the presumptive otic placode, but is expressed in the adjacent neurectoderm. Fgfr4, which encodes the likely receptor for both FGF19 and FGF15, is expressed in the neurectoderm of both species, and is also expressed in the mesoderm only in chick. These results suggest the hypotheses that during otic induction, FGF19 signals in either an autocrine fashion to the mesoderm or a paracrine fashion to the neurectoderm, whereas FGF15 signals in an autocrine fashion to the neurectoderm. Thus, the FGFs that signal to the neurectoderm are the best potential candidates for redundancy with FGF15 during mouse otic development.

Fibroblast growth factor receptor 4 (FGFR4) mediates signaling to the prolactin but not the FGFR4 promoter.

Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of activities. Signaling of the 23 members of the FGF family is mediated through FGFR1-4. We show that FGF-19, which selectively binds FGFR4, can induce prolactin (PRL) but not growth hormone expression. FGF-19 also stimulated MAPK activation, an effect that was abrogated by a soluble dominant negative (dn) form of FGFR4. The response of the pituitary PRL promoter to FGF maps to an Ets-Pit1 binding site. We have previously shown that the hematopoietic zinc finger-containing transcription factor Ikaros (Ik) regulates FGFR4 as part of an overlapping site with that for an Ets-type factor in the FGFR4 promoter. Thus, we examined whether FGF-19 might regulate its own receptor through the Ets-Ik element in the FGFR4 promoter. Ets stimulated and dn-Ets inhibited basal FGFR4 and PRL promoter activity. In contrast, Ets enhanced FGF-19-induced PRL activation but failed to confer an effect for FGF-19 on the FGFR4 promoter. We conclude that FGFR4 mediates FGF-19 signaling to the PRL promoter. Our data also suggest a possible functional role for Ik in sorting Ets signals to the FGFR4 promoter, as distinct from the PRL promoter, where Ets partners with Pit1.