Ayumi Miyake

Structure and Expression of the Rat mRNA Encoding a Novel Member of the Fibroblast Growth Factor Family

We isolated the cDNA encoding a novel member of the fibroblast growth factor (FGF) family from rat embryos by homology-based polymerase chain reaction. The FGF-related cDNA encodes a protein of 215 amino acids (∼24 kDa), which has a conserved ∼120-amino acid core with ∼30-60% amino acid sequence identity with the FGF family. This protein with a hydrophobic amino terminus appears to be a secreted protein. The cDNA was translated in a coupled in vitro transcription-translation system. The molecular mass of the translation product was observed to be ∼26 kDa. The expression of the FGF-related mRNA in the rat embryo and adult tissues was determined by Northern analysis and in situ hybridization. The mRNA was expressed in several discrete regions of the embryo. In adult tissues, the mRNA was preferentially expressed in the lung. The expression profile of the FGF-related mRNA was different from those of other FGF family mRNAs. As this protein is the 10th documented protein related to FGFs, we tentatively term this protein FGF-10.

Fibroblast Growth Factor (FGF)-23 Inhibits Renal Phosphate Reabsorption by Activation of the Mitogen-activated Protein Kinase Pathway

The homeostasis of the plasma phosphate level is essential for many biological processes including skeletal mineralization. The reabsorption of phosphate in the kidney is a major determinant of the plasma levels of phosphate. Phosphatonin is a hormone-like factor that specifically inhibits phosphate uptake in renal proximal epithelial cells. Recent studies on tumor-induced osteomalacia suggested that phosphatonin was potentially identical to fibroblast growth factor (FGF)-23. However, as purified recombinant FGF-23 could not inhibit phosphate uptake in renal proximal epithelial cells, the mechanism of action of FGF-23 remains to be elucidated. Therefore, we examined the mechanism of action of FGF-23 in cultured renal proximal epithelial cells, opossum kidney cells. FGF-23 was found to require heparin-like molecules for its inhibitory activity on phosphate uptake. FGF-23 binds to the FGF receptor 3c, which is mainly expressed in opossum kidney cells, with high affinity. An inhibitor for tyrosine kinases of the FGF receptor, SU 5402, blocked the activity of FGF-23. FGF-23 activated the mitogen-activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGF. Inhibitors of the MAPK pathway, PD98059 and SB203580, also blocked the activity of FGF-23. The present findings have revealed a novel MAPK-dependent mechanism of the regulation of phosphate uptake by FGF signaling.

Preferential neurotrophic activity of fibroblast growth factor-20 for dopaminergic neurons through fibroblast growth factor receptor-1c

Degeneration of dopaminergic neurons of the substantia nigra causes Parkinsons disease. Therefore, neurotrophic factors for dopaminergic neurons are of substantial clinical interest. Fibroblast growth factor (FGF)‐20 preferentially expressed in the substantia nigra pars compacta (SNPC) of the rat brain significantly enhanced the survival of midbrain dopaminergic neurons. Here we examined the mechanism of action of FGF‐20 on dopaminergic neurons. FGF‐20 slightly enhanced the survival of total neurons of the midbrain, indicating that it preferentially enhanced the survival of dopaminergic neurons. FGF receptor (FGFR)‐1c was found to be expressed abundantly in dopaminergic neurons in the SNPC but at much lower levels in neurons of other midbrain regions by in situ hybridization. FGF‐20 was also found to bind FGFR‐1c with high affinity with the BIAcore system. Furthermore, FGF‐20 activated the mitogen‐activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGFs. Both the FGFR‐1 inhibitor SU5402 and the MAPK pathway inhibitor PD98059 also significantly inhibited the activation of the MAPK pathway by FGF‐20 and the neurotrophic activity of FGF‐20. The present findings indicate that the activation of the MAPK pathway by FGF‐20 signaling through FGFR‐1c plays important roles in the survival of dopaminergic neurons in the SNPC.

RAT OLIGODENDROCYTES AND ASTROCYTES PREFERENTIALLY EXPRESS FIBROBLAST GROWTH FACTOR RECEPTOR-2 AND -3 MRNAS

Fibroblast growth factors (FGFs) exert various effects on glial cells as well as on neurons in the brain. The mRNAs for four FGF receptors (FGFR‐1‐FGFR‐4) are expressed in the brain. Although FGFR‐1 and ‐4 mRNAs are preferentially expressed in neurons, FGFR‐2 and ‐3 mRNAs are preferentially expressed in glial cells. However, the glial cells that express these receptors remained to be identified. In this study, we found that oligodendrocytes and astrocytes in the brain preferentially expressed FGFR‐2 and FGFR‐3 mRNAs, respectively. The isoforms of immunoglobulin‐like domain III (IIIb and IIIc) of the receptors have crucial roles in ligand binding. We also determined the isoforms of FGFR‐2 and FGFR‐3 expressed in glial cells to be of type IIIc. The expression of FGFR‐2 IIIc and FGFR‐3 IIIc with different ligand specificities might play important roles in the various effects of FGFs on oligodendrocytes and astrocytes.

Neurotrophic Activity of Neudesin, a Novel Extracellular Heme-binding Protein, Is Dependent on the Binding of Heme to Its Cytochrome b5-like Heme/Steroid-binding Domain

Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme-binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402 nm. The addition of exogenous hemin extensively increased the amount of hemin-bound neudesin. In contrast, neudesinΔHBD, a mutant lacking the heme-binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin-hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The neurotrophic activity of neudesin was enhanced by the binding of Fe(III)-protoporphyrin IX, but neither Fe(II)-protoporphyrin IX nor protoporphyrin IX alone. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti-neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme-affinity column at all. Neudesin is the first extracellular heme-binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme-binding proteins.

Rat FGF receptor-4 mRNA in the brain is expressed preferentially in the medial habenular nucleus

The fibroblast growth factor (FGF) receptor family consists of four members, FGFR-1, FGFR-2, FGFR-3 and FGFR-4, that are closely related receptor tyrosine kinases. We examined the expression of rat FGFR-4 mRNA in the brain by in situ hybridization and compared it with that of the mRNAs for other FGF receptors. In contrast with FGFR-1, FGFR-2 and FGFR-3 mRNAs which are expressed widely in the brain, the FGFR-4 mRNA in the brain is expressed preferentially in the medial habenular nucleus neurons. The present finding indicates that FGFR-4 has a function specific to the medial habenular nucleus.

Oral administration of soluble β‐glucans extracted from Grifola frondosa induces systemic antitumor immune response and decreases immunosuppression in tumor‐bearing mice

Maitake D (MD)‐Fraction is a highly purified soluble β‐glucan derived from Grifola frondosa (an oriental edible mushroom). Intraperitoneal (i.p.) injection of MD‐Fraction has been reported to inhibit tumor growth via enhancement of the host immune system. In this study, we demonstrated that oral administration of MD‐Fraction as well as i.p. injection significantly inhibited tumor growth in murine tumor models. After oral administration, MD‐Fraction was not transferred to the blood in its free form but was captured by antigen‐presenting cells such as macrophages and dendritic cells (DCs) present in the Peyers patch. The captured MD‐Fraction was then transported to the spleen, thereby inducing the systemic immune response. Our study showed that MD‐Fraction directly induced DC maturation via a C‐type lectin receptor dectin‐1 pathway. The therapeutic response of orally administered MD‐Fraction was associated with (i) induced systemic tumor‐antigen specific T cell response via dectin‐1‐dependent activation of DCs, (ii) increased infiltration of the activated T cells into the tumor and (iii) decreased number of tumor‐caused immunosuppressive cells such as regulatory T cells and myeloid‐derived suppressor cells. Our preclinical study suggests that MD‐Fraction is a useful oral therapeutic agent in the management of patients with cancer.

Expression of Fgf23 in Activated Dendritic Cells and Macrophages in Response to Immunological Stimuli in Mice

Fibroblast growth factors (Fgfs) are polypeptide growth factors with diverse biological activities. While several studies have revealed that Fgf23 plays important roles in the regulation of phosphate and vitamin D metabolism, the additional physiological roles of Fgf23 remain unclear. Although it is believed that osteoblasts/osteocytes are the main sources of Fgf23, we previously found that Fgf23 mRNA is also expressed in the mouse thymus, suggesting that it might be involved in the immune system. In this study we examined the potential roles of Fgf23 in immunological responses. Mouse serum Fgf23 levels were significantly increased following inoculation with Escherichia coli or Staphylococcus aureus or intraperitoneal injection of lipopolysaccharide. We also identified activated dendritic cells and macrophages that potentially contributed to increased serum Fgf23 levels. Nuclear factor-kappa B (NF-κB) signaling was essential for the induction of Fgf23 expression in dendritic cells in response to immunological stimuli. Moreover, we examined the effects of recombinant Fgf23 protein on immune cells in vitro. Fgfr1c, a potential receptor for Fgf23, was abundantly expressed in macrophages, suggesting that Fgf23 might be involved in signal transduction in these cells. Our data suggest that Fgf23 potentially increases the number in macrophages and induces expression of tumor necrosis factor-α (TNF-α), a proinflammatory cytokine. Collectively, these data suggest that Fgf23 might be intimately involved in inflammatory processes.

Fgf21 is essential for haematopoiesis in zebrafish

Fibroblast growth factors (Fgfs) function as key secreted signalling molecules in many developmental events. The zebrafish is a powerful model system for the investigation of embryonic vertebrate haematopoiesis. Although the effects of Fgf signalling on haematopoiesis in vitro have been reported, the functions of Fgf signalling in haematopoiesis in vivo remain to be explained. We identified Fgf21 in zebrafish embryos. Fgf21‐knockdown zebrafish embryos lacked erythroid and myeloid cells but not blood vessels and lymphoid cells. The knockdown embryos had haemangioblasts and haematopoietic stem cells. However, the knockdown embryos had significantly fewer myeloid and erythroid progenitor cells. In contrast, Fgf21 had no significant effect on cell proliferation and apoptosis in the intermediate cell mass. These results indicate that Fgf21 is a newly identified factor essential for the determination of myelo‐erythroid progenitor cell fate in vivo.

Fgf4 is required for left-right patterning of visceral organs in zebrafish.

Fgf signaling plays essential roles in many developmental events. To investigate the roles of Fgf4 signaling in zebrafish development, we generated Fgf4 knockdown embryos by injection with Fgf4 antisense morpholino oligonucleotides. Randomized LR patterning of visceral organs including the liver, pancreas, and heart was observed in the knockdown embryos. Prominent expression of Fgf4 was observed in the posterior notochord and Kupffers vesicle region in the early stages of segmentation. Lefty1, lefty2, southpaw, and pitx2 are known to play crucial roles in LR patterning of visceral organs. Fgf4 was essential for the expression of lefty1, which is necessary for the asymmetric expression of southpaw and pitx2 in the lateral plate mesoderm, in the posterior notochord, and the expression of lefty2 and lefty1 in the left cardiac field. Fgf8 is also known to be crucial for the formation of Kupffers vesicle, which is needed for the LR patterning of visceral organs. In contrast, Fgf4 was required for the formation of cilia in Kupffers vesicle, indicating that the role of Fgf4 in the LR patterning is quite distinct from that of Fgf8. The present findings indicate that Fgf4 plays a unique role in the LR patterning of visceral organs in zebrafish.