Department of Molecular Cell Biology and Molecular Medicine
Institute of Advanced Medicine
Wakayama Medical University
TEL : 073-441-0607
FAX : 073-441-0864
|教授||坂口 和成 Kazushige Sakaguchi, MD., Ph.D.|
|准教授||京 雪楓 Xuefeng Jing, MD., Ph.D.|
|講師||片山 圭一 Kei-ichi Katayama, DVM.,Ph.D.|
|非常勤講師||澤田 貴宏 Takahiro Sawada, Ph.D..|
|特別研究員||武下 愛 Ai Takeshita, DVM., Ph.D.|
|研究生||河上 和紀 Kazuki Kawakami|
|研究補助員||﨑山 真由子 Mayuko Sakiyama|
|研究補助員||池田 裕加里 Yukari Ikeda|
|秘書||北川 二美 Futami Kitagawa|
Focus of Our Research
Cell surface receptors receive extracellular signals via interaction with a specific ligand, and transduce the signals from the cell membrane to other parts of the cell. Many types of receptors have been identified through molecular cloning, and most of their functions have been clarified. For intracellular signal transduction, cytoplasmic signaling molecules are required. These molecules relay signals to downstream via conformational change and/or phosphorylation, resulting in a change in cell metabolism, cytoskeletal modulation, up- or down-regulation of gene transcription, etc.
We have so far focused on several receptors in certain biological systems to investigate how these receptors interact with each other to fulfill their functions, and how the signal transduction pathway from the cell surface receptors can be modulated under physiological and pathological conditions.
Fibroblast growth factor receptors (FGFRs) and calcium/phosphorus metabolism
The first focus is on the fibroblast growth factor receptor (FGFR) signaling system. Using a parathyroid cell line (PT-r cells) that was established by us (1), we were among the first groups that proved the requirement of heparan sulfate for FGFR to bind to FGF and to be properly activated (Fig. 1) (2-5). We also discovered that regulation of parathyroid cell proliferation by calcium is mediated by the aFGF autocrine system.
A, The 2-loop FGFR isoform without heparan sulfate; B, The 2-loop FGFR isoform interacting with the external heparan sulfate proteoglycan; C, The 2-loop FGFR isoform interacting with heparin; D, The 2-loop FGFR isoform interacting with its own heparan sulfate moiety; E, The 3-loop FGFR isoform.
We are currently working on how FGF23 affects the function of parathyroid cells. FGF23 belongs to a family of FGF ligands, is known to be an endocrine hormone that is produced from the osteoblast/osteocyte lineage, and requires Klotho alpha as a co-receptor to bind to FGFRs. FGF23 stimulates phosphaturia and suppresses activation of vitamin D in the kidney, and appears to affect secretion and/or synthesis of parathyroid hormone in the parathyroid glands. Since the parathyoids, the kidney and the bone interact with each other by producing several factors related with regulation of calcium and phosphorus in a body, we have to be careful in analyzing the specific function of each factor. One way of resolving the problem is to knockout a specific molecule from a specific cell type in vivo, and study the effect under in vivo and in vitro conditions. We have created a Klotho Flox mouse line and a FGFR1-4 quadruple Flox mouse line, from which Klotho or FGFRs can be conditionally knocked out in a cell-type specific fashion.
Interaction of FGFR with EphA4, and neural stem cell proliferation and differentiation
FGFRs have been known to play important roles in proliferation and differentiation of neural stem cells. We screened a human brain cDNA library for binding partners of the FGFR cytoplasmic domain using the yeast two-hybrid screening system, and identified EphA4 as one of such partners. Both FGFR and Eph belong to a superfamily of receptor tyrosine kinases. We found that the EphA4-FGFR interaction upregulates two pathways (Fig. 2); (1) an FRS2alpha (fibroblast growth factor receptor substrate 2alpha; a docking protein) -mediated signal transduction pathway that culminates in proliferation and self-renewal of neural stem cells, and (2) another pathway via ephexin1, a guanine nucleotide exchange factor, that regulates Rho family small G proteins for cytoskeletal modulation (7-10). Injection of a ligand of EphA4, ephrin-A1, into the lateral ventricle of Parkinson-model rats is effective in repairing the degenerated brain and remedying the abnormal behavior(10).
Our current focus in this project is how these receptor molecules affect the formation of brain cortex in vivo. We are studying two types of the brain stem cell-specific conditional KO mice of EphA4 that delete the molecule at different developmental stages.
GH-IGF1 axis and body size determination
Growth hormone (GH) is a classical hormone secreted from the pituitary, and functions to increase body size after birth mainly through inducing insulin-like growth factor1 (IGF1) from the liver and other local tissues. The signal transduction pathway known as a GH-IGF1 axis had been studied extensively, and the molecular mechanisms appeared to be clarified completely a decade ago. However, we have recently discovered a mechanism to challenge this hypothesis. We have identified EphA4 as an essential binding partner of GHR (Fig. 3). EphA4 binds to GHR and augments activation of the JAK2/STAT5B pathway. In addition, EphA4 directly binds to and phosphorylates STAT5B. Through these interactions, EphA4 transcriptionally enhances IGF1 production in liver cells as well as in others. Epha4 KO mice show short stature in an Epha4 gene dose-dependent fashion, and respond well to IGF1 but not to GH treatment (11). We are currently working on the molecular details of this signaling pathway.
Fig. 3 (refer to http://www.cell.com/cell-reports/fulltext/S2211-1247(12)00261-6)
- Kazushige Sakaguchi, Arthur Santora, Mark Zimering, Francesco Curcio, Gerald D. Aurbach, and Maria Luisa Brandi: Functional epithelial cell line cloned from rat parathyroid glands. Proc. Natl. Acad. Sci. U.S.A. 84:3269-3273, 1987
- Masaki Yanagishita, Maria Luisa Brandi, and Kazushige Sakaguchi: Characterization of proteoglycans synthesized by a rat parathyroid cell line. J. Biol. Chem. 264:15714-15720, 1989
- Yasuhiro Takeuchi, Kazushige Sakaguchi, Masaki Yanagishita, Gerald D. Aurbach, and Vincent C. Hascall: Extracellular calcium regulates distribution and transport of heparan sulfate proteoglycans in a rat parathyroid cell line. J. Biol. Chem. 265:13661-13668, 1990
- Kazushige Sakaguchi, Masaki Yanagishita, Yasuhiro Takeuchi, and Gerald D. Aurbach: Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line. J. Biol. Chem. 266:7270-7278, 1991
- Kazushige Sakaguchi: Acidic fibroblast growth factor autocrine system as a mediator of calcium-regulated parathyroid cell growth. J. Biol. Chem. 267:24554-24562, 1992
- Kazushige Sakaguchi, Matthew V. Lorenzi, Donald P. Bottaro and Toru Miki: The acidic domain and first immunoglobulin-like loop of FGF receptor-2 modulate downstream signaling through glycosaminoglycan modification. Mol. Cell. Biol. 19: 6754-6764, 1999
- Hideyuki Yokote, Koji Fujita, Xuefeng Jing, Takahiro Sawada, Sitai Liang, Li Yao, Xiaomei Yan, Yueqiang Zhang, Joseph Schlessinger, and Kazushige Sakaguchi: Trans-activation of EphA4 and FGF-receptors mediated by direct interactions between their cytoplasmic domains. Proc. Natl. Acad. Sci. USA 102: 18866-18871, 2005
- Yueqiang Zhang, Takahiro Sawada, Xuefeng Jing, Hideyuki Yokote, Xiaomei Yan, and Kazushige Sakaguchi: Regulation of ephexin1, a guanine nucleotide exchange factor of Rho family GTPases, by fibroblast growth factor receptor-mediated tyrosine phosphorylation. J. Biol. Chem. 282:31103-31112, 2007
- Takahiro Sawada, Xuefeng Jing, Yueqiang Zhang, Emi Shimada, Hideyuki Yokote, Masayasu Miyajima, and Kazushige Sakaguchi: Ternary complex formation of EphA4, FGFR and FRS2a・plays an important role in the proliferation of embryonic neural stem/progenitor cells. Genes Cells 15: 297-311, 2010
- Xuefeng Jing, Hideto Miwa, Takahiro Sawada, Ichiro Nakanishi, Tomoyoshi Kondo, Masayasu Miyajima, Kazushige Sakaguchi: Ephrin-A1-mediated dopaminergic neurogenesis and angiogenesis in a rat model of Parkinson’s disease. PLoS ONE 7(2): e32019. doi:10.1371/journal.pone.0032019, 2012
- Xuefeng Jing, Masayasu Miyajima, Takahiro Sawada, Qingfa Chen, Keiji Iida, Kenryo Furushima, Daiki Arai, Kazuo Chihara, and Kazushige Sakaguchi: Crosstalk of humoral and cell-cell contact-mediated signals in postnatal body growth. Cell Reports 2: 652-665, 2012