βKlotho Is Required for Fibroblast Growth Factor (FGF) 21 Signaling through FGF Receptor (FGFR) 1c and FGFR3c

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Molecular Endocrinology 22(4):1006–1014 Copyright © 2008 by The Endocrine Society doi: 10.1210/me.2007-0313 ␤Klotho Is Required for Fibroblast Growth Factor (FGF) 21 Signaling through FGF Receptor (FGFR) 1c and FGFR3c Masashi Suzuki, Yuriko Uehara, Kaori Motomura-Matsuzaka, Junko Oki, Yoshinori Koyama, Miho Kimura, Masahiro Asada, Akiko Komi-Kuramochi, Syuichi Oka, and Toru Imamura Signaling Molecules Research Laboratory (M.S., Y.U., K.M.-M., J.O., M.K., M.A., A.K.-K., S.O., T.I.), and Institute for Biological Resources and Functions (Y.K.), National Institute of Advanced Industrial Science and Technology, Ibaraki 305-8566, Japan; and Department of Applied Biological Science (Y.U., T.I.), Tokyo University of Science, Noda, Chiba 278-8510, Japan Fibroblast growth factor (FGF) 21, a structural relative of FGF23 that regulates phosphate homeostasis, is a regulator of insulin-independent glucose transport in adipocytes and plays a role in the regulation of body weight. It also regulates ketogenesis and adaptive responses to starvation. We report that in a reconstituted receptor activation assay system using BaF3 cells, which do not endogenously express any type of FGF receptor (FGFR) or heparan sulfate proteoglycan, FGF21 alone does not activate FGFRs and that ␤Klotho is required for FGF21 to activate two specific FGFR subtypes: FGFR1c and FGFR3c. Coexpression of ␤Klotho and FGFR1c on BaF3 cells enabled FGF21, but not FGF23, to activate receptor signaling. Con- versely, coexpression of FGFR1c and Klotho, a protein related to ␤Klotho, enabled FGF23 but not FGF21 to activate receptor signaling, indicating that expression of ␤Klotho/Klotho confers target cell specificity on FGF21/FGF23. In all of these cases, heparin enhanced the activation but was not essential. In 3T3-L1 adipocytes, up-regulation of glucose transporter (GLUT) expression by FGF21 was associated with expression of ␤Klotho, which was absent in undifferentiated 3T3-L1 fibroblasts. It is thus suggested that ␤Klotho expression is a crucial determinant of the FGF21 specificity of the target cells upon which it acts in an endocrine fashion. (Molecular Endocrinology 22: 1006–1014, 2008) T onstrated that systematic administration of FGF21 reduces blood glucose and triglycerides to near normal levels in diabetic rodents and that transgenic mice overexpressing FGF21 were resistant to diet-induced obesity. It also has been reported that FGF21 regulates ketogenesis and lipolysis (7, 8). The clear therapeutic potential of FGF21 for the treatment of diabetes or other metabolic diseases has made elucidation of the FGF21 signaling pathway a pressing issue. However, the signaling pathway via which FGF21 acts has remained unclear. For instance, although it stimulates tyrosine phosphorylation of FGF receptor (FGFR) 1 and FGFR2 as well as phosphorylation of FGFR substrate 2␣ (FRS2␣) and MAPK (Erk1/Erk2) in 3T3-L1 adipocytes, it has no effect in preadipocytes (6). In addition, the observation that FGF21 has little or no ability to stimulate proliferation of BaF3 cells expressing any of the FGFR subtypes (9) suggests that an unknown factor/mechanism may underlie FGF21-dependent activation of FGFRs. Interestingly, two research groups have recently demonstrated that Klotho, the product of a gene shown to be mutated in a mouse strain characterized by premature aging, functions as a coreceptor for FGF23 (10, 11). They observed that Klotho⫺/⫺ and Fgf23⫺/⫺ mice exhibit very similar phenotypes, which included elevated levels of calcium, phosphate, and 1,25-dihydroxyvitamin D in serum, ectopic calcification, generalized organ HE FIBROBLAST GROWTH factor (FGF) family is comprised of 22 structurally related proteins in mammals that have a wide variety of functions in organogenesis, tissue remodeling, nervous system control, and angiogenesis (1, 2). In addition, recent analyses of the FGF19 subfamily, which consists of FGF19 (the human ortholog for mouse FGF15), FGF21, and FGF23, has revealed these mediators to function as metabolic regulators. For instance, FGF23 is a critical regulator of phosphate and vitamin D homeostasis (3), whereas FGF15/FGF19 secreted from the distal small intestine regulates bile acid homeostasis (4). Fgf21 was originally identified as a new FGF family gene in mouse embryo using homology-based PCR (5). Thereafter, Kharitonenkov et al. (6) showed that FGF21 treatment increases expression of GLUT1 mRNA and stimulates glucose incorporation into differentiated mouse 3T3-L1 adipocytes, indicating the new FGF to be a glucose regulator. They further dem- First Published Online January 10, 2008 Abbreviations: FBS, Fetal bovine serum; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FRS2, fibroblast growth factor receptor substrate 2; GLUT, glucose transporter. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. Suzuki et al. • ␤Klotho Is Required for FGF21 Signaling relative mRNA expression (fold) 3T3-L1 adipocytes 4 2 3T3-L1 fibroblasts 0 0 5 10 FGF21 (µg/ml) 2 mRNA expression (copies: x 107/µg RNA) 15 FGFR1 FGFR2 FGFR3 FGFR4 1 0 2 1 0 0 4 8 12 16 20 0 4 8 12 days after differentiation induction 16 20 C (copies: x 105/µg RNA) We observed that FGF21 treatment leads to an increase in the expression of glucose transporter 1 (GLUT1) mRNA in 3T3-L1 adipocytes but not in undifferentiated fibroblasts (Fig. 1A) as previously reported (6). FGF signaling is thought to be transduced, at least in part, through activation of one or more FGFR tyrosine kinases (15). Kharitonenkov and colleagues (6, 16) showed that FGF21 activates both FGFR1 and FGFR2 tyrosine kinase and suggested they may function as FGF21 receptors. To test the possibility that a difference in FGFR levels underlies the difference in the sensitivity of 3T3-L1 adipocytes and fibroblasts to FGF21, we used quantitative RT-PCR to measure FGFR mRNA expression during 3T3-L1 differentiation. As shown in Fig. 1B, 3T3-L1 fibroblasts expressed both FGFR1 and FGFR2 mRNA, and the levels of both gradually declined as the cells differentiated into adipocytes. These data suggest that the level of FGFR1 or -R2 expression cannot, by itself, account for the unresponsiveness of 3T3-L1 fibroblasts to FGF21. 3T3-L1 fibroblasts express little or no FGFR3 and FGFR4 mRNA. Among the 22 members of the FGF family, FGF21, FGF15/19 and FGF23 comprise the FGF19 subfamily (2). Recently the senescence-related molecule Klotho was reported to function as a coreceptor for FGF23 (10, 11). In vitro pull-down assays revealed that FGF23 binds to Klotho, and Klotho binds to FGFR1c (10, 11) and other FGFRs (10) independently of heparin (11). To test the possibility that Klotho functions as a coreceptor for FGF21, we assessed expression of Klotho mRNA during 3T3-L1 differentiation. Using real-time q (...truncated)