The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1–4). We demonstrated that Klotho and βKlotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Here we show that, like FGF21, FGF19 also requires βKlotho. Both FGF19 and FGF21 can signal through FGFR1–3 bound by βKlotho and increase glucose uptake in adipocytes expressing FGFR1. Additionally, both FGF19 and FGF21 bind to the βKlotho-FGFR4 complex; however, only FGF19 signals efficiently through FGFR4. Accordingly, FGF19, but not FGF21, activates FGF signaling in hepatocytes that primarily express FGFR4 and reduces transcription of CYP7A1 that encodes the rate-limiting enzyme for bile acid synthesis. We conclude that the expression of βKlotho, in combination with particular FGFR isoforms, determines the tissue-specific metabolic activities of FGF19 and FGF21. The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1–4). We demonstrated that Klotho and βKlotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Here we show that, like FGF21, FGF19 also requires βKlotho. Both FGF19 and FGF21 can signal through FGFR1–3 bound by βKlotho and increase glucose uptake in adipocytes expressing FGFR1. Additionally, both FGF19 and FGF21 bind to the βKlotho-FGFR4 complex; however, only FGF19 signals efficiently through FGFR4. Accordingly, FGF19, but not FGF21, activates FGF signaling in hepatocytes that primarily express FGFR4 and reduces transcription of CYP7A1 that encodes the rate-limiting enzyme for bile acid synthesis. We conclude that the expression of βKlotho, in combination with particular FGFR isoforms, determines the tissue-specific metabolic activities of FGF19 and FGF21. The FGF19 2The abbreviations used are:FGFfibroblast growth factorFGFRFGF receptorERKextracellular signal-regulated kinaseTBSTris-buffered salinesiRNAsmall interfering RNARTreverse transcription subfamily of ligands, which consists of FGF15 (the mouse ortholog of human FGF19), FGF19, FGF21, and FGF23, has emerged as a novel group of endocrine factors that regulate diverse metabolic processes in adulthood (1Itoh N. Ornitz D.M. Trends Genet. 2004; 20: 563-569Abstract Full Text Full Text PDF PubMed Scopus (879) Google Scholar). FGF15/19 expression is induced upon feeding in intestinal epithelial cells in response to bile acid released into the intestinal lumen. FGF15/19 then acts on hepatocytes to reduce bile acid synthesis through suppressing transcription of CYP7A1, which encodes the rate-limiting enzyme for bile acid synthesis (2Inagaki T. Choi M. Moschetta A. Peng L. Cummins C.L. McDonald J.G. Luo G. Jones S.A. Goodwin B. Richardson J.A. Gerard R.D. Repa J.J. Mangelsdorf D.J. Kliewer S.A. Cell Metab. 2005; 2: 217-225Abstract Full Text Full Text PDF PubMed Scopus (1364) Google Scholar). FGF15/19 also acts on the gall bladder and stimulates its filling with bile. Thus, FGF15/19 functions as an essential component in a postprandial negative feedback loop for bile acid synthesis and release (2Inagaki T. Choi M. Moschetta A. Peng L. Cummins C.L. McDonald J.G. Luo G. Jones S.A. Goodwin B. Richardson J.A. Gerard R.D. Repa J.J. Mangelsdorf D.J. Kliewer S.A. 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Specifically, FGF21 stimulates lipolysis in adipocytes to release fatty acids, which in turn converted to ketones in the liver. FGF21 was originally identified as a hormone that stimulates glucose uptake in adipocytes (6Kharitonenkov A. Shiyanova T.L. Koester A. Ford A.M. Micanovic R. Galbreath E.J. Sandusky G.E. Hammond L.J. Moyers J.S. Owens R.A. Gromada J. Brozinick J.T. Hawkins E.D. Wroblewski V.J. Li D.S. Mehrbod F. Jaskunas S.R. Shanafelt A.B. J. Clin. Investig. 2005; 115: 1627-1635Crossref PubMed Scopus (1648) Google Scholar). However, unlike insulin, FGF21 reduces fat storage because it stimulates lipolysis (4Inagaki T. Dutchak P. Zhao G. Ding X. Gautron L. Parameswara V. Li Y. Goetz R. Mohammadi M. Esser V. Elmquist J.K. Gerard R.D. Burgess S.C. Hammer R.E. Mangelsdorf D.J. Kliewer S.A. Cell Metab. 2007; 5: 415-425Abstract Full Text Full Text PDF PubMed Scopus (1207) Google Scholar, 5Badman M.K. Pissios P. Kennedy A.R. Koukos G. Flier J.S. Maratos-Flier E. Cell Metab. 2007; 5: 426-437Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar). The FGF23 gene was identified as the gene mutated in patients with autosomal dominant hypophosphatemic rickets (7White K.E. Evans W.E. O'Rlordan J.L.H. Speer M.C. Econs M.J. Lorenz-Deplereux B. Grabowski M. Meitinger T. Storm T.M. Nat. Genet. 2000; 26: 345-348Crossref PubMed Scopus (1297) Google Scholar). Autosomal dominant hypophosphatemic rickets patients carry a missense mutation in the FGF23 gene that confers resistance to proteolytic inactivation of FGF23 protein, resulting in increased blood FGF23 levels. Because of its inhibitory activity on phosphate reabsorption and vitamin D biosynthesis in the kidney, high FGF23 blood levels in autosomal dominant hypophosphatemic rickets patients result in phosphate wasting and defects in bone mineralization (8Quarles L.D. Am. J. Physiol. 2003; 285: E1-E9Crossref PubMed Scopus (287) Google Scholar, 9Schiavi S.C. Kumar R. 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Eliseen-kova A.V. Xu C. Neubert T. Zhang F. Linhardt R.J. Yu X. White K.E. Inagaki T. Kliewer S.A. Yamamoto M. Kurosu H. Ogawa Y. Kuro-o M. Lanske B. Razzaque M.S. Mohammadi M. Mol. Cell. Biol. 2007; 27: 3417-3428Crossref PubMed Scopus (434) Google Scholar, 13Harmer N.J. Pellegrini L. Chirgadze D. Fernandez-Recio J. Blundell T.L. Biochemistry. 2004; 43: 629-640Crossref PubMed Scopus (106) Google Scholar). The weak heparin binding affinity of the FGF19 family members enables them to avoid being captured in extracellular matrices and thus to function as endocrine factors. On the other hand, this weak heparin binding activity reduces the capacity of heparin/heparan sulfate to promotes direct interaction between FGFs and FGFRs (14Mohammadi M. Olsen S.K. Ibrahimi O.A. Cytokine Growth Factor Rev. 2005; 16: 107-137Crossref PubMed Scopus (563) Google Scholar). Indeed, attempts to demonstrate a direct interaction between FGFRs and the FGF19 family proteins in vitro have failed. These observations imply that FGF19 subfamily members require additional cofactors, besides heparin/heparan sulfates, to stably bind to their cognate FGFRs in their target tissues. We and others identified the Klotho protein as a cofactor necessary for FGF23 binding to FGFRs and for efficient activation of FGF signaling (15Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1474) Google Scholar). The klotho gene was originally identified in mice as an aging-suppressor gene that extends life span when overexpressed and accelerates the development of aging-like phenotypes when disrupted (17Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. Iwasaki H. Iida A. Shiraki-Iida T. Nishikawa S. Nagai R. Nabeshima Y. Nature. 1997; 390: 45-51Crossref PubMed Scopus (2841) Google Scholar, 18Kurosu H. Yamamoto M. Clark J.D. Pastor J.V. Nandi A. Gurnani P. McGuinness O.P. Chikuda H. Yamaguchi M. Kawaguchi H. Shimomura I. Takayama Y. Herz J. Kahn C.R. Rosenblatt K.P. Kuro-o M. Science. 2005; 309: 1829-1833Crossref PubMed Scopus (1411) Google Scholar). The klotho gene encodes a single-pass transmembrane protein and is expressed in limited tissues, most notably in the distal convoluted tubules in the kidney (17Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. Iwasaki H. Iida A. Shiraki-Iida T. Nishikawa S. Nagai R. Nabeshima Y. Nature. 1997; 390: 45-51Crossref PubMed Scopus (2841) Google Scholar). The Klotho protein physically interacts with FGFR1c, 3c, and 4 as well as with FGF23 itself (14Mohammadi M. Olsen S.K. Ibrahimi O.A. Cytokine Growth Factor Rev. 2005; 16: 107-137Crossref PubMed Scopus (563) Google Scholar) to stabilize FGF23-FGFR interactions. Forced expression of Klotho conferred responsiveness to FGF23 upon various cell types (15Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar). The fact that Klotho is essential for efficient activation of FGF signaling by FGF23 may explain why Klotho-deficient mice and FGF23-deficient mice show many overlapping phenotypes, including hyperphosphatemia, hypervitaminosis D, and multiple aging-like symptoms (19Shimada T. Kakitani M. Yamazaki Y. Hasegawa H. Takeuchi Y. Fujita T. Fukumoto S. Tomizuka K. Yamashita T. J. Clin. Investig. 2004; 113: 561-568Crossref PubMed Scopus (1287) Google Scholar, 20Razzaque M.S. Sitara D. Taguchi T. St-Arnaud R. Lanske B. FASEB J. 2006; 20: 720-722Crossref PubMed Scopus (302) Google Scholar). Furthermore, we showed that βKlotho, a Klotho family member protein, functions as a cofactor necessary for FGF21 binding to FGFRs and effective activation of FGF signaling (21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar). βKlotho shares 41% amino acid identity with Klotho and is expressed in adipose tissue, liver, and pancreas (22Ito S. Kinoshita S. Shiraishi N. Nakagawa S. Sekine S. Fujimori T. Nabeshima Y. Mech. Dev. 2000; 98: 115-119Crossref PubMed Scopus (263) Google Scholar). βKlotho also physically interacts with multiple FGFRs and significantly increases the affinity of FGF21 for the FGFRs in a manner similar to FGF23, Klotho, and FGFRs (21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. 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Thus, the genetic evidence strongly suggests that FGF15/19, FGFR4, and βKlotho are essential components in the negative regulation of bile acid synthesis. In this report, we provide molecular and cellular evidence indicating that FGF19, like its subfamily member FGF21, requires βKlotho to stably bind to FGFRs and effectively activate FGF signaling. The salient difference between FGF19 and FGF21 activities lies in their distinct receptor binding specificity and the tissue-specific distribution of their cognate receptors. These findings provide new insights into the mechanism by which the FGF19 subfamily of ligands exert distinct metabolic activities in different target organs. Expression Vectors—Expression vectors for βKlotho and FGFRs designed with a V5 epitope tag at their C terminus were generated by polymerase chain reaction of cDNAs as described previously (15Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar). Cell Culture and Transfection—Culture of the mouse 3T3-L1 preadipocytes and induction of adipocyte differentiation were described previously (21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar). Human embryonic kidney cells (HEK293), rat hepatoma cells (H4IIE), and rat myoblastic cells (L6) (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and penicillin/streptomycin. Transfection of expression vectors was performed 36 h prior to the experiments using Lipofectamine (Invitrogen) and FuGENE HD (Roche Applied Science) for HEK293 cells and L6 cells, respectively, according to the manufacturer's protocols. Preparation of FGF19, FGF21, and FGF23—Human recombinant FGF19, FGF21, and FGF23 (R179Q) were expressed in Escherichia coli, refolded in vitro, and purified by affinity, ion exchange, and size exclusion chromatographies as previously described (25Plotnikov A.N. Hubbard S.R. Schlessinger J. Mohammadi M. Cell. 2000; 101: 413-424Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Egr-1 Promoter Assay—The human early growth response-1 (Egr-1) gene promoter containing ERK-responsive elements (–580 to +30) (16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1474) Google Scholar, 26Bauer I. Hohl M. Al-Sarraj A. Vinson C. Thiel G. Arch Biochem. Biophys. 2005; 438: 36-52Crossref PubMed Scopus (30) Google Scholar) was PCR-amplified from human genomic DNA and subcloned into the pEGFP-N1 vector (Clontech, Mountain View, CA). HEK293 cells were transfected with the reporter plasmid using the FuGENE transfection reagent and selected in medium containing 0.8 mg/ml G418 (Sigma-Aldrich). Clones of stable transformants were isolated and plated on 96-well plates, stimulated with FGF2 (Upstate Biotechnology, Lake Placid, NY) for 24 h, and lysed with phosphate-buffered saline containing 1% Triton X-100. Fluorescence of the cell lysates was measured using a microplate reader (FLUOstar OPTIMA, BMG Labtechnologies, Inc., Durham, NC) in the fluorescence mode (excitation, 485 nm; emission, 520 nm). Background signal was determined as fluorescence from the well without cells and subtracted to obtain net fluorescence generated from cells. A clone (E2-7) that showed robust response to FGF2 was cotransfected with human βKlotho and puromycin-resistant vectors and selected in the medium containing 0.5 mg/ml G418 and 0.6 μg/ml puromycin (InvivoGen, San Diego, CA). Clones of the double-transfectants were isolated and stimulated with FGF21 and subjected to the enhanced green fluorescent protein assay as described. A clone (Eβ2) that showed robust response to FGF21 was used for testing FGF19 activity. Immunoblot Analysis of FGF Signaling—Cells cultured on multi-well plates were serum-starved overnight and then treated for 10 min with human recombinant FGF19, FGF21, FGF23 (R179Q), or FGF2. The cells were snap-frozen in liquid nitrogen, lysed in a buffer containing inhibitors for phosphatases and proteases, and processed for immunoblot analysis using antibodies against phospho-FRS2α (Cell Signaling Technology, Beverly, MA), phospho-44/42 mitogen-activated protein kinase (ERK1/2) (Cell Signaling), and ERK1/2 (Cell Signaling) as previously described (15Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar). The signal intensity was quantified using an image analysis software (ImageQuant, Molecular Dynamics, Sunnyvale, CA). Male 129sv mice at 8 weeks of age were administered either FGF19 (1 μgg–1 body weight), FGF21 (0.3 μgg–1 body weight), or vehicle (10 mm HEPES, pH 7.4, 150 mm NaCl) by injection into the inferior vena cava. Liver, perigonadal fat pads, kidneys, and hind limb muscles were excised 15, 17, 19, and 21 min, respectively, after the injection. The tissues were flash-frozen in liquid nitrogen, homogenized in the lysis buffer, and subjected to immunoblotting. All of the animal experiments were approved by the Institutional Animal Care and Research Advisory Committee of The University of Texas Southwestern Medical Center at Dallas. FGF19 Pull-down—Cell lysates were prepared from HEK293 cells transfected with FGFR alone or cotransfected with FGFR and βKlotho and incubated with anti-V5-agarose beads (Sigma-Aldrich) at 4 °C for 3 h. The beads were washed four times with Tris-buffered saline (TBS) containing 1% Triton X-100 and then incubated with FGF19 (1 μg/ml) at 4 °C for 3 h. There-after, the beads were washed three times with Krebs-Ringer-HEPES buffer (15Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar) containing 1% Triton X-100 followed by three washes with the same buffer lacking Triton X-100. Bead-bound proteins were eluted with Laemmli sample buffer and subjected to immunoblot analysis using antibodies against V5 tag (Invitrogen), βKlotho (R & D Systems, Minneapolis, MN), or FGF19 (R & D Systems). Knock-down of βKlotho Expression by RNA Interference— 3T3-L1 adipocytes were transfected with siRNA duplexes by electroporation as previously described and then used for immunoblot analysis and for glucose uptake assay (21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar). H4IIE cells were transfected by electroporation as well. Briefly, the cells were harvested using trypsin, washed twice with phosphate-buffered saline, suspended in Opti-MEM (Invitrogen) (1 × 107 cells/ml), mixed with 10 nmol/107 cells of siRNA oligonucleotides (see supplemental Table S1), and electroporated with a gene pulser system (0.24 kV and 960 microfarads capacitance) (Bio-Rad). After electroporation, the cells were incubated at 4 °C for 10 min before reseeding onto 6-well plates. Thirty hours after transfection, the cells were serum-starved overnight and used for immunoblot analysis and quantitative RT-PCR. Glucose Uptake Assay—3T3-L1 adipocytes transfected with siRNA were stimulated with FGF19 (1 μg/ml), FGF21 (1 μg/ml), or FGF2 (0.1 μg/ml) in Dulbecco's modified Eagle's medium with 0.1% free fatty acid-free bovine serum albumin (Sigma-Aldrich) for 18 h at 37 °C and subjected to the glucose uptake assay as described previously (21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar). Quantitative RT-PCR—Total RNA was isolated using the RNeasy kit (Qiagen). For quantification of CYP7A1 and SHP mRNA, H4IIE cells transfected with siRNA were stimulated with FGF19, FGF21, or vehicle for 18 h before RNA isolation. 4 μg of total RNA was treated with 0.2 unit of DNase I (Promega, Madison, WI) and then reverse-transcribed into first-strand cDNA using the ThemoScript RT-PCR system (Invitrogen) according to the manufacturer's protocol. The specific primers used to quantify gene expression were listed in supplemental Table S2. Quantitative RT-PCR reactions contained 25 ng of cDNA, 150 nm of each primer, and 5 μl of SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA) in a total volume of 10 μl. All of the reactions were performed in triplicate on an Applied Biosystems Prism 7900HT sequence detection system, and relative mRNA levels were calculated by the comparative threshold cycle method using cyclophilin as the internal control. Immunoprecipitation—Liver (200 mg) and white adipose tissue (1000 mg) were homogenized in 1 ml of homogenization buffer (20 mm HEPES, pH 7.4, 100 mm NaCl, and 0.5 mm EDTA) containing protease inhibitors. The homogenates were incubated for 30 min at 4 °C after the addition of Triton X-100 (final, 1.2% w/v) and then centrifuged twice for 12 min at 18,000 × g to remove debris. The supernatant of liver and white adipose tissue were precleared with 40 μl of protein G-Sepharose or protein A-Sepharose (Amersham Biosciences) conjugated with 20 μg of normal goat or rabbit IgG for 3 h at 4 °C, respectively. The precleared lysates of liver and white adipose tissues, respectively, were incubated with 20 μl of protein A-Sepharose conjugated with 20 μgof anti-FGFR4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or normal goat IgG and with anti-FGFR1 antibody (Santa Cruz) or normal rabbit IgG for 3 h at 4 °C. The beads were washed four times with TBS containing 1% Triton X-100 and three times with TBS. Bead-bound proteins were eluted with Laemmli sample buffer and subjected to immunoblot analysis using anti-βKlotho, anti-FGFR1 (R & D Systems), and anti-FGFR4 (R & D Systems) antibodies. FGF19 Requires βKlotho to Activate FGF Signaling—Like FGF21 and FGF23, FGF19 failed to robustly activate FGF signaling in HEK293 cells as measured by induction of FRS2α and ERK phosphorylation. Forced expression of Klotho in HEK293 cells caused a selective response to FGF23 but not to FGF19 or FGF21. Conversely, forced expression of βKlotho conferred responsiveness to both FGF21 and FGF19 but not FGF23 (Fig. 1A). Because activation of FGF signaling with FGF23 increases promoter activity and expression of the Egr-1 (early growth response-1) gene in Klotho-expressing cells (16Urakawa I. Yamazaki Y. Shimada T. Iijima K. Hasegawa H. Okawa K. Fujita T. Fukumoto S. Yamashita T. Nature. 2006; 444: 770-774Crossref PubMed Scopus (1474) Google Scholar), we tested whether FGF19 and FGF21 also increase Egr-1 promoter activity in βKlotho-expressing cells. Consistent with the results shown in Fig. 1A, FGF19 and FGF21 activated the Egr-1 promoter only in βKlotho-expressing HEK293 cells in a dose-dependent manner, whereas FGF2 activated it independently of βKlotho expression (Fig. 1B). These observations indicate that FGF19 requires βKlotho to activate FGF signaling. We have shown that βKlotho and Klotho enhance binding affinity of FGF21 and FGF23, respectively, to their cognate FGFRs (15Kurosu H. Ogawa Y. Miyoshi M. Yamamoto M. Nandi A. Rosenblatt K.P. Baum M.G. Schiavi S. Hu M.C. Moe O.W. Kuro-o M. J. Biol. Chem. 2006; 281: 6120-6123Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar). We therefore tested whether βKlotho also enhances binding of FGF19 to its cognate FGFRs. As previously reported, βKlotho binds to FGFR1c and FGFR4 more avidly than does to FGFR2c and FGFR3c (Fig. 1C) and does not interact with “b” isoforms of FGFRs (21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar). In the absence of βKlotho, FGF19 did not bind to FGFR1c and FGFR4 and bound poorly to FGFR2c and 3c. In the presence of βKlotho, binding of FGF21 to FGFR1c and FGFR4 was significantly increased, whereas binding to FGFR2c and FGFR3 was only slightly increased (Fig. 1C). These observations are reminiscent of our previous findings for FGF21 (21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar) and indicate that FGF19 also requires βKlotho for binding to FGFR1c and FGFR4 and for robust activation of FGF signaling. Adipocytes Respond to Both FGF19 and FGF21—Because differentiated 3T3-L1 adipocytes express βKlotho endogenously and respond to FGF21 (21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar), we tested whether FGF19 also activates FGF signaling in these cells. Dose-responsiveness of FRS2α and ERK1/2 phosphorylation induced by FGF19 was comparable with that induced by FGF21 (Fig. 2A). We observed significant attenuation of FGF19- and FGF21-induced FRS2α and ERK1/2 phosphorylation by knocking down βKlotho expression using two independent siRNAs (Fig. 2, B and C), indicating that endogenous βKlotho is required for both FGF19 and FGF21 to activate FGF signaling in adipocytes. Quantification of mRNA levels of mouse FGFR1–4 revealed that 3T3-L1 adipocytes predominantly express FGFR1 (Fig. 2D). These observations indicate that activation of FGF signaling by FGF19 and FGF21 in these cells is mediated through the βKlotho-FGFR1 complex. Because FGF21 increases glucose uptake in adipocytes independently of insulin (6Kharitonenkov A. Shiyanova T.L. Koester A. Ford A.M. Micanovic R. Galbreath E.J. Sandusky G.E. Hammond L.J. Moyers J.S. Owens R.A. Gromada J. Brozinick J.T. Hawkins E.D. Wroblewski V.J. Li D.S. Mehrbod F. Jaskunas S.R. Shanafelt A.B. J. Clin. Investig. 2005; 115: 1627-1635Crossref PubMed Scopus (1648) Google Scholar, 21Ogawa Y. Kurosu H. Yamamoto M. Nandi A. Rosenblatt K.P. Goetz R. Eliseenkova A.V. Mohammadi M. Kuro-o M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7432-7437Crossref PubMed Scopus (488) Google Scholar), we next tested whether FGF19 exerts similar activity upon adipocytes. Indeed, FGF19 significantly increased glucose uptake within 18 h, which was comparable with that induced by FGF21 (Fig. 2E). These activities of FGF19 and FGF21 are also dependent on βKlotho because they were abolished by knocking down βKlotho expression using two independent siRNAs (Fig. 2E). Hepatocytes Respond to FGF19 but Not FGF21—Because the rat hepatoma cell line H4IIE also expresses βKlotho endogenously, we tested whether FGF19 and FGF21 activate FGF signaling in these cells. The dose-response characteristics of FGF19-induced phosphorylation for FRS2α and ERK1/2 in H4IIE cells were similar to that observed in adipocytes (Fig. 3A). In addition, we noted significant attenuation of FGF19-induced FRS2α and ERK1/2 phosphorylation by knocking down βKlotho expression using two independent siRNAs (Fig. 3, B and C), confirming that endogenous βKlotho is required for FGF19 to activate FGF signaling in hepatocytes as well as in adipocytes. However, in contrast to adipocytes, H4IIE hepatocytes are ∼100-fold less sensitive to FGF21 in terms of induction of FRS2α and ERK1/2 phosphorylation (Fig. 3A). Quantification of mRNA levels of rat FGFR1–4 demonstrated that H4IIE hepatocytes predominantly express FGFR4 (Fig. 3D), indicating that FGF19 can activate FGF signaling through