Differential specificity of endocrine FGF19 and FGF21 to FGFR1 and FGFR4 in complex with KLB

Abstract

Background: Recent studies suggest that betaKlotho (KLB) and endocrine FGF19 and FGF21 redirect FGFR signaling to regulation of metabolic homeostasis and suppression of obesity and diabetes. However, the identity of the predominant metabolic tissue in which a major FGFR-KLB resides that critically mediates the differential actions and metabolism effects of FGF19 and FGF21 remain unclear.

Methodology/principal findings: We determined the receptor and tissue specificity of FGF21 in comparison to FGF19 by using direct, sensitive and quantitative binding kinetics, and downstream signal transduction and expression of early response gene upon administration of FGF19 and FGF21 in mice. We found that FGF21 binds FGFR1 with much higher affinity than FGFR4 in presence of KLB; while FGF19 binds both FGFR1 and FGFR4 in presence of KLB with comparable affinity. The interaction of FGF21 with FGFR4-KLB is very weak even at high concentration and could be negligible at physiological concentration. Both FGF19 and FGF21 but not FGF1 exhibit binding affinity to KLB. The binding of FGF1 is dependent on where FGFRs are present. Both FGF19 and FGF21 are unable to displace the FGF1 binding, and conversely FGF1 cannot displace FGF19 and FGF21 binding. These results indicate that KLB is an indispensable mediator for the binding of FGF19 and FGF21 to FGFRs that is not required for FGF1. Although FGF19 can predominantly activate the responses of the liver and to a less extent the adipose tissue, FGF21 can do so significantly only in the adipose tissue and adipocytes. Among several metabolic and endocrine tissues, the response of adipose tissue to FGF21 is predominant, and can be blunted by the ablation of KLB or FGFR1.

Conclusions: Our results indicate that unlike FGF19, FGF21 is unable to bind FGFR4-KLB complex with affinity comparable to FGFR1-KLB, and therefore, at physiological concentration less likely to directly and significantly target the liver where FGFR4-KLB predominantly resides. However, both FGF21 and FGF19 have the potential to activate responses of primarily the adipose tissue where FGFR1-KLB resides.

Figure 1. Receptor specificity of FGF1, FGF19 and FGF21.

(A) Comparative binding properties of FGF19 and FGF1. Parental T-Rex 293 cells and cells expressing FGFR1-KLB, FGFR1, FGFR4-KLB, FGFR4 and KLB upon 30 ng/ml Tet induction for FGFRs were incubated with 2 ng/ml ¹²⁵I-labeled FGF1 (white rectangle) or ¹²⁵I-FGF19 (black rectangle) as indicated. Cell surface bound radioactivity was determined by γ–counter. Data are shown as mean ± s.d. of three independent experiments. (B) Covalent Affinity chemical cross-linking was performed as described . Cross-linking bands were indicated by bidirectional arrows. (C) Receptor binding properties of FGF21 revealed by competition binding with ¹²⁵I-FGF19. FGF21, FGF19 and FGF1 at 1 µg/ml were incubated in the presence of 2 ng/ml ¹²⁵I-FGF19 with parental cells and cells expressing FGFR1-KLB, FGFR1, FGFR4-KLB, FGFR4 and KLB. Specific cell surface binding and statistic analyses were determined as above.

Figure 2. Analyses of binding kinetic and affinity constant.

T-Rex 293 cells expressing inducible FGFR1 and constitutive KLB (A) and hepatoma cells HR4 expressing FGFR4 and reduced level of KLB (B) were maintained in DMEM medium with 5% FBS. Binding kinetic analysis of radiolabeled FGF19 to the FGFR-KLB complexes on cell surfaces were done as described in the Materials and Methods. The binding affinity constant values were determined by the Scatchard plot.

Figure 3. Dose-dependent differential binding of FGF21, FGF19 and FGF1 to FGFR1-KLB and FGFR4-KLB.

(A) Competition ability of FGF21 and FGF1 with FGF19 to bind FGFR-KLB. Graded concentrations as indicated for FGF21 (open square), FGF19 (filled triangle) and FGF1 (filled circle) were added together with 2 ng/ml ¹²⁵I-FGF19 to the cells expressing FGFR1-KLB (solid line) or FGFR4-KLB (dot line) after 30 ng/ml Tet induction overnight, the remaining specific bindings of ¹²⁵I-FGF19 were determined as described in Figure 1A. (B) Competition ability of FGF21 with FGF1 to bind FGFR1-KLB. Graded concentrations as indicated for FGF21 (open square), FGF19 (not shown) and FGF1 (filled circle) were added together with 2 ng/ml ¹²⁵I-FGF1 to the cells expressing FGFR1-KLB (solid line) or FGFR4-KLB (Inset), the remaining specific bindings of ¹²⁵I-FGF1 under these conditions were determined as described above.

Figure 4. Dose-dependent differential binding of FGF21, FGF19 and FGF1 to KLB or FGFR4 alone.

(A) Interaction of FGF21, FGF19 and FGF1 with KLB. Graded concentrations as indicated for FGF21 (open square), FGF19 (not shown) and FGF1 (filled circle) were added together with 2 ng/ml ¹²⁵I-FGF19 to the cells expressing KLB alone, and the remaining specific bindings of ¹²⁵I-FGF1 under these conditions were then determined. (B) Competitive binding of FGF21 and FGF1 with FGF19 to FGFR4 alone. Graded concentrations as indicated for FGF21 (open square), FGF19 (not shown) and FGF1 (filled circle) were added together with 2 ng/ml ¹²⁵I-FGF19 to the cells expressing FGFR4 alone, and the remaining specific bindings of ¹²⁵I-FGF1 under these conditions were then determined.

Figure 5. Differential activation of FGFRs and downstream MAPKs by FGF19 and FGF21.

(A) Tyrosine phosphorylation of FGFR and activation of ERK1/2 in engineered T-Rex 293 cells . Expression of FGFR1 and FGFR4 was induced with 30 ng/ml Tet overnight. Cells were stimulated with 100 ng/ml FGF21, FGF19 and FGF1 in presence of 1 µg/ml heparin, cell lysates in 1×SDS sample buffer were used for immunoblot analyses using antibodies as indicated. The identity of FGFR1 and FGFR4 was pre-determined by their respective antibodies (not showed). (B) Responses of adipocytes and hepatocyte-like cells to the stimulation of FGF19 and FGF21. Mature 3T3-L1 adipocytes and HR4 hepatoma cells after overnight serum-starvation were treated by FGF21, FGF19 and FGF1 at the concentrations as indicated, and cell lysates were used for immunoblot analyses for MAPK activation as described above. The average relative activation level of Erk1/2 for each cell type under different stimulation condition is expressed as the percentage to the peak activation of Erk1/2 treated with FGF1 after normalized as ratio of pErk1/2 to total Erk1/2.

Figure 6. Dose-dependent activity potential of FGF19 and FGF21.

Cells expressing endogenous FGFR4-KLB and FGFR1-KLB as indicated were stimulated by different concentrations of FGF19 and FGF21, respectively, for 10 minutes in 37°C culture. Cell lystes were then used to determine the pERK1/2 levels under these conditions by western-blotting. The pErk1/2 level for each cell type under each stimulation condition is expressed as normalized arbitral units of ratio of pErk1/2 to total Erk1/2. The half maximal effective concentration (EC50) is then calculated in the logarithmic plot.

Figure 7. Differential tissue-specific responses to FGF21 and FGF19 as measured by the expression level of early responsive gene c-Fos.

Thirty age- and weight-matched mice (2×3×5) were fasted for 24 hrs with water freely available, and then injected intraperitoneally with FGF21, FGF19 (0.5 mg/Kg body weight) and PBS vesicle control as indicated. After 20 min, liver and adipose tissue (A) and several other endocrine and metabolic tissues (B) were isolated and processed for RNA purification. Quantitative PCR was used to assess the expression of c-Fos in response to different treatments (n = 5).

Figure 8. Differential tissue-specific responses to FGF21 and FGF19 as measured by the activation level of Erk1/2.

Endocrine FGF treatment and tissue collection in mice were as described in Figure 7. The pErk1/2 levels in response to FGF21 in different tissues from wild-type and KLB−/− mice (A) as compared to PBS control, or in the adipose tissue and liver from the FGFR1 floxed (Flox) and CN mice (B) as compared to FGF19 and PBS were determined by immunoblot analyses. Data are representatives of 4–6 mice for each treatment scheme in each genotype group. The average relative activation level of Erk1/2 for each type of tissue is expressed as percentage to the peak activation in WAT of the Flox mice treated with FGF21, after normalized as ratio of pErk1/2 to total Erk1/2 (A) or ratio of pErk1/2 to β–Actin (B).

Figure 9. Tissue-specific expression of FGFRs and KLB.

Tissues as mentioned in Figure 8 were isolated from healthy C57/BL6, and total RNA extracted was used for qPCR analyses of the expression of FGFR1–4 and KLB. Normalized expression levels were expressed as folds of difference relative to that of β-Actin detected in each sample, and multiplied by 10⁴ for graphic presentation. Data were presented as the mean of triplicates ± S.D. Inset: small scale plot on y-axis of expression levels in skeletal muscle.