Faculty Bibliography

Inflammation plays a central role in the etiology of diabetic nephropathy, a global health issue. We observed a significant reduction in the renal expression of fibroblast growth factor 1, a known mitogen and insulin sensitizer, in patients with diabetic nephropathy and in mouse models implying that fibroblast growth factor 1 possesses beneficial anti-inflammatory and renoprotective activities in vivo. To test this possibility, we investigated the effects of chronic i.p. administration of fibroblast growth factor 1 into both the streptozotocin-induced type 1 diabetes and db/db type 2 diabetes models. Indeed, recombinant fibroblast growth factor 1 significantly suppressed renal inflammation (i.e., cytokines, macrophage infiltration), glomerular and tubular damage, and renal dysfunction in both type 1 and type 2 diabetes mice. Fibroblast growth factor 1 was able to correct the elevated blood glucose levels in type 2 but not in type 1 diabetic mice, suggesting that the anti-inflammatory effect of fibroblast growth factor 1 was independent of its glucose-lowering activity. The mechanistic study demonstrated that fibroblast growth factor 1-mediated inhibition of the renal inflammation in vivo was accompanied by attenuation of the nuclear factor kappaB and c-Jun N-terminal kinase signaling pathways, further validated in vitro using cultured glomerular mesangial cells and podocytes. Thus, fibroblast growth factor 1 holds great promise for developing new treatments for diabetic nephropathy through countering inflammatory signaling cascades in injured renal tissue.

Upon ligand engagement, the single-pass transmembrane receptor tyrosine kinases (RTKs) dimerize to transmit qualitatively and quantitatively different intracellular signals that alter the transcriptional landscape and thereby determine the cellular response. The molecular mechanisms underlying these fundamental events are not well understood. Considering recent insights into the structural biology of fibroblast growth factor signaling, we propose a threshold model for RTK signaling specificity in which quantitative differences in the strength/longevity of ligand-induced receptor dimers on the cell surface lead to quantitative differences in the phosphorylation of activation loop (A-loop) tyrosines as well as qualitative differences in the phosphorylation of tyrosines mediating substrate recruitment. In this model, quantitative differences on A-loop tyrosine phosphorylation result in gradations in kinase activation, leading to the generation of intracellular signals of varying amplitude/duration. In contrast, qualitative differences in the pattern of tyrosine phosphorylation on the receptor result in the recruitment/activation of distinct substrates/intracellular pathways. Commensurate with both the dynamics of the intracellular signal and the types of intracellular pathways activated, unique transcriptional signatures are established. Our model provides a framework for engineering clinically useful ligands that can tune receptor dimerization stability so as to bias the cellular transcriptome to achieve a desired cellular output.

FGF23 is a bone-derived phosphaturic hormone, the excess or deficiency of which leads to a variety of diseases. Molecular mechanisms governing FGF23 production are poorly understood. The extra-large Ga-subunit (XLalphas) is a variant of the stimulatory G protein alpha-subunit (Gsalpha), which mediates the actions of PTH, another phosphaturic hormone shown to stimulate FGF23 production. XLalphas can mimic the cellular actions of Gsa when overexpressed, but its unique actions, as well as its roles in mineral metabolism, remain unclear. We found that XLalphas is expressed in osteocytes and osteoblasts during early postnatal development. At postnatal day 10, XLalphas knockout (XLKO) mice exhibited hyperphosphatemia (11.43+/-0.43 vs. 10.23+/- 0.21 mg/dl in WT) and hypocalcemia (1.41+/- 0.02 vs. 1.46+/- 0.01 mmol/l in WT), as well as significantly increased 1,25(OH)2D levels (224.82 3.49 vs. 142.51+/- 4.02 pmol/L in WT). Quantitative RT-PCR analysis of whole kidneys from XLKO pups revealed a 2.6-fold increase in Cyp27b1 and a 1.9-fold increase in Cyp24a1 mRNA levels compared to WT littermates. We also observed increased expression of sodium phosphate co-transporter Npt2a in XLKO renal brush-border membranes. Consistent with these findings, serum FGF23 levels were significantly reduced in XLKO pups (204.40+/- 10.50 vs. 398.68+/- 10.14 pg/mL in WT). FGF23 mRNA levels in XLKO femurs were also significantly reduced (34.79+/- 5.98% of WT levels), along with a reduction in FGFR1 mRNA levels (31.05+/- 2.83% of WT levels). XLKO bones showed reduced total and phospho-FRS2a, as well as phospho-ERK1/2 levels. Injection of a stable FGF23 mutant (FGF23R176Q/R179Q) into XLKO mice normalized Pi levels (saline-injected WT: 12.07+/- 0.10 mg/mL; saline-injected KO: 13.64+/- 0.16 mg/mL; FGF23-injected WT: 10.53+/- 0.17 mg/mL; FGF23-injected KO: 11.62+/- 0.23 mg/mL), and significantly decreased renal Cyp27b1 mRNA levels. CRISPR/Cas9-mediated knockout of XLalphas in a murine osteocytic cell line (Ocy454) significantly decreased FGF23 (14.81+/- 2.11% of control) and FGFR1 mRNA levels (26.79+/- 2.63% of control), as well as the level of phospho-ERK1/2. Moreover, transcriptional activation of FGFR1 expression through CRISPR/Cas9 Synergistic Activation Mediator resulted in elevated FGF23 mRNA levels in WT Ocy454 cells (3.56+/- 0.29 fold over control) and rescued the reduced FGF23 expression in XLKO Ocy454 cells (3.45+/- 0.27 fold over control). Our findings indicate that XLalphas promotes FGF23 production in bone by maintaining FGFR1 expression and MAPK signaling

Aims: Fibroblast growth factor 1 (FGF1), a heparin/heparan sulfate-binding growth factor, is a potent cardioprotective agent against myocardial infarction (MI). The impact of heparin, the standard of care for MI patients entering the emergency room, on cardioprotective effects of FGF1 is unknown, however. Methods and results: To address this, a rat model of MI was employed to compare cardioprotective potentials (lower infarct size and improve post-ischemic function) of native FGF1 and an engineered FGF1 (FGF1DeltaHBS) with reduced heparin-binding affinity when given at the onset of reperfusion in the absence or presence of heparin. FGF1 and FGF1DeltaHBS did not alter heparin's anticoagulant properties. Treatment with heparin alone or native FGF1 significantly reduced infarct size compared to saline (P < 0.05). Surprisingly, treatment with FGF1DeltaHBS markedly lowered infarct size compared to FGF1 (P < 0.05). Both native and modified FGF1 restored contractile and relaxation function (P < 0.05 versus saline or heparin). Furthermore, FGF1DeltaHBS had greater improvement in cardiac function compared to FGF1 (P < 0.05). Heparin negatively impacted the cardioprotective effects (infarct size, post-ischemic recovery of function) of FGF1 (P < 0.05) but not of FGF1DeltaHBS. Heparin also reduced the biodistribution of FGF1, but not FGF1DeltaHBS, to the left ventricle. FGF1 and FGF1DeltaHBS bound and triggered FGFR1-induced downstream activation of ERK1/2 (P < 0.05); yet, heparin co-treatment decreased FGF1-produced ERK1/2 activation, but not that activated by FGF1DeltaHBS. Conclusion: These findings demonstrate that modification of the heparin-binding region of FGF1 significantly improves the cardioprotective efficacy, even in the presence of heparin, identifying a novel FGF ligand available for therapeutic use in ischemic heart disease.

Fibroblast growth factor 23 (FGF23) is the causative factor of X-linked hypophosphatemia (XLH), a genetic disorder effecting 1:20,000 that is characterized by excessive phosphate excretion, elevated FGF23 levels and a rickets/osteomalacia phenotype. FGF23 inhibits phosphate reabsorption and suppresses 1alpha,25-dihydroxyvitamin D (1,25D) biosynthesis, analytes that differentially contribute to bone integrity and deleterious soft tissue mineralization. As inhibition of ligand broadly modulates downstream targets, balancing efficacy and unwanted toxicity is difficult when targeting the FGF23 pathway. We demonstrate that a FGF23 c-tail-Fc fusion molecule selectively modulates the phosphate pathway in vivo by competitive antagonism of FGF23 binding to the FGFR/ alpha klotho receptor complex. Repeated injection of FGF23 c-tail Fc in Hyp mice, a pre-clinical model of XLH, increases cell surface abundance of kidney NaPi transporters, normalizes phosphate excretion and significantly improves bone architecture in the absence of soft tissue mineralization. Repeated injection does not modulate either 1,25D or calcium in a physiologically relevant manner in either a wild-type or disease setting. These data suggest that bone integrity can be improved in models of XLH via the exclusive modulation of phosphate. We posit that the selective modulation of the phosphate pathway will increase the window between efficacy and safety risks, allowing increased efficacy to be achieved in the treatment of this chronic disease

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic form of isolated gonadotropin-releasing hormone (GnRH) deficiency caused by mutations in > 30 genes. Fibroblast growth factor receptor 1 (FGFR1) is the most frequently mutated gene in CHH and is implicated in GnRH neuron development and maintenance. We note that a CHH FGFR1 mutation (p.L342S) decreases signaling of the metabolic regulator FGF21 by impairing the association of FGFR1 with beta-Klotho (KLB), the obligate co-receptor for FGF21. We thus hypothesized that the metabolic FGF21/KLB/FGFR1 pathway is involved in CHH Genetic screening of 334 CHH patients identified seven heterozygous loss-of-function KLB mutations in 13 patients (4%). Most patients with KLB mutations (9/13) exhibited metabolic defects. In mice, lack of Klb led to delayed puberty, altered estrous cyclicity, and subfertility due to a hypothalamic defect associated with inability of GnRH neurons to release GnRH in response to FGF21. Peripheral FGF21 administration could indeed reach GnRH neurons through circumventricular organs in the hypothalamus. We conclude that FGF21/KLB/FGFR1 signaling plays an essential role in GnRH biology, potentially linking metabolism with reproduction.

The epithelial fibroblast growth factor 9 (FGF9) subfamily specifically binds and activates the mesenchymal "c" splice isoform of FGF receptors 1-3 (FGFR1-3) to regulate organogenesis and tissue homeostasis. The unique N and C termini of FGF9 subfamily ligands mediate a reversible homodimerization that occludes major receptor binding sites within the ligand core region. Here we provide compelling X-ray crystallographic, biophysical, and biochemical data showing that homodimerization controls receptor binding specificity of the FGF9 subfamily by keeping the concentration of active FGF9 monomers at a level, which is sufficient for a normal FGFR "c" isoform binding/signaling, but is insufficient for an illegitimate FGFR "b" isoform binding/signaling. We show that deletion of the N terminus or alanine substitutions in the C terminus of FGF9 skews the delicate ligand equilibrium toward active FGF9 monomers causing off-target binding and activation of FGFR b isoforms. Our study is the first to implicate ligand homodimerization in the regulation of ligand-receptor specificity.

The recent discovery of metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis has expanded the functions of this classically known mitogen. To dissect the molecular basis for this functional pleiotropy, we engineered an FGF1 partial agonist carrying triple mutations (FGF1DeltaHBS) that diminished its ability to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1DeltaHBS exhibited a severely reduced proliferative potential, while preserving the full metabolic activity of wild-type FGF1 in vitro and in vivo. Hence, suboptimal FGFR activation by a weak FGF1-FGFR dimer is sufficient to evoke a metabolic response, whereas full FGFR activation by stable and sustained dimerization is required to elicit a mitogenic response. In addition to providing a physical basis for the diverse activities of FGF1, our findings will impact ongoing drug discoveries targeting FGF1 and related FGFs for the treatment of a variety of human diseases.

Neonatal micropenis and cryptorchidism raise the suspicion of congenital hypogonadotropic hypogonadism (CHH), a rare genetic disorder caused by GnRH deficiency. Low plasma testosterone levels and low gonadotropins during minipuberty provide a clinical diagnostic clue, yet these tests are seldomly performed in general practice. We report a male neonate with no family history of reproductive disorders who was born with micropenis and cryptorchidism. Hormonal testing at age 2.5 months showed low testosterone (0.3 nmol/L) and undetectable gonadotropins (luteinizing hormone and follicle-stimulating hormone both < 0.5 U/L), suggestive of CHH. Genetic testing identified a de novo, heterozygous mutation in fibroblast growth factor receptor 1 (FGFR1 p.L630P). L630 resides on the ATP binding cleft of the FGFR1 tyrosine kinase domain, and L630P is predicted to cause a complete loss of receptor function. Cell-based assays confirmed that L630P abolishes FGF8 signaling activity. Identification of a loss-of-function de novo FGFR1 mutation in this patient confirms the diagnosis of CHH, allowing for a timely hormonal treatment to induce pubertal development. Therefore genetic testing can complement clinical and hormonal assessment for a timely diagnosis of CHH in childhood.

Receptor tyrosine kinase (RTK) signaling is tightly regulated by protein allostery within the intracellular tyrosine kinase domains. Yet the molecular determinants of allosteric connectivity in tyrosine kinase domain are incompletely understood. By means of structural (X-ray and NMR) and functional characterization of pathogenic gain-of-function mutations affecting the FGF receptor (FGFR) tyrosine kinase domain, we elucidated a long-distance allosteric network composed of four interconnected sites termed the 'molecular brake', 'DFG latch', 'A-loop plug', and 'alphaC tether'. The first three sites repress the kinase from adopting an active conformation, whereas the alphaC tether promotes the active conformation. The skewed design of this four-site allosteric network imposes tight autoinhibition and accounts for the incomplete mimicry of the activated conformation by pathogenic mutations targeting a single site. Based on the structural similarity shared among RTKs, we propose that this allosteric model for FGFR kinases is applicable to other RTKs.