Human FGF-21 Is a Substrate of Fibroblast Activation Protein

FGF-21 is a key regulator of metabolism and potential drug candidate for the treatment of type II diabetes and other metabolic disorders. However, the half-life of active, circulating, human FGF-21 has recently been shown to be limited in mice and monkeys by a proteolytic cleavage between P171 and S172. Here, we show that fibroblast activation protein is the enzyme responsible for this proteolysis by demonstrating that purified FAP cleaves human FGF-21 at this site in vitro, and that an FAP-specific inhibitor, ARI-3099, blocks the activity in mouse, monkey and human plasma and prolongs the half-life of circulating human FGF-21 in mice. Mouse FGF-21, however, lacks the FAP cleavage site and is not cleaved by FAP. These findings indicate FAP may function in the regulation of metabolism and that FAP inhibitors may prove useful in the treatment of diabetes and metabolic disorders in humans, but pre-clinical proof of concept studies in rodents will be problematic.     

成纤维细胞生长因子21的研究进展*

成纤维细胞生长因子21（fibroblast growth factor 21,FGF21）是一种主要的脂肪代谢调节因子,主要在肝脏中表达;FGF21有助于肝脏的脂肪代谢以及生酮反应,可以促进脂肪细胞摄取葡萄糖,促进胰岛素分泌,延缓肿瘤的发展等功能。近年来研究过程中发现,FGF21可以用于糖尿病和降血脂等其他代谢疾病治疗。主要对FGF21的特点,作用机理及其分子机制进行了概括,并对FGF21在糖尿病治疗和降血脂方面的研究进行了综述。     

Intact Fibroblast Growth Factor 23 Concentrations in Hypophosphatemic Disorders

ObjectiveEvaluation of circulating fibroblast growth factor 23 (FGF23) concentrations plays a key role in the differential diagnosis of patients presenting with hypophosphatemia. FGF23 concentrations obtained by different immunoassays are not comparable and subsequently, differences in the clinical performance of the assays might arise. In this study, we evaluated the clinical performance of the Medfrontier FGF23 Intact immunoassay (MedFrontier, Minaris Medical Co, Ltd, Tokyo, Japan) in clinically relevant hypophosphatemic conditions.MethodsIntact FGF23 (iFGF23) was measured in serum samples from 61 patients with FGF23-dependent hypophosphatemia (42-tumor induced osteomalacia [TIO] and 19-X-linked hypophosphatemia [XLH]); 8 patients with FGF23-independent hypophosphatemia (6-Fanconi Syndrome and 2-Vitamin D dependent rickets); 10 normophosphatemic patients; 15 chronic kidney disease (CKD) stage-2/3 and 20 CKD stage-4/5 patients; and a healthy control population. Disease-specific differences in measured iFGF23 concentrations and FGF23 concentration association with phosphate concentrations were reported.ResultsiFGF23 concentrations were significantly elevated in 90% and 84% of TIO and XLH hypophosphatemia patients as compared to healthy controls (both TIO and XLH, P = .0001). There was no significant correlation between iFGF23 and phosphate concentrations (P = .74 and P = .86) for TIO and XLH, respectively. Patients with CKD showed a significant increase in serum iFGF23 as the estimated glomerular filtration rate decreased (ρ = -0.79, P ≤ 0.0001).ConclusionsThis study evaluated the clinical performance of the MedFrontier iFGF23 assay in a large cohort of XLH and TIO Caucasian and Asian patients. The clinical sensitivity of this iFGF23 assay is appropriate for clinical use.     

Of Worms and Men: An Evolutionary Perspective on the Fibroblast Growth Factor (FGF) and FGF Receptor Families

FGFs (fibroblast growth factors) play major roles in a number of developmental processes. Recent studies of several human disorders, and concurrent analysis of gene knock-out and properties of the corresponding recombinant proteins have shown that FGFs and their receptors are prominently involved in the development of the skeletal system in mammals. We have compared the sequences of the nine known mammalian FGFs, FGFs from other vertebrates, and three additional sequences that we extracted from existing databases: two human FGF sequences that we tentatively designated FGF10 and FGF11, and an FGF sequence from C?norhabditis elegans. Similarly, we have compared the sequences of the four FGF receptor paralogs found in chordates with four non-chordate FGF receptors, including one recently identified in C. elegans. The comparison of FGF and FGF receptor sequences in vertebrates and nonvertebrates shows that the FGF and FGF receptor families have evolved through phases of gene duplications, one of which may have coincided with the emergence of vertebrates, in relation with their new system of body scaffold. Received: 6 April 1996 / Accepted: 5 July 1996     

Rational Design of a Fibroblast Growth Factor 21-Based Clinical Candidate,LY2405319

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.     

血清成纤维细胞生长因子2、21、23与妊娠期糖尿病患者新生儿结局的关系

摘要 目的：探讨血清成纤维细胞生长因子2（FGF2）、成纤维细胞生长因子21（FGF21）、成纤维细胞生长因子23（FGF23）与妊娠期糖尿病（GDM）患者新生儿结局的关系。方法：选取2021年1月～2022年12月期间于我院产检的妊娠24～28周孕妇147例,均进行口服葡萄糖耐量试验（OGTT）,根据OGTT结果分为GDM组（n=86）和非GDM组（n=61）。其中GDM组根据新生儿结局分为不良组（n=21）和良好组（n=65）。对比非GDM组、GDM组的血清FGF2、FGF21、FGF23水平及新生儿结局情况。对比不良组和良好组的血清FGF2、FGF21、FGF23水平。单因素及多因素Logistic回归分析影响GDM患者新生儿结局的影响因素。结果：GDM组的血清FGF2、FGF21、FGF23水平均高于非GDM组（P<0.05）。GDM组的不良新生儿结局总发生率高于非GDM组（P<0.05）。不良组的血清FGF2、FGF21、FGF23水平均高于良好组（P<0.05）。单因素分析显示,GDM患者不良新生儿结局与年龄、孕前体质量指数（BMI）、分娩前BMI、空腹血糖（FPG）、餐后2 h血糖（2hPG）、空腹胰岛素（FINS）、胰岛素抵抗指数（HOMA-IR）有关（P<0.05）。多因素分析结果显示,年龄偏高、FPG偏高、孕前BMI偏高、2hPG偏高、分娩前BMI偏高、HOMA-IR偏高、FGF2偏高、FINS偏高、FGF21偏高、FGF23偏高均是GDM患者不良新生儿结局的危险因素（P<0.05）。结论：GDM患者血清FGF2、FGF21、FGF23水平升高,其与年龄、孕前BMI、分娩前BMI、FPG、2hPG、FINS、HOMA-IR偏高均是导致GDM患者不良新生儿结局的危险因素。     

Fibroblast Growth Factor Signaling Is Essential for Self-renewal of Dental Epithelial Stem Cells

A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.     

Fibroblast growth factor 21 (FGF21) inhibits chondrocyte function and growth hormone action directly at the growth plate

Fibroblast growth factor 21 (FGF21) modulates glucose and lipid metabolism during fasting. In addition, previous evidence indicates that increased expression of FGF21 during chronic food restriction is associated with reduced bone growth and growth hormone (GH) insensitivity. In light of the inhibitory effects on growth plate chondrogenesis mediated by other FGFs, we hypothesized that FGF21 causes growth inhibition by acting directly at the long bones' growth plate. We first demonstrated the expression of FGF21, FGFR1 and FGFR3 (two receptors known to be activated by FGF21) and β-klotho (a co-receptor required for the FGF21-mediated receptor binding and activation) in fetal and 3-week-old mouse growth plate chondrocytes. We then cultured mouse growth plate chondrocytes in the presence of graded concentrations of rhFGF21 (0.01-10 μg/ml). Higher concentrations of FGF21 (5 and 10 μg/ml) inhibited chondrocyte thymidine incorporation and collagen X mRNA expression. 10 ng/ml GH stimulated chondrocyte thymidine incorporation and collagen X mRNA expression, with both effects prevented by the addition in the culture medium of FGF21 in a concentration-dependent manner. In addition, FGF21 reduced GH binding in cultured chondrocytes. In cells transfected with FGFR1 siRNA or ERK 1 siRNA, the antagonistic effects of FGF21 on GH action were all prevented, supporting a specific effect of this growth factor in chondrocytes. Our findings suggest that increased expression of FGF21 during food restriction causes growth attenuation by antagonizing the GH stimulatory effects on chondrogenesis directly at the growth plate. In addition, high concentrations of FGF21 may directly suppress growth plate chondrocyte proliferation and differentiation.     

Expression and Characterization of Fibroblast Growth Factor 8 from Mexican Axolotl, Ambystoma mexicanum

Fibroblast growth factor (FGF) has been known to regulate the proliferation and differentiation of a variety of cell types via interaction with a specific FGF receptor on the cell surface. In the present study, Fgf8 cDNA of Mexican axolotl, Ambystoma mexicanum, was expressed in Escherichia coli as an MBP-FGF8 fusion protein. The cell proliferation activity of the recombinant FGF8 (rFGF8) was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MTT) assay. The addition of rFGF8 to the culture medium enhanced proliferation of BALB/c 3T3 and BHK21 cells about 1.4--1.5 fold. To analyze the binding activity of rFGF8 to the cell surface, cell surface enzyme linked immunosorbent assay was developed. Comparison of the structure of basic FGF with the computer-simulated structure of FGF8 suggested that Tyr-58, Glu-132, Tyr-139, and Leu-179 might be the potential receptor binding sites. Amino acid substitution muteins of FGF8 were constructed by PCR-derived directed mutagenesis and the muteins were overexpressed in E. coli. The rFGF8 muteins were purified and their binding activities were analyzed. Substitution of Tyr-58 or Glu-132 or Leu-179 of the FGF8 with alanine reduced the binding affinity, while substitution of Tyr-139 with alanine did not alter the binding affinity. These results imply that Tyr-58, Glu-132, and Leu-179 of FGF8 might be involved in its binding to the cell surface.     

Fibroblast Growth Factor 2 as an Antifibrotic: Antagonism of Myofibroblast Differentiation and Suppression of Pro-Fibrotic Gene Expression

Fibrosis is a pathological condition that is characterized by the replacement of dead or damaged tissue with a nonfunctional, mechanically aberrant scar, and fibrotic pathologies account for nearly half of all deaths worldwide. The causes of fibrosis differ somewhat from tissue to tissue and pathology to pathology, but in general some of the cellular and molecular mechanisms remain constant regardless of the specific pathology in question. One of the common mechanisms underlying fibroses is the paradigm of the activated fibroblast, termed the “myofibroblast,” a differentiated mesenchymal cell with demonstrated contractile activity and a high rate of collagen deposition. Fibroblast growth factor 2 (FGF2), one of the members of the mammalian fibroblast growth factor family, is a cytokine with demonstrated antifibrotic activity in non-human animal, human, and in vitro models. FGF2 is highly pleiotropic and its receptors are present on many different cell types throughout the body, lending a great deal of variety to the potential mechanisms of FGF2 effects on fibrosis. However, recent reports demonstrate that a substantial contribution to the antifibrotic effects of FGF2 comes from the inhibitory effects of FGF2 on connective tissue fibroblasts, activated myofibroblasts, and myofibroblast progenitors. FGF2 demonstrates effects antagonistic towards fibroblast activation and towards mesenchymal transition of potential myofibroblast-forming cells, as well as promotes a gene expression paradigm more reminiscent of regenerative healing, such as that which occurs in the fetal wound healing response, than fibrotic resolution. With a better understanding of the mechanisms by which FGF2 alters the wound healing cascade and results in a shift away from scar formation and towards functional tissue regeneration, we may be able to further address the critical need of therapy for varied fibrotic pathologies across myriad tissue types.     

Inhibition of lipolysis may contribute to the acute regulation of plasma FFA and glucose by FGF21 in ob/ob mice

FGF21 is a unique member of the fibroblast growth factors (FGFs) and a novel hormone that regulates glucose, lipid, and energy homeostasis. The beneficial effects of FGF21 reported thus far have mostly been from chronic treatments. In order to better understand the mechanism for FGF21 action, we evaluated the acute effects of FGF21 in vivo and in vitro. Here we report that a single injection of FGF21 acutely reduced plasma free fatty acid levels similar to its acute effects on plasma glucose in ob/ob mice. In vitro, FGF21 inhibited lipolysis in adipocytes during a short treatment and reduced total lipase activity. These results demonstrate the potential importance of adipocyte lipolysis to the observed acute improvements in plasma parameters.     

Fibroblast growth factor family aberrations in cancers: clinical and molecular characteristics

Fibroblast growth factor ligands and receptors (FGF and FGFR) play critical roles in tumorigenesis, and several drugs have been developed to target them. We report the biologic correlates of FGF/FGFR abnormalities in diverse malignancies. The medical records of patients with cancers that underwent targeted next generation sequencing (182 or 236 cancer-related genes) were reviewed. The following FGF/FGFR genes were tested: FGF3, 4, 6, 7, 10, 12, 14, 19, 23 and FGFR1, 2, 3, and 4. Of 391 patients, 56 (14.3%) had aberrant FGF (N = 38, all amplifications) and/or FGFR (N = 22 including 5 mutations and one FGFR3-TACC3 fusion). FGF/FGFR aberrations were most frequent in breast cancers (26/81, 32.1%, p = 0.0003). In multivariate analysis, FGF/FGFR abnormalities were independently associated with CCND1/2, RICTOR, ZNF703, RPTOR, AKT2, and CDK8 alterations (all P < 0.02), as well as with an increased median number of alterations (P < 0.0001). FGF3, FGF4, FGF19 and CCND1 were co-amplified in 22 of 391 patients (5.6%, P < 0.0001), most likely because they co-localize on the same chromosomal region (11q13). There was no significant difference in time to metastasis or overall survival when comparing patients harboring FGF/FGFR alterations versus those not. Overall, FGF/FGFR was one of the most frequently aberrant pathways in our population comprising patients with diverse malignancies. These aberrations frequently co-exist with anomalies in a variety of other genes, suggesting that tailored combination therapy may be necessary in these patients.