Cell signaling is a process by which cells interact with each other and their environment through hormones, neurotransmitters, and growth factors that bind to specific receptors [1]. Growth factors are grouped according to their structure, function, and the type of cell or tissue that they regulate. Among the many growth factors are the fibroblast growth factor (FGF) family, transforming growth factor β (TGF-β) family, vascular endothelial growth factor (VEGF) family, epidermal growth factor (EGF) family, hepatocyte growth factor (HGF) family, platelet-derived growth factor (PDGF) family, insulin growth factor (IGF) family and neurotrophins family [2].

The fibroblast growth factor (FGF) family consists of cell signaling molecules that play major roles in cell proliferation, migration, morphogenesis, differentiation, tissue repair, and regeneration. FGF, together with other growth factors such as epidermal growth factor (EGF), and tumor necrosis factor (TNF)-α, are part of the MAP kinase pathways. These growth factors are activated by the Transforming growth factors (TGF-β). The cross-talk between these intracellular pathways is known to affect gene expression. For example, in wild-type mice with injured gut, there is dysregulation of the microbiota, which in turn leads to the upregulation of TGFβ1 expression. The upregulated TGFβ1 promotes FGF2 expression and FGF2 cross-talk with interleukin 17 (IL-17). The FGF2-IL-17 cross-talk leads to the expression of an array of repair-associated genes in intestinal epithelial cells [3].

FGFs are comprised of 22 members and are further grouped into 7 subfamilies based on their signaling events and functions. It is established that the 22 members share 13-71% sequence similarity in mammals and each subfamily possesses a wide range of biological functions [4,5]. The FGF1 subfamily comprises of FGF1 and FGF2, FGF4 subfamily comprises of FGF4, FGF5 and FGF6, FGF7 subfamily consists of FGF3, FGF7, and FGF10, FGF8 subfamily comprises FGF8, FGF17, and FGF18, FGF9 subfamily comprises of FGF9, FGF16, and FGF20, FGF11 subfamily comprises of FGF11, FGF12, FGF13, and FGF14, FGF19 comprises of FGF15/FGF19, FGF21, and FGF23 (Figure 1). FGF15 and FGF19 are orthologs with FGF15 associated with mice and FGF19 found in humans and primates [6]. Based on their mode of triggering their functions, FGFs have been divided into paracrine, intracrine, and endocrine. FGF1, FGF4, FGF7, and FGF9 subfamilies are paracrine FGFs, the FGF11 subfamily is intracrine FGFs and FGF15/19 are endocrine FGFs. Canonical FGFs also known as paracrine FGFs are tightly bound to heparin and heparin sulfate (HS) glycosaminoglycan cofactor and this association promotes FGF signaling by stabilizing the interaction of FGFs with their receptor as well as limiting diffusion through the extracellular matrix. Endocrine FGFs on the other hand have a lower binding affinity to heparin and heparin sulfate glycosaminoglycan but interact with Klotho proteins as cofactors. Intracrine FGFs are not secreted but are cofactors for voltage-gated sodium channels [7-9]. FGF signaling is triggered through the tyrosine kinase receptor, which consists of three immunoglobulin-like domains: I, II, and III. Ligand binding is specified by immunoglobin-like II and III, and the linker is located between them while immunoglobin-like I and an acidic amino acid residue (Acidic box) located between domains I and II are known to inhibit ligand binding [7,10-14].

Paracrine fibroblast growth factors include FGF1 subfamily, FGF4 subfamily, FGF7 subfamily, FGF8 subfamily, and FGF9 subfamily. These fibroblast growth factors exhibit mitogenic, angiogenic, differentiation, chemotactic, and anti-apoptotic properties. Therefore, they can function on stem cells and various cell types of mesenchymal and epithelial origins. They are also candidates for wound healing and tissue repair [15-17].

Paracrine fibroblast growth factors function locally via interaction and activation of cell surface tyrosine kinase FGF receptors (FGFRs) through a high-affinity interaction with heparin or heparan sulfate [15]. FGFRs are transmembrane proteins consisting of four genes (FGFR1, FGFR2, FGFR3 and FGFR4). Heparin-dependent FGF-FGFR binding causes receptor dimerization and trans-autophosphorylation of the tyrosine receptor. FGFRs contain an extracellular domain, a transmembrane domain (TMD), and an intracellular tyrosine kinase domain. The extracellular domain comprises three immunoglobulin (Ig)- like domains (I, II, and III), an acidic region, and a heparin-binding motif. The transmembrane domain is known to anchor the FGFs in the cell membrane and promote its dimerization. Activated Fibroblast Growth Factor Receptors (FGFR) induce the main downstream intracellular signaling pathways [Rat Sarcoma Virus (RAS)/mitogen-activated protein kinase (MAPK)], phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)–protein kinase B (AKT), and phospholipase C gamma (PLCγ) ) and Signal Transducer and Activator of Transcription (STAT) [16] as depicted in Figure 2. Various gene expressions are controlled by RAS-MAPK signaling pathway via phosphorylation of E26 transformation-specific (ETS) transcription factors that stimulate DNA interaction and gene expressions. Phosphorylation events in embryogenesis play various physiological roles in the adult state, including regulating angiogenesis, wound healing, and organ development [17]. In contrast to the paracrine FGFs, intracrine FGFs (FGF11, FGF12, FGF13, FGF14) do not possess the N-terminal signal peptide, which is why they function as intracellular proteins [18].

Fibroblast growth factor 1 (FGF1), an acidic fibroblast growth factor, is a member of the FGF1 subfamily. FGF1 has been correlated with various biological actions such as development, angiogenesis, and adipogenesis [9]. It has been indicated that FGF1 does not have classical secretory signal peptides; instead, it is exported via direct translocation through the cell membrane [10]. Furthermore, FGF1 has also been found in the nucleus of some cells such as NIH/3T3 cells [7]. The nuclear FGF1 has various functions, including regulating the cell cycle, cell differentiation, survival, and apoptosis [12]. Amongst all FGFs, FGF1 is the only isoform that can activate all FGFR splice variants [13]. The second member in the FGF1 subfamily is the basic FGF or FGF2. FGF2 plays a role in fibroblasts and other mesoderm-derived cell types, such as vascular endothelial cells, smooth muscle cells, osteoblasts, and chondrocytes. Recombinant FGF2 has medical applications in skin wound-healing processes [19]. FGF4, FGF5, and FGF6 play an essential role in various cellular processes, particularly embryonic development and tissue repair [20]. These fibroblast growth factors are involved in cell proliferation, differentiation and tissue morphogenesis [21]. FGF4 is highly expressed in the ectoderm, mesoderm, and tail bud [22]. FGF4 has multiple functions in developmental processes, such as cardiac valve leaflet formation and limb development [15]. FGF4 application is emerging as it has a unique role in oncogenesis, tumor progression, and resistance to anti-tumor therapy in multiple types of cancer [23]. FGF5 has a negative effect on controlling the hair follicle growth cycle [15]. Functional studies also reveal FGF5 overexpression in prostate cancer (PCa) and benign prostatic hyperplasia (BPH). The development of new FGF5 inhibitors could potentially target the PCa and BPH [24]. FGF6 plays a part in myogenesis, and it has been indicated that the mitogen is upregulated after muscle injury [16,25]. FGF6 and FGF9 are known adipokines that regulate uncoupling protein 1 (UCP1) through a transcriptional network that is dissociated from brown adipogenesis and acts to modulate systemic energy metabolism. Further studies on FGF6 and FGF9 could lead to the development of therapeutic strategies to tackle obesity, type 2 diabetes, and cardiovascular disease [26].

The fibroblast growth factor 7 subfamily consists of FGF3, FGF7, FGF10, and FGF22 [17]. The members of this subfamily are secreted specifically by mesenchyme, and in the overlying epithelium, they activate the FGFR1b and FGFR2b isoforms. As a result, the signaling of these members is important to the development of essentially all organs, glands, and limbs [18].

Metabolic FGFs regulate signaling pathways including RAS/MAPK, PI3K–AKT, PLCγ, and STAT, as shown in Figure 3. These FGFs regulate pathways that maintain the homeostasis of bile acids, lipids, glucose, energy, and minerals [27,28]. FGF15/19 triggers its metabolic effect by primarily interacting with both FGFR1 and FGFR4 and needs β-Klotho as a cofactor. FGF15/19 is released by epithelial cells in the small intestines and plays a regulatory role in bile acid, lipid, and carbohydrate metabolism [29-31]. To gain better insights into FGF15/19’s role in metabolism, studies using FGF15 knockout and liver-associated β-Klotho mice revealed abnormal glucose homeostasis and elevated gluconeogenesis levels in the fed state. Potthoff et al work on glucose metabolism, also shows that FGF15/19 inhibits hepatic gluconeogenesis [32]. Interestingly, these effects on glucose metabolism are independent of insulin activity. FGF19 mimics insulin effect by promoting protein and glycogen synthesis, independent of insulin. FGF15/19 controls bile acid synthesis by downregulating cholesterol 7α-hydroxylase gene (CYP7A1), a key rate-limiting enzyme in the process. Mice lacking FGF15, FGFR4, and β-Klotho show impaired expression of bile acid synthesis after feeding. [33-35]. Various studies demonstrate the possibility of using FGF19 for the treatment of diabetes and obesity [36].

FGF21 on the other hand exerts its activity by interacting with FGFR1c and β-Klotho. FGF21 expression is influenced by nutritional conditions (such as fasting, refeeding, and overfeeding) and the organism’s stress levels. FGF21 administration is known to reduce plasma glucose levels. However, β-Klotho or FGFR1c knockout mice did not respond to FGF21 treatment. It remains unclear whether different types of adipocytes play a role in FGF21’s glucose-lowering effects [30,37,38].

FGF23, the most recently discovered endocrine FGF, has been identified as a causative factor in tumor-induced osteomalacia, characterized by phosphate wasting. FGF23 interacts with α-Klotho and FGFR1c, FGFR3c and FGFR4. FGF23 regulates calcium homeostasis and phosphates homeostasis in the kidney and parathyroid glands. FGF23 plays a crucial role in the pathophysiology of disorders like chronic kidney disease and various bone-related conditions. Mice with an FGF23 knockout exhibit phosphate retention, which leads to imbalances in phosphate metabolism [39-43].

Abnormal signaling of FGFs are related to various diseases such as metabolic diseases, limb abnormalities, mitochondrial diseases, and cancer [36].

Klotho proteins are essential cofactors of endocrine FGFs, stabilizing their interaction with FGFs receptors. Klotho proteins are known to promote cognition, reduce oxidative stress, and enhance synaptic plasticity. Klotho is abundantly expressed in the kidney, as well as in the choroid plexus in the brain, and sex organs such as the ovaries, testis, and placenta [44,45].

The overexpression of Klotho proteins increases mice's lifespan while mice with the Klotho knockout gene result in the acceleration of aging phenotypes [46]. The Klotho gene comprises five exons and is found in the plasma membrane and Golgi apparatus. The Klotho protein has a short intracellular domain of approximately 10 amino acids, which appears to have no known function. The extracellular domain on the other hand has two internal repeats, KL1 and KL2. Four basic amino acids serve as a linker between the two internal repeats. α-Klotho, β-klotho, and γ-Klotho are the three subtypes of the Klotho transmembrane proteins. α-Klotho and β-Klotho are vital for the formation of endocrine FGFs-FGFR complexes. α-Klotho shares 42% amino acid sequence homology with β-Klotho. The expression of β-Klotho predominantly occurs in the liver and adipose tissue while α-Klotho is predominantly expressed in the distal tubule cells. β-Klotho plays a key role in metabolic activities such as glucose uptake, bile acid synthesis, and fatty acid metabolism independent of α-Klotho. α-Klotho on the other hand regulates phosphate absorption and the activity of 1,25-dihydroxyvitamin D₃. α and β-Klotho crystal structures have been elucidated. α-Klotho co-crystallized with FGF23 and the ligand binding domain of FGFR1c revealed that α-Klotho has a long thread termed receptor binding arm which interacts directly with FGFR1c. A groove created between α-Klotho and FGFR1c is occupied by FGF23 with a C-terminus oriented towards α-Klotho and an N-terminus oriented towards FGFR1c. Similar to α-Klotho, β-Klotho crystalized with and without FGF21 shows that β-Klotho interacts with the C-terminus of FGF21. However, certain structural regions were not able to be resolved such as the receptor binding arm [47].

FGF signaling plays crucial roles in metabolism, development, and homeostasis, but the malfunction of FGF/FGF receptor signaling is implicated in various human diseases such as cancer, dwarfism, chronic kidney disease (CKD), obesity, and insulin resistance [48,49]. For example, FGF19 possesses potent pharmacological benefits in administration against obesity, diabetes, and fatty liver disease. However, the involvement of FGF19 in tumorigenesis is the main challenge in translating FGF19-based pharmacotherapy from the bench to the clinic. In mice, ectopic expression of FGF19 controls hepatocyte proliferation, hepatocellular dysplasia, and neoplasia, while upregulated FGF19 expression is related to tumor progression and poor prognosis in patients with hepatocellular carcinoma (HCC) [20,50,51]. In lung diseases, there is an abnormal expression of FGFs/FGFRs. In human fetal congenital cystic adenomatoid malformation, there is a 4-fold increase in expression than the normal fetal lung. FGF1/FGFR is elevated in idiopathic pulmonary fibrosis (IPF), which is projected to lead to the pathogenesis of lung fibrosis. Due to the implications of FGFs in human diseases, various efforts are being made to dissect the structural basis underpinning metabolic and mitogenic activity.

The FGF family has been extensively studied and revealed to be the class of proteins involved in wound healing, tissue repair, and regeneration. Since FGF proteins are prone to degradation, exploring the mechanisms behind this degradation will be crucial for realizing their full potential in therapeutic applications [52]. After understanding the FGF degradation mechanism, stable FGF variants can be designed and developed using computational and site-directed mutagenesis. Additionally, during FGF-based therapeutic applications, chemical environments, release methods, such as hydrogels and nanoparticles that enhance or maintain FGF protein stability should be investigated. The Thallapuranam group has recently developed stable FGF variants with enhanced biological activity, which have shown promise in wound healing applications [53]. An example of this stable version is a variant of FGF1, Q54P/K126N/R136E, which was attained by a 3 amino acid change on the wild-type FGF1. The wild-type FGF1 without heparin has a melting temperature (Tm) of ~41^oC while the Q54P/K126N/R136E FGF1 mutant has a Tm of ~60^oC in the absence of heparin. When subjected to chemical denaturation with urea, wild-type FGF1 exhibited a Cm (the concentration at which 50% of the protein unfolds) of ~1.9M, while the Q54P/K126N/R136E mutant had a Cm of ~3.97M. In another study by the same group, proline in the heparin-binding site was substituted with glutamic acid, and positively charged arginine was replaced by glutamic acid. Compared to wild-type FGF1, the P135E/R136E variant had an increased Tm of ~53°C.39 [53,54]. In another study, to investigate the role of salt bridges in FGF1 structure, charge reversal at the heparin-binding pocket was carried out by substituting aspartic acid with arginine at positions 82 and 84. After subjecting the D82R/D84R double mutant to thermal denaturation, the mutant had a Tm of ~52.6 [55]. These results reveal the enhanced stability of FGF1 using computational and site-directed mutagenesis. Since the FGF19 subfamily is involved in diverse biological activities, its members offer new avenues for treating various human metabolic diseases. For example, FGF19 plays a crucial role in regulating bile acid cycles and systemic metabolism [56,57]. Current studies indicate that the FGF21 agonist is a potential therapeutic candidate for treating type 2 diabetes and obesity [58,59]. An example is Efruxifermin (Fc-FGF21), a fusion protein of the human IgG1 Fc domain combined with human FGF21. Phase 1 studies reported individuals with type 2 diabetes showed improved glycemic control [60]. FGF23 plays a key role in regulating phosphate metabolism and is implicated in conditions such as inherited hypophosphatemia rickets, familial tumoral calcinosis, X-linked hypophosphatemia, and the progression of chronic kidney disease [59]. Exciting future prospects involving mitogenic and metabolic FGFs are awaited and with a better understanding of the mechanistic steps in their regulation will pave the way for the design of potent therapeutics against a plethora of dilapidating FGF-regulated diseases. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This work was supported by the Department of Energy (DE-FG02-01ER15161), the National Institutes of Health/National Cancer Institute NIH/NCI (1 RO1 CA 172631) and the NIH through the COBRE program (P30 GM103450), and the Arkansas Biosciences Institute (ABI-TKSK-2016-17). Research reported in this publication was also supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM154267.