KEY POINTS
- Soluble klotho (sKL) and heparan sulfate (HS) act as independent fibroblast growth factor receptor (FGFR) co-receptors for FGF23.
- sKL promotes fibroblast growth factor (FGF)23 binding to various FGFR isoforms and thereby might mediate FGF23 responsiveness in many tissues. The physiological relevance of this molecular mechanism is currently not clear but might include the promotion of FGFR1c-mediated renal phosphate excretion. By facilitating FGF23 binding to FGFR4, sKL might protect from the pathologic actions of elevated FGF23.
- sKL blocks the binding between specific paracrine FGFs and FGFRs and thereby might protect from various pathologic effects caused by paracrine FGFs, such as fibrosis.
- sKL seems to be much more than a circulating co-receptor for FGFRs. It has a spectrum of different functions and activities which might explain the pleiotropic tissue-protective actions of sKL. This includes its role as a binding partner and modulator of other ligand/receptor complexes, glycoproteins, glycolipids and gangliosides, as well as intrinsic glycosidase activity which can modify many cell surface molecules. These different activities seem to be highly regulated by the posttranslational modification of sKL.
- HS promotes FGF23 binding mainly to FGFR4 and thereby might promote the pathologic actions of elevated FGF23, such as cardiac hypertrophy in chronic kidney disease. The biological relevance of the regulation of FGF23 by HS under physiologic conditions is currently unknown.
INTRODUCTION
The mammalian fibroblast growth factor (FGF) family comprises 18 structurally related, secreted proteins that have diverse functions in development, metabolism, and disease [1]. The biological effects of all FGF family members are mediated by binding to one of the FGF receptor isoforms (FGFR1–4) which belong to the superfamily of receptor tyrosine kinases [2]. Alternative splicing in the juxtamembrane region of FGFR1–3 produces b- and c-variants that differ in their FGF-binding spectrum. FGFs are classified as paracrine and endocrine FGFs based on their mechanism of action. Paracrine FGFs, such as FGF1 and FGF2, bind heparan sulfate (HS) which is required for the subsequent binding and activation of FGFRs and the initiation of signal transduction events [3,4]. The three members of the subfamily of endocrine FGFs (i.e. FGF19, FGF21 and FGF23) have intrinsically low binding affinity for HS [5–8], which allows them to escape the extracellular environment, enter the circulation, and act as hormones [9]. Instead of HS, endocrine FGFs utilize a family of transmembrane proteins, known as α-klotho for FGF23 (called klotho from here on) and β-klotho for FGF19 and FGF21, to promote efficient FGFR binding [7].
FGF23 is a hormone that is secreted by osteocytes, and targets cells by binding to FGFR1c and klotho, resulting in the induction of Ras/mitogen activated protein kinase (MAPK) signaling [10]. In the parathyroid gland and kidney, FGF23 inhibits parathyroid hormone secretion and reduces production of active vitamin D, respectively [11–13]. By targeting proximal tubular epithelial cells, FGF23 reduces surface expression of the sodium/phosphate cotransporters NaPi-2a and NaPi-2c and thereby renal phosphate reabsorption [13,14]. The combined net effect of FGF23's physiologic actions is an increase in renal phosphate excretion and a reduction in systemic phosphate levels [15]. In patients with chronic kidney disease (CKD), phosphate excretion and the renal effects of FGF23 are diminished due to a loss of functional kidney mass and reduced klotho expression, resulting in highly elevated serum levels of phosphate and FGF23. Beginning in the early stages of CKD, serum FGF23 levels rise progressively to maintain normal phosphate levels but fail to promote renal phosphate excretion as nephron function and FGF23 responsiveness decline [16,17]. In patients with end-stage renal disease (ESRD) who depend on renal replacement therapy, FGF23 levels can reach 1000-fold above normal values [18]. Elevated FGF23 can contribute to various pathologic alterations [19,20], including pathologic cardiac remodeling [21–23] and to the high cardiovascular mortality that is observed in the majority of CKD patients [24]. Mechanistically, it has been shown that elevated FGF23 can directly target cardiac myocytes in a klotho-independent manner, and activate FGFR4 and the phospholipase Cγ (PLCγ)/calcineurin/nuclear factor of activated T cells (NFAT) signaling cascade, thereby inducing hypertrophic cell growth [21–23,25–27].
Surprisingly, recent studies have revealed that contrary to the established view, HS can be part of the FGF23:FGFR signaling complex and modulate FGF23-induced signaling and effects [28▪▪,29▪▪]. Furthermore, there is increasing experimental evidence that klotho can exist as a circulating co-receptor for FGF23:FGFR that mediates FGF23 effects in cells that do not express klotho [28▪▪,29▪▪]. Here, we discuss the roles of klotho and HS as soluble and circulating FGFR co-receptors, and how they might affect the physiologic and pathologic actions of FGF23.
FIBROBLAST GROWTH FACTOR 23 AND SOLUBLE KLOTHO
Klotho is a single-pass transmembrane that consists of two characteristic extracellular domains (KL1 and KL2) and a short cytoplasmic tail. Klotho can be glycosylated [30,31,32▪▪], and its ectodomain can be cleaved by proteases, generating a soluble fragment (soluble klotho, or sKL) that can be released from the kidney and detected in the circulation [30,33–36]. The dominant (and potentially only) form of sKL includes the entire ectodomain consisting of KL1 and KL2 with a molecular weight of about 130 kDa. Several in vitro studies have shown that membrane-associated klotho and sKL share a common function, in that both mediate FGF23-induced Ras/MAPK signaling [25,28▪▪,29▪▪,32▪▪,37–42]. A recent analysis of the crystal structure revealed that FGF23, FGFR1c and sKL form a protein complex, where sKL binds directly to both FGF23 and FGFR1c [28▪▪]. By doing so, sKL functions as a molecular scaffold that brings FGFR1c and FGF23 in close proximity, thereby conferring stability of the complex and promoting FGF23/FGFR-mediated signaling. sKL does not only exist in a complex with FGF23 and FGFRs, but can also bind FGFRs in the absence of FGF23 [29▪▪,43,44] as well as FGF23 in the absence of FGFRs [5,43]. Recent studies have identified two short motifs in the C-terminus of FGF23 that act as klotho binding sites [45▪,46▪]. Each site can independently bind FGF23, but both together act synergistically to achieve optimal FGFR binding. Overall, sKL can increase FGFR binding affinity of FGF23 in two ways: by first binding to FGFR and serving as a soluble FGFR co-receptor, or by first binding to FGF23 and serving as a circulating FGF23 binding partner. Either way, sKL mediates FGF23-FGFR binding in an FGFR isoforms-specific manner, with highest FGF23-sKL affinities for FGFR1c and FGFR4, lower affinity for FGFR3c, and no binding to FGFR2c (Fig. 1) [29▪▪,37,43,44]. Furthermore, in the absence of sKL, FGF23 can bind FGFR4 with low affinity [29▪▪,47,48], supporting the FGFR4-mediated actions of FGF23 elevations that have been reported in cells lacking klotho [22,49].
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FIGURE 1: sKL and HS facilitate FGF23 binding to specific FGFR isoforms. sKL and HS serve as independent co-receptors for FGF23, and they promote FGF23 binding to specific FGFR isoforms (FGFR1c, 2c, 3c, 4). In the absence of sKL and HS, FGF23 binds with low affinity to FGFR4. The presence of HS increases the binding affinity of FGF23 for FGFR4. In the presence of sKL, FGF23 binds with high affinity to FGFR1c and FGFR4, and with lower affinity to FGFR3c. FGF23, fibroblast growth factor 23; FGFR, fibroblast growth factor receptor; HS, heparan sulfate; sKL, soluble klotho.
Combined, these experimental studies suggest that sKL acts as a circulating, nonenzymatic scaffold protein and an on-demand bona fide co-receptor for FGF23 that facilitates the interaction between FGF23 and membrane-bound FGFRs on cell types that do not express klotho or that have lost klotho expression [19]. However, the biological relevance of these findings is not clear. Biochemical binding assays and cell culture studies use proteins at concentrations that are usually supra-physiological, and the formation of these protein complexes might not occur on the surface of cells or in the circulation. Since sKL has high affinity for FGFR1c [29▪▪], which is the major FGFR isoform that mediates the phosphaturic actions of FGF23 in the kidney [50–52], renal effects of sKL would be expected. Indeed, it has been shown that elevating serum sKL levels in wildtype mice by adeno-associated viral (AAV) overexpression of sKL in the liver or by injection of recombinant sKL protein increases Ras/MAPK signaling and reduces NaPi-2a expression in the kidney resulting in increased renal phosphate excretion and hypophosphatemia [28▪▪,38]. A recent study using a similar AAV approach confirmed that the elevation of sKL reduced serum phosphate levels in wildtype mice [53]. Furthermore, a single injection of recombinant sKL protein in healthy rats increased renal phosphate excretion and reduced serum phosphate levels within a few hours [54]. When conducted in mouse models with CKD or genetic klotho deficiency, sKL elevations by AAV delivery reduced renal NaPi-2a expression as well as serum phosphate to normal levels [39]. Frequent injections of the rat sKL protein into mice with genetic klotho deficiency also resulted in increased urinary phosphate excretion [55]. Similar effects on lowering serum phosphate levels have been observed in a mouse model of CKD receiving continuous infusions of recombinant sKL protein via osmotic minipumps [56]. Combined, these animal studies suggest that sKL can compensate for the reduction or loss of klotho that occurs in the context of CKD (Fig. 2). By binding to remaining FGFR1c molecules on proximal tubular epithelial cells, sKL might generate high-affinity FGF23 binding sites resulting in increased renal responsiveness to FGF23 and renal phosphate excretion even if functional kidney mass progressively declines (Fig. 3). It is possible that even in the healthy kidney circulating sKL binds to FGFR1c molecules on proximal tubular epithelial cells that are not in a complex with endogenous klotho, thereby generating additional high-affinity FGF23 binding sites and resulting in increased renal responsiveness to FGF23 in the absence of systemic FGF23 elevations. This hypothesis is supported by an in vivo study showing that sKL's interaction with FGF23 and FGFRs is required to mediate sKL effects, as an sKL mutant lacking the FGFR binding domain does not show phosphaturic activity when injected into mice [28▪▪]. This study also suggests that sKL only has FGF23-dependent effects [28▪▪]. The lack of a klotho-independent role of FGF23 in regulating phosphate homeostasis is also supported by other studies showing that mice lacking both FGF23 and klotho have the same phenotype as mice lacking FGF23 or klotho. This includes changes in mineral homeostasis, such as alterations in serum and urinary levels of phosphate and in renal expression of NaPi-2a/c, and the development of a bone phenotype [57,58].
FIGURE 2: sKL and HS might have various effects in CKD. In CKD, FGF23 responsiveness of the kidney is decreased, in part due to a reduction in renal klotho expression, and renal phosphate (Pi) excretion is impaired. This results in an elevation of serum Pi levels and increased production of FGF23 by the bone. The highly elevated serum FGF23 levels can directly target the heart and contribute to pathologic cardiac remodeling. The elevation of sKL, for example by injection of the recombinant sKL protein, might have various effects in CKD. It might restore FGF23 responsiveness of the kidney and thereby renal Pi excretion, leading to a reduction in systemic Pi levels. It might also block the direct pathologic actions of FGF23 on the heart. Furthermore, sKL might promote the autocrine effects of FGF23 in the bone, resulting in even higher FGF23 production. The increase in systemic HS levels, for example by heparin infusions during the hemodialysis process, might have various effects in CKD. It might aggravate the pathologic actions of FGF23 on the heart. Whether HS also promotes the autocrine effects of FGF23 in the bone and/or the effects of FGF23 on the kidney and renal Pi excretion is currently not known. Overall, it appears that sKL might have mainly protective effects in CKD, while HS might contribute to CKD-associated pathologies. CKD, chronic kidney disease; FGF23, fibroblast growth factor 23; FGFR, fibroblast growth factor receptor; HS, heparan sulfate; sKL, soluble klotho.
FIGURE 3: sKL has various targets and functions. sKL promotes FGF23 binding to specific FGFR isoforms resulting in distinct effects in specific tissues (green labeling). By binding FGFR1c on proximal tubular epithelial cells, sKL functions like klotho and increases FGF23-regulated phosphate excretion. By increasing the binding of FGF23 to FGFR4, sKL potentially alters FGFR4 activation and signaling and blocks the pathologic actions of FGF23 on the heart. sKL can also have more widespread actions (red labeling). sKL blocks the binding of paracrine FGFs to different FGFR isoforms and thereby inhibits various actions of paracrine FGFs, such as the induction of cell proliferation. sKL can also directly bind and block other ligand:receptor complexes, such as TGFβ1:TGFβR, and thereby inhibit their various actions, such as the induction of fibrosis. sKL might also have systemic actions by acting on many different, if not all, cell types (yellow labeling). sKL can act like a lectin and bind glycoproteins, glycolipids and monogangliosides on cell membranes. sKL might also have glycosidase activity and change the glycosylation pattern of many membrane molecules. Lectin and enzymatic activities of sKL could modulate the function and abundance of a wide spectrum of cell surface molecules, including receptors and transporters. CKD, chronic kidney disease; FGF23, fibroblast growth factor 23; FGFR, fibroblast growth factor receptor; HS, heparan sulfate; sKL, soluble klotho.
Of note, since experimental elevations of sKL are accompanied by increases in FGF23 production in bone [38,39,41], the observed sKL effects on renal phosphate excretion could be indirect and mediated by sKL-induced elevations in circulating FGF23 levels rather than sKL targeting FGFR1c in the proximal tubular cells and generating novel FGF23 binding sites (Fig. 2). A positive association between sKL and FGF23 was first indicated in a patient with hypophosphatemic rickets and unexplained elevations in FGF23, who turned out to carry a mutation in the klotho gene locus resulting in high serum sKL levels [59]. Furthermore, studies in cultured osteoblasts lacking endogenous klotho have shown that the co-treatment with sKL and FGF23 elevates FGF23 expression [60,61], suggesting that in the presence of sKL, FGF23 can increase its own expression in bone via a positive feedback loop, similar to what has been shown in cancer cells that express klotho [62]. Clearly, in vivo studies involving the tracing of sKL and analyzing the potential binding of sKL to renal FGFR1c will be required to determine if sKL can indeed target the kidney and induce the proposed FGFR1c-mediated effects on phosphate excretion.
Binding assays detected that sKL can also bind FGFR4 [29▪▪], which might have important implications for pathologies that are associated with elevated FGF23 levels and reduced klotho expression, such as CKD. In the absence of klotho, elevated FGF23 can directly bind FGFR4 with low affinity [29▪▪] and induce cardiac hypertrophy [22]. It was recently found that sKL directly inhibits the pathologic actions of FGF23 on cultured cardiac myocytes [29▪▪]. Although this finding might explain previous in vivo studies showing that elevating sKL levels in animal models of CKD has cardio-protective effects [41,56,63–65], the precise mechanism remains unknown (Fig. 2). Since sKL increases the affinity of FGF23 for FGFR4, sKL does not simply block the cardiac actions of FGF23 but rather promotes FGF23 binding to its FGFR4-expressing target cells in the heart (Fig. 3). As hypothesized before [19,41,66], sKL might induce a switch in FGF23/FGFR4-mediated signaling, from PLCγ/calcineurin/NFAT to Ras/MAPK signaling [25], and thereby from FGF23-induced pathologic to protective cardiac events. Alternatively, since sKL has higher affinity for FGFR1c than for FGFR4 [29▪▪], sKL might force FGF23 into FGFR1c binding and cardio-protective signaling and away from klotho-independent, pro-hypertrophic calcineurin/NFAT signaling that is mediated by FGFR4 [22,25]. Combined, these experimental findings should set the stage for preclinical CKD studies with the goal of testing potential cardio-protective effects of sKL. However, injection studies in rodents will require the production of large amounts of recombinant sKL protein with high bioactivity and stability which is currently challenging to do.
It has been shown that sKL binding to FGFRs not only enhances the affinity of FGFRs for FGF23, but concomitantly suppresses FGFR binding to certain paracrine FGFs [29▪▪,67,68] (Fig. 3). The physiologic consequences of sKL's inhibitory action on paracrine FGF signaling are not clear, but studies in cultured proximal tubular epithelial cells have shown that sKL binding to FGFR1c inhibits FGF2-induced signaling and thereby renal fibrosis [69,70]. Furthermore, the presence of sKL might dedicate all FGFRs in FGF23 target cells exclusively to FGF23 and avoid interference from paracrine FGFs. FGFR1c is widely expressed, and in many tissues, including the kidney, FGFR1c has the highest expression levels among all FGFR isoforms [71]. By having high binding affinity for FGFR1c, sKL might ensure proper inhibition of FGFR1c signaling driven by paracrine FGFs that is dominant in many cell types. Thereby sKL might not only mediate FGFR1c responsiveness to FGF23, but at the same time inhibit FGFR-mediated actions of paracrine FGFs on cells. This mechanism might ensure that in proximal tubular epithelial cells, the site of klotho-regulated phosphate reabsorption, FGF23 but none of the paracrine FGFs regulate FGFR1c-mediated phosphate uptake.
FIBROBLAST GROWTH FACTOR 23-INDEPENDENT EFFECTS OF SOLUBLE KLOTHO
An alternative model for sKL's function in mineral metabolism and its role as a hormone with pleiotropic, tissue-protective and antiaging effects is based on its intrinsic enzymatic activity as a glycosidase [10,54,72–74]. sKL might be capable of modulating the function and abundance of various molecules present on the cell surface, including receptors, ion channels, and transporters, by changing their glycosylation patterns [75] (Fig. 3). The KL1 and KL2 domains share amino acid sequence homology with β-glycosidases of bacteria and plants. However, the essential glutamate residue at the β-glycosidase active center is replaced with asparagine and alanine in KL1 and KL2, respectively. Indeed, a recent structural analysis of sKL combined with an in vitro assay to detect glycosidase, sialidase, and β-glucuronidase activities confirmed that the active sites of KL1 and KL2 are not accessible for substrates and that sKL lacks enzymatic activity [28▪▪]. Nevertheless, strong experimental evidence for FGF23-independent effects of sKL on regulating phosphate homeostasis comes from a study in FGF23 knockout mice, where a single injection of recombinant sKL protein increased renal phosphate excretion and reduced serum phosphate levels [54]. sKL might directly regulate NaPi-2a expression on proximal tubular epithelial cells in an FGFR-independent manner, but the mechanism is unknown. It is possible that although sKL has no glycosidase activity, it can still bind sialic acid via KL1 and KL2, thereby functioning like a lectin and targeting glycoproteins, glycolipids and monogangliosides on cell membranes [76] (Fig. 3). Since the binding affinity of each KL domain for sialic acid is low [77], sKL preferentially interacts with lipid raft domains where gangliosides are enriched. The association with sKL might then affect overall lipid raft dynamics and composition, thereby regulating the localization and activity of a variety of raft-associated molecules [78,79].
Interestingly, a recent study focusing on the production of recombinant sKL protein identified a structure−function relationship in sKL [32▪▪]. When expressed and purified from a cell line with low sialylation activity, sKL had high affinity for FGF23:FGFR1c and low glucuronidase activity. In contrast, when purified from a different cell line with high sialylation activity, sKL showed low affinity for FGF23:FGFR1c and high glucuronidase activity. This finding suggests that sKL can act as both an FGF23:FGFR1c co-receptor and a glucuronidase, and that these two functions might be structurally divergent. Posttranslational modifications might determine not only the degree but also the type of sKL bioactivity, and glycosylation of sKL might expose the active sites of KL1 and KL2 that are usually masked [28▪▪]. Such a mechanism would add another level of complexity to the regulation and to the functional variability of sKL. This study also indicates that sKL stability depends on its posttranslational modifications, including sialylation and N-glycosylation [32▪▪].
It has been shown that besides FGF23:FGFR, sKL can also directly bind other ligand/receptor complexes, such as insulin:IGF1:IGF1R, TGFβ1:TGFβR, AngII:AT1R, and Wnt:Frizzled [19,75] (Fig. 3). Klotho seems to have antifibrotic effects in the kidney [69,80], and one of the proposed mechanisms is the inhibition of TGFβ1 signaling [81]. It has been shown that a small domain within KL1 can bind to TGFβR and inhibit receptor activation on fibroblasts [82▪,83]. When injected as a peptide into mouse models of kidney injury, this KL1 fragment ameliorated renal fibrosis and preserved kidney function [82▪]. Furthermore, a different part of the KL1 domain seems to directly interact with Wnt and thereby blocks its binding to and the activation of its receptors Frizzled and LRP5/6 [84▪,85]. When injected as a peptide into mouse models of diabetic nephropathy, this KL1 fragment protects from glomerular injury and interstitial fibrosis [84▪].
FIBROBLAST GROWTH FACTOR 23 AND HEPARAN SULFATE
Paracrine FGFs bind HS, a linear glycosaminoglycan (GAG) composed of repeating disaccharide units of glucosamine and uronic acid which are highly sulfated and thereby negatively charged (3, 4). HS directly interacts with both FGFs and FGFRs and promotes the formation of a stable 1:1:1 FGF:FGFR:HS ternary complex as well as FGFR:FGFR dimerization, leading to the formation of a symmetric 2:2:2 FGF:FGFR:HS complex and the induction of downstream signaling [86–89]. Binding to HS also sequesters paracrine FGFs around cells, provides an extracellular reservoir for FGF storage, and enhances FGF stability and half-life (3). Overall, HS acts as a key mediator for the biological activity of all paracrine FGFs [2–4,67].
Surprisingly, the structural analysis of the FGF23:FGFR1c:sKL complex revealed the presence of HS [28▪▪]. Other studies have shown that FGF23 can directly bind HS in the absence of FGFRs [5,6], and that the preformed complex of FGF23 and HS can bind to FGFRs [29▪▪]. It is also known that HS can bind FGFRs in an isoform-specific manner in the absence of FGFs [90–92]. Therefore, it is possible that FGFR:HS complexes can serve as high-affinity docking sites for FGF23, which needs to be tested experimentally. The biological relevance of these findings is currently not clear. Previous studies in cell culture models suggest that in the absence of klotho, HS significantly increases the mitogenic response to FGF23 treatments [47,48]. Furthermore, in vitro studies in proximal tubular epithelial cells have shown that in order for FGF23 to achieve maximum effects on Ras/MAPK signaling and the inhibition of phosphate reabsorption, the co-treatment with HS is required [93,94]. Vice-versa, removal of GAGs from cultured proximal tubular epithelial cells causes a loss of FGF23 responsiveness [47]. These studies suggest that GAGs are part of the FGF23:FGFR1c:klotho signaling complex in the kidney that regulates phosphate excretion (Fig. 4). However, by using FGF23 mutant forms devoid of HS binding, it has been shown that HS is dispensable for the phosphaturic activity of FGF23 in mice (5), suggesting that HS is not a component of the endocrine FGF23/FGFR1c signal transduction unit. Clearly, while the crystal structure strongly suggests the presence of HS in the FGF23:FGFR1c:sKL complex [28▪▪], more in vivo studies are needed to determine the physiologic relevance of this complex. For example, is should be studied if HS elevations can increase FGF23-medited phosphate excretion by the kidney (Fig. 2). Furthermore, in vitro studies have shown that HS can mediate FGF23-induced Ras/MAPK signaling in cultured osteoblasts in the absence of klotho [42]. The in vivo relevance of this finding and HS-mediated FGF23/FGFR effects on bone need to be tested experimentally.
FIGURE 4: HS has various targets and functions. HS promotes FGF23 binding to specific FGFR isoforms resulting in distinct effects in specific tissues (green labeling). By increasing the binding of FGF23 to FGFR4, HS promotes the pathologic actions of FGF23 on the heart. Whether by binding FGFR1c:klotho complexes on proximal tubular epithelial cells, HS can increase FGF23 binding and phosphate excretion is currently unknown. HS can also have more widespread actions (red labeling). HS is required for the binding of paracrine FGFs to different FGFR isoforms and thereby promotes the various actions of paracrine FGFs, such as cell proliferation. Furthermore, heparin (a mixture of HS variants) has well established systemic effects (yellow labeling). Heparin directly binds antithrombin and accelerates its anticoagulation activity thereby blocking the formation of blood clots. CKD, chronic kidney disease; FGF23, fibroblast growth factor 23; FGFR, fibroblast growth factor receptor; HS, heparan sulfate; sKL, soluble klotho.
Overall, it appears that although the affinity of FGF23 for HS is much lower compared to paracrine FGFs, HS can still act as a co-factor for FGF23/FGFR binding. This effect of HS might depend on the FGFR isoform. Surprisingly, a recent binding study revealed that in the absence of sKL, HS increases the affinity of FGF23 for FGFR4 but not for FGFR1c [29▪▪] (Fig. 1). This study also indicates that HS might not only mediate the dimerization of the signaling unit, leading to the formation of a symmetric 2:2:2:2 FGF23−FGFR−sKL−HS quaternary complex and subsequent activation of Ras/MAPK signaling, as previously reported [28▪▪], but also support the preceding step by promoting FGF23 binding to FGFR [29▪▪]. The finding that HS specifically increases FGF23 binding to FGFR4 [29▪▪] might have important clinical implications, since FGFR4 mediates the pathologic actions of FGF23 on cardiac myocytes [22,25] (Fig. 4).
Heparin is a linear GAG structurally similar to HS, but with a higher degree of sulfation, that is used as an experimental proxy for HS in some of the studies described earlier. Heparin is also used clinically as a highly efficient anticoagulant drug [95]. Heparin forms a complex with antithrombin and accelerates its mode of action by 1000 times (Fig. 4). It has been recently shown that heparin promotes the direct actions of FGF23 on cardiac myocytes by enhancing contractility, dysregulating intracellular calcium, promoting arrhythmogenicity, and increasing hypertrophic cell growth [29▪▪]. Combined, these cellular alterations might result in accelerated pathologic cardiac remodeling, as supported by mouse models with systemic FGF23 elevations where frequent heparin injections aggravated the cardiac phenotype [29▪▪] (Fig. 2). ESRD patients frequently receive heparin infusions to prevent blood clotting during the hemodialysis process. Since hemodialysis does not reduce serum FGF23 levels [96], these patients are exposed to constant systemic elevations of both FGF23 and heparin. Hemodialysis does not reduce cardiovascular risk of ESRD patients, and it is possible that heparin infusions contribute to the extremely high mortality rates of hemodialysis patients. Based on the current experimental evidence, this hypothesis should be tested in clinical ESRD studies.
Unfractionated heparin, used clinically, is a mixture of polymorphic polysaccharide chains obtained after the purification of vertebrate organs, such as porcine intestine, and the manufacturing processes has not changed substantially since its introduction in the 1930s. Future studies should determine what components of heparin affect FGF23:FGFR binding and if those overlap with heparin's anticoagulation activity. To do so, the synthesis of specific HS variants is required, which has been challenging [97]. FGFs and FGFRs both can bind HS, but the different FGF and FGFR isoforms differ in their HS binding affinity and prefer to bind specific HS variants [4,6,98,99], which affects the FGF:FGFR binding specificity [90,100,101] and thereby FGF bioactivity [3,67]. It is possible that the presence of specific HS variants determines if a target tissue can recognize and respond to a certain FGF isoform [102,103]. Cell culture studies have shown that FGF23 has increased bioactivity in the presence of highly sulfated HS variants and in the presence of a specific sugar backbone [47]. Therefore, FGF23 responsiveness might be dependent on the specific GAG make-up of a cell type or tissue, and it is possible that by binding to specific HS variants, FGF23 can target specific tissues. Furthermore, FGF23 binding to HS in the extracellular environment might result in high local concentrations of FGF23 and in tissue levels that might be even higher than FGF23 concentrations in the circulation.
CONCLUSION
Recent studies have found that sKL and HS act as independent co-receptors for FGF23/FGFR binding. HS increases the binding affinity of FGF23 for FGFRs to a lower extent than sKL. Both co-receptors can bind FGFRs in the absence of FGF23 or bind FGF23 in the absence of FGFRs. By doing so, sKL and HS can activate FGF23/FGFR signaling, but they differ in their FGFR specificity. While sKL mediates FGF23 binding to various FGFR isoforms, HS seems to mainly facilitate the FGF23 interaction with FGFR4. Current experimental findings indicate that sKL and HS might have major effects in scenarios of FGF23 elevation, such as CKD, by reducing and aggravating tissue injury, respectively. However, these findings still need to be evaluated in preclinical and clinical studies.
It is possible that more than one sKL variant as well as more than one mode of sKL action exist. Many previous studies in the field ignored this complexity and simply used sKL proteins that were commercially available, thereby ignoring potential differences in the effects of variants and posttranslational modifications, as well as the extremely low half-life of sKL. Overall, it appears that sKL has FGF23-dependent and FGF23-independent actions, and that some of these effects are caused by an intrinsic enzymatic activity in sKL whereas others are based on sKL binding to specific targets. Similarly, HS exists in different variants with complex structures that differ in sizes and charges. Clearly, more experimental studies are needed to better characterize the various functions of FGF23's co-receptors sKL and HS which exist in many different forms.
Acknowledgements
All figures were created with BioRender.com.
Financial support and sponsorship
C.F. was supported by grants R01HL145528 and R01DK125459 from the NIH, and by the UAB-UCSD O’Brien Core Center for Acute Kidney Injury Research, the AMC21 program of the Department of Medicine at UAB, and the Tolwani Innovation Award from the Division of Nephrology at UAB.
Conflicts of interest
C.F. has served as a consultant for Bayer and Calico Labs, and he is an inventor on two pending patents (PCT/US2019/049211; PCT/US19/49161) aimed to produce and detect bioactive sKL. He is a co-founder of a startup biotech company (Alpha Young LLC) that has the goal to further develop and commercialize these products and assays. He is currently the CSO of Alpha Young LLC. He has a patent on FGFR inhibition (European Patent No. 2723391). M.T. and Q.L. have declared that no conflict of interest exists.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
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