Part One Fibroblast Growth Factor 23 And Osteoporosis: Evidence From Bench To Bedside

Abstract

Osteoporosis is a chronic debilitating disease caused by imbalanced bone remodeling processes that impair the structural integrity of bone. Over the last ten years, the association between fibroblast growth factor 23 (FGF23) and osteoporosis has been studied in both pre-clinical and clinical investigations. FGF23 is a bone-derived endocrine factor that regulates mineral homeostasis via the fibroblast growth factor receptors (FGFRs)/αKlotho complex. These receptors are expressed in the kidney and the parathyroid gland. Preclinical studies have supported the link between the local actions of FGF23 on the bone remodeling processes. In addition, clinical evidence regarding the effects of FGF23 on bone mass and fragility fractures suggests potential diagnostic and prognostic applications of FGF23 in clinical contexts, particularly in the elderly and patients with chronic kidney disease. However, inconsistent findings exist and there are areas of uncertainty requiring exploration. This review comprehensively summarizes and discusses preclinical and clinical reports on the roles of FGF23 on osteoporosis, with an emphasis on the local action, as opposed to the systemic action, of FGF23 on the bone. Current gaps in knowledge and future research directions are also suggested to encourage further rigorous research in this important field.

Keywords

FGF23; osteoblast; osteoclast; biochemical markers; bone remodeling; osteoporosis

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Introduction

Osteoporosis is a chronic debilitating disease caused by an imbalance in bone remodeling processes that favor bone resorption over bone formation. The structural integrity of the bone is maintained through intricate and interrelated activities of bone-forming osteoblasts, bone-resorbing osteoclasts, physicochemical conditions that modulate local matrix mineralization, and systemic mineral homeostasis. Over the last ten years, the association between fibroblast growth factor 23 (FGF23) and osteoporosis has been studied [1–4]. FGF23 is a bone-derived endocrine factor that regulates phosphate and vitamin D homeostasis via the fibroblast growth factor receptors (FGFRs)/αKlotho complex. These receptors are expressed in the kidney and the parathyroid gland [5–7]. The FGF23-mediated mechanism interacts with the classical calcium/phosphate regulating processes driven by parathyroid hormone (PTH) and calcitriol (active vitamin D). In addition to the systemic effects of FGF23, preclinical studies have revealed mechanistic insights into the local actions of FGF23 on bone remodeling processes [8,9]. Moreover, accumulating evidence from clinical studies reported the association between FGF23, bone remodeling, and fragility fracture in the elderly, either with or without a decline in renal function. These recent advances in the insights regarding FGF23's effects on mineral homeostasis and bone remodeling suggest potential clinical applications of FGF23 in the clinical context. However, inconsistent findings exist and there are areas of uncertainty requiring exploration. Thus, this review comprehensively summarizes preclinical and clinical reports of the roles of FGF23 on osteoporosis and chronic kidney disease–mineral and bone disease (CKD-MBD), with an emphasis on the local actions, as opposed to systemic actions, of FGF23 on the bone. The potential use of FGF23 as a biomarker for osteoporosis, CKD-MBD, and fragility fracture prediction is also discussed. Finally, current gaps in knowledge and future research directions are also suggested to encourage further rigorous research in this important field.

Regulation of FGF23 Expression

FGF23 is mainly produced by osteoblasts and osteocytes [10–12]. In those cell types, FGF23 expression is stimulated by the calcitriol (via the vitamin D receptor [VDR] signaling pathway) and hyperphosphatemia (via the sensing of extracellular phosphate levels mediated by type III sodium-dependent phosphate transporter [PiT2]) [9,13] and FGFR1c [14]. There are two possible mechanistic links of high extracellular phosphate (Pi) regulating FGF23 secretion via FGFR1c and PiT2 actions. For the first hypothesized mechanism, Pi-dependent stimulation of the ERK/MAPK pathway via FGFR1c can promote GALANT3 expression, preventing intact FGF23 (iFGF23) cleavage by O-glycosylation [14]. However, the mechanisms of FGFR1c activation by extracellular phosphate remain unclear. Another study proposed the mechanism in which high extracellular phosphate acts on osteocytes/osteoblasts via the PiT2 sensors, increasing iFGF23 levels in FAM20C (promoting FGF23 cleavage by phosphorylation at cleavage site) and GALANT3 independent mechanisms [13]. Furthermore, there was no significant change in Pi-dependent FGF23 secretion when the ERK/MAPK pathway was inhibited. It is possible to conclude that the physiological link between Pi-dependent activation and FGF23 secretion is mostly dependent on PiT2 action through other downstream signaling pathways other than ERK/MAPK. As discussed, the mechanistic understanding of Pi-dependent FGF23 regulation via PiT2 sensing remains to be explored.

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FGF23 Effects on Mineral Homeostasis

Circulating FGF23 acts on the FGFRs/αKlotho complex (particularly those containing the FGFR subtypes 1, 3, or 4) on renal proximal tubular cells [6], and parathyroid cells [7,15]. In the kidney, increasing FGF23 levels causes hypophosphatemia and decreased calcitriol levels. In the proximal tubules, FGF23 reduces phosphate reabsorption by suppressing the expression of sodium-dependent phosphate co-transporter type II a (NaPi-2a) [5,6,9]. Vitamin D metabolism is also under tight regulation by FGF23, which downregulates 1α-hydroxylase and upregulates 24-hydroxylase in the proximal tubular cells [5,6,9]. Consequently, hypophosphatemia and decreased calcitriol levels inhibit bone-derived FGF23 secretion as a negative feedback control by diminishing VDR and extracellular phosphate signaling in the bone. In the parathyroid gland, PTH regulation is another important pathway for bone-mineral homeostasis. The suppression of PTH by FGF23 is primarily mediated by a Klotho-dependent mechanism through the MAPK/ERK signaling pathway [7,15] and a Klotho-independent mechanism via the calcineurin signaling pathway [16]. Even though FGF23 significantly inhibits PTH production and secretion, the dominant regulators of PTH levels remain at circulating free (ionized) calcium and calcitriol levels, which are monitored by the calcium-sensing receptor (CaSR) and the VDR in the parathyroid gland [17,18]. Subsequently, low calcitriol levels and hypocalcemia induced by increasing FGF23 levels indirectly stimulate PTH synthesis and release in the parathyroid via VDR and CaSR, overriding the inhibitory action of FGF23. Thus, elevated PTH can counterbalance the calcium-lowering effect of FGF23 by increasing calcium resorption from bone [5]. The physiological actions of FGF23 on mineral homeostasis are summarized in Figure 1 and Supplementary Materials Table S1.

Figure 1. FGF23 regulation and its interaction with the traditional paradigm on mineral homeostasisFGF23 is mainly produced by osteoblasts and osteocytes, which is stimulated by calcitriol via the VDRsignaling pathway and hyperphosphatemia via sensing of extracellular phosphate levels mediated by PiT2 (dominant pathway) and FGFRlc. In proximal tubular cells, FGF23 binding with the theaKlotho/FGFR complex causes hypophosphatemia by inhibiting NaPi-2a and decreasing calcitriol levels by suppressing the vitamin D activation process. Decreased calcitriol also reduces calcium and phosphate absorption from the Gl tract. In parathyroid cells, PTH secretion is dominantly controlled by circulating calcium via CaSR, which overrides the inhibitory effect of FGF23. Increased PTHinduces calcium resorption from bone and also causes hypophosphatemia by inhibiting NaPi-2aactivity. Consequently, hypophosphatemia and decreased calcitriol levels will act as the negative feedback control of FGF23 production by diminishing VDR, PiT2, and FGFRlc signaling. This figure was generated with publication licensed by BioRender, Toronto, ON, Canada (Agreement number: VZ237SO181, 19 November 2021). Abbreviations: CaSR, Calcium-sensing receptor; EP, ExtracellularaKlotho; ERK1/2, Extracellular signal-regulated kinases; FL, Full-length aKlotho; FGF23, Fibroblast growth factor 23; FGFR, Fibroblast growth factor receptor; GALNT3, polypeptide N-acetyl.

Given the fact that human mineral homeostasis is mostly dependent on the kidney loss of renal function in CKD and ESRD has a significant impact on mineral homeostasis, including hyperphosphatemia, hypocalcemia, and lowered calcitriol. Subsequently, FGF23levels rise early and steadily with the progression of kidney function in the early stages of CKD as a physiologic compensation to maintain normal phosphorus balance by enhancing urinary phosphate excretion in conjunction with indirectly increased parathyroid hormone levels and decreasing gut phosphorus and calcium absorption through decreased calcitriol synthesis. In the late stages, this compensatory mechanism may become maladaptive from the loss of renal functions, resulting in a progressive increased FGF23 level, decreased calcitriol levels, hypocalcemia, and associated consequences such as CKD-MBD from secondary hyperparathyroidism. Excess FGF23 levels in CKD/ESRD also contribute directly to bone remodeling and mineralization. The details of FGF23's effects on the pathogenesis of CKD-MBD will be discussed later.

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Local Effects of FGF23 on Osteoblast and Bone Formation

Previous experimental studies have demonstrated that bone-derived FGF23 affects bone remodeling. Under physiologic conditions, an in vitro study of mouse osteoblasts showed that increased FGF23 protein expression was associated with peaked osteoblastic activity and increased ALP bone nodules. During the same period, increased OPN expression was also observed that might relate to increased FGF23 [10]. This study also showed that increasing calcitriol caused FGF23 upregulation and matrix mineralization inhibition in a dose-dependent fashion. This result raises the possibility that FGF23 regulates bone mineralization by controlling OPN expression. These findings were consistent with results from another in vitro study which showed that treating mouse MSCs with a physiologic concentration of FGF23 promoted osteoblast differentiation and activity by up-regulating OC and ALP, and OPN in a dose-dependent manner [12]. Inhibition of the FGF23-FGFRs-αKlotho pathway caused diminished osteoid nodule formation, reduced expression of osteoblast markers, and OPN. This finding supports the existence of an auto-/paracrine effect of FGF23 via the FGFRs-Klotho complex.

According to recent preclinical evidence, auto-/paracrine effects of FGF23 on bone mineralization have been uncovered in Klotho and a calcitriol-independent manner [19]. FGF23 action on FGFR3 indirectly regulated bone mineralization by suppressing tissue nonspecific alkaline phosphatase (TNAP), whereas calcitriol-VDR action directly increased OPN transcription in osteoblasts. This study revealed the mechanistic insight of FGF23's impacts on bone mineralization through two key players: pyrophosphate (PPi) and inorganic phosphate, but not directly via OPN transcription. According to well-established evidence, PPi is synthesized intracellularly by ectonucleotide pyrophosphatase/phosphodiesterase 1 and 3 (ENPP1 and ENPP3) [20], then transported to the extracellular matrix (ECM) via the transmembrane protein ANK [21]. PPi can inhibit hydroxyapatite crystal formation and deposit on type I collagen [22,23]. Increased levels of PPi in the ECM, therefore, inhibit bone mineralization through the same mechanism as OPN activity [24]. Consequently, PPi in the ECM is hydrolyzed into inorganic phosphate by tissue nonspecific alkaline phosphatase (TNAP) [25]. Inorganic phosphate is a major component of hydroxyapatite crystals and an established stimulator of OPN secretion by an unknown mechanism [26]. In this study, the supraphysiological concentration of FGF23 showed an inhibitory effect on TNAP activity via the FGFR3-ERK pathway, thereby increasing extracellular PPi and decreasing extracellular inorganic phosphate concentration, consequently decreasing OPN expression regardless of Klotho status. Additionally, the ALP activity of differentiated osteoblasts from both wild-type and Klotho knock-out mice was significantly suppressed by FGF23 in a dose-dependent manner. Nevertheless, the Klotho-independent FGF23 action was found only under the supra physiologic condition (20 times of physiological FGF23 level). The aforementioned study showed that treating mice MSCs with physiologic concentrations of FGF23 promoted osteoblast differentiation and activity by dose-dependently up-regulating OC and ALP, whereas the Klotho knock-out model did not alter the expression of osteoblastic biomarkers in the same experiment [12]. These contradictory results of FGF23's effects on osteoblast differentiation and activity could be explained by the following explanations. Increased osteoblast differentiation and activity at physiological FGF23 levels may be a response to the remodeling balance shifting toward bone formation as a result of decreased bone mineralization. Another possibility is that FGF23 has bimodal effects in physiological and supraphysiological conditions. In physiological circumstances, FGF23 may act via the FGFRs-Klotho complex to promote osteoblast differentiation and activity, whereas supraphysiological FGF23 levels decrease bone formation by inhibiting bone mineralization and osteoblast activities in a Klotho-independent pathway. These results suggest possible roles of FGF23 in bone formation, as illustrated in Figure 2 and summarized in Supplementary Materials Table S2.

Figure 2. Regulation of FGF23 and its autocrine/ paracrine effects on bone formation. In supraphysiologic conditions, FGF23 acts directly on FGFR3 in a Klotho-independent manner, thereby inhibiting bone formation. Increased FGF23 suppresses differentiated osteoblast activity and TNAP transcription, which subsequently causes PPi accumulation in the ECM and inhibits matrix miners. realization. In physiological conditions, the actions of FGF23 on canonical receptors (FGFRs-Klothocomplex) also downregulate TNAP, decreasing matrix mineralization. However, the upregulation of osteoblastic markers in these conditions may be caused by the shifting of remodeling balance toward bone formation or the direct action of FGF23 via canonical receptors. The symbol "?" and dash lines denote issues of controversy and unknown mechanisms, respectively. This figure was generated with publication licensed by BioRender, Toronto, ON, Canada (Agreement number: VC237SOKSX19 November 2021). Abbreviations: BALP, Specific bone Alkaline phosphatase; FGF23, Fibroblast growth factor 23; Pi, inorganic phosphate; PPi, Pyrophosphate; Runx2, Runt-related transcription factor 2; TNAP, Tissue nonspecific alkaline phosphatase; OC, Osteocalcin.

Local Effects of FGF23 on Osteoclast and Bone Resorption

Two studies evaluated the association between FGF23 and osteoclasts [9,27]. From the first study [9], stimulating VDR signaling by adding calcitriol to the culture media in the VDR knock-out mouse chondrocyte/osteoblast co-culture model resulted in decreasedFGF23 and an absence of RANKL expression without any alteration of other osteoblast markers (OC, Runx2, and OPG). In contrast, the wild-type model in the same experiment resulted in increased RANKL expression and non-significant increased FGF23. FGF23 expression was unchanged either in the wild-type or VDR knock-out osteoblast culture these findings suggest that chondrocyte VDR signaling regulates FGF23 expression, not osteoblast VDR. A decrease in osteoclastogenesis due to the lack of VDR signaling, thereby resulting in an increasing ratio between total bone volume and trabecular bone in this study, might be mediated by FGF23. However, the hypothesis that FGF23 may be a downstream effector of the chondrocyte VDR signaling pathway which influences RANKL expression requires further confirmation. The second in-vitro study directly investigated the association between FGF23 and osteoclasts in human monocyte-derived osteoclast cultures treated with FGF23 [27]. This study showed that biphasic physiological FGF23 effects via FGFR inhibited the early stages of osteoclastogenesis in human monocytes but marginally increased osteoclast-mediated bone resorption. However, the mechanistic link between local FGF23 actions on osteoclast biology remains unclear. These results suggest possible roles of FGF23 in bone remodeling by regulating osteoclastogenesis and osteoclast-mediated bone resorption, as illustrated in Figure 3 and summarized in Supplementary Materials Table S2. The mechanistic link between increased FGF23 and bone remodeling reported in these preclinical studies led to clinical investigations into the association between FGF23 and osteoporosis in the elderly, CKD, or end-stage renal disease (ESRD) patients, who commonly have elevated levels of FGF23, as discussed later.

Figure 3. Regulation of FGF23 and its autocrine/paracrine effects on osteoclast and bone resorption. A decrease in osteoclastogenesis by downregulating RANKL expression may be mediated by FGF23 via an unknown mechanism. The biphasic physiological effects of FGF23 via FGFR on human monocyte-derived osteoclast cultures inhibit the early stages of osteoclastogenesis from osteoclast progenitors but substantially inhibited osteoclast-mediated bone resorption. However, the hypothesis that FGF23 may influence RANKL expression, osteoclastogenesis, and osteoclast-mediated bone resorption requires further confirmation. The symbol “?” and dash lines denote issues of controversy and unknown mechanisms, respectively. This figure was generated with publication licensed by BioRender, Toronto, ON, Canada (Agreement number: NS237SOEQK, 19 November 2021). Abbreviations: FGF23, Fibroblast growth factor 23; RANK, Receptor activator of nuclear factor-κB; RANKL, Receptor activator of nuclear factor-κB ligand.

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Role of FGF23 in Postmenopausal and Age-Related Osteoporosis Pathogenesis

Osteoporosis is primarily caused by an imbalance in the remodeling process. In the early stages of postmenopausal osteoporosis, a deficiency of estrogen induces an increase in RANKL expression, which results in increased osteoclast numbers and activity as well as concurrent suppression of osteoblast functions. Subsequently, increased net bone resorption outpacing bone formation caused rapid loss of mainly trabecular bone mass. As previously discussed, FGF23 inhibits bone mineralization by suppressing TNAP and resulting in PPi accumulation in both physiological and supraphysiological conditions. Thus, increased FGF23 could accentuate the net negative remodeling by inhibiting bone formation. Although some studies found that physiological concentration of increased FGF23 was associated with increased osteoblast differentiation and bone nodule formation, these might not be direct actions of FGF23 on osteoblasts but could be a compensatory response to the inhibition of bone formation. The auto-/paracrine inhibitory effect of FGF23 is also confirmed by the study that indicated that excessive levels of FGF23 decrease ALP activity in differentiated osteoblasts, whereas the absence of FGF23 signaling results in a Klotho-independent, cell-autonomous increase in ALP activity. Moreover, FGF23 may regulate the bone remodeling balance by inhibiting the early stages of osteoclastogenesis from osteoclast progenitors and promoting osteoclast-mediated bone resorption.

In the second longer phase (age-related osteoporosis), the impaired bone quality is the gradual loss of minerals from bones with aging, which are also influenced by other age-related conditions, such as deconditioning or frailty, vitamin D deficiency, and secondary hyperparathyroidism. An imbalance of systemic mineral homeostasis in the elderly particularly impaired renal function, hyperphosphatemia, and additionally led to an increase in FGF23 secretion, resulting in a progressive decrease in bone mineralization. Nevertheless, bone fragility and fragility fracture in osteoporosis are influenced by multiple risk factors including genetic features, the level of weight-bearing physical activity, nutrition, smoking, body mass index (BMI), concurrent diseases, and medications. The reported clinical evidence for an independent association between FGF23 and bone fragility in postmenopausal and age-related osteoporosis has been comprehensively summarized below.

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The Association between FGF23 and Bone Fragility in the Elderly

As discussed previously, preclinical reports have demonstrated the possible effect of FGF23 on osteoporosis pathogenesis. Even though increased FGF23 showed a promising inhibitory effect on bone mineralization in preclinical studies, there was not enough supporting clinical evidence to establish a robust causal relationship between FGF23 and BMD, which is a major predictor of bone fragility. The large cohort of males with osteoporosis revealed only a weak association between FGF23 and bone mineral density (BMD) that diminished when adjusting for potential confounders, including, age, height, weight, and smoking [2]. Another study in men with osteoporosis also showed that femoral neck BMD was not significantly changed in different FGF23 quartiles [28]. In contrast, the studies in post-menopausal women found that FGF23 had a strong negative correlation with BMD [1] and a significant association with femoral BMD after adjusting for PTH, 25(OH)D, and leptin [4]. Furthermore, another cross-sectional study in osteoporosis patients, in which the majority are women, demonstrated a weak independent association between high FGF23 and deceased trabecular bone microarchitecture but not cortical bone [3]. However, this evidence was insufficient to prove that elevating FGF23 had a clinically significant effect on decreasing BMD in women. The paucity of an independent association between FGF23 level and age-related osteoporosis suggests that an autocrine/paracrine effect of FGF23 does not play a significant role in the pathogenesis of age-related osteoporosis.

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Wachiranun Sirikul 1 , Natthaphat Siri-Angkul 2,3,4, Nipon Chattipakorn 2,3,4 and Siriporn C. Chattipakorn 2,4,5,

1 Department of Community Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; wachiranun.sir@cmu.ac.th

2 Neurophysiology Unit, Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; natthaphat.s@cmu.ac.th (N.S.-A.); nipon.chat@cmu.ac.th (N.C.)

3 Cardiac Electrophysiology Unit, Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

4 Center of Excellence in Cardiac Electrophysiology Research, Chiang Mai University, Chiang Mai 50200, Thailand

5 Department of Oral Biology and Diagnostic Sciences, Faculty of Dentistry, Chiang Mai University, Chiang Mai 50200, Thailand