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Molecular mechanisms underlying the complications of X-Linked Hypophosphatemia: Comparison
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Please note this is a comparison between Version 2 by Lindsay Dong and Version 3 by Lindsay Dong.

X-linked hypophosphatemia (XLH) is characterized by mutations in the PHEX gene, leading to elevated serum levels of FGF23, decreased production of 1,25 dihydroxyvitamin D3 (1,25D), and hypophosphatemia. Those affected with XLH manifest impaired growth and skeletal and dentoalveolar mineralization as well as increased mineralization of the tendon–bone attachment site (enthesopathy), all of which lead to decreased quality of life.

  • : XLH
  • rickets
  • mineralization
  • growth
  • enthesopathy
  • osteocyte
  • canaliculi
  • lacunae

1. Introduction

X-Linked Hypophosphatemia (XLH) is the most common inheritable form of rickets (1:20,000) caused by an X-linked dominant inactivating mutation of the Phosphate Regulating Endopeptidase Homolog, X-Linked (PHEX) gene [1][2][3][4]. PHEX-inactivating mutations lead to high circulating levels of fibroblast growth factor 23 (FGF23), which results in hypophosphatemia, impaired production of the active form of vitamin D, 1,25 dihydroxyvitamin D3 (1,25D) [5], and a mild elevation in serum parathryoid hormone (PTH) levels. Children with XLH manifest symptoms by 1–2 years of age, including rickets, bone deformities, and dental caries, while adults develop osteomalacia, osteoarthritis, and painful mineralizations of the bone–tendon attachment site (enthesis), which is called enthesopathy [6][7]. Both affected males and females present with similar biochemical characteristics and as children do not differ in height [8].

1.1. Phosphate Regulating Hormones: 1,25 Dihydroxyvitamin D3 (1,25D) and FGF23

Vitamin D3 (cholecalciferol) is synthesized from 7-dehydroxycholesterol in the skin when exposed to UV-B light. In the liver, cholecalciferol is hydroxylated to form 25-hydroxyvitamin D3 (25(OH)D), which is the predominant circulating form of vitamin D. Bound to the vitamin D binding protein, 25(OH)D is transported to the kidney, and in the proximal renal tubule, 25(OH)D is further hydroxylated to form the biologically active form of vitamin D, 1,25D, by vitamin D 1-alpha-hydroxylase (CYP27B1) [9][10]. The biological effects of 1,25D are mediated through the vitamin D receptor (VDR), which is a nuclear steroid receptor. 1,25D binds to VDR, upon which VDR forms an obligate heterodimer with retinoic X receptor (RXR) and binds to target genes via two cysteine-rich zinc finger structures [9][11][12]. 1,25D acts to regulate calcium and phosphate homeostasis by promoting bone resorption as well as intestinal absorption and renal reabsorption of calcium and phosphate [9]. 1,25D also acts to decrease parathyroid hormone gene transcription and parathyroid cell proliferation, thus leading to decreased serum levels of PTH and increased renal reabsorption of phosphate [13][14]. FGF23 is a ligand hormone synthesized predominantly in osteocytes and osteoblasts to regulate phosphate homeostasis [15]. FGF23 decreases serum phosphate levels by directly increasing renal phosphate wasting and indirectly decreasing phosphate gastrointestinal absorption [16][17]. FGF23 increases phosphate renal excretion by downregulating the expression of sodium–phosphate co-transporters NPT2a and NPT2c in the renal proximal tubules [18][19]. This phosphate-regulating hormone also decreases the expression of CYP27B1 and enhances the expression of CYP24A1, which catabolizes 1,25D, resulting in decreased serum levels of 1,25D. This decrease in 1,25D synthesis in turn leads to the suppression of intestinal absorption and renal reabsorption of phosphate [20][21].

1.2. Treatment of XLH

Conventional therapy for XLH patients includes a combination of exogenous 1,25D (calcitriol) and phosphate supplements. Excessive phosphate supplementation leads to secondary or tertiary hyperparathyroidism; thus, 1,25D therapy is added to phosphate supplementation not only to increase serum phosphate levels but to also prevent secondary/tertiary hyperparathyroidism [6]. Recently, a humanized antibody targeting FGF23, burosumab (Crysvita), was approved for the treatment of children and adults with XLH. Burosumab increases and sustains serum phosphate levels in the low–normal range while maintaining normal serum and urine calcium and serum PTH levels [22][23][24]. Although one of the advantages of burosumab is its ability to block FGF23-specific actions in addition to improving phosphate homeostasis, it is not able to sustain increased serum 1,25D levels during long-term treatment [22][25][26]. Clinical trials have demonstrated that burosumab significantly improves rachitic growth plate changes and mildly improves growth compared to traditional supplementation treatment [26]. In adults with XLH, burosumab improved pseudofracture healing, decreased osteomalacia, and improved quality of life [22][27]. Data on the effects of burosumab on chronic complications of XLH like enthesopathy and osteoarthritis are unknown.

2. Impairment of Growth and Growth Plate Maturation

2.1. Growth in XLH

Individuals with XLH exhibit short stature and disproportionate growth characterized by a longer torso and shorter long bones [28][29]. The rickets observed in XLH is associated with delayed ambulation, deformities of the weight-bearing lower limbs (varus or valgus deformities), and waddling gait [30]. Pediatric patients with XLH demonstrate normal length/height at birth, but growth velocity decreases during infancy/early childhood by one year of age and then progressively decelerates to a nadir in early childhood and remains abnormally low thereafter [31][32][33]. The final height of adults with XLH is significantly shorter than unaffected individuals, where affected adults have an average z score of −1.9 [28]. Growth plate abnormalities are evident in rapidly growing long bones of children affected with XLH, including the distal femur, radius and ulna and proximal and distal tibia, and distal radius and ulna. Radiographs of long bones demonstrate widened growth plates with irregular metaphyseal margins. There may also be fraying and/or cupping of the metaphyses. The degree of growth retardation correlates with the severity of the growth plate abnormalities [34].

2.2. Growth Plate Abnormalities in XLH

Growth plate maturation is essential to longitudinal bone growth. During this process, proliferative chondrocytes differentiate into prehypertrophic chondrocytes and then finally hypertrophic chondrocytes [35]. Hypertrophic chondrocytes are characterized by the expression of type X collagen and a widespread increase in cell volume. They promote the vascularization of the cartilage template by secreting molecules such as vascular endothelial growth factor (VEGF), mineralization of the adjacent skeletal matrix, and osteoblast differentiation from nearby perichondrial cells [36]. Upon terminal differentiation, hypertrophic chondrocytes undergo apoptosis, allowing for osteoblasts to invade and promote mineralization of the primary spongiosa [37].

2.3. Molecular Mechanisms of Impaired Growth in XLH

Investigations into the pathophysiology of the growth plate abnormalities of XLH have demonstrated that hypophosphatemia leads to the impaired apoptosis of terminally differentiated hypertrophic chondrocytes, resulting in the expansion of the hypertrophic chondrocyte layer of the growth plate [38][39][40][41]. Normalization of serum calcium and phosphate levels in mice lacing the vitamin D receptor (VDR) prevented rickets, while Hyp mice and mice fed a low-phosphate diet (both of which have low serum phosphate) continue to have expansion of the hypertrophic chondrocyte layer and impaired hypertrophic chondrocyte apoptosis. Hypophosphatemia is present in all of these murine models of rickets, demonstrating that phosphate is critical for normal growth plate maturation and hypertrophic chondrocyte apoptosis [38]. Raf kinases, including A-, B-, and C-Raf, activate MEK/ERK signaling. Proliferative chondrocytes primarily express A-Raf and B-Raf, where the ablation of either Raf isoform does not alter normal growth plate maturation [42]. Hypertrophic chondrocytes predominantly express C-Raf [43], where the deletion of C-Raf in chondrocytes leads to an expansion of the hypertrophic layer of the growth plate with an impaired induction of p-ERK1/2, hypertrophic chondrocyte apoptosis, and vascular invasion at the chondroosseous junction [44]. In cultured primary hypertrophic chondrocytes, the deletion of C-Raf alone is not sufficient to impair phosphate-induced ERK1/2 phosphorylation [45]. However, ablation of all three Raf isoforms in primary chondrocytes led to impaired phosphate-induced p-ERK1/2 in vitro. Corresponding with this, deletion of all three Raf isoforms in the chondrocytes of mice leads to a significant expansion of the hypertrophic chondrocyte layer[45]. In the growth plate, periarticular chondrocytes secrete parathyroid hormone-related peptide (PTHrP), which acts through the PTH/PTHrP receptor (PPR) on proliferating chondrocytes to stimulate their proliferation and inhibit their differentiation [35][46][47]. Studies demonstrated that the impaired chondrocyte differentiation and decreased p-ERK1/2 observed in the chondrocytes of embryonic murine metatarsal treated with low phosphate media requires PTHrP action [48]. Furthermore, mice haploinsufficient for PTHrP fed a low-phosphate diet did not have an expansion of the hypertrophic chondrocyte layer and decreased p-ERK1/2 and hypertrophic chondrocyte apoptosis as seen in WT mice fed a low-phosphate diet [48]. These studies indicate that PTHrP action contributes to the impaired chondrocyte differentiation and growth plate abnormalities seen with hypophosphatemia and thus in mice and humans with XLH. In vitro studies in primary murine hypertrophic chondrocytes showed that PTH pretreatment impairs mitochondrial phosphate-induced ERK1/2 phosphorylation and the subcellular redistribution of apoptosis-regulating proteins p-BAD and BAD, supporting the hypothesis that PTH action impairs hypertrophic chondrocyte apoptosis [48].  In addition to PTHrP, 1,25D has also been shown to play a role in the growth plate abnormalities of Hyp mice. Deletion of the sodium-dependent phosphate transporter 2a (NPT2a), which enables phosphate resorption in the renal proximal tubules, in mice leads to hypophosphatemia, which in turn results in suppressed PTH levels and high serum 1,25D levels with hypercalcemia [49]. In contrast to Hyp mice, NPT2aKO mice develop an expansion of the hypertrophic chondrocyte layer of the growth plate at 2 weeks of age, where this growth plate expansion normalizes by 5 weeks of age despite persistent hypophosphatemia [41]. This improvement in growth plate maturation corresponds with increased serum levels of 1,25D. Ablation of the vitamin D receptor (VDR) in NPT2aKO mice resulted in persistent expansion of the hypertrophic chondrocyte layer, thus demonstrating that enhanced 1,25D action can compensate for hypophosphatemia during growth plate maturation and prevent rickets [41]. In further support for a role for 1,25D actions in growth plate maturation, the treatment of primary murine hypertrophic chondrocytes with 1,25D further enhanced phosphate-induced pERK1/2 and the treatment of Hyp mice with 1,25D alone improved growth and growth plate morphology more than treatment with an antibody targeting FGF23 (FGF23Ab), indicating 1,25D and phosphate play roles in enabling hypertrophic chondrocyte apoptosis. The results of these studies imply that the impaired 1,25D action due to high circulating levels of FGF23 in Hyp mice combined with hypophosphatemia contribute to the impaired growth observed in these mice.

3. Skeletal Mineralization, Microarchitecture, and Biomechanics in XLH

Mice and humans with XLH have poorly mineralized bones. Histomorphometric analyses show that both Hyp cortical and trabecular bone have a significant increase in osteoid volume with dramatically impaired bone formation rate and mineralization apposition rate [50]. Micro-CT analyses demonstrate that Hyp femurs have significantly decreased whole distal femur bone volume fraction (BV/TV) and impaired cortical microarchitecture, including decreased cortical thickness (Ct.Th) and cortical area fraction (Ct.Ar/Tt.Ar) as well as increased cortical porosity [51]. Moreover, Hyp femurs have an increase in circumference with increased total cross-sectional area (Tt.Ar) and medullary area (Ma.Ar) compared to WT [52]. Trabecular microarchitecture is also severely abnormal, with Hyp femurs having significantly reduced trabecular bone volume fraction (BV/TV), which is associated with decreased trabecular number (Tb.N) and increased trabecular spacing (Tb.Sp) [52]. Consistent with the micro-CT analyses, histomorphometric analyses demonstrate that Hyp femurs have very few trabeculae. Interestingly, mice lacking the sodium phosphate transporter 2a (NPT2a), which have hypophosphatemia, low serum FGF23 levels, and high 1,25D levels, have mild abnormalities in cortical microarchitecture but severe compromise in trabecular structure. Like Hyp bones, NPT2aKO bones have decreased trabecular BV/TV and increased trabecular spacing, suggesting that phosphate may play an important role in regulating trabecular structure. Micro-CT evaluation of inferred biomechanical parameters showed a significant decrease in polar moments of inertia (pMOI), Imin, and Imax) in Hyp bones. Consistent with this and the dramatically impaired skeletal mineralization, biomechanical testing demonstrated that Hyp femurs have increased elasticity with extremely decreased strength and toughness [51].

4. Osteocyte Perilacunar and Canalicular Organization

4.1. Osteocytes

Osteocytes are the most abundant bone cell in the skeleton [53]. These bone cells not only act as mechanosensors for the skeleton [54][55], they also secrete sclerostin, which binds to low-density lipoprotein receptor-related protein (Lrp)5/6 in order to antagonize Wnt signaling and block bone formation [56]. Osteocytes also serve as endocrine cells by secreting FGF23 to regulate phosphate and 1,25D homeostasis [57]. Throughout the calcified bone matrix, osteocytes are embedded in cave-like structures called lacunae (15–20 µm) and are interconnected by long dendritic cell extensions termed canaliculi (approximately 250–300 nm in diameter) [58]. The lacunae, together with the canaliculi, form the lacuno-canalicular network (LCN). The interstitial fluid flows through the canaliculi in response to the bone matrix deformations resulting from mechanical loads on the bone [59]. The interconnected canaliculi and the gap junctions allow for communication with neighboring osteocytes and other adjacent cells by carrying oxygen, nutrients, and small molecules [60]. Thus, this network is essential in maintaining bone quality [61].

4.2. Osteocyte LCN Remodeling

Qing et al. demonstrated that osteocytes, like osteoclasts, can also remodel the mineralized extracellular matrix in a process called perilacunar remodeling [62]. This study reported that lactating mice have increase lacunar size, suggesting that the increased demand for calcium during lactation leads to enhanced perilacunar matrix resorption. Gene array and gene expression analysis demonstrated that osteocytes from lactating mice, as compared to those from virgin mice, have an increased expression of genes traditionally expressed by osteoclasts to enable bone resorption, including Tartrate Resistant Acid Phosphatase (TRAP), cathepsin K (CTSK), ATPase H+ transporting V0 subunit D2 (ATP6v0d2), ATPase H+ transporting V1 subunit G1 (ATP6v1g1), carbonic anhydrase (CAR) 1 and 2, and Na+/H+ exchanger domain containing 2 (NHDEC2). Corresponding with the restoration of lacunar size and the decrease in calcium demand during post-weaning, the expression of these matrix resorption genes returned to virgin levels after lactation stopped [62]. Deletion of the PTH receptor (PTHR1) in mice prevented the increase in lacunar size and enhanced osteocyte staining for TRAP and cathepsin K during lactation [62].

4.3. Regulation of Osteocyte LCN Organization in Hyp Mice

It has long been shown that there are perilacunar halos of osteoid in the bones of Hyp mice [63]. More recently, it was reported that Hyp bones also have decreased osteocyte number and increased osteocyte apoptosis, corresponding with dramatically impaired whole-bone biomechanics [51]. The calvaria and tibiae of Hyp mice have enlarged lacunae and impaired canalicular organization, with Hyp bones being characterized by very sparse and few canaliculi with decreased canalicular branching and connectivity compared to WT control [61]. Administration of daily 1,25D or the FGF23Ab to Hyp mice suppresses osteocyte cell death, restores lacunar size, and improves canalicular morphology, suggesting 1,25D and phosphate play roles in regulating osteocyte perilacunar and canalicular remodeling [61].  The role of 1,25D in maintaining LCN morphology is corroborated by the increased osteocyte lacunar volume [64] and poor canalicular structure [65] seen in vitamin D-deficient human cortical bone as compared to vitamin D sufficient controls. In order to study the role of 1,25D in regulating osteocyte-mediated perilacunar remodeling and canalicular organization, lacuno-canalicular (LCN) organization was analyzed in bones from mice lacking the VDR in osteocytes (VDRf/f;DMP1Cre+).  Mice null for sodium phosphate transporter 2a (NPT2aKO) have hypophosphatemia and high serum 1,25D levels with low FGF23 levels, therefore LCN remodeling was analyzed in the mice to determine if a physiological increase in 1,25D can compensate for low serum phosphate in regulating LCN remodeling.  Like Hyp mice, tibial and calvarial osteocytes from VDRf/f;DMP1Cre+ and NPT2aKO mice have enlarged osteocyte lacunae and impaired canalicular organization compared to respective controls. These studies show that 1,25D acts directly on osteocytes to modulate LCN organization and that hypophosphatemia independent of 1,25D action plays a role in regulating LCN remodeling[52]. 

5. Enthesopathy

5.1. The Enthesis

The region where the tendon inserts into bone, known as the enthesis, is a specialized tissue that is critical for joint movement [66]. The enthesis allows for the transmission of contractile forces from muscle to bone [67][68]. Fibrocartilaginous entheses attach to bone via a transitionary layer of fibrocartilage [69] and consist of four different zones: the bony eminence, mineralized fibrocartilage, unmineralized fibrocartilage and tendon [69][70]. This region has a characteristic gradation in mineral concentration and collagen orientation [71][72]. There is a linear increase in mineral volume fraction between tendon and bone as well as a decrease in the alignment of collagen fibers between tendon and bone [72]. Scleraxis (Scx) is a bHLH transcription factor that is a marker for tendon and ligament progenitors [73]. The deletion of scleraxis in cells expressing Prx1 (limb progenitor cells) resulted in abnormal morphology, impaired biomechanical properties, and disorganized collagen fiber orientation in supraspinatus entheses [73], thus demonstrating that Scx is necessary for normal enthesis organization. In addition, regulators of chondrogenesis including bone morphogenic proteins (BMPs) and Sox9 have been implicated in enthesis development [74][75][76]. When BMP4 is deleted in Scx-expressing cells, formation of the bony ridge onto which the deltoid tendon inserts into is impaired [76]. SOX9, a BMP target gene, is necessary for chondrogenesis and is expressed in chondroprogenitor cells as well as in enthesis cells [74][77] and in the bony eminences onto which the entheses insert [78][79]. Lineage tracing studies demonstrated that enthesis cells are descendants of both Scx+ and Sox9+ progenitor cells [74][80]. When SOX9 was deleted in Scx+ cells, enthesis development and organization were compromised [77], indicating that Sox9 plays a role in enthesis formation [79].

5.2. Enthesopathy in XLH

Enthesopathy is an abnormal mineralization of the tendon–bone insertion, which results in pain, impaired movement, and altered gait. This complication is observed in a majority of adults affected with XLH [81] and can lead to a significant impairment of quality of life [81][82][83]. In a survey of 39 patients with XLH, 49% of patients exhibited enthesopathy at the pelvis, 56% at the knees, 74% at the ankles, and 41% at the spine [81]. The conventional treatment of XLH consists of daily doses of oral phosphate and active vitamin D analogss such as calcitriol [82][84]. While there are limited clinical data examining the effects of conventional therapy on XLH enthesopathy development, Gjorup et al. demonstrated that those affected with XLH who were treated consistently with conventional therapy during childhood had a decreased incidence of vertebral enthesopathy compared to XLH patients who received intermittent or no therapy as a child [85]. 

5.3. Molecular Pathogenesis of XLH Enthesopathy

The molecular regulation of enthesopathy development in XLH is poorly understood. The Hyp mouse model of the XLH mutation has been used to investigate the pathogenesis of enthesopathy [81]. Achilles entheses in Hyp mice demonstrate an expansion of hypertrophic-appearing chondrogenic cells that are positive for Safranin O (SafO, stain for cartilage proteoglycans) and alkaline phosphatase activity (ALP, marker of mineralization) [74][81][86]. Lineage-tracing studies showed that post-natal enthesis cells of both wild-type and Hyp mice originate from Scx and Sox9-expressing progenitors [74], with Hyp entheses having an expansion of SafO/ALP+ cells that express Sox9 by P14 [74]. Corresponding with the chondrogenic characteristics of the enthesopathy cells, the hypertrophic-appearing cells in Hyp entheses also demonstrate an increased expression of BMP signaling marker p-Smad 1/5/8, BMP signaling target IHH, and IHH signaling targets PTCH and Runx2 by P14 [74]. These data support a pathogenic role for BMP/IHH signaling in XLH enthesopathy development. Achilles entheses from Hyp mice treated with either daily 1,25D or an anti-FGF23 targeting antibody, both of which increase 1,25D action, starting P2 (prior to enthesopathy development in Hyp mice) attenuated enthesopathy development, with treated Hyp entheses having a decreased expansion of SafO/ALP+ cells and decreased BMP/IHH signaling compared to untreated Hyp entheses [74]. In contrast, treatment of Hyp mice with phosphate and calcitriol starting P30 (after enthesopathy has developed) did not attenuate the expansion of ALP+ cells observed in Hyp entheses [87].
 

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