Alterations in Vitamin D Metabolite, Parathyroid Hormone and Fibroblast Growth Factor-23 Concentrations in Sclerostin-Deficient Mice Permit the Maintenance of a High Bone Mass

Abstract

Humans with mutations of the sclerostin (Sost) gene, and knockout animals in which the Sost gene has been experimentally deleted, exhibit an increase in bone mass. We review the mechanisms by which Sost knockout mice are able to accrete increased amount of calcium and phosphorus required for the maintenance of a high bone mass. Recently published information from our laboratory, shows that bone mass is increased in Sost-deficient mice through an increase in osteoblast, and a decrease in osteoclast activity, which is mediated by activation of β-catenin and an increase in prostacyclin synthesis in osteocytes and osteoblasts. The increases in calcium and phosphorus retention required for enhanced bone mineral accretion are brought about by changes in the vitamin D endocrine system, parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF-23). Thus, in Sost knockout mice, concentrations of serum 1,25-dihydroxyvitamin D (1,25(OH)₂D) are increased and concentrations of PTH and FGF-23 are decreased thereby allowing a positive calcium and phosphorus balance. Additionally, in the absence of Sost expression, urinary calcium is decreased, either through a direct effect of sclerostin on renal calcium handling, or through its effect on the synthesis of 1,25(OH)₂D. Adaptations in vitamin D, PTH and FGF-23 physiology occur in the absence of sclerostin expression and mediate increased calcium and phosphorus retention required for the increase in bone mineralization.

1. Introduction

Sclerosteosis and van Buchem’s disease are due mutations in the SOST gene

Patients with inactivating mutations of the sclerostin (SOST) gene, and mice in which the sclerostin (Sost) gene has been deleted, both develop a high bone mass phenotype [1–6]. Patients with sclerosteosis, and those with a milder variant, van Buchem’s disease, have exceptionally dense skeletons with a bone mass that is several times greater than normal [1–3]. Brunkow et al [7] showed that “sclerosteosis is an autosomal recessive sclerosing bone dysplasia characterized by progressive skeletal overgrowth. The majority of affected individuals have been reported in the Afrikaaner population of South Africa, where a high incidence of the disorder occurs as the result of a founder effect”. They described two independent mutations in a novel gene, SOST. Affected Afrikaaner patients carry a nonsense mutation in the gene near the 5′ portion of the gene, whereas an unrelated person of Senegalese origin was found to have a splice mutation within the single intron of the gene. The SOST gene encodes a protein, sclerostin, that is related to the cystine-knot-containing proteins including Dan, Cerebrus, Gremlin, PDRC and Caronte, all of which antagonize bone morphogenetic proteins [7–10]. Sclerostin is expressed by osteocytes and functions by antagonizing BMP 6, BMP 4, and most importantly, Wnt signaling [11–27]. Since patients and animals with mutations or deletions of the sclerostin gene have a high bone mass, we asked how mineral homeostasis was maintained in the face of such high demands for calcium and phosphorus. To do so, we generated a Sost knockout mouse, and examined its bone phenotype and circulating concentrations of calciotropic and phosphotropic hormones and peptides.

2. Deletion of the Sost gene in mice results in a high bone mass phenotype

We recently reported on the generation of mice in which the Sost gene was inactivated [5]. Similar to previously published models [4, 6], these mice had high bone mass with significant increases in bone mineral content and bone mineral density throughout the skeleton, including the spine and femur. Micro-CT determinations of cortical bone showed that bone cortical area was significantly increased; similar determinations performed on trabecular bone showed significant increases in trabecular bone volume fraction and trabecular thickness. Quantitative bone histomorphometry revealed increased osteoblast surface and decreased osteoclast surface. Osteoblast activity was enhanced, as mineralizing surfaces per unit bone, mineral appositional rate, and bone formation rate per trabecular volume were substantially increased. Thus, increased bone formation in our Sost knockout model was due to increased bone formation and decreased bone resorption. These same Sost knockout mice also repaired surgically-induced bone fractures more efficiently and quickly than wild-type mice [26].

3. Enhanced bone formation and repair in Sost knockout mice are associated with increased Wnt and prostacyclin signaling

Previous work from other laboratories has shown that sclerostin inhibits BMP and Wnt signaling [11–27]. In recently published observations, we observed increased Wnt signaling in osteoblasts in healing fractures of Sost knockout mice [26], as well as in cultures of primary osteocytes derived from Sost knockout mice [27]. To further understand the mechanism of action of sclerostin in bone, we performed differential gene expression/microarray experiments in osteocytes derived from Sost knockout and wild-type mice [27]. We observed increased expression of the mRNA for Ptgis, the enzyme responsible for the synthesis of prostacyclin (PGI₂), a prostaglandin metabolite that has previously been shown to have anabolic actions in bone [27]. We examined the expression of various prostaglandin metabolites in osteocytes derived from Sost knockout and wild-type mice and demonstrated that PGI₂ (assayed as its stable metabolite, PG 6-keto F_1α) was the only prostaglandin metabolite produced in increased amounts in Sost knockout versus wild-type mice. The increase in prostacyclin production by osteocytes was associated with an increase in Ptgis mRNA and Ptgis protein expression in osteocytes of Sost knockout mice. It is likely that the increase in Ptgis expression in osteocytes is due to the enhanced binding of LEF1 to a promoter element in the Ptgis gene, as shown by chromatin immunoprecipitation experiments. We verified the effects of prostacyclin on osteoblast function by adding prostacyclin to cultured osteoblasts and showing increases in alkaline phosphatase expression and mineralization of bone matrix.

4. Regulation of Calcium Homeostasis in the Intestine and Kidney in Sclerostin Deficiency

How does the organism increase calcium and phosphorus accretion and retention to permit increased bone formation and bone mass? What are the adaptations that occur in the vitamin D endocrine, PTH and FGF-23 metabolic pathways that allow calcium and phosphorus absorption to increase in the intestine? What changes occur in the renal excretion of calcium and phosphorus?

Mineral balance in normals states

To place the discussion regarding sclerostin-induced metabolic changes in context, we will briefly review the mechanisms by which calcium is absorbed and excreted. In states of neutral calcium balance, the amount of calcium excreted by the kidney is equivalent to the net amount of dietary calcium absorbed by the intestine (net calcium absorbed = calcium absorbed – calcium secreted) [28]. The kidney, intestine, and vitamin D-parathyroid hormone (PTH)-endocrine system, play important roles in the adaptation to variations in dietary calcium and phosphorus intakes [29–34].

Mechanisms of intestinal calcium absorption

Dietary calcium is absorbed mostly in the proximal intestine where increases in circulating and tissue 1,25(OH)₂D enhance active calcium transport processes in response to decreased calcium intake [35, 36]. Mediators of Ca²⁺ transport in the enterocyte include apically situated, transient receptor potential cation channels, subfamily V, type 5 and 6 channels (TRPV5, TRPV6), which mediate the increase in Ca²⁺ uptake from the lumen into the cell; and the baso-lateral plasma membrane calcium (PMCA) pump that increases the rate of extrusion of Ca²⁺ across the basolateral membrane [30, 33, 37–44]. Intra-cellular Ca²⁺ binding proteins such as calbindin D_9K and D_28K facilitate the movement of Ca²⁺ across the cell [40, 45].

Mechanisms of renal calcium absorption

The kidney reabsorbs most (>98%) of the Ca²⁺ filtered at the glomerulus in both the proximal and distal tubule; the amounts of Ca²⁺ reabsorbed in these segments are regulated by the volume status and sodium excretion, and calciotropic hormones, PTH and 1,25(OH)₂D [29, 31–34, 46–56]. About 50% of total plasma calcium is free and is filtered by the glomerulus [57, 58]. About 70% of filtered Ca²⁺ is reabsorbed in the proximal tubule, mainly by paracellular processes [59] that are linked with sodium (Na⁺) reabsorption [57, 60–64]. Ca²⁺ and Na⁺ reabsorption are not dissociated following the administration of peptide hormones, diuretics or by changes in pH [61, 62, 65–67]. A small amount of transcellular Ca²⁺ transport occurs in the proximal tubule and is mediated by the Na-K ATPase, Na⁺- Ca²⁺ exchanger and the plasma membrane Ca²⁺ pump [68–70]. Twenty to 25 percent of filtered Ca²⁺ is reabsorbed in the thick ascending loop of Henle primarily by paracellular mechanisms [57, 71–82]. The furosemide-sensitive Na-K-Cl co-transporter, NKCC2 [83, 84], contributes to the driving force for paracellular Ca²⁺ transport. Mutations of NKCC2 which cause the common form of Bartter’s syndrome are often associated with calciuria [85]. Activation of the baso-lateral membrane Ca²⁺-sensing receptor (CaSR) inhibits reabsorption of Ca²⁺ in the thick ascending limb [86, 87] and its deletion in the kidney results in a reduced capacity to excrete Ca²⁺ [88]. In the distal convoluted tubule (primarily DCT2) and connecting tubule (together abbreviated as DT), 5–10% of filtered Ca²⁺ is reabsorbed via trans-cellular pathways [89–91] by active transport processes against electrical and concentration gradients. Mediators of Ca²⁺ transport in the renal DT include apically situated, transient receptor potential cation channels, subfamily V, type 5 and 6 channels (TRPV5, TRPV6), which mediate the increase in Ca²⁺ uptake from the lumen into the cell [34, 92–94] and the baso-lateral plasma membrane calcium (PMCA) pump [31, 32, 46], Na⁺-Ca²⁺ exchanger (NaCX) [95, 96] and the Na⁺-Ca²⁺-K⁺ exchanger (NaCKX) [97] which increase the rate of extrusion of Ca²⁺ across the basolateral membrane. Intra-cellular Ca²⁺ binding proteins such as calbindin D_9K and D_28K facilitate the movement of Ca²⁺ across the cell [46, 98, 99]. Ca²⁺ reabsorption in this segment of the nephron is regulated by a variety of hormones such as PTH [49–51, 97, 100, 101], calcitonin [102, 103], and 1,25(OH)₂D₃ [52, 53, 55, 56, 94, 104–109].

Metabolic transformation of 25-hydroxyvitamin D₃ in the kidney

The kidney is also the major site for metabolic transformation of 25(OH)D to 1,25(OH)₂D and 24,25(OH)₂D [110–114]. The 25(OH)D-1α-hydroxylase [115] and the 25(OH)D-24-hydroxylase [116] are expressed in the proximal and distal tubule of the kidney and are reciprocally regulated by PTH [35, 117], growth factors such as insulin-like growth factor (IGF) [118, 119] and fibroblast growth factor 23 (FGF-23) [120], 1,25(OH)₂D₃ itself [121], and perhaps by Ca²⁺ directly [122]. Adaptations in hydroxylase activity, which occur with changes in calcium concentrations are PTH-dependent, whereas those which occur following alterations in serum Pi concentrations [123] are driven by growth factors such as IGF [118, 119] and FGF-23 [120]. 1,25(OH)₂D₃, synthesized in the kidney by 25(OH)D-1α-hydroxylase, not only increases calcium and phosphate absorption in the intestine [110, 111, 124] and the mobilization of calcium and phosphate from bone [125, 126], but also increases the renal reabsorption of Ca²⁺ in the distal tubule by increasing TRPV5 and TRPV6 [94, 127], calbindin D [55, 128–130], and PMCA [108, 131, 132] expression. Figure 1 summarizes current information regarding changes that occur in response to decreasing dietary calcium intake. As depicted, the vitamin D-PTH-endocrine system plays a central role in adaptations to alterations in dietary calcium intake [29, 30, 133].

Figure 1.

Current understanding of adaptations to dietary calcium.

Alterations in vitamin D metabolism, PTH and FGF-23 in sclerostin deficiency – definition of a role for sclerostin in calcium physiology

Several changes in the concentrations of calcium and phosphorus regulating hormones were observed and Sost-deficient mice (Figure 2) [5]. First, concentrations of serum 1,25(OH)₂D concentrations were significantly increased in Sost deficiency without attendant hypercalcemia. The increases in 1α,25(OH)₂D concentrations in Sost deficient mice would be expected increase the absorption of calcium and phosphorus in the intestine and kidney, thus providing increased amounts of mineral for bone formation. The decrease in absolute urinary calcium excretion and renal fractional excretion of calcium in Sost^−/− mice are likely due to the effects of 1α,25(OH)₂D on renal calcium reabsorption, although a direct effect of sclerostin on renal calcium handling cannot be eliminated. An increase in renal 25(OH)D-1α hydroxylase (Cyp27b1) mRNA and protein expression in Sost^−/− mice, suggests that the increase in serum 1α,25(OH)₂D concentrations are due to increased 1α,25(OH)₂D synthesis and not due to change in 1α,25(OH)₂D clearance. When recombinant sclerostin was added to cultures of proximal tubular cells the expression of the messenger RNA for Cyp27b1, the 1α-hydroxylase cytochrome P450, was diminished. Serum 24, 25(OH)₂D concentrations were diminished in Sost^−/− mice, whereas PTH concentrations were similar in knockout and wild-type mice, consistent with previous studies in humans [134].

Figure 2.

(A) Serum concentrations of 1α,25-dihydroxyvitamin D in WT (n = 11) and sost KO mice (n = 9). (B) Serum concentrations of 24,25-dihydroxyvitamin D in WT (n = 11) and sost KO mice (n = 7). (C) Serum concentrations of intact fibroblast growth factor 23 in WT (n = 8) and sost KO mice (n = 6). (D) Serum phosphorus concentrations in WT (n = 13) and sost KO mice (n = 11). (E) Serum calcium concentrations in WT (n = 19) and sost KO mice (n = 18). (F) Serum parathyroid hormone concentrations in WT (n = 13) and sost KO mice (n = 14). From Ryan et al. Proc Natl Acad Sci U S A. 2013 April 9;110(15):6199–6204.

As noted in Figure 2, FGF-23 concentrations were decreased and serum Pi was increased. The decrease in FGF-23 concentrations could contribute to the increase in 1α,25(OH)₂D concentrations. Despite reductions in FGF-23, urinary Pi excretion was unchanged. The data suggest that sclerostin might modulate the concentrations of factors that alter phosphorus homeostasis.

Thus, adaptations to reductions in calcium intake and resultant downstream alterations in hormones (see Figure 1 for current understanding) should be amended to include changes in sclerostin expression (Figure 2). In the modified scheme, reduced sclerostin expression, which occurs as a result of increases in PTH [135–138], would enhance renal Ca²⁺ reabsorption directly or through changes in 1,25(OH)₂D synthesis. The change in 1,25(OH)₂D synthesis might be direct or mediated through changes in FGF-23 concentrations.

Conclusions

Sclerostin changes bone mass through alterations in osteocyte and osteoblast Wnt and prostacyclin signaling pathways. To respond to alterations in bone mass, 1,25(OH)₂D and FGF-23 concentrations are altered as is the renal excretion of calcium thereby resulting in a positive calcium and phosphorus balance.

Figure 3.

Proposed physiological adaptations to dietary calcium. The sequence of changes in hormones demonstrated the effects of PTH on sclerostin concentrations, and the additional effects of sclerostin on tubular Ca transport and 1α,25(OH)₂D synthesis.

Highlights.

• Sost deficiency is associated with increased bone mass and osteoblast activity.

• Increased serum 1,25(OH)₂D enhances Ca and P balance in sclerostin deficiency.

• Reduced PTH and FGF-23 concentrations permit phosphorus retention.

• Hormonal changes facilitate enhanced mineral retention in sclerostin deficiency.