Clemens Bergwitz

Regulation of Phosphate Homeostasis by PTH, Vitamin D, and FGF23

In contrast to the regulation of calcium homeostasis, which has been extensively studied over the past several decades, relatively little is known about the regulation of phosphate homeostasis. Fibroblast growth factor 23 (FGF23) is part of a previously unrecognized hormonal bone-parathyroid-kidney axis, which is modulated by PTH, 1,25(OH)(2)-vitamin D (1,25(OH)(2)D), dietary and serum phosphorus levels. Synthesis and secretion of FGF23 by osteocytes are positively regulated by 1,25(OH)(2)D and serum phosphorus and negatively regulated, through yet unknown mechanisms, by the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and by dentin matrix protein 1 (DMP1). In turn, FGF23 inhibits the synthesis of 1,25(OH)(2)D, and it may negatively regulate the secretion of parathyroid hormone (PTH) from the parathyroid glands. However, FGF23 synergizes with PTH to increase renal phosphate excretion by reducing expression of the renal sodium-phosphate cotransporters NaPi-IIa and NaPi-IIc in the proximal tubules. Most insights gained into the regulation of phosphate homeostasis by these factors are derived from human genetic disorders and genetically engineered mice, which are reviewed in this paper.

SLC34A3 Mutations in Patients with Hereditary Hypophosphatemic Rickets with Hypercalciuria Predict a Key Role for the Sodium-Phosphate Cotransporter NaPi-IIc in Maintaining Phosphate Homeostasis

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare disorder of autosomal recessive inheritance that was first described in a large consanguineous Bedouin kindred. HHRH is characterized by the presence of hypophosphatemia secondary to renal phosphate wasting, radiographic and/or histological evidence of rickets, limb deformities, muscle weakness, and bone pain. HHRH is distinct from other forms of hypophosphatemic rickets in that affected individuals present with hypercalciuria due to increased serum 1,25-dihydroxyvitamin D levels and increased intestinal calcium absorption. We performed a genomewide linkage scan combined with homozygosity mapping, using genomic DNA from a large consanguineous Bedouin kindred that included 10 patients who received the diagnosis of HHRH. The disease mapped to a 1.6-Mbp region on chromosome 9q34, which contains SLC34A3, the gene encoding the renal sodium-phosphate cotransporter NaP(i)-IIc. Nucleotide sequence analysis revealed a homozygous single-nucleotide deletion (c.228delC) in this candidate gene in all individuals affected by HHRH. This mutation is predicted to truncate the NaP(i)-IIc protein in the first membrane-spanning domain and thus likely results in a complete loss of function of this protein in individuals homozygous for c.228delC. In addition, compound heterozygous missense and deletion mutations were found in three additional unrelated HHRH kindreds, which supports the conclusion that this disease is caused by SLC34A3 mutations affecting both alleles. Individuals of the investigated kindreds who were heterozygous for a SLC34A3 mutation frequently showed hypercalciuria, often in association with mild hypophosphatemia and/or elevations in 1,25-dihydroxyvitamin D levels. We conclude that NaP(i)-IIc has a key role in the regulation of phosphate homeostasis.

Genetic evidence of serum phosphate-independent functions of FGF-23 on bone

Maintenance of physiologic phosphate balance is of crucial biological importance, as it is fundamental to cellular function, energy metabolism, and skeletal mineralization. Fibroblast growth factor-23 (FGF-23) is a master regulator of phosphate homeostasis, but the molecular mechanism of such regulation is not yet completely understood. Targeted disruption of the Fgf-23 gene in mice (Fgf-23−/−) elicits hyperphosphatemia, and an increase in renal sodium/phosphate co-transporter 2a (NaPi2a) protein abundance. To elucidate the pathophysiological role of augmented renal proximal tubular expression of NaPi2a in Fgf-23−/− mice and to examine serum phosphate–independent functions of Fgf23 in bone, we generated a new mouse line deficient in both Fgf-23 and NaPi2a genes, and determined the effect of genomic ablation of NaPi2a from Fgf-23−/− mice on phosphate homeostasis and skeletal mineralization. Fgf-23−/−/NaPi2a−/− double mutant mice are viable and exhibit normal physical activities when compared to Fgf-23−/− animals. Biochemical analyses show that ablation of NaPi2a from Fgf-23−/− mice reversed hyperphosphatemia to hypophosphatemia by 6 weeks of age. Surprisingly, despite the complete reversal of serum phosphate levels in Fgf-23−/−/NaPi2a−/−, their skeletal phenotype still resembles the one of Fgf23−/− animals. The results of this study provide the first genetic evidence of an in vivo pathologic role of NaPi2a in regulating abnormal phosphate homeostasis in Fgf-23−/− mice by deletion of both NaPi2a and Fgf-23 genes in the same animal. The persistence of the skeletal anomalies in double mutants suggests that Fgf-23 affects bone mineralization independently of systemic phosphate homeostasis. Finally, our data support (1) that regulation of phosphate homeostasis is a systemic effect of Fgf-23, while (2) skeletal mineralization and chondrocyte differentiation appear to be effects of Fgf-23 that are independent of phosphate homeostasis.

Brittle IV mouse model for osteogenesis imperfecta IV demonstrates postpubertal adaptations to improve whole bone strength.

The Brtl mouse model for type IV osteogenesis imperfecta improves its whole bone strength and stiffness between 2 and 6 months of age. This adaptation is accomplished without a corresponding improvement in geometric resistance to bending, suggesting an improvement in matrix material properties.

Residues in the Membrane-spanning and Extracellular Loop Regions of the Parathyroid Hormone (PTH)-2 Receptor Determine Signaling Selectivity for PTH and PTH-related Peptide

The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1–36), and to the nondiscriminating peptide [Ile5,Trp23]PTHrP-(1–36) (the Phe23 → Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand.

A G Protein-coupled Receptor from Zebrafish Is Activated by Human Parathyroid Hormone and Not by Human or Teleost Parathyroid Hormone-related Peptide IMPLICATIONS FOR THE EVOLUTIONARY CONSERVATION OF CALCIUM-REGULATING PEPTIDE HORMONES

Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a full-length zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47–52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R(43), a 5′ splice variant that lacked 17 amino acids in the amino-terminal extracellular domain, showed cAMP accumulation when challenged with [Tyr34]hPTH(1–34)-amide (hPTH) (EC50, 1.64 ± 0.95 nm) and [Ile5,Trp23,Tyr36]hPTHrP-(1–36)-amide ([Ile5, Trp23]hPTHrP) (EC50, 46.8 ± 12.1 nm) but not when stimulated with [Tyr36]hPTHrP-(1–36)-amide (hPTHrP), [Trp23,Tyr36]hPTHrP-(1–36)-amide ([Trp23]hPTHrP), or [Ala29,Glu30,Ala34,Glu35,Tyr36]fugufish PTHrP-(1–36)amide (fuguPTHrP). FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands.

Cloning and characterization of the vitamin D receptor from Xenopus laevis.

The Vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the effects of 1,25-dihydroxyvitamin D3 on mineral ion homeostasis. Although the mammalian and avian VDRs have been extensively studied, little is known about the VDR in lower vertebrate species. To address this, we have isolated the Xenopus laevis VDR (xVDR) complementary DNA. Overall, the xVDR shares 79%, 73%, 73%, and 75% identity at the amino acid level with the chicken, mouse, rat, and human VDRs, respectively. The amino acid residues and subdomains important for DNA binding, hormone binding, dimerization, and transactivation are mostly conserved among all VDR species. The xVDR polypeptide can heterodimerize with the mouse retinoid X receptor α, bind to the rat osteocalcin vitamin D response element (VDRE), and induce vitamin D-dependent transactivation in transfected mammalian cells. Northern analysis reveals two xVDR messenger RNA species of 2.2 kb and 1.8 kb in stage 60 Xenopus tissues. In the adult, xVDR expressi...

Cellular mechanism of decreased bone in Brtl mouse model of OI: imbalance of decreased osteoblast function and increased osteoclasts and their precursors.

The Brtl mouse, a knock‐in model for moderately severe osteogenesis imperfecta (OI), has a G349C substitution in half of type I collagen α1(I) chains. We studied the cellular contribution to Brtl bone properties. Brtl cortical and trabecular bone are reduced before and after puberty, with BV/TV decreased 40–45%. Brtl ObS/BS is comparable to wildtype, and Brtl and wildtype marrow generate equivalent number of colony‐forming units (CFUs) at both ages. However, OcS/BS is increased in Brtl at both ages (36–45%), as are TRACP+ cell numbers (57–47%). After puberty, Brtl ObS/BS decreases comparably to wildtype mice, but osteoblast matrix production (MAR) decreases to one half of wildtype values. In contrast, Brtl OcS falls only moderately (∼16%), and Brtl TRACP staining remains significantly elevated compared with wildtype. Consequently, Brtl BFR decreases from normal at 2 mo to one half of wildtype values at 6 mo. Immunohistochemistry and real‐time RT‐PCR show increased RANK, RANKL, and osteoprotegerin (OPG) levels in Brtl, although a normal RANKL/OPG ratio is maintained. TRACP+ precursors are markedly elevated in Brtl marrow cultures and form more osteoclasts, suggesting that osteoclast increases arise from more RANK‐expressing precursors. We conclude that osteoblasts and osteoclasts are unsynchronized in Brtl bone. This cellular imbalance results in declining BFR as Brtl ages, consistent with reduced femoral geometry. The disparity in cellular number and function results from poorly functioning osteoblasts in addition to increased RANK‐expressing precursors that respond to normal RANKL/OPG ratios to generate more bone‐resorbing osteoclasts. Interruption of the stimulus that increases osteoclast precursors may lead to novel OI therapies.

A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc

The present study describes two novel compound heterozygous mutations, c.410C>T(p.T137M) (T137M) on the maternal and g.4225_50del on the paternal allele of SLC34A3, in a previously reported male with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and recurrent kidney stones (Chen C, Carpenter T, Steg N, Baron R, Anast C. Pediatrics 84: 276-280, 1989). For functional analysis in vitro, we generated expression plasmids encoding enhanced green fluorescence protein (EGFP) concatenated to the NH2 terminus of wild-type or mutant human type IIc Na-Pi cotransporter (NaPi-IIc), i.e., EGFP-hNaPi-IIc, EGFP-[M137]hNaPi-IIc, or EGFP-[Stop446]hNaPi-IIc. The V446Stop mutant showed complete loss of expression and function when assayed for apical patch expression in opossum kidney (OK) cells and sodium-dependent 33P uptake into Xenopus laevis oocytes. Conversely, EGFP-[M137]hNaPi-IIc was inserted into apical patches of OK cells and into oocyte membranes. However, when quantified by confocal microscopy, surface fluorescence was reduced to 40% compared with wild-type. After correction for surface expression, the rate of 33P uptake by oocytes mediated by EGFP-[M137]hNaPi-IIc was decreased by an additional 60%. The resulting overall reduction of function of this NaPi-IIc mutant to 16%, taken together with complete loss of expression and function of g.4225_50del(V446Stop), thus appears to be sufficient to explain the phenotype in our patient. Furthermore, the stoichiometric ratio of 22Na and 33P uptake was increased to 7.1 +/- 3.65 for EGFP-[M137]hNaPi-IIc compared with wild-type. Two-electrode studies indicate that EGFP-[M137]hNaPi-IIc is nonelectrogenic but displayed a significant phosphate-independent inward-rectified sodium current, which appears to be insensitive to phosphonoformic acid. M137 thus may uncouple sodium-phosphate cotransport, suggesting that this amino acid residue has an important functional role in human NaPi-IIc.

Defective O-Glycosylation due to a Novel Homozygous S129P Mutation Is Associated with Lack of Fibroblast Growth Factor 23 Secretion and Tumoral Calcinosis

BACKGROUND Homozygous mutations in fibroblast growth factor (FGF23) have recently been described as the genetic cause of one form of hyperphosphatemic tumoral calcinosis (HFTC). However, it remained unclear to date how these mutations lead to loss of biologically active FGF23 in the circulation. METHODS We here report a novel homozygous mutation, c.385T>C in FGF23 exon 2, which changes codon 129 from serine to proline (S129P) in a previously described individual affected by HFTC. The S129P mutation as well as two known FGF23 mutations, S71G and S129F, were introduced into an expression vector encoding wild-type (wt) human (h) FGF23 to yield [P129]hFGF23, [F129]hFGF23, and [G71]hFGF23; whole lysates, glycoprotein fractions, and conditioned media from HEK293 and COS-7 cells expressing these constructs were subjected to Western blot analysis using affinity-purified goat anti-hFGF23(51-69) and anti-hFGF23(206-222) antibodies. RESULTS We detected 25- and 32-kDa protein species in total lysates of HEK293 cells expressing wt-hFGF23. The 32-kDa band, representing O-glycosylated hFGF23, was not detectable in the glycoprotein fraction of lysates from HEK293 cells expressing [P129]hFGF23, and in comparison with wt-FGF23 only small amounts of [P129]hFGF23 were secreted into the medium. Similar results were obtained for cells expressing [G71]hFGF23 and [F129]hFGF23. CONCLUSION Our data for the first time directly show that FGF23 mutations associated with HFTC impair O-glycosylation in vitro resulting in poor secretion of the mutant hormone thereby explaining the characteristic hyperphosphatemic phenotype of homozygous carriers in vivo.