Bone as a source of FGF23: regulation by phosphate?
Introduction
Fibroblast growth factor 23 (FGF23) is involved in a number of disorders of phosphate regulation. Missense mutations in FGF23 have been identified in families with the phosphate wasting disorder autosomal dominant hypophosphatemic rickets (ADHR) [1]. Oncogenic osteomalacia (OOM) patients have elevated serum FGF23 concentrations [2], with high FGF23 protein expression in the tumors [3], [4]. The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) has been identified as the gene mutated in patients with the phosphate wasting disorder X-linked Hypophosphatemic Rickets (XLH) [5]. It appears likely therefore that FGF23 and PHEX both play a role in the regulation of phosphate.
FGF23 expression has been detected in several tissues to date, including human heart, liver, thyroid/parathyroid, small intestine, testis, skeletal muscle, and lymph node, and in several mouse tissues such as bone, thymus, and the ventrolateral thalamic nucleus of the brain [1], [6], [7], [8]. The principal site of secretion of circulating FGF23 is unknown. It has been reported that in mice, FGF23 mRNA expression may be highest in bone [8]. Recently, FGF23 expression has also been reported in human bone, and elevated serum FGF23 in patients with fibrous dysplasia of bone was associated with renal phosphate wasting and with fibrous dysplasia tissue mass [9].
There is evidence that FGF23 may reduce serum phosphate concentrations. Mice implanted with cells secreting FGF23 have been reported to become hypophosphatemic, with low serum 1,25-dihydroxyvitamin D3 concentrations and osteomalacia [6] and injection of mice with FGF23 has resulted in reduced serum phosphate and serum 1,25-dihydroxyvitamin D3 concentrations [10], [11]. Suppression of renal sodium phosphate co-transporter (NaPi) activity by FGF23 has been reported in vivo with normal and mutant FGF23 [10], [11]. In vitro, FGF23 has been reported to have an inhibitory effect on phosphate uptake in cultured renal cells under some conditions [12], [13]. Although there is evidence that FGF23 may play a role in phosphate regulation, the mechanism of action of FGF23 is unknown, as are the conditions and factors by which FGF23 expression is regulated.
The physiological role of PHEX and its relation to FGF23 are also unclear. PHEX is expressed predominantly in teeth and bone [14] and plays an important role in the mineralization of osteoblasts. Although there was early evidence that the endopeptidase PHEX may cleave FGF23 [12], recent studies have demonstrated that it is unlikely that FGF23 is a direct substrate of PHEX [8]. Similarly, it has been postulated that inactive PHEX in XLH patients would result in elevated serum FGF23 concentrations; however, several studies using different assays have not demonstrated consistent elevation of FGF23 concentration above normal controls in all XLH patients [2], [15], [16]. As circulating FGF23 is not always elevated in XLH, this suggests that local effects in organs such as bone may be important for the XLH phenotype and is consistent with earlier evidence of intrinsic osteoblast defects in the mouse model, Hyp [17]. Further, mineralization defects in osteoblasts from Hyp mice, which have mutations in PHEX, have been partially corrected by osteoblast-targeted PHEX expression [18].
The expression of FGF23 mRNA in human tissues, OOM tumor tissue, and cell lines was examined in this study. Because the expression of FGF23 mRNA was detected in bone and in primary human osteoblast-like bone cells, the regulation of FGF23 and PHEX mRNA by extracellular phosphate and mineralizing conditions was investigated in the bone cells.
Section snippets
Tissue samples
Normal tissue was obtained from surgically derived samples snap-frozen in liquid nitrogen. These were stored in a tissue bank at −70°C. Bone tissue was obtained from biopsies of femoral heads from subjects undergoing hip replacement.
Cell culture
Cultures of primary human osteoblast-like bone cells, or fetal bone cells (FBC), were grown from bone chips as previously described [19]. Cultures of SV40-transformed FBC (SV40-FBC) [20] had previously been established, and have phenotypic properties similar to FBC,
FGF23 mRNA expression in tissues and cell lines
FGF23 mRNA was detected in human bone, kidney, and liver tissues by RT-PCR (Fig. 1). Huh7 cells (Fig. 1) and HEK293 cells (Fig. 3) were also found to express FGF23 mRNA. FGF23 mRNA was not detected in primary human keratinocytes (Fig. 1). A human renal proximal tubule cell line, IHKE-1 cells, as well as OK3B2 opossum kidney cells were also found to express FGF23 mRNA by RT-PCR confirmed by sequencing (results not shown). All cDNA samples amplified with the primers for the housekeeping gene
Discussion
In this study, FGF23 mRNA expression was demonstrated in several human tissues and cell lines. In the normal human tissues studied, FGF23 mRNA expression was highest in bone, followed by kidney medulla, liver, thyroid, and kidney cortex tissue. There was no detectable expression in parathyroid tissue. The expression in human bone tissue is consistent with expression studies in mice that have reported FGF23 expression to be highest in bone [8]. The four bone samples had differing levels of FGF23
Acknowledgments
We thank Ms. Sutharshani Sivagurunathan and Dr. Meloni Muir for the initial culture of the primary osteoblast-like bone cells. Ms. Jan Shaw is thanked for statistical analysis of data. This work was supported by the NHMRC Australia (AEN, RSM, BGR), GlaxoSmithKline (MM), and by Eli Lilly (AEN, RSM, BGR).
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Current address: Garvan Institute of Medical Research, Sydney, NSW 2010, Australia.