Mineralized tissue cells are a principal source of FGF23
Introduction
The discovery of fibroblast growth factor (FGF) 23, the 22nd member of the FGF family, has provided significant new understanding of regulation of systemic Pi homeostasis [1]. FGF23 was identified originally as a phosphaturic factor in autosomal dominant hypophosphatemic rickets (ADHR) [2] and thereafter in tumor-induced osteomalacia (TIO) [3], renal phosphate wasting in McCune–Albright syndrome/fibrous dysplasia (FD) [4], familial tumoral calcinosis [5], and possibly X-linked hypophosphatemic rickets (XLH) [6]. FGF23 is essential for maintenance of phosphate homeostasis and/or 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) metabolism [3], [7], [8], [9], [10], [11], [12], [13]. However, much remains to be understood about the mechanisms underlying FGF23 activities, as highlighted by the fact that not all the phenotypic characteristics of the FGF23-deficient mice seem consistent with those observed in FGF23 overloading [3], [8], [9], [10], [11]. Notably, in spite of an increase in serum phosphate levels in fgf23−/− mice, hypomineralization of certain bones is seen [12], [13]. An unexpected variance in serum FGF23 levels in humans has also been reported in hypophosphatemic disorders as well as normal conditions [14]. Elucidation of the cellular source of FGF23 and its regulatory mechanism(s) appear to be essential to address these apparent inconsistencies.
FGF23 mRNA was initially identified in a wide variety of tissues, including brain, thymus, thyroid, small intestine, testis, heart, liver and lymph node [2], [3], [15]. More recent real-time quantitative RT–PCR data suggest that FGF23 mRNA is most abundantly expressed in bone amongst tissues examined in normal mice [7]. Reports of FGF23 mRNA expression in osteoblastic cells are discrepant. No detectable expression was reported in primary bone cell cultures from mouse calvaria and limb bud cells, the mouse osteoblastic cell line, MC3T3-E1, or the human osteosarcoma cell line, SaOS2 [2]. However, immortalized mouse osteoblastic cells from the simian virus 40 (SV40) transgenic mice were found to express FGF23 mRNA but the levels did not change significantly during culture [7]. On the other hand, FGF23 mRNA expression was seen in SV40-transformed human fetal bone cells, and levels were up-regulated in relation to matrix mineralization and increased extracellular phosphate [16]. Although odontoblasts and cementoblasts share some features with osteoblasts, and dental defects are observed in some hypophosphatemic disorders [17], little information is available concerning whether FGF23 is also expressed during odontogenesis.
Levels of FGF23 mRNA are increased concomitant with mineralization defects in immortalized osteoblastic cells from SV40-transgenic Hyp (the homologue of XLH) mice, while interestingly SV40-transgenic Hyp osteogenic cells can differentiate normally [7]. FGF23 mRNA/protein is observed in osteoblasts and in newly formed osteocytes in healing fracture callus and fibrous dysplasia (FD) bone with a lower detection level in lining cells and osteocytes in normal conditions [4]. Moreover, Hyp mice crossed with FGF23-deficient mice are reported to be indistinguishable from FGF23-null mice, both in terms of serum phosphate levels and in skeletal phenotype [13]. Taken together, these observations suggest that expression levels of FGF23 are correlated with normal and pathophysiological bone formation and metabolism.
Recent data have implicated FGF23 as a counter-regulatory phosphaturic hormone for vitamin D. For example, 1,25(OH)2D3 up-regulates FGF23 in bone (especially in osteoblastic cells, which express the 1,25(OH)2D3 receptor) [18], [19], but the phenotype of vitamin D receptor-null mice supports the lack of a direct effect of 1,25(OH)2D3 on bone mineralization [20]. Thus, a novel vitamin D–FGF23 system appears to exist and be associated with bone mineralization, but details of expression and regulation of FGF23 during normal bone formation and metabolism are still fragmentary. We have therefore determined the distribution of FGF23 in normal rat bone and tooth formation in vivo and its expression profile during normal osteoblast development and matrix mineralization in vitro.
Section snippets
Animals
Timed-pregnant or young adult (8 weeks old) male Wistar rats were housed and handled to minimize pain or discomfort to animals according to protocols approved by Research Facilities for Laboratory Animal Science, Natural Science Center for Basic Research and Development, Hiroshima University. Rats for histological studies were anesthetized with an intraperitoneal injection of sodium pentobarbital just before sampling. To obtain calvaria cells (see below), both fetuses and their mothers were
FGF23 expression in young adult and fetal rats
Given conflicting reports concerning FGF23 mRNA expression [2], [3], [4], [7], [15], [16], we first used real-time quantitative RT–PCR to determine the tissue expression profile and to ask whether mineralized tissues express FGF23 mRNA in both fetal (21-day-old) and young adult (8-week-old) rats (Fig. 1). FGF23 mRNA was detected in all tissues examined including calvaria and femur where levels were much higher (> 30-fold) than in other tissues, such as spleen, thymus, small intestine, liver,
Discussion
Our data suggest that the levels of circulating FGF23 under normal physiological conditions may be dependent on its expression in mineralized tissue in young adult rats and that matrix-forming cells as well as osteoclasts in mineralized tissues including tooth may be the primary source of FGF23, a source dependent on the development stage of cells, matrix mineralization and possibly mineral metabolism.
In addition to the high expression of FGF23 mRNA in bone versus other tissues tested (data
Acknowledgments
We thank Hiroko Hatano for her technical assistance. This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (16591828 to YY).
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