Abstract

To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal.

Highlights

  • Inorganic phosphate (Pi) has an essential role in cell signaling and metabolism and is tightly regulated by parathyroid hormone (PTH), 1,25-dihydroxy vitamin D (1,25-D), and fibroblast growth factor 23 (FGF23) to avoid excess or deficiency [1]

  • Transgene expression was below endogenous mPit1 mRNA levels in primary bone cells (PBCs) obtained from these mice (S1A Fig) and serum Pi, urine Pi/urine creatinine, and phosphate excretion index (PEI) at P80 were unchanged compared to wild-type mice (WT) littermates (S1C and S1D Fig)

  • PBC cultures treated with adenovirus encoding cre-recombinase to excise the LSL-cassette in vitro and to activate the chicken beta actin/rabbit beta-globin gene (CAG) promotor showed several orders of magnitude higher expression of HA-hPIT1 when compared to untreated H11-CAG-LSL-HA-hPIT1tg PBC (S1B Fig)

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Summary

Introduction

Inorganic phosphate (Pi) has an essential role in cell signaling and metabolism and is tightly regulated by parathyroid hormone (PTH), 1,25-dihydroxy vitamin D (1,25-D), and fibroblast growth factor 23 (FGF23) to avoid excess or deficiency [1]. Because of lack of suitable in vitro models, it remains unclear whether PIT1 or PIT2 regulate PTH, 1,25-D or FGF23. Hypomorphic Pit ablation is viable and reduces femur length, but no significant changes of Pi or calcium metabolism were observed [12]. Along with inappropriately low intact FGF23 levels in these mice, these findings suggest that Pit is upstream of and positively regulating FGF23. Based on these findings, it is possible that PIT1 and PIT2 compensate for each other preventing significant changes in the respective single gene ablation models

Methods
Results
Conclusion

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