FGF23 induction in phosphate-responsive single cells

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Abstract. Inorganic phosphate is necessary for intracellular signaling, the formation of DNA-RNA backbones, energy storage and production in the form of ATP, as well as maintaining a mineralized skeleton. However, the mechanisms by which mammals adapt to changes in phosphate to affect hormone production and bone mineralization are currently unknown. This proposal seeks to identify biocomponents involved in transmitting signals to modulate blood concentrations of Fibroblast growth factor-23 (FGF23), the key hormone in phosphate homeostasis. FGF23 requires the expression of its co-receptor αKlotho to normalize blood phosphate by promoting phosphate excretion from the kidney and reducing 1,25(OH)2 vitamin D (1,25D) to suppress phosphate absorption in the intestine. In the mammalian musculoskeletal system, too little phosphate results in severe skeletal deformities including rickets and osteomalacia, dental abnormalities (abscesses) and fractures/pseudofractures. We have shown these manifestations arise in autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets (ARHR, type 1 due to DMP1 mutations, and type 3 due to FAM20c mutations), and X-linked hypophosphatemia (XLH; mouse model Hyp). Phosphate retention can result from the disorder hyperphosphatemic familial tumoral calcinosis (hfTC), characterized by severe tissue and vascular calcifications. We and others demonstrated that heterogeneous loss of function mutations in FGF23 itself, GALNT3, and KLOTHO are responsible for low iFGF23 and the elevated serum phosphate in these patients. FGF23 is produced in bone osteoblasts and osteocytes, and in response to increased blood phosphate concentrations intact bioactive FGF23 (‘iFGF23’) is dose-dependently secreted over hours and days, consistent with necessary transcriptional activity. Further, human subjects that undergo phosphate loading also have significant increases in circulating FGF23 over days. Finally, VDR-deficient mice have low serum levels of phosphate and FGF23, but when placed on a phosphate-rich ‘‘rescue’’ diet serum iFGF23 levels are elevated, indicating that phosphate can increase FGF23 independently of 1,25D. High serum phosphate leads to mineralization of blood vessels and brain, causing cardiovascular disease, the primary cause of death in chronic kidney disease (CKD). This is a critical outcome, as elevated FGF23 is independently associated with a >6-fold increased odds for CKD patient mortality. Thus, our central hypothesis is: changes in extracellular phosphate cause transcriptional reprogramming in osteoblasts and osteocytes to control FGF23 production. The studies in this exploratory proposal will take advantage of single-cell responses to changes of blood phosphate in vivo and use FGF23 as a ‘molecular tag’ in an unbiased manner. We expect the findings from this work to begin to elucidate novel mechanisms controlling FGF23 under normal conditions and during metabolic bone diseases.