Bone Abstracts

Objectives: As osteoblasts mature, they acquire the expression of multiple molecules involved in phosphate metabolism, including dentin matrix protein 1 (DMP1) and fibroblast growth factor 23 (FGF23). This suggests that osteoblasts and osteocytes may sense and respond to alterations in the phosphate availability in their microenvironment. We previously reported that increased extracellular inorganic phosphate (Pi) triggered signal transduction in various cell types to alter gene expression and demonstrated the involvement of a type III sodium/phosphate (Na⁺/Pi) co-transporter Pit1 and FGF receptor. Pit2, another type III Na⁺/Pi co-transporter, is ubiquitously expressed similarly to Pit1, and loss-of-function mutations in PiT2 cause familial idiopathic basal ganglia calcification. Here we aimed to investigate the role of Pit2 in responsiveness of osteoblasts to extracellular Pi.

Methods: We applied CRISPR/Cas9 to an osteoblastic cell line MC3T3-E1 (subclone #4) to generate Pit2-knockout (KO) cells. Reduced Pi uptake in Pit2-KO cells was confirmed using ³²P-orthophosphate. Then, Pit2-KO and control cells were cultured for 8 weeks in the medium containing 3 mM of Pi and 50 (μg/ml of ascorbic acid, and mineralization was evaluated by alizarin red staining. Temporal change in gene expression was analyzed real-time PCR. Acute effects of increased Pi were also examined by incubating cells in the presence of 1, 4, or 7 mM Pi for 48 hours.

Results: After 8 weeks of culture in the presence of 3 mM Pi, both Pit2-KO and control cells were mineralized. However, the expression of Pit1, osteopontin and Fgf23 was increased in control cells but not in Pit2-KO cells during the culture. As to the acute effects of Pi, 48-hour treatment with 7 mM Pi increased the expression of Dmp1 and Fgf2 and reduced that of alkaline phosphatase (Alpl) in both Pit2-KO and controls cells.

Discussion: Impaired induction of the expression of Pit1, osteopontin and Fgf23 in Pit2-KO cells cultured in the presence of 3 mM Pi for 8 weeks suggests that the effects of chronic elevation of Pi may be attenuated by reduced Pi uptake. On the other hand, the responsiveness to acute elevation of Pi was retained in Pit2-KO cells, implying the dispensability of Pit2 for Pi sensing.

Disclosure The authors declared no competing interests.