Sodium-Dependent Phosphate Transporters in Osteoclast Differentiation and Function

Abstract

Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.

Redox‐Dependent Bone Alkaline Phosphatase Dysfunction Drives Part of the Complex Bone Phenotype in Mice Deficient for Memo1

“…For culture of osteoclast‐like cells, femoral marrow cells were harvested from Memo cKO and control mice, treated with 4OH‐tamoxifen 1 μM (Sigma) and colony stimulating‐factor 1 30 ng/mL (CSF1; PeproTech) for 24 hours, then nonadherent cells were differentiated as described . Differentiation was verified by TRAP activity as described .…”

“…Differentiation was verified by TRAP activity as described . To determine osteoclast resorptive function, 45 Ca release from calcium phosphate‐coated wells was assessed in 96‐well plates from the fifth to the sixth day of differentiation and normalized by TRAP activity as described …”

“…For culture of osteoclast-like cells, femoral marrow cells were harvested from Memo cKO and control mice, treated with 4OH-tamoxifen 1 mM (Sigma) and colony stimulating-factor 1 30 ng/mL (CSF1; PeproTech) for 24 hours, then nonadherent cells were differentiated as described. (19) Differentiation was verified by TRAP activity as described. (20) To determine osteoclast resorptive function, 45 Ca release from calcium phosphate-coated wells was assessed in 96-well plates from the fifth to the sixth day of differentiation and normalized by TRAP activity as described.…”

“…(20) To determine osteoclast resorptive function, 45 Ca release from calcium phosphate-coated wells was assessed in 96-well plates from the fifth to the sixth day of differentiation and normalized by TRAP activity as described. (19,21) Calvaria osteoblasts from Memo cKO and control mice were isolated on postnatal day 1 or 2 by serial digestion using collagenase II (Worthington) in MEM Hank's Medium (Sigma) as described previously. (22) Digestions II to IV were pooled, cultured for 4 days in alpha-MEM þ FCS 10% þ penicillin/streptomycin 1%, then frozen.…”

Clinical aspects of the phosphate transporters NaPi-IIa and NaPi-IIb: mutations and disease associations

“…Prior studies had suggested expression also in osteoblasts [101], osteoclasts [50], and odontoblasts [64]; however, the expression in osteoclasts appears to be very low and of little physiological relevance [1] and overall these studies performed in rodents have not been replicated in humans.…”