Phosphate transport in osteoclasts: A functional and immunochemical characterization

Phosphate transport in osteoclasts: A functional and immunochemical characterization. Osteoclasts are polarized cells involved in bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the active process of bone resorption. We hypothesize that osteoclasts may possess specific Pi-transport system(s) for transcellular movement of Pi released from bone into the resorption cavity. We have previously reported the existence of a Na-dependent Pi cotransporter in the avian osteoclast, which provides a model culture system for the fully differentiated phenotype capable of bone resorption. In whole cell Pi-uptake studies, the rate of Pi transport was sensitive to both ouabain and 2,4-DNP, an inhibitor of aerobic ATP production. When these osteoclasts were exposed to bone particles, there was an immediate stimulation of Pi transport, independent of de novo protein synthesis. The stimulatory effect of bone particles was inhibited by peptides with the Arg-Gly-Asp-Ser (RGDS) motif, an effect which implicates integrins and cell-matrix interaction in the regulation of Pi transport. We performed Western blots on both whole cell lysates and membrane fractions using a polyclonal antibody to the N-terminal of NaPi-2 (the rat variant) and found a single ∼100kDa protein; the non-immune serum was used as control. Immunofluorescence studies using the same N-terminal antibody to NaPi-2 detected the protein in discrete vesicles. There was an induction of the protein in membrane fractions isolated from osteoclasts cultured in the presence of bone particles. Our preliminary studies indicate that a Na-Pi cotransporter may exist in the avian osteoclast, immunologically related to the NaPi-2 family, and which may be regulated through integrin-mediated pathways in the presence of bone. We also hypothesize that there may be a redistribution of vesicular pools containing the Na-Pi cotransporter toward discrete plasma membrane sites on the polarized osteoclast for transcellular movement of Pi during active bone resorption

Chemistry of magnetic covalent organic frameworks (MagCOFs): from synthesis to separation applications

This review aims to explore the design, development and applications of magnetically supported covalent organic frameworks (MCOFs) in extraction of metal ions, nitro explosives and chemical pollutants.</jats:p

Na+^{+}-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions.

We previously demonstrated that inhibition of Na-dependent phosphate (P(i)) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/P(i) cotransporters in the osteoclast plasma membrane provide P(i) for ATP synthesis and that the osteoclast may utilize part of the P(i) released from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/P(i) cotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/P(i) cotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/P(i) cotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/P(i) cotransporters in the osteoclast

Na⁺-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions

We previously demonstrated that inhibition of Na-dependent phosphate (Pi) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/Picotransporters in the osteoclast plasma membrane provide Pifor ATP synthesis and that the osteoclast may utilize part of the Pireleased from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/Picotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/Picotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/Picotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/Picotransporters in the osteoclast.</jats:p