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

Abstract

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+/H+ Exchanger Regulatory Factor 1 (NHERF1) Directly Regulates Osteogenesis

“…Although described in osteoclasts (27), NHERF1 expression was not previously shown to occur in primary osteoblasts that produce bone matrix. We now show that NHERF1 is prominently expressed in osteoblasts that mediate matrix production (Fig.…”

Intestinal Npt2b Plays a Major Role in Phosphate Absorption and Homeostasis

“…29,30 Bone histomorphometric analysis did not identify differences between WT and Npt2b Ϫ/Ϫ mice (Table 1). These observations mirror those observed in the Npt2c Ϫ/Ϫ mouse where differences in bone changes relative to WT were not found.…”

“…10 Taken together, these data suggest that the type II transporters play a minor role in bone remodeling or may compensate for each other when one is absent. It has been proposed that the type III phosphate cotransporter Pit1, which also is expressed in osteoclasts and osteoblasts, 29 may be the critical phosphate transporter in bone.…”

Identification of a Novel Transport-independent Function of PiT1/SLC20A1 in the Regulation of TNF-induced Apoptosis

“…A scaffolding role is also revealed by the Drosophila Na,K-ATPase, which seems to have evolved from having primarily a pumping function to a more specialized, pump-independent role as a scaffold protein for the formation and functioning of epithelial junctions (50). PiT1 has been shown to co-localize with the actin cytoskeleton in murine osteoclasts (65), and its absence could therefore affect the whole coupling of the plasma membrane to the cytoskeleton and indirectly modify the cell response to exogenous stimuli. PiT1 could also play a role as a scaffolding protein, via its large intracellular domain, and gather the proteins involved in signaling or the dampening of this signaling.…”