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Introduction
The coupling of osteogenesis and osteoclastogenesis is mediated through intercellular signaling during bone remodeling (1). Receptor activator of nuclear factor-κB ligand (RANKL) is a central player in osteoblast-induced osteoclast differentiation (2). RANKL is expressed in osteoblasts and osteocytes and binds the receptor activator of nuclear factor-κB on the surface of osteoclast precursors to induce osteoclast differentiation through the NF-κB pathway (2). Osteoprotegerin (OPG) functions as a soluble decoy receptor of RANKL, and thereby inhibits osteoclastogenesis (2).
Runt-related transcription factor 2 (RUNX2) is a master transcription factor in osteogenesis (3). The targeted disruption of RUNX2 results in a complete loss of bone formation owing to the arrest of osteoblast maturation (4). Transgenic mice overexpressing RUNX2 in osteoblasts show increased osteoclast differentiation and dramatically enhanced bone resorption (5). The dual role of RUNX2 in osteogenesis and osteoclastogenesis indicates its involvement in integrating signals between osteoblasts and osteoclasts (6). RUNX2 transactivates many essential genes in osteogenesis (7,8), yet its role in osteoclastogenesis remains unclear. There are putative RUNX2 binding sites in the RANKL promoter sequence (9), however, RUNX2 does not significantly affect RANKL expression in osteoblasts (10). It has also been reported that RUNX2 may regulate osteoclastogenesis through RANKL trafficking (6). This observation suggests that RUNX2 may target genes required for the regulation of the intracellular trafficking of RANKL.
A previous genomic analysis reported that the forced expression of RUNX2 upregulated several genes related to intracellular trafficking (11); one of these genes encoded for lysosomal-associated protein transmembrane 5 (LAPTM5). LAPTM5 is a transmembrane protein that resides in lysosomes and functions as a regulator of protein trafficking (12). The transport of LAPTM5-positive vesicles from the Golgi to the lysosome is modulated by its binding to the E3 ubiquitin ligase NEDD4, a HECT-type E3 ligase that belongs to the Nedd4 family (13). LAPTM5 is involved in the negative regulation of antigen receptor expression as a mediator of lysosomal degradation (14–16). The role of LAPTM5 in lysosomal function and its possible regulation by RUNX2 led to the hypothesis that LAPTM5 may be a modulator of RANKL trafficking in osteoblasts, the assessment of which could further the understanding of the coupling of osteogenesis and osteoclastogenesis. In the present study, this hypothesis was initially tested by analyzing RUNX2 binding sites in the...