FKBP12: A partner of Snx10 required for vesicular trafficking in osteoclasts

Abstract

Osteoclasts employ highly specialized intracellular trafficking controls for bone resorption and organelle homeostasis. The sorting nexin Snx10 is a (Phosphatidylinositol 3-phosphate) PI3P-binding protein, which localizes to osteoclast early endosomes. Osteoclasts from humans and mice lacking functional Snx10 are severely dysfunctional. They show marked impairments in endocytosis, extracellular acidification, ruffled border formation, and bone resorption, suggesting that Snx10 regulates membrane trafficking. To better understand how SNx10 regulates vesicular formation and trafficking in osteoclasts, we set out on a search for Snx10 partners. We performed a yeast two-hybrid screening and identified FKBP12. FKBP12 is expressed in receptor activator of nuclear factor kB ligand-stimulated RAW264.7 monocytes, coimmunoprecipitates with Snx10, and colocalizes with Snx10 in osteoclasts. We also found that FKBP12, Snx10, and early endosome antigen 1 (EEA1) are present in the same subcellular fractions obtained by centrifugation in sucrose gradients, which confirms localization of FKBP12 to early endosomes. Taken together, these results indicate that Snx10 and FKBP12 are partners and suggest that Snx10 and FKBP12 are involved in the regulation of endosome/lysosome homeostasis via the synthesis. These findings may suggest novel therapeutic approaches to control bone loss by targeting essential steps in osteoclast membrane trafficking.

Keywords: osteoclast; resorption; vesicular trafficking.

Figure 1:. Y2H screening identifies FKBP12 as a partner of Snx10

The Predicted Biological Score (PBS) indicates interaction reliability and is primarily based on the comparison between the number of independent prey fragments found for an interaction and the chance of finding them at random (background noise). A PBS of A indicates the highest confidence. FKBP12 had 9 hits representing 3 independent clones: 1. Clone 15-641 (4 hits), 2. Clone 21-451 (2 hits) and 3. Clone 20-477 (3 hits). These numbers indicate the position of the 5p and 3p prey fragment ends, relative to the position of the ATG start codon (A=0). The degree of identity of all these fragments relative to the reference gene (FKBP12) is 100%.

Figure 2:. FKBP12 and Snx10 are expressed in the same (lighter) sucrose gradient fractions.

Collected gradient fractions 1–12 of differentiated RAW264.7 cells were analyzed by SDS-PAGE and Western blotting for the distribution of organelle markers: early endosome antigen 1 (EEA1; EE marker), FKBP12 and Snx10. Fractions 1-3 contained the majority of the FKBP12 signal.

Figure 3.. FKBP12 and Snx10 coprecipitate in osteoclast protein extracts

FKBP12 is expressed in unstimulated and RANKL-stimulated RAW264.7 cells (2a). Snx10 interacts with FKBP12 in RANKL-stimulated RAW264.7 cells (2b). Immunoprecipitation was done using an α-FKBP12 antibody. Immunoprecipitates were subjected to western blot analysis using an α-Snx10 antibody. The black arrow (2b) indicates a 25KD band corresponding to Snx10.

Figure 4.. Immunofluorescent analysis confirms of Snx10 and FKBP12 co-localize to osteoclasts

RANKL and M-CSF stimulated mouse bone marrow mononuclear cells were fixed and stained with Snx10 (orange) and FKBP12 (green) antibodies. DAPI staining indicate nucleus. The superimposed image (overlay) indicates that both proteins colocalize to endosomes.