Rab3D Regulates a Novel Vesicular Trafficking Pathway That Is Required for Osteoclastic Bone Resorption

Abstract

Rab3 proteins are a subfamily of GTPases, known to mediate membrane transport in eukaryotic cells and play a role in exocytosis. Our data indicate that Rab3D is the major Rab3 species expressed in osteoclasts. To investigate the role of Rab3D in osteoclast physiology we examined the skeletal architecture of Rab3D-deficient mice and found an osteosclerotic phenotype. Although basal osteoclast number in null animals is normal the total eroded surface is significantly reduced, suggesting that the resorptive defect is due to attenuated osteoclast activity. Consistent with this hypothesis, ultrastructural analysis reveals that Rab3D^−/− osteoclasts exhibit irregular ruffled borders. Furthermore, while overexpression of wild-type, constitutively active, or prenylation-deficient Rab3D has no significant effects, overexpression of GTP-binding-deficient Rab3D impairs bone resorption in vitro. Finally, subcellular localization studies reveal that, unlike wild-type or constitutively active Rab3D, which associate with a nonendosomal/lysosomal subset of post-trans-Golgi network (TGN) vesicles, inactive Rab3D localizes to the TGN and inhibits biogenesis of Rab3D-bearing vesicles. Collectively, our data suggest that Rab3D modulates a post-TGN trafficking step that is required for osteoclastic bone resorption.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We thank Robert Raffaniello (State University of New York-Health Science Center at Brooklyn, N.Y.) for generously providing Rab3D antibodies and Romano Regazzi (Institut de Biologie Cellulaire et de Morphologie, Lausanne, Switzerland) for the myc-Rab3DQ81L expression vector. All flow cytometry experiments were carried out in the Western Australian Lotteries Commission Flow Cytometry Unit with the assistance of Matthew Wikstrom. Confocal microscopy was carried out at the Western Australian Lotteries Commission Confocal Microscopy Unit at the UWA Department of Pharmacology. Special thanks go to Paul Rigby and Verity Smuts for the assistance with the confocal and scanning electron microscopy and to all members of the Zheng and Jahn laboratories for their insightful discussions.

This research was supported by grants from the National Health and Medical Research Council of Australia and National Institutes of Health awarded to Ming-Hao Zheng (ID9736447), F. Patrick Ross (AR48812 and AR46852), and Steven L. Teitelbaum (AR32788, AR46523, and AR48853).