Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans
Liesbeth Van Wesenbeeck, Paul R. Odgren, Fraser P. Coxon, Annalisa Frattini, Pierre Moens, Bram Perdu, Carole A. MacKay, Els Van Hul, Jean-Pierre Timmermans, Filip Vanhoenacker, Ruben Jacobs, Barbara Peruzzi, Anna Teti, Miep H. Helfrich, Michael J. Rogers, Anna Villa, Wim Van Hul
Liesbeth Van Wesenbeeck, Paul R. Odgren, Fraser P. Coxon, Annalisa Frattini, Pierre Moens, Bram Perdu, Carole A. MacKay, Els Van Hul, Jean-Pierre Timmermans, Filip Vanhoenacker, Ruben Jacobs, Barbara Peruzzi, Anna Teti, Miep H. Helfrich, Michael J. Rogers, Anna Villa, Wim Van Hul
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Research Article Bone biology

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

Abstract

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

Authors

Liesbeth Van Wesenbeeck, Paul R. Odgren, Fraser P. Coxon, Annalisa Frattini, Pierre Moens, Bram Perdu, Carole A. MacKay, Els Van Hul, Jean-Pierre Timmermans, Filip Vanhoenacker, Ruben Jacobs, Barbara Peruzzi, Anna Teti, Miep H. Helfrich, Michael J. Rogers, Anna Villa, Wim Van Hul

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Figure 6

Subcellular localization of Plekhm1 in HEK 293 cells.

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Subcellular localization of Plekhm1 in HEK 293 cells. HEK293 cells were ...
HEK293 cells were transfected with plasmids, then fixed 24 hours later and analyzed by confocal microscopy. (A) Both Plekhm1-EGFP and Plekhm1-dsRedM displayed a diffuse cytoplasmic distribution, but were also clearly associated with intracellular vesicles, which frequently became abnormally enlarged. (B–E) HEK293 cells were cotransfected with Plekhm1-dsRedM and EGFP-Rab5 (B), EGFP-Rab6 (C), EGFP-Rab7 (D), or EGFP-Rab9 (E). Plekhm1 did not colocalize with either Rab5 or Rab6, but partially colocalized with Rab9 and completely colocalized to intracellular vesicles with Rab7. Interestingly, Plekhm1 showed more pronounced localization to these Rab7-positive vesicles when cotransfected with EGFP-Rab7. (F and G) HEK293 cells were transfected with EGFP-Rab7 and Plekhm1-dsRedM mutants corresponding to the mutations found in the osteopetrotic patient (hplekhm1; F) and the ia rat (rplekhm1; G). Neither mutant form of Plekhm1 colocalized with Rab7. Panels represent 1-μm xy optical sections. Scale bars: 10 μm.