PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis
Toshifumi Fujiwara, … , Stavros C. Manolagas, Haibo Zhao
Toshifumi Fujiwara, … , Stavros C. Manolagas, Haibo Zhao
Published October 20, 2016
Citation Information: JCI Insight. 2016;1(17):e86330. https://doi.org/10.1172/jci.insight.86330.
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Research Article Bone biology

PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis

Abstract

Mutations of the Plekhm1 gene in humans and rats cause osteopetrosis, an inherited bone disease characterized by diminished bone resorption by osteoclasts. PLEKHM1 binds to RAB7 and is critical for lysosome trafficking. However, the molecular mechanisms by which PLEKHM1 regulates lysosomal pathways remain unknown. Here, we generated germline and conditional Plekhm1-deficient mice. These mice displayed no overt abnormalities in major organs, except for an increase in trabecular bone mass. Furthermore, loss of PLEKHM1 abrogated the peripheral distribution of lysosomes and bone resorption in osteoclasts. Mechanistically, we indicated that DEF8 interacts with PLEKHM1 and promotes its binding to RAB7, whereas the binding of FAM98A and NDEL1 with PLEKHM1 connects lysosomes to microtubules. Importantly, suppression of these proteins results in lysosome positioning and bone resorption defects similar to those of Plekhm1-null osteoclasts. Thus, PLHKEM1, DEF8, FAM98A, and NDEL1 constitute a molecular complex that regulates lysosome positioning and secretion through RAB7.

Authors

Toshifumi Fujiwara, Shiqiao Ye, Thiago Castro-Gomes, Caylin G. Winchell, Norma W. Andrews, Daniel E. Voth, Kottayil I. Varughese, Samuel G. Mackintosh, Yunfeng Feng, Nathan Pavlos, Takashi Nakamura, Stavros C. Manolagas, Haibo Zhao

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Figure 6

Loss of Plekhm1 has no effects on TFEB expression, lysosome biogenesis, autophagic, and endocytic degradation pathways in mature osteoclasts.

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Loss of Plekhm1 has no effects on TFEB expression, lysosome biogenesis, ...
(A) qPCR detection of mRNA expression of Tfeb and its downstream target genes, Clcn7, Dpp7, Ostm1, and Tcirg1, during osteoclast differentiation in wild-type and Plekhm1–/– (KO) cultures (mean ± SD, n = 3). m, bone marrow monocytes; pOC, preosteoclasts; OC, mature osteoclasts. (B) Western blot detection of protein expression of TFEB during osteoclast differentiation in WT and KO cultures. The lysate of 293-T cells served as a positive control. Actin served as a loading control. The data are representatives of 3 independent experiments. (C) Western blot detection of LAMP-2 expression during osteoclast differentiation. Tubulin served as a loading control. The experiment was repeated 3 times. (D) Mature osteoclasts were either untreated (FBS) or underwent serum and amino acid starvation in HBSS buffer for 2 hours. In another set of experiment, osteoclasts were treated with DMSO, 250 nm of mTOR inhibitor Torin1, and 50 μM of lysosome inhibitor chloroquin (CQ) for 2 hours. The protein levels of phospho-mTOR, LC3-I, and LC3-II were detected by Western blots. The ratio of LC3-II/LC3-I was measured by densitometry of Western blots using NIH ImageJ software. The experiment was repeated twice. (E and F) Receptor degradation assays of c-Fms (E) and EGF receptor (EGF-R) (F) in osteoclasts stimulated with cytokines for the indicated times, as detected by Western blotting and densitometry quantification using NIH ImageJ software. Actin served as loading controls. The experiments were repeated twice.