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 4

Loss of Plekhm1 attenuates bone resorption without affecting osteoclast differentiation.

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Loss of Plekhm1 attenuates bone resorption without affecting osteoclast ...
(A) Tartrate-resistant acid phosphatase (TRAP) staining of wild-type and Plekhm1–/– (KO) osteoclasts cultured on plastic dishes and bovine cortical bone slices. Representative images are from 6 samples/group/culture of 3 independent cultures from different mice. Scale bar: 200 μm. (B) Number of TRAP+ osteoclasts with more than 3 nuclei/well of a 48-well plate (n = 6). (C) Protein expression of osteoclast markers, cathepsin K (CTSK) and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), as detected by Western blotting during osteoclast differentiation. Actin served as a loading control. m, bone marrow monocytes; pOC, preosteoclasts; OC, mature osteoclasts. The experiment was repeated 3 times. (D) mRNA expression of osteoclast marker genes, Acp5 (also known as TRAP), Ctsk, Nfactc1, calcitonin receptor (CalcR), in mature osteoclasts, as measured by quantitative real-time PCR (n = 3). (E) Resorption pit staining of WT and KO osteoclasts cultured on bone slices. White arrows point out the tiny pits dug by KO osteoclasts. Each image is a representative of 6 bone slices/group/culture of at least 3 independent cultures from different mice. Scale bar: 40 μm. (F) The amount of medium CTx-I, collagen fragments released by osteoclasts during bone resorption, was measured by ELISA (n = 5). **P < 0.01 versus WT by Student t test. Data in B, D, and F are presented as scatter dot plots. The mean and SD of each group are overlaid onto each column of dots.