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.
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
(A and B) Histomorphometry analyses of osteoclast and osteoblast parameters of the femurs of 2-month-old male wild-type, germline knockout (KO), control (con), and conditional knockout (cKO) mice. N.OC, number of osteoclasts; OC.Pm, osteoclast perimeter; N.OB, number of osteoblasts; OB.Pm, osteoblast perimeter; MAR, bone mineral apposition rate; BFR, bone formation rate. The data are presented as scatter dot plots (WT, n = 13; KO, n = 12–13; con, n = 15; cKO, n = 12). The mean and SD of each group are overlaid onto each column of dots. **P < 0.01 versus WT or con by Student’s t test. (C and F) Alkaline phosphatase staining of 14-day cultures of osteoblasts. (D and G) Alizarin red staining of bone nodules of 21-day cultures of osteoblasts. Images are representatives of 3 wells/genotype from 3 independent experiments. Original magnification, ×1. (E and H) qPCR detection of Runx2 and osteocalcin (Ocn) mRNA expression during osteoblast differentiation (n = 3). (C–E) Osteoblasts from calvarias in a 6-well plate. (F–H) Osteoblasts from bone marrow stromal cells in a 12-well plate.