Rab32 and Rab38 maintain bone homeostasis by regulating intracellular traffic in osteoclasts.

Osteoclasts play a crucial role in bone homeostasis by forming resorption pits on bone surfaces, resulting in bone resorption. The osteoclast expression of Rab38 protein is highly induced during differentiation from macrophages. Here we generated mice with double knockout (DKO) of Rab38 and its paralogue, Rab32, to investigate the roles of these proteins in osteoclasts. Bone marrow-derived macrophages from Rab32/38 DKO mice differentiated normally into osteoclasts in vitro. However, DKO osteoclasts showed reduced bone resorption activity. These osteoclasts also demonstrated defective secretion of tartrate-resistant acid phosphatase and cathepsin K into culture medium. Furthermore, the plasma membrane localization of a3, an osteoclast-specific a subunit of V-ATPase, was abrogated in DKO mice, substantiating the reduced resorption activity. In vivo, Rab32- and Rab38-positive cells were attached to the bone surface. Eight-week-old DKO mice showed significantly thickened trabecular bones in micro-CT and histomorphometry analysis, as well as reduced serum levels of cross-linked C-telopeptide of type I collagen, indicating diminished bone resorption in vivo. In DKO male mice over 10 weeks of age, hyperostosis appeared at the talofibular syndesmosis, the distal junction of the tibia and fibula. Furthermore, middle-aged mice (10 to 12 months of age) exhibited kyphosis, which is not usually observed in wild-type male mice until around 24 months of age. These results indicate that Rab32 and Rab38 contribute to osteoclast function by supporting intracellular traffic, thereby maintaining normal bone homeostasis.Key words: Rab32, Rab38, osteoclast, lysosome-related organelle, secretory lysosome.

Characterization of Rab32- and Rab38-positive lysosome-related organelles in osteoclasts and macrophages.

Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts.

Influences of menopause, aging, and gender on the cleavage of type II collagen in cartilage in relationship to bone turnover.

OBJECTIVE: It is unclear whether there are changes in cartilage turnover after menopause, although these are well documented in bone. The aim of this study was to explore the possibility that menopause might change cartilage turnover as well as bone turnover. We also examined age and gender to estimate the independent influences of these parameters together with menopause on cartilage and bone turnover. DESIGN: Serum samples were collected from 140 healthy volunteers, 69 men (mean age +/- SD, 42.8 +/- 13.8 y) and 71 women (44.4 +/- 10.5 y) with self-reported pre- or postmenopausal status who had no swelling or pain in their spine and joints. Body mass index was also recorded. The serum concentration of a biomarker of cartilage type II collagen (C2C) degradation and concentrations of serum bone alkaline phosphatase and urine cross-linked type I collagen N-telopeptide, both accepted biomarkers of bone turnover, were measured together. Analyses of covariance were performed to test the mean differences of biomarkers by gender and by menopause status with age adjustment. RESULTS: In men there were no significant correlations between age and the biomarkers. In women C2C concentration decreased with age. Cross-linked type I collagen N-telopeptide and bone alkaline phosphatase levels were both increased after menopause, whereas C2C showed no detectable changes. C2C was not significantly related to body mass index. CONCLUSIONS: This study suggests that there are fundamental differences in the degradation of uncalcified C2C and bone type I collagen degradation and bone assembly in response to menopause and aging.