Leucine Repeat Rich Kinase 1 Controls Osteoclast Activity by Managing Lysosomal Trafficking and Secretion

SIMPLE SUMMARY: Osteoporosis, an age-related disease, develops in part because the rate of new bone formation does not catch up with the increased bone destruction caused by osteoclasts. A full understanding of how osteoclasts function is essential to identify novel drug targets for osteoporosis treatment. Previously, we demonstrated that mice lacking leucine rich repeat kinase 1 (LRRK1) had high bone mass due to defective resorption caused by dysfunctional osteoclasts. Little is known about how LRRK1 regulates osteoclast activity. Here, we found that LRRK1-deficient osteoclasts had an altered distribution of the acidic vacuoles/lysosomes, unlike the wild-type osteoclast. The lysosomes remained in the cytoplasm away from the extracellular lacunae of the bone. The apparent F-actin ring was large, but the sealing zone was weak. Albeit CTSK and v-ATPase were comparably expressed in LRRK1 deficient osteoclasts, CTSK and v-ATPase, two lysosome-associated proteins, were not stationed on the ruffled border. Our data indicate the LRRK1 positively controls osteoclast activity via regulating lysosomal distribution, acid secretion, and protease exocytosis. ABSTRACT: We previously demonstrated that mice with targeted deletion of the leucine repeat rich kinase 1 (Lrrk1) gene were osteopetrotic due to the failure of osteoclasts to resorb bone. To determine how LRRK1 regulates osteoclast activity, we examined the intracellular and extracellular acidification with an acidotropic probe, acridine orange, in live osteoclasts on bone slices. We examined lysosome distribution in osteoclasts by localization of LAMP-2, cathepsin K, and v-ATPase by immunofluorescent staining with specific antibodies. We found that both vertical and horizontal cross-sectional images of the wild-type (WT) osteoclasts showed orange-staining of the intracellular acidic vacuoles/lysosomes dispersed to the ruffled border. By contrast, the LRRK1 deficient osteoclasts exhibited fluorescent orange staining in the cytoplasm away from the extracellular lacunae because of an altered distribution of the acidic vacuoles/lysosomes. In addition, WT osteoclasts displayed a peripheral distribution of LAMP-2 positive lysosomes with a typical actin ring. The clustered F-actin constitutes a peripheral sealing zone and a ruffled border which was stretched out into a resorption pit. The LAMP-2 positive lysosomes were also distributed to the sealing zone, and the cell was associated with a resorption pit. By contrast, LRRK1-deficient osteoclasts showed diffused F-actin throughout the cytoplasm. The sealing zone was weak and not associated with a resorption pit. LAMP-2 positive lysosomes were also diffuse in the cytoplasm and were not distributed to the ruffled border. Although the LRRK1-deficient osteoclast expressed normal levels of cathepsin K and v-ATPase, the lysosomal-associated cathepsin K and v-ATPase were not accumulated at the ruffled border in Lrrk1 KO osteoclasts. Our data indicate that LRRK1 controls osteoclast activity by regulating lysosomal distribution, acid secretion, and protease exocytosis.