Multinucleated osteoclasts are specialised cells with the capacity to resorb bone. To perform this activity, the osteoclast has a unique membrane area, the ruffled border, where protons are released to lower the pH. The membrane of the ruffled border is formed by fusion of lysosomal vacuoles and thus resembles the membrane of a lysosome. The composition of the lysosomal membrane is different from all other membranes due to an extreme high carbohydrate content, characteristic phospholipids, the presence of cholesterol, vacuolar proton ATPase and the membrane proteins LAMP-1 and 2. The latter proteins constitute 50% of the total amount of proteins of the membrane. Since it is known that the expression of LAMP-2 is very high in the ruffled border membrane, we investigated whether this protein plays a role in the formation of osteoclasts and/or in resorption. Bone marrow cells and osteoblasts were isolated from 6 weeks old wild type and LAMP-2 deficient male mice (approved by the Animal Welfare Committee, VU University Amsterdam). The bone marrow cells were cultured with M-CSF and RANKL for 6 days or co-cultured with osteoblasts for 21 days. Gene expression of RANKL was measured by qPCR. The presence of RANKL at the cell-membrane was investigated by FACS analysis.
We found in the cultures with M-CSF and RANKL more osteoclasts in the LAMP-2 deficient cultures. These osteoclasts contained also more nuclei per cell. Surprisingly, in co-cultures of osteoblasts isolated from LAMP-2 deficient mice with bone marrow cells, osteoclast formation was completely absent. Immunohistochemical staining of osteoblasts showed a comparable expression of RANKL between wild-type and LAMP-2 deficient osteoblasts. However, FACS revealed that RANKL expression on the plasma membrane was strongly reduced (WT 51% and LAMP-2 deficient cells 19%). Our data strongly suggest that LAMP-2 plays a crucial role in intracellular transportation of RANKL to the plasma membrane.
Disclosure: The authors declared no competing interests.