Lysosomes are acidic vesicles containing degradative enzymes, including cysteine proteases such as cathepsins. While lysosomal acid and enzymes degrade cytoplasmic contents and mediate bone resorption by osteoclasts, their function in the osteoblast lineage is unknown. We previously observed that mice with targeted Efnb2-deficiency in osteocytes have bone fragility due to high mineral and collagen contents, and that EphrinB2 (Efnb2)-deficient Ocy454 osteocyte-like cells have low lysosome activity. We therefore hypothesized that reduced lysosome activity in osteoblasts and osteocytes would lead to collagen and mineral accumulation in vivo and in vitro.
Using second harmonic generation microscopy it was found that bone of Efnb2-deficient mice had significantly (~10%) thicker collagen fibres than controls. Although mineralisation of these bones was high, wide-angle X-ray scattering detected no difference in mineral crystal length compared to controls, indicating that their greater mineralisation was due to greater mineral crystal numbers. Immunohistochemistry for a lysosomal marker (Lysosomal-associated Membrane Protein 1 (LAMP1)), revealed 34% fewer LAMP1-positive osteocytes than controls in mice with Efnb2-deficiency. This highlighted the association between EphrinB2 and lysosome deficiency in osteocytes in vivo.
Effects of lysosome activity inhibitors were tested in 7-day differentiated Ocy454 cells, to the stage of expressing osteoblast (Collagen1a1, Osterix) markers. Cells were exposed to a broad lysosomal activity inhibitor (bafilomycin A1) or an inhibitor against cysteine proteases (E-64) for 48 hours. Mineral accumulation was significantly greater with both treatments (by 58% and 74%, respectively), suggesting that lysosomal enzymes produced by late osteoblasts limit mineral accumulation in surrounding tissue.
These data suggest that EphrinB2 in osteoblasts and osteocytes limits mineral accrual and collagen fibre width by producing lysosomal enzymes, including cysteine proteases. This indicates that defects in lysosome production and activity in the osteoblast lineage may permit excess mineral and collagen accrual, and thereby impair bone material quality and bone strength.