Oral Presentation ANZBMS-MEPSA-ANZORS 2022

EphrinB2 and lysosomes in osteocytes are essential for collagen arrangement in the extracellular matrix (#15)

Martha Blank 1 2 , Mriga Dutt 3 , Benjamin L Parker 3 , Cameron J Nowell 4 , T. John Martin 1 2 , Natalie A Sims 1 2
  1. Bone Cell Biology and Disease Unit, St. Vincent’s Institute of Medical Research, Melbourne, Victoria, Australia
  2. Department of Medicine, The University of Melbourne, St. Vincent’s Hospital, Melbourne, Victoria, Australia
  3. Department of Anatomy and Physiology, The University of Melbourne, Melbourne, Victoria, Australia
  4. Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia

Lysosomes are acidic intracellular vesicles. They contain enzymes that degrade collagen matrix in bone, and are essential for osteoclast-mediated bone resorption. However, their importance in osteocytes is unknown. We recently observed lysosome deficiency in cultured osteocytes with EphrinB2 (Efnb2) knockdown, and that Efnb2-deficiency in osteocytes (Dmp1Cre.Efnb2) caused a phenotype of fragile bones with greater mineral and collagen content than controls. Here we sought to identify the lysosomal enzymes affected by Efnb2-deficiency in osteocytes and determine how they regulate the collagen matrix.

To investigate how Efnb2-deficiency influences lysosomal collagen-degrading enzymes in osteocytes, we performed proteomics on cultured Efnb2-deficient and vector control Ocy454 cells. Multiple collagen-degrading proteins were lower in Efnb2-deficient cells compared to controls. This included matrix metallopeptidases 2 (MMP2) and 14 (MMP14), procathepsin L (CTSL), cathepsin B (CTSB) and cathepsin D (CTSD).

Next, we assessed whether the absence of these enzymes effects the ability of osteocytes to remodel collagen in a 3D culture system. Control osteocytes caused significant collagen contraction, to 25% of the original area at day 15 and only 15% at day 21. In contrast, collagen containing Efnb2-deficient Ocy454 cells showed no matrix contraction at day 15, and less contraction than controls (30% of original area) at day 21.

To determine how the absence of collagen-degrading enzymes in osteocytes affects the collagen matrix in vivo, we assessed collagen fiber architecture in tibial cortical bone from 12 week old Dmp1Cre.Efnb2 mice by second harmonic generation imaging. Dmp1Cre.Efnb2 bones had thicker collagen fiber bundles with less parallel orientation than control bones.

These data indicate that the absence of collagen-degrading enzymes in osteocytes resulting from EphrinB2-deficiency limits osteocyte-dependent collagen contraction in vitro and collagen arrangement in vivo. This suggests a novel role for osteocyte-derived lysosomes to prevent aggregation of collagen in their surrounding matrix to preserve optimum bone quality and strength.