Oral Virtual Presentation (Virtual only) ESA-SRB-ANZBMS 2021

EphrinB2 limits mineralisation in osteocytes in an mTOR-dependent manner by downregulating the lysosomal master transcriptional regulator TFEB (#14)

Martha Blank 1 2 , Naomi Ling 3 , Emma McGowan 1 , William Smiles 4 , Blessing Crimeen-Irwin 1 , Mriga Dutt 5 , Benjamin L Parker 5 , 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. Metabolic Signalling Unit, St. Vincent’s Institute of Medical Research, Melbourne, Victoria, Australia
  4. Neurometabolism Unit, St. Vincent's Institute of Medical Research, Melbourne, Victoria, Australia
  5. Department of Anatomy and Physiology, The University of Melbourne, Melbourne, Victoria, Australia

Lysosomes are acidic vesicles that degrade cytoplasmic contents, and while they are essential for osteoclast-mediated bone resorption, their function in osteocytes is unknown. We recently observed that EphrinB2 (Efnb2) deletion in osteocytes leads to hypermineralised, fragile bones and reduced osteocyte lysosome content. Osteocyte-mediated mineralisation is limited by RhoA-associated protein kinase, downstream of the mammalian Target Of Rapamycin (mTOR) signalling pathway. Lysosome biogenesis is controlled by Transcription Factor EB (TFEB), a substrate of mTOR. Here, we sought to define whether EphrinB2 limits lysosome numbers and mineralisation through an mTOR-dependent molecular pathway.

To determine the cause of lysosome deficiency, we measured TFEB in Efnb2-deficient Ocy454 osteocyte-like cells and found that TFEB mRNA and protein levels were reduced, by 50% and 66%, respectively. Proteomic analysis confirmed that multiple known TFEB targets, including lysosomal hydrolases, membrane and acidification proteins were significantly downregulated in Efnb2-deficient osteocytes.

We next defined the upstream and downstream targets of mTOR signalling modified by Efnb2 deficiency in osteocytes. Western blotting revealed no difference in upstream AMPK activity (pT172), but AKT activity (pT308) was doubled in Efnb2-knockdown cells compared to control. mTOR has two downstream signalling complexes, mTORC1 and mTORC2. While we found no change in active RhoA, an mTORC2 target, in hypermineralising Efnb2-knockdown cells compared to controls, mTORC1 activation (p70S6K (pT389)) was doubled. Furthermore, when we inhibited mTORC1 activity with rapamycin, this reduced mineral deposition by ~95% in wildtype Ocy454 cells. This suggests that EphrinB2 loss elevates AKT activity, leading to higher mTORC1 activity and greater mineralisation.

These data indicates that EphrinB2 may restrain mineralisation by increasing TFEB activity and lysosome numbers in osteocytes through limiting AKT and mTORC1 activity. This suggests a novel role for TFEB and lysosomes in osteocyte function, specifically in the control of mineralisation and bone strength.