Identifying genes that are responsive to low magnitude mechanical signals

1. Identifying genes that are responsive to low magnitude mechanical signals Elizabeth Russell Robert J. Pignolo

2. Decreased maintenance of bone mass Mechanical loading & Bone Mass

3. Adaptation to mechanical loading at cortical and cancellous sites Adaptation to daily, cyclic, axial loading of a long bone results in: (1) Inhibition of bone loss (2) Elevated bone mineral content (3) Greater effects at cancellous versus cortical sites (4) Variation depending on the term and level of loading

4. Distal tibia of untrained (L) limb shows extensive loss of trabecular bone Skeletal stress, bone remodeling, and muscle mass II Eser P et al. Bone 34:869-80 (2004); Shields RK et al.Arch Phys Med Rehabil 87:1376-81 (2006)

5. Osteocytes are mechanosensors

6. lining cells      osteocytes osteoclast Quiescent bone experiences stress Resorption cavity weakens trebecula and causes a local elevation of strain Osteoblasts are recruited by osteocytes to form bone During bone formation process, some of the osteoblasts are entrapped in the bone matrix, where they differentiate to new osteocytes After repair, remaining osteoblasts become lining cells, covering the new bone surface

7. Resorption cavity Bone resorption Osteoclasts Perturbations Stimuli from other osteocytes Mechanical loading Bone architecture Bone formation Osteoblasts Recruitment stimuli Distribution of strain Sensation of rate of strain Osteocytes Enhanced external load intensity (amplitude and frequency) and resorption cavities induce bone formation

8. Mechanosensitive cells • MSCs, osteoblasts, osteoclasts, osteocytes and cells of the vasculature • Early mechanosensing mechanisms: • may involve ion channels, integrins, connexins, caveolar and noncaveolar lipid rafts, shape alteration at the membrane or cytoskeleton • G-proteins, MAPKs, and nitric oxide implicated in downstream intracellular signaling • Low magnitude mechanical signals (LMMS) have been shown to increase the number of MSCs, as well as their potential to differentiate into osteoblasts versus adipocytes Rubin J et al Gene 2006; 367:1–16; Luu, YK et al J Bone Miner Res 2009;24:50–61

9. Osteocyte dysfunction with aging • With age the skeleton becomes less responsive to loads • Osteocyte damage or apoptosis in the young skeleton leads to osteoclastic bone resorption followed by formation, but in the aged skeleton can lead to empty lacunae or micropetrosis where the lacuna fills in with mineral • Changes in peri-lacunar mineral density, elastic modulus of the peri-lacunar matrix, and in the size of lacunae and canaliculi affect mechanosensation by the aging osteocyte • Even if the osteocyte remained viable for decades, its mechanoresponsiveness would be compromised Bonewald, L Working Group on Skeletal Aging, ASBMR Annual Meeting (2009)

10. Noninvasive device to achieve low-magnitude mechanical stimulation • Extremely low magnitude mechanical signals (LMMS): • Improve both quantity and quality of trabecular bone • Are anabolic to trabecular bone in children • Increase bone and muscle mass in the weight-bearing skeleton of young adult females with low BMD • Increase spinal trabecular bone and keep visceral fat at baseline levels in young women with osteopenia • Currently being tested in a 2-year, double-blind, randomized, placebo-controlled clinical trial in 200 elderly women and men J Bone Miner Res 2002;17:349–357; J Bone Miner Res 2004;19:360 –369; J Bone Miner Res 2006;21:1464–1474;Luu, YK et al J Bone Miner Res 2009;24:50–61

11. Identifying vibration-induced bone-enhancing (Vibe) genes Cells responsive to mechanical signals LMMS x 10 min; 37oC x 1 hr Isolation of RNA Isolation of secreted protein Acetone precipitation 1D gel separation Trypsin digestion Microarray Analysis Mass Spectroscopy Analysis

12. Identifying vibration-induced bone-enhancing (Vibe) genes

13. Microarray analysis: Genes downregulated by LMMS • ZNF578 • Unknown • Unknown • Unknown • Unknown • Unknown • Unknown • LOC22 1442 • Unknown • SNORD 25

14. Microarray analysis: Genes upregulated by LMMS • Unknown • Unknown • Unknown • Unknown • Unknown • Unknown • Unknown • Unknown

15. -LMMS +LMMS E S S E S S 75kD 70kD 45kD 30kD E- E+ Mass spectroscopy analysis: Secreted proteins responsive to LMMS

16. Mass spectroscopy analysis: Secreted proteins upregulated in response to LMMS

17. Identification of LMMS- responsive secretory proteins I

18. Identification of LMMS- responsive secretory proteins II

19. Mass spectroscopy analysis: Secreted proteins downregulated in response to LMMS

20. Identification of LMMS- responsive secretory proteins

21. Identification of LMMS-responsive genes LMMS-responsive genes not induced in senescent cells Differentially expressed LMMS-responsive genes Identifying vibration-induced bone-enhancing (Vibe) genes

22. -LMMS +LMMS E S S E S S 75kD 70kD 45kD 30kD E+ S+ S+ Mass spectroscopy analysis: Secreted proteins responsive to LMMS

23. Differentially expressed LMMS-responsive genes

24. Identifying vibration-induced bone-enhancing (Vibe) genes Identification of LMMS-responsive genes LMMS-responsive genes not induced in senescent cells Differentially expressed LMMS-responsive genes Functional effects: - Enhanced Ob differentiation - Increased mineral apposition rate Vibe Genes