Identifying genes that are responsive to low magnitude mechanical signals Elizabeth Russell Robert J. Pignolo
Identifying genes that are responsive to low magnitude mechanical signals
Elizabeth Russell
Robert J. Pignolo
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
Adaptation to mechanical loading at cortical and cancellous sites
Eser P et al. Bone 34:869-80 (2004); Shields RK et al. Arch Phys Med Rehabil 87:1376-81 (2006)
Distal tibia of untrained (L)limb shows extensive loss of trabecular bone
Skeletal stress, bone remodeling, and muscle mass II
During bone formation process, some of the osteoblasts are entrapped in the bone matrix, where they differentiate to new osteocytes
Resorption cavity weakens trebecula and causes a local elevation of strain
Osteoblasts are recruited by osteocytes to form bone
After repair, remaining osteoblasts become lining cells, covering the new bone surface
Quiescent bone experiences stress
lining cells
osteocytes
osteoclast
Stimuli from other osteocytes
Osteoclasts
OsteocytesDistribution
of strain
Mechanicalloading
Resorptioncavity
Perturbations
Recruitmentstimuli
Sensation ofrate of strain
OsteoblastsBone
architectureBone
formation
Boneresorption
Enhanced external load intensity (amplitude and frequency) and resorption cavities induce bone formation
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
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)
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
Identifying vibration-induced bone-enhancing (Vibe) genes
Cells responsiveto mechanical signals
Isolation of RNA Isolation of secreted protein
MicroarrayAnalysis
Mass SpectroscopyAnalysis
LMMS x 10 min; 37oC x 1 hr
Acetone precipitation1D gel separationTrypsin digestion
Identifying vibration-induced bone-enhancing (Vibe) genes
Early passage MSCs Senescent MSCs
+ LMMS 3 Samples 3 Samples
- LMMS 3 Samples 3 Samples
-2.3
-2.25
-2.2
-2.15
-2.1
-2.05
-2
-1.95
-1.9
1 2 3 4 5 6 7 8 9 10
Transcripts downregulated by LMMS
Rel
ativ
e ex
pre
ssio
n
1. ZNF5782. Unknown3. Unknown4. Unknown5. Unknown6. Unknown7. Unknown8. LOC22 14429. Unknown10.SNORD 25
Microarray analysis: Genes downregulated by LMMS
1. Unknown2. Unknown3. Unknown4. Unknown5. Unknown6. Unknown7. Unknown8. Unknown
Microarray analysis: Genes upregulated by LMMS
1.9
1.95
2
2.05
2.1
2.15
2.2
2.25
2.3
2.35
2.4
1 2 3 4 5 6 7 8
Transcripts upregulated by LMMS
Rel
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Mass spectroscopy analysis: Secreted proteins responsive to LMMS
75kD
70kD
45kD
30kD
E ES SS S-LMMS +LMMS
E- E+
0
5
10
15
20
25
30
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46
Secreted proteins upregulated by LMMS
Rel
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Mass spectroscopy analysis: Secreted proteins upregulated in response to LMMS
Identification of LMMS- responsive secretory proteins Icollagen alpha-2(I) chain precursor [Homo sapiens] 3.017009
unknown [Homo sapiens] 5.135734
unknown [Homo sapiens] 3.807923
plasminogen activator inhibitor-1 precursor [Homo sapiens] 9.86036
unknown [Homo sapiens] 2.875161
unnamed protein product [Homo sapiens] 28.17117
unnamed protein product [Homo sapiens] 3.220077
unnamed protein product [Homo sapiens] 4.024024
unnamed protein product [Homo sapiens] 2.280781
unnamed protein product [Homo sapiens] 3.702703
unnamed protein product [Homo sapiens] 2.951952
keratin, type II cytoskeletal 5 [Homo sapiens] 4.024775
cathepsin B preproprotein [Homo sapiens] 3.018018
periostin isoform 4 [Homo sapiens] 3.542342
Collagen alpha 1 chain precursor variant [Homo sapiens] 8.045045
unnamed protein product [Homo sapiens] 3.824324
unnamed protein product [Homo sapiens] 6.441441
tissue inhibitor of metalloproteinases [Homo sapiens] 12.51
unnamed protein product [Homo sapiens] 2.280781
unnamed protein product [Homo sapiens] 17.87
Identification of LMMS- responsive secretory proteins IIunnamed protein product [Homo sapiens] 4.0225
unnamed protein product [Homo sapiens] 7.15
unnamed protein product [Homo sapiens] 1.3422
unknown [Homo sapiens] 6.25
unnamed protein product [Homo sapiens] 5.36
unnamed protein product [Homo sapiens] 12.51
unknown [Homo sapiens] 11.61
annexin A2 isoform 2 [Homo sapiens] 4.8288
keratin [Homo sapiens] 2.8153
unnamed protein product [Homo sapiens] 8.04
unnamed protein product [Homo sapiens] 3.57
complement C4-A preproprotein [Homo sapiens] 3.57
unnamed protein product [Homo sapiens] 6.25
fibrillin-1 precursor [Homo sapiens] 4.47
unnamed protein product [Homo sapiens] 3.57
unnamed protein product [Homo sapiens] 4.47
unnamed protein product [Homo sapiens] 4.47
unnamed protein product [Homo sapiens] 4.47
serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1 [Homo sapiens] 2.68
-14
-12
-10
-8
-6
-4
-2
0
1 2 3 4 5 6 7 8 9 10
Secreted proteins downregulated by LMMS
Rel
ativ
e E
xpre
ssio
n
Mass spectroscopy analysis: Secreted proteins downregulated in response to LMMS
Identification of LMMS- responsive secretory proteins
transforming growth factor, beta-induced, 68kDa, isoform CRA_b [Homo sapiens] -2.00861
transferrin, isoform CRA_d [Homo sapiens] -1.86434
hemoglobin subunit alpha [Homo sapiens] -1.86555
Chain B, Human 1gg1 Fc Fragment, 2.5 Angstrom Structure -12.22
unnamed protein product [Homo sapiens] -2.48045
alpha 2 macroglobulin variant [Homo sapiens] -7.77
aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit A [Coprococcus catus GD/7] -3.74157
unnamed protein product [Homo sapiens] -3.33
haptoglobin-related protein precursor [Homo sapiens] -3.33
unnamed protein product [Homo sapiens] -2.22
Identifying vibration-induced bone-enhancing (Vibe) genes
Identification ofLMMS-responsive genes
Differentially expressedLMMS-responsive genes
LMMS-responsive genes notinduced in senescent cells
Mass spectroscopy analysis: Secreted proteins responsive to LMMS
75kD
70kD
45kD
30kD
E ES SS S-LMMS +LMMS
S+E+ S+
0
5
10
15
20
25
30
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
Secreted proteins upregulated by LMMS
Rel
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Differentially expressed LMMS-responsive genes