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

Identifying genes that are responsive to low magnitude mechanical signals

Elizabeth Russell

Robert J. Pignolo

Mechanical loading & Bone Mass

Decreased maintenance of bone mass

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

Osteocytes are mechanosensors

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


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

ativ

e ex

pre

ssio

n

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

ativ

e ex

pre

ssio

n

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


plasminogen activator inhibitor-1 precursor [Homo sapiens] 9.86036


unnamed protein product [Homo sapiens] 28.17117






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



tissue inhibitor of metalloproteinases [Homo sapiens] 12.51



Identification of LMMS- responsive secretory proteins IIunnamed protein product [Homo sapiens] 4.0225







annexin A2 isoform 2 [Homo sapiens] 4.8288

keratin [Homo sapiens] 2.8153



complement C4-A preproprotein [Homo sapiens] 3.57


fibrillin-1 precursor [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


haptoglobin-related protein precursor [Homo sapiens] -3.33



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

ativ

e ex

pre

ssio

n

Differentially expressed LMMS-responsive genes


Identification ofLMMS-responsive genes

Differentially expressedLMMS-responsive genes

LMMS-responsive genes notinduced in senescent cells

Functional effects:- Enhanced Ob differentiation- Increased mineral apposition rate

VibeGenes

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