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Quantitative proteomic analysis of dexamethasone-induced effects on osteoblast differentiation, proliferation, and apoptosis in MC3T3-E1 cells using SILAC

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Abstract

Summary

The impairment of osteoblast differentiation is one cause of the glucocorticoid-induced osteoporosis (GCOP). The quantitative proteomic analysis of the dexamethasone (DEX)-induced effects of osteoblast differentiation, proliferation, and apoptosis using stable-isotope labeling by amino acids in cell culture (SILAC) demonstrated drastic changes of some key proteins in MC3T3-E1 cells.

Introduction

The impairment of osteoblast differentiation is one of the main explanations of GCOP. SILAC enables accurate quantitative proteomic analysis of protein changes in cells to explore the underlying mechanism of GCOP.

Methods

Osteoprogenitor MC3T3-E1 cells were treated with or without 10−6 M DEX for 7 days, and the differentiation ability, proliferation, and apoptosis of the cells were measured. The protein level changes were analyzed using SILAC and liquid chromatography-coupled tandem mass spectrometry.

Results

In this study, 10−6 M DEX inhibited both osteoblast differentiation and proliferation but induced apoptosis in osteoprogenitor MC3T3-E1 cells on day 7. We found that 10−6 M DEX increased the levels of tubulins (TUBA1A, TUBB2B, and TUBB5), IQGAP1, S100 proteins (S100A11, S100A6, S100A4, and S100A10), myosin proteins (MYH9 and MYH11), and apoptosis and stress proteins, while inhibited the protein levels of ATP synthases (ATP5O, ATP5H, ATP5A1, and ATP5F1), G3BP-1, and Ras-related proteins (Rab-1A, Rab-2A, and Rab-7) in MC3T3-E1 cells.

Conclusions

Several members of the ATP synthases, myosin proteins, small GTPase superfamily, and S100 proteins may participate in functional inhibition of osteoblast progenitor cells by GCs. Such protein expression changes may be of pathological significance in coping with GCOP.

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Abbreviations

ACTA:

Actin, alpha skeletal muscle

ACTB:

Actin, cytoplasmic 1

ALP:

Alkaline phosphatase

ANXA1:

Annexin A1

ANXA8:

Annexin A8

ATP5A1:

ATP synthase subunit alpha, mitochondrial precursor

ATP5F1:

ATP synthase B chain, mitochondrial precursor

ATP5H:

ATP synthase D chain, mitochondrial

ATP5O:

ATP synthase O subunit, mitochondrial precursor

BAX:

Apoptosis regulator BAX

DEX:

Dexamethasone

G3BP1:

Ras GTPase-activating protein-binding protein 1

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GCOP:

GC-induced osteoporosis

GC:

Glucocorticoid

HSPA4:

Heat shock 70 kDa protein 4

HSP90AA1:

Heat shock protein HSP 90-alpha

IQGAP1:

Ras GTPase-activating-like protein 1

INTS3:

Integrator complex subunit 3

RAB:

Ras-related protein

SILAC:

Stable-isotope labeling by amino acids in cell culture

TUBA1A:

Tubulin alpha-1 chain

TUBB2B:

Tubulin beta-2B chain

TUBB5:

Tubulin beta-5 chain

LC-MS/MS:

Liquid chromatography-coupled tandem mass spectrometry

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MYH9:

Myosin 9

MYBBP1A:

Myb-binding protein 1A

MYH11:

Isoform 1 of myosin 11

PDCD6IP:

Programmed cell death 6-interacting protein

PDCD6:

Programmed cell death 6

PIGOK:

Protein Interrogation of Gene Ontology and KEGG databases

PTRF:

Polymerase I and transcript release factor

PPIB:

Peptidylprolyl isomerase B

VCP:

Transitional endoplasmic reticulum ATPase

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Acknowledgements

This work is supported by the NIH (Grant DE 018385), the National Natural Science Foundation of China (30950019), and the Medicine and Health Research Fund of Zhejiang Province, China (2009B166). We thank Chantal M. Sottas for the technical support.

Conflicts of interest

None.

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Authors and Affiliations

  1. Population Council, 1230 York Avenue, New York, NY, 10065, USA

    D. Hong, C. Wang & R.-S. Ge

  2. Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, 317000, China

    D. Hong & H.-X. Chen

  3. Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY, 10021, USA

    H.-Q. Yu & H.-T. Deng

  4. The Second Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China

    Q.-Q. Lian & R.-S. Ge

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  1. D. Hong
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  2. H.-X. Chen
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  3. H.-Q. Yu
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  4. C. Wang
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  5. H.-T. Deng
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  6. Q.-Q. Lian
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  7. R.-S. Ge
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Corresponding authors

Correspondence to H.-T. Deng or R.-S. Ge.

Additional information

Dun Hong and Hai-Xiao Chen contributed equally to this work.

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Hong, D., Chen, HX., Yu, HQ. et al. Quantitative proteomic analysis of dexamethasone-induced effects on osteoblast differentiation, proliferation, and apoptosis in MC3T3-E1 cells using SILAC. Osteoporos Int 22, 2175–2186 (2011). https://doi.org/10.1007/s00198-010-1434-8

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  • DOI: https://doi.org/10.1007/s00198-010-1434-8

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