Osteoporosis International

, Volume 22, Issue 7, pp 2175–2186

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

Original Article

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.

Keywords

Apoptosis Dexamethasone Osteoblast differentiation Proliferation Proteomics SILAC 

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

Copyright information

© International Osteoporosis Foundation and National Osteoporosis Foundation 2010

Authors and Affiliations

  1. 1.Population CouncilNew YorkUSA
  2. 2.Orthopedic DepartmentTaizhou Hospital, Wenzhou Medical CollegeLinhaiChina
  3. 3.Proteomics Resource CenterThe Rockefeller UniversityNew YorkUSA
  4. 4.The Second Affiliated HospitalWenzhou Medical CollegeWenzhouChina

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