Osteoporosis International

, Volume 22, Issue 7, pp 2175–2186 | Cite as

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

Notes

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.

References

  1. 1.
    Canalis E, Bilezikian JP, Angeli A et al (2004) Perspectives on glucocorticoid-induced osteoporosis. Bone 34:593–598PubMedCrossRefGoogle Scholar
  2. 2.
    Rubin MR, Bilezikian JP (2002) Clinical review 151: the role of parathyroid hormone in the pathogenesis of glucocorticoid-induced osteoporosis: a re-examination of the evidence. J Clin Endocrinol Metab 87:4033–4041PubMedCrossRefGoogle Scholar
  3. 3.
    Bouvard B, LeGrand C, Audran M et al (2010) Glucocorticoid-induced osteoporosis: a review. Clin Rev Bone Miner Metab 8:15–26CrossRefGoogle Scholar
  4. 4.
    Smith E, Frenkel B (2005) Glucocorticoids inhibit the transcriptional activity of LEF/TCF in differentiating osteoblasts in a glycogen synthase kinase-3beta-dependent and -independent manner. J Biol Chem 280:2388–2394PubMedCrossRefGoogle Scholar
  5. 5.
    Ohnaka K, Tanabe M, Kawate H et al (2005) Glucocorticoid suppresses the canonical Wnt signal in cultured human osteoblasts. Biochem Biophys Res Commun 329:177–181PubMedCrossRefGoogle Scholar
  6. 6.
    Leclerc N, Noh T, Cogan J et al (2008) Opposing effects of glucocorticoids and Wnt signaling on Krox20 and mineral deposition in osteoblast cultures. J Cell Biochem 103:1938–1951PubMedCrossRefGoogle Scholar
  7. 7.
    Camps M, Nichols A, Arkinstall S (2000) Dual specificity phosphatases: a gene family for control of MAP kinase function. FASEB J 14:6–16PubMedGoogle Scholar
  8. 8.
    Canalis E (2008) Notch signaling in osteoblasts. Sci Signal 1:pe17PubMedCrossRefGoogle Scholar
  9. 9.
    Iu MF, Kaji H, Sowa H et al (2005) Dexamethasone suppresses Smad3 pathway in osteoblastic cells. J Endocrinol 185:131–138PubMedCrossRefGoogle Scholar
  10. 10.
    Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386PubMedCrossRefGoogle Scholar
  11. 11.
    Jacob RJ, Cramer R (2006) PIGOK: linking protein identity to gene ontology and function. J Proteome Res 5:3429–3432PubMedCrossRefGoogle Scholar
  12. 12.
    Marenholz I, Heizmann CW, Fritz G (2004) S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). Biochem Biophys Res Commun 322:1111–1122PubMedCrossRefGoogle Scholar
  13. 13.
    Engelbrecht Y, de Wet H, Horsch K et al (2003) Glucocorticoids induce rapid up-regulation of mitogen-activated protein kinase phosphatase-1 and dephosphorylation of extracellular signal-regulated kinase and impair proliferation in human and mouse osteoblast cell lines. Endocrinology 144:412–422PubMedCrossRefGoogle Scholar
  14. 14.
    Lian JB, Shalhoub V, Aslam F et al (1997) Species-specific glucocorticoid and 1, 25-dihydroxyvitamin D responsiveness in mouse MC3T3–E1 osteoblasts: dexamethasone inhibits osteoblast differentiation and vitamin D down-regulates osteocalcin gene expression. Endocrinology 138:2117–2127PubMedCrossRefGoogle Scholar
  15. 15.
    Smith E, Redman RA, Logg CR et al (2000) Glucocorticoids inhibit developmental stage-specific osteoblast cell cycle. Dissociation of cyclin A-cyclin-dependent kinase 2 from E2F4-p130 complexes. J Biol Chem 275:19992–20001PubMedCrossRefGoogle Scholar
  16. 16.
    O’Brien CA, Jia D, Plotkin LI et al (2004) Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology 145:1835–1841PubMedCrossRefGoogle Scholar
  17. 17.
    Jaglin XH, Poirier K, Saillour Y et al (2009) Mutations in the beta-tubulin gene TUBB2B result in asymmetrical polymicrogyria. Nat Genet 41:746–752PubMedCrossRefGoogle Scholar
  18. 18.
    Keays DA, Tian G, Poirier K et al (2007) Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans. Cell 128:45–57PubMedCrossRefGoogle Scholar
  19. 19.
    Plauchu H, Encha-Razavi F, Hermier M et al (2001) Lissencephaly type III, stippled epiphyses and loose, thick skin: a new recessively inherited syndrome. Am J Med Genet 99:14–20PubMedCrossRefGoogle Scholar
  20. 20.
    Shi X, Yang X, Chen D et al (1999) Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling. J Biol Chem 274:13711–13717PubMedCrossRefGoogle Scholar
  21. 21.
    Yang X, Ji X, Shi X et al (2000) Smad1 domains interacting with Hoxc-8 induce osteoblast differentiation. J Biol Chem 275:1065–1072PubMedCrossRefGoogle Scholar
  22. 22.
    Oster G, Wang H (2000) Reverse engineering a protein: the mechanochemistry of ATP synthase. Biochim Biophys Acta 1458:482–510PubMedCrossRefGoogle Scholar
  23. 23.
    Tsai B, Ye Y, Rapoport TA (2002) Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat Rev Mol Cell Biol 3:246–255PubMedCrossRefGoogle Scholar
  24. 24.
    Ye Y, Shibata Y, Yun C et al (2004) A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature 429:841–847PubMedCrossRefGoogle Scholar
  25. 25.
    Sakaguchi M, Sonegawa H, Murata H et al (2008) S100A11, an dual mediator for growth regulation of human keratinocytes. Mol Biol Cell 19:78–85PubMedCrossRefGoogle Scholar
  26. 26.
    Fullen DR, Reed JA, Finnerty B et al (2001) S100A6 expression in fibrohistiocytic lesions. J Cutan Pathol 28:229–234PubMedCrossRefGoogle Scholar
  27. 27.
    Ilg EC, Schafer BW, Heizmann CW (1996) Expression pattern of S100 calcium-binding proteins in human tumors. Int J Cancer 68:325–332PubMedCrossRefGoogle Scholar
  28. 28.
    Holt GE, Schwartz HS, Caldwell RL (2006) Proteomic profiling in musculoskeletal oncology by MALDI mass spectrometry. Clin Orthop Relat Res 450:105–110PubMedCrossRefGoogle Scholar
  29. 29.
    Luo X, Sharff KA, Chen J et al (2008) S100A6 expression and function in human osteosarcoma. Clin Orthop Relat Res 466:2060–2070PubMedCrossRefGoogle Scholar
  30. 30.
    Marenholz I, Lovering RC, Heizmann CW (2006) An update of the S100 nomenclature. Biochim Biophys Acta 1763:1282–1283PubMedCrossRefGoogle Scholar
  31. 31.
    Rescher U, Gerke V (2008) S100A10/p11: family, friends and functions. Pflugers Arch 455:575–582PubMedCrossRefGoogle Scholar
  32. 32.
    Yao XL, Cowan MJ, Gladwin MT et al (1999) Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity. J Biol Chem 274:17202–17208PubMedCrossRefGoogle Scholar
  33. 33.
    Siddappa R, Licht R, van Blitterswijk C et al (2007) Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering. J Orthop Res 25:1029–1041PubMedCrossRefGoogle Scholar
  34. 34.
    Kemppainen RJ, Behrend EN (1998) Dexamethasone rapidly induces a novel ras superfamily member-related gene in AtT-20 cells. J Biol Chem 273:3129–3131PubMedCrossRefGoogle Scholar
  35. 35.
    Chen YX, Li ZB, Diao F et al (2006) Up-regulation of RhoB by glucocorticoids and its effects on the cell proliferation and NF-kappaB transcriptional activity. J Steroid Biochem Mol Biol 101:179–187PubMedCrossRefGoogle Scholar
  36. 36.
    Briggs MW, Sacks DB (2003) IQGAP1 as signal integrator: Ca2+, calmodulin, Cdc42 and the cytoskeleton. FEBS Lett 542:7–11PubMedCrossRefGoogle Scholar
  37. 37.
    Mateer SC, Wang N, Bloom GS (2003) IQGAPs: integrators of the cytoskeleton, cell adhesion machinery, and signaling networks. Cell Motil Cytoskeleton 55:147–155PubMedCrossRefGoogle Scholar
  38. 38.
    Parker F, Maurier F, Delumeau I et al (1996) A Ras-GTPase-activating protein SH3-domain-binding protein. Mol Cell Biol 16:2561–2569PubMedGoogle Scholar
  39. 39.
    Tocque B, Delumeau I, Parker F et al (1997) Ras-GTPase activating protein (GAP): a putative effector for Ras. Cell Signal 9:153–158PubMedCrossRefGoogle Scholar
  40. 40.
    Gallouzi IE, Parker F, Chebli K et al (1998) A novel phosphorylation-dependent RNase activity of GAP-SH3 binding protein: a potential link between signal transduction and RNA stability. Mol Cell Biol 18:3956–3965PubMedGoogle Scholar
  41. 41.
    Soncini C, Berdo I, Draetta G (2001) Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease. Oncogene 20:3869–3879PubMedCrossRefGoogle Scholar
  42. 42.
    Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117PubMedCrossRefGoogle Scholar
  43. 43.
    Nuoffer C, Davidson HW, Matteson J et al (1994) A GDP-bound of rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments. J Cell Biol 125:225–237PubMedCrossRefGoogle Scholar
  44. 44.
    Tisdale EJ, Bourne JR, Khosravi-Far R et al (1992) GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. J Cell Biol 119:749–761PubMedCrossRefGoogle Scholar
  45. 45.
    Bucci C, Thomsen P, Nicoziani P et al (2000) Rab7: a key to lysosome biogenesis. Mol Biol Cell 11:467–480PubMedGoogle Scholar
  46. 46.
    Mukhopadhyay A, Funato K, Stahl PD (1997) Rab7 regulates transport from early to late endocytic compartments in Xenopus oocytes. J Biol Chem 272:13055–13059PubMedCrossRefGoogle Scholar
  47. 47.
    Press B, Feng Y, Hoflack B et al (1998) Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment. J Cell Biol 140:1075–1089PubMedCrossRefGoogle Scholar
  48. 48.
    Vonderheit A, Helenius A (2005) Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol 3:e233PubMedCrossRefGoogle Scholar
  49. 49.
    McMichael BK, Wysolmerski RB, Lee BS (2009) Regulated proteolysis of nonmuscle myosin IIA stimulates osteoclast fusion. J Biol Chem 284:12266–12275PubMedCrossRefGoogle Scholar
  50. 50.
    Soulez M, Rouviere CG, Chafey P et al (1996) Growth and differentiation of C2 myogenic cells are dependent on serum response factor. Mol Cell Biol 16:6065–6074PubMedGoogle Scholar
  51. 51.
    Tamama K, Sen CK, Wells A (2008) Differentiation of bone marrow mesenchymal stem cells into the smooth muscle lineage by blocking ERK/MAPK signaling pathway. Stem Cells Dev 17:897–908PubMedCrossRefGoogle Scholar
  52. 52.
    Autret A, Martin SJ (2009) Emerging role for members of the Bcl-2 family in mitochondrial morphogenesis. Mol Cell 36:355–363PubMedCrossRefGoogle Scholar
  53. 53.
    Missotten M, Nichols A, Rieger K et al (1999) Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein. Cell Death Differ 6:124–129PubMedCrossRefGoogle Scholar
  54. 54.
    Chatellard-Causse C, Blot B, Cristina N et al (2002) Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization. J Biol Chem 277:29108–29115PubMedCrossRefGoogle Scholar

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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