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Ascorbate-induced osteoblast differentiation recruits distinct MMP-inhibitors: RECK and TIMP-2

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Abstract

The bone formation executed by osteoblasts represents an interesting research field both for basic and applied investigations. The goal of this work was to evaluate the molecular mechanisms involved during osteoblast differentiation in vitro. Accordingly, we demonstrated that, during the osteoblastic differentiation, TIMP-2 and RECK presented differential expressions, where RECK expression was downregulated from the 14th day in contrast with an increase in TIMP-2. Concomitantly, our results showed a temporal regulation of two major signaling cascades during osteoblast differentiation: proliferation cascades in which RECK, PI3 K, and GSK-3β play a pivotal role and latter, differentiation cascades with participation of Ras, Rho, Rac-1, PKCα/β, and TIMP-2. Furthermore, we observed that phosphorylation level of paxillin was downregulated while FAK125 remained unchangeable, but active during extracellular matrix (ECM) remodeling. Concluding, our results provide evidences that RECK and TIMP-2 are involved in the control of ECM remodeling in distinct phases of osteoblast differentiation by modulating MMP activities and a multitude of signaling proteins governs these events.

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Abbreviations

ALP:

Alkaline phosphatase

ECM:

Extracellular matrix

MMP:

Matrix metalloproteinase

MT1-MMP:

MT1-matrix metalloproteinase

TIMP-2:

Tissue inhibitors of metalloproteinases

RECK:

Reversion-inducing-cysteine-rich protein with Kazal Motifs

FAK:

Focal adhesion kinase

p-NPP:

p-Nitrophenyl-phosphate

FBS:

Fetal bovine serum

DTT:

Dithiothreitol

SDS:

Sodium dodecyl sulfate

PMSF:

Phenyl-methyl sulphonyl fluoride-serine-protease enzyme inhibitor

PVDF:

Polyvinylidene fluoride

PTK:

Protein tyrosine kinase

PTP:

Protein tyrosine phosphatase

cdk:

Cyclin-dependent kinase

Rb:

Retinoblastoma

MAPK:

Mitogen-activated protein kinase

LIMK:

LIM kinases

ATCC:

American type culture collection

PBS:

Phosphate-buffered saline

TBS:

Tris-buffered saline

ECL:

Enhanced chemoluminescence

FGF:

Fibroblast growth factor

PI3K:

Phosphoinositide-3 kinase

GSK-3β:

Glycogen synthase kinase 3β

PKC:

Protein kinase C

MDA:

Malondialdehyde

MPO:

N-methyl-2-phenylindole

CDNB:

1-Chloro-2,4-dinitrobenzene

GST:

Glutathione-S-transferase

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Acknowledgements

We are grateful to Dr. Claudia L. Soraggi, Ms. Denise B. Ciampi and Rodrigo A. da Silva for the technical support. We thank Prof. Hernandes F. Carvalho for critical suggestions. WFZ is supported by a PhD scholarship from Fundação de Amparo à Pesquisa do Estado de São Paulo (grant no. 04/14906-2).

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Authors and Affiliations

  1. Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Cidade Universitária, 13083-970, Campinas, Sao Paulo, Brazil

    Willian F. Zambuzzi, Claudia L. Yano, Alexandre D. M. Cavagis & Carmen V. Ferreira

  2. Universidade Metodista de Piracicaba (UNIMEP), Piracicaba, Sao Paulo, Brazil

    Alexandre D. M. Cavagis

  3. Kinome Profiling Unit, Department of Cell Biology, University Medical Center Groningen, University of Groningen Groningen, Groningen, The Netherlands

    Maikel P. Peppelenbosch

  4. Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense (UFF), Niterói, Rio de Janeiro, Brazil

    José Mauro Granjeiro

  5. Núcleo de Terapia Celular e Molecular (Nucell), Universidade de São Paulo (USP), Sao Paulo, Sao Paulo, Brazil

    José Mauro Granjeiro

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  1. Willian F. Zambuzzi
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  2. Claudia L. Yano
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Correspondence to Willian F. Zambuzzi.

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Zambuzzi, W.F., Yano, C.L., Cavagis, A.D.M. et al. Ascorbate-induced osteoblast differentiation recruits distinct MMP-inhibitors: RECK and TIMP-2. Mol Cell Biochem 322, 143–150 (2009). https://doi.org/10.1007/s11010-008-9951-x

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