NFAT3 and TGF-β-SMAD3 regulate the expression of miR-140 in osteoarthritisReportar como inadecuado

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Arthritis Research and Therapy

, 15:R197

First Online: 21 November 2013Received: 04 February 2013Accepted: 12 November 2013


IntroductionMicroRNAs miRNAs down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 WWP2 gene, decreases the expression of genes that play detrimental roles in osteoarthritis OA. As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.

MethodsGene expression in human chondrocytes was determined by quantitative polymerase chain reaction qPCR and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs siRNAs. Binding sites of the miR-140 regulatory sequence rsmiR-140 were identified by mutagenesis and chromatin immunoprecipitation ChIP in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.

ResultsIn contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 NMP4, myc-associated zinc MAZ, nuclear factor of activated T-cells NFAT, and mothers against decapentaplegic homolog 3 SMAD3. Silencing NFAT3 P ≤0.01 and SMAD3 P ≤0.05 differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 P ≤0.003 and P ≤0.05, respectively. NFAT3 activation increased and transforming growth factor-β TGF-β decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 activator and SMAD3 repressor directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression.

ConclusionsThis is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.

AbbreviationsDMEMDulbecco’s modified Eagle’s medium

ACRAmerican College of Rheumatology

ADAMTSA disintegrin and metalloproteinase with a thrombospondin type 1 motif

BMP2Bone morphogenetic protein 2

bpBase pair

BSABovine serum albumin

ChIPChromatin immunoprecipitation

DAPI4’,6-diamidino-2-phenylindole, dihydrochloride

FAKFocal adhesion kinase

FCSFetal calf serum

GAPDHGlyceraldehyde 3-phosphate dehydrogenase

IGFBP5Insulin-like growth factor-binding protein-5

IgGImmunoglobulin G

MAZMyc-associated zinc


MMPMatrix metalloproteinase

NFATNuclear factor of activated T-cells

NF-κBNuclear factor kappa B

NMP4Nuclear matrix transcription factor 4


PBSPhosphate-buffered saline

PCRPolymerase chain reaction

PTK2Protein tyrosine kinase

qPCRQuantitative PCR

rsmiR-140miR-140 regulatory sequence

SEMStandard error of the mean

siRNASmall interfering RNA

SIS3Specific inhibitor of SMAD3 phosphorylation

SMAD3Mothers against decapentaplegic homolog 3

SREBF2Sterol regulatory element binding factor-2

TGF-βTransforming growth factor beta

TSSTranscription start sites

WWP2WW domain containing E3 ubiquitin protein ligase 2.

Electronic supplementary materialThe online version of this article doi:10.1186-ar4387 contains supplementary material, which is available to authorized users.

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Autor: Ginette Tardif - Jean-Pierre Pelletier - Hassan Fahmi - David Hum - Yue Zhang - Mohit Kapoor - Johanne Martel-Pelletier


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