Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells

Alexandra Karystinou, Anke Jozefien Roelofs, Anna Neve, Francesco P Cantatore, Henning Wackerhage, Cosimo De Bari

Research output: Contribution to journalArticle

32 Citations (Scopus)
7 Downloads (Pure)

Abstract

Introduction
The control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs.

Methods
Expression of total YAP, its paralogue transcriptional co-activator with PDZ-binding motif (TAZ), and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis, C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally, YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry.

Results
YAP, but not TAZ, was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants, however, was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1, Id2 and Id3 in response to BMP-2. In developing mouse limbs, Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage, suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo.

Conclusions
Our findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology.

Original languageEnglish
Article number147
Number of pages14
JournalArthritis Research & Therapy
Volume17
Early online date30 May 2015
DOIs
Publication statusPublished - 30 May 2015

Fingerprint

Chondrogenesis
Mesenchymal Stromal Cells
Proteins
Bone Morphogenetic Protein 2
Down-Regulation
Reverse Transcription
Cartilage
Western Blotting
Genes
Polymerase Chain Reaction
Alcian Blue
Rheumatology
Tissue Engineering
Hindlimb

Keywords

  • mesenchymal stem cells
  • YAP
  • TAZ
  • chondrogenesis
  • transforming growth factor beta
  • bone morphogenetic proteins
  • Smad signalling
  • cartilage repair
  • osteparthritis

Cite this

Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells. / Karystinou, Alexandra; Roelofs, Anke Jozefien; Neve, Anna; Cantatore, Francesco P; Wackerhage, Henning; De Bari, Cosimo.

In: Arthritis Research & Therapy, Vol. 17, 147, 30.05.2015.

Research output: Contribution to journalArticle

@article{01dbd9ef0a494746bcecc11f079590f1,
title = "Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells",
abstract = "IntroductionThe control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs. MethodsExpression of total YAP, its paralogue transcriptional co-activator with PDZ-binding motif (TAZ), and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis, C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally, YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry. ResultsYAP, but not TAZ, was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants, however, was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1, Id2 and Id3 in response to BMP-2. In developing mouse limbs, Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage, suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo. ConclusionsOur findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology.",
keywords = "mesenchymal stem cells, YAP , TAZ, chondrogenesis, transforming growth factor beta, bone morphogenetic proteins, Smad signalling, cartilage repair, osteparthritis",
author = "Alexandra Karystinou and Roelofs, {Anke Jozefien} and Anna Neve and Cantatore, {Francesco P} and Henning Wackerhage and {De Bari}, Cosimo",
note = "This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Acknowledgements The authors would like to thank Dr Marius Sudol for the hYAP plasmids (obtained through Addgene), Dr Pete Zammit for the pMSCV-IRES-eGFP plasmid, Dr Robert Judson for subcloning the hYAP cDNAs into the pMSCV-IRES-eGFP plasmid, Dr Lynda Erskine for the provision of mouse embryo samples, and Professor Jimmy Hutchison and the Orthopaedics Department at the Aberdeen Royal Infirmary for the provision of human tissue samples. The authors are also grateful to Denise Tosh and Susan Clark for excellent technical support. This work was funded by Arthritis Research UK (grant 19429).",
year = "2015",
month = "5",
day = "30",
doi = "10.1186/s13075-015-0639-9",
language = "English",
volume = "17",
journal = "Arthritis Research & Therapy",
issn = "1478-6354",
publisher = "BioMed Central",

}

TY - JOUR

T1 - Yes-associated protein (YAP) is a negative regulator of chondrogenesis in mesenchymal stem cells

AU - Karystinou, Alexandra

AU - Roelofs, Anke Jozefien

AU - Neve, Anna

AU - Cantatore, Francesco P

AU - Wackerhage, Henning

AU - De Bari, Cosimo

N1 - This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Acknowledgements The authors would like to thank Dr Marius Sudol for the hYAP plasmids (obtained through Addgene), Dr Pete Zammit for the pMSCV-IRES-eGFP plasmid, Dr Robert Judson for subcloning the hYAP cDNAs into the pMSCV-IRES-eGFP plasmid, Dr Lynda Erskine for the provision of mouse embryo samples, and Professor Jimmy Hutchison and the Orthopaedics Department at the Aberdeen Royal Infirmary for the provision of human tissue samples. The authors are also grateful to Denise Tosh and Susan Clark for excellent technical support. This work was funded by Arthritis Research UK (grant 19429).

PY - 2015/5/30

Y1 - 2015/5/30

N2 - IntroductionThe control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs. MethodsExpression of total YAP, its paralogue transcriptional co-activator with PDZ-binding motif (TAZ), and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis, C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally, YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry. ResultsYAP, but not TAZ, was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants, however, was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1, Id2 and Id3 in response to BMP-2. In developing mouse limbs, Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage, suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo. ConclusionsOur findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology.

AB - IntroductionThe control of differentiation of mesenchymal stromal/stem cells (MSCs) is crucial for tissue engineering strategies employing MSCs. The purpose of this study was to investigate whether the transcriptional co-factor Yes-associated protein (YAP) regulates chondrogenic differentiation of MSCs. MethodsExpression of total YAP, its paralogue transcriptional co-activator with PDZ-binding motif (TAZ), and individual YAP transcript variants during in vitro chondrogenesis of human MSCs was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). YAP expression was confirmed by western blotting. To determine the effect of high YAP activity on chondrogenesis, C3H10T1/2 MSC-like cells were transduced with human (h)YAP and treated in micromass with bone morphogenetic protein-2 (BMP-2). Chondrogenic differentiation was assessed by alcian blue staining and expression of chondrocyte-lineage genes. BMP signalling was determined by detection of pSmad1,5,8 by western blotting and expression of BMP target genes by quantitative RT-PCR. Finally, YAP and pYAP were detected in mouse embryo hindlimbs by immunohistochemistry. ResultsYAP, but not TAZ, was downregulated during in vitro chondrogenesis of human MSCs. One of the YAP transcript variants, however, was upregulated in high-density micromass culture. Overexpression of hYAP in murine C3H10T1/2 MSCs inhibited chondrogenic differentiation. High YAP activity in these cells decreased Smad1,5,8 phosphorylation and expression of the BMP target genes Inhibitor of DNA binding/differentiation (Id)1, Id2 and Id3 in response to BMP-2. In developing mouse limbs, Yap was nuclear in the perichondrium while mostly phosphorylated and cytosolic in cells of the cartilage anlage, suggesting downregulation of Yap co-transcriptional activity during physiological chondrogenesis in vivo. ConclusionsOur findings indicate that YAP is a negative regulator of chondrogenic differentiation of MSCs. Downregulation of YAP is required for chondrogenesis through derepression of chondrogenic signalling. Therapeutic targeting of YAP to promote cartilage repair and prevent secondary osteoarthritis is an exciting prospect in rheumatology.

KW - mesenchymal stem cells

KW - YAP

KW - TAZ

KW - chondrogenesis

KW - transforming growth factor beta

KW - bone morphogenetic proteins

KW - Smad signalling

KW - cartilage repair

KW - osteparthritis

U2 - 10.1186/s13075-015-0639-9

DO - 10.1186/s13075-015-0639-9

M3 - Article

VL - 17

JO - Arthritis Research & Therapy

JF - Arthritis Research & Therapy

SN - 1478-6354

M1 - 147

ER -