Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes

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Abstract

To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2-binding proteins, and CCN2-CCN2 and CCN2-CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (Kd) were 1.17 × 10 -9 m for CCN2 and CCN2, and 1.95 × 10-9 m for CCN2 and CCN3. Ectopically overexpressed green fluorescent protein-CCN2 and Halo-CCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2-CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3. To identify proteins that regulate CCN2 activity in chondrocytes, yeast two-hybrid screenings identified CCN2 and CCN3, and their binding domains were estimated. Direct binding between CCN2 and CCN3 was confirmed in vitro and in vivo. CCN2 enhanced, whereas CCN3 inhibited, the expression of cartilaginous matrix genes; and the combined treatment with CCN2 and CCN3 abolished the inhibitory effect by CCN3.

Original languageEnglish
Pages (from-to)3584-3597
Number of pages14
JournalFEBS Journal
Volume279
Issue number19
DOIs
Publication statusPublished - Oct 2012

Fingerprint

Connective Tissue Growth Factor
Chondrocytes
Aggrecans
Phenotype
Yeast
Antibodies
Screening
Yeasts
Blocking Antibodies
Surface Plasmon Resonance
von Willebrand Factor
Surface plasmon resonance
Green Fluorescent Proteins
Gene Library
Dimers
Carrier Proteins
Proteins
Genes
Fluorescence
Cells

Keywords

  • ACAN
  • CCN2/CTGF
  • CCN3/NOV
  • chondrocyte
  • dimerization

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

@article{efa8570ffac3443db7002eb28f3eed0c,
title = "Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes",
abstract = "To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2-binding proteins, and CCN2-CCN2 and CCN2-CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (Kd) were 1.17 × 10 -9 m for CCN2 and CCN2, and 1.95 × 10-9 m for CCN2 and CCN3. Ectopically overexpressed green fluorescent protein-CCN2 and Halo-CCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2-CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3. To identify proteins that regulate CCN2 activity in chondrocytes, yeast two-hybrid screenings identified CCN2 and CCN3, and their binding domains were estimated. Direct binding between CCN2 and CCN3 was confirmed in vitro and in vivo. CCN2 enhanced, whereas CCN3 inhibited, the expression of cartilaginous matrix genes; and the combined treatment with CCN2 and CCN3 abolished the inhibitory effect by CCN3.",
keywords = "ACAN, CCN2/CTGF, CCN3/NOV, chondrocyte, dimerization",
author = "Mitsuhiro Hoshijima and Takako Hattori and Eriko Aoyama and Takashi Nishida and Takashi Yamashiro and Masaharu Takigawa",
year = "2012",
month = "10",
doi = "10.1111/j.1742-4658.2012.08717.x",
language = "English",
volume = "279",
pages = "3584--3597",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "19",

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TY - JOUR

T1 - Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes

AU - Hoshijima, Mitsuhiro

AU - Hattori, Takako

AU - Aoyama, Eriko

AU - Nishida, Takashi

AU - Yamashiro, Takashi

AU - Takigawa, Masaharu

PY - 2012/10

Y1 - 2012/10

N2 - To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2-binding proteins, and CCN2-CCN2 and CCN2-CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (Kd) were 1.17 × 10 -9 m for CCN2 and CCN2, and 1.95 × 10-9 m for CCN2 and CCN3. Ectopically overexpressed green fluorescent protein-CCN2 and Halo-CCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2-CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3. To identify proteins that regulate CCN2 activity in chondrocytes, yeast two-hybrid screenings identified CCN2 and CCN3, and their binding domains were estimated. Direct binding between CCN2 and CCN3 was confirmed in vitro and in vivo. CCN2 enhanced, whereas CCN3 inhibited, the expression of cartilaginous matrix genes; and the combined treatment with CCN2 and CCN3 abolished the inhibitory effect by CCN3.

AB - To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2-binding proteins, and CCN2-CCN2 and CCN2-CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (Kd) were 1.17 × 10 -9 m for CCN2 and CCN2, and 1.95 × 10-9 m for CCN2 and CCN3. Ectopically overexpressed green fluorescent protein-CCN2 and Halo-CCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2-CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3. To identify proteins that regulate CCN2 activity in chondrocytes, yeast two-hybrid screenings identified CCN2 and CCN3, and their binding domains were estimated. Direct binding between CCN2 and CCN3 was confirmed in vitro and in vivo. CCN2 enhanced, whereas CCN3 inhibited, the expression of cartilaginous matrix genes; and the combined treatment with CCN2 and CCN3 abolished the inhibitory effect by CCN3.

KW - ACAN

KW - CCN2/CTGF

KW - CCN3/NOV

KW - chondrocyte

KW - dimerization

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U2 - 10.1111/j.1742-4658.2012.08717.x

DO - 10.1111/j.1742-4658.2012.08717.x

M3 - Article

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AN - SCOPUS:84866344934

VL - 279

SP - 3584

EP - 3597

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

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