Repression of chondrogenesis through binding of notch signaling proteins HES-1 and HEY-1 to N-box domains in the COL2A1 enhancer site

Arthritis Rheum. 2008 Sep;58(9):2754-63. doi: 10.1002/art.23730.


Objective: Notch signaling is implicated in the repression of mesenchymal stem cell (MSC) chondrogenic differentiation. The purpose of this study was to examine the mechanism of this repression and how it is modulated to permit chondrogenesis.

Methods: Notch intracellular domain (NICD) protein levels were monitored via Western blotting throughout chondrogenic differentiation of human MSCs in pellet cultures. Overexpression of Notch signaling components and their effect on chondrogenesis was achieved by transfecting plasmids coding for NICD, HES-1, and HERP-2/HEY-1. COL2A1 and AGGRECAN expression was monitored via quantitative polymerase chain reaction analysis. Chromatin immunoprecipitation (ChIP) was used to test whether HES-1 and HEY-1 bind putative N-box domains in intron 1 of COL2A1.

Results: High levels of NICD proteins were reduced during chondrogenesis of human MSCs, and this was mediated by transforming growth factor beta3 (TGFbeta3). COL2A1 gene expression was repressed following overexpression of NICD (2-fold) and HES-1 (3-fold) and was markedly repressed by overexpression of HEY-1 (80-fold). HEY-1 repressed AGGRECAN expression 10-fold, while NICD and HES-1 had no effect. We identified 2 putative N-box domains adjacent to, and part of, the SOX9 enhancer binding site located in intron 1 of COL2A1. ChIP studies showed that endogenous HES-1 and HEY-1 bound to these sites. Transducin-like enhancer, the HES-1 corepressor protein, was displaced during chondrogenic differentiation and following TGFbeta3 treatment.

Conclusion: These results reveal novel mechanisms by which Notch signaling represses gene expression. Notch signaling proteins act on the SOX9 binding site in the COL2A1 enhancer and prevent SOX9-mediated transcriptional activation of COL2A1 and, thus, chondrogenic differentiation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Blotting, Western
  • Bone Morphogenetic Protein 6 / pharmacology
  • Carbamates / pharmacology
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cell Differentiation / physiology
  • Cells, Cultured
  • Chondrogenesis / drug effects
  • Chondrogenesis / physiology*
  • Chromatin Immunoprecipitation
  • Collagen Type II / metabolism*
  • Dipeptides / pharmacology
  • Fibroblast Growth Factor 2 / pharmacology
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism*
  • Humans
  • Mesenchymal Stem Cells
  • Receptors, Notch / genetics
  • Receptors, Notch / metabolism
  • Regulatory Sequences, Nucleic Acid / genetics
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • Time Factors
  • Transcription Factor HES-1
  • Transforming Growth Factor beta3 / pharmacology


  • Basic Helix-Loop-Helix Transcription Factors
  • Bone Morphogenetic Protein 6
  • COL2A1 protein, human
  • Carbamates
  • Cell Cycle Proteins
  • Collagen Type II
  • Dipeptides
  • HEY1 protein, human
  • Homeodomain Proteins
  • L 685458
  • Receptors, Notch
  • Repressor Proteins
  • Transcription Factor HES-1
  • Transforming Growth Factor beta3
  • Fibroblast Growth Factor 2
  • HES1 protein, human