The function of CD146 in human annulus fibrosus cells and mechanism of the regulation by TGF-β

J Orthop Res. 2022 Jul;40(7):1661-1671. doi: 10.1002/jor.25190. Epub 2021 Oct 18.


The mouse outer annulus fibrosus (AF) was previously shown to contain CD146+ AF cells, while in vitro culture and exposure to transforming growth factor-beta (TGF-β) further increased the expression of CD146. However, neither the specific function of CD146 nor the underlying mechanism of TGF-β upregulation of CD146+ AF cells have been elucidated yet. In the current study, CD146 expression and its role in cultured human AF cells was investigated studying the cells' capacity for matrix contraction and gene expression of functional AF markers. In addition, TGF-β pathways were blocked by several pathway inhibitors and short hairpin RNAs (shRNAs) targeting SMAD and non-SMAD pathways to investigate their involvement in TGF-β-induced CD146 upregulation. Results showed that knockdown of CD146 led to reduction in AF cell-mediated collagen gel contraction, downregulation of versican and smooth muscle protein 22α (SM22α), and upregulation of scleraxis. TGF-β-induced CD146 upregulation was significantly blocked by inhibition of TGF-β receptor ALK5, and partially inhibited by shRNA against SMAD2 and SMAD4 and by an Protein Kinase B (AKT) inhibitor. Interestingly, the inhibition of extracellular signal-regulated kinases (ERK) pathway induced CD146 upregulation. In conclusion, CD146 was shown to be crucial to maintain the cell contractility of human AF cells in vitro. Furthermore, TGF-β upregulated CD146 via ALK5 signaling cascade, partially through SMAD2, SMAD4, and AKT pathway, whereas, ERK was shown to be a potential negative modulator. Our findings suggest that CD146 can potentially be used as a functional marker in AF repair strategies.

Keywords: AKT; ALK5; CD146 regulation; SMADs; TGF-β; annulus fibrosus; cell contractility.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Annulus Fibrosus* / metabolism
  • CD146 Antigen* / metabolism
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Humans
  • Proto-Oncogene Proteins c-akt
  • Receptor, Transforming Growth Factor-beta Type I
  • Smad2 Protein
  • Smad4 Protein
  • Transforming Growth Factor beta* / metabolism


  • CD146 Antigen
  • MCAM protein, human
  • SMAD2 protein, human
  • SMAD4 protein, human
  • Smad2 Protein
  • Smad4 Protein
  • Transforming Growth Factor beta
  • Proto-Oncogene Proteins c-akt
  • Extracellular Signal-Regulated MAP Kinases
  • Receptor, Transforming Growth Factor-beta Type I
  • TGFBR1 protein, human