Potential role for Ext1-dependent heparan sulfate in regulating P311 gene expression in A549 carcinoma cells

Biochim Biophys Acta Gen Subj. 2018 Jun;1862(6):1472-1481. doi: 10.1016/j.bbagen.2018.03.024. Epub 2018 Mar 24.

Abstract

Background: Exostosin-1 (EXT1), a member of the EXT protein family, is indispensable for synthesis of heparan sulfate (HS) chains that bind to and modulate the signaling efficiency of numerous growth factor activities. We have previously shown that Ext1 mutated mouse embryonic fibroblasts produce short sulfated HS chains which dramatically influence tumor cell behavior in a 3-dimensional (3D) heterospheroid system composed of tumor cells and fibroblasts.

Methods: In this study, we have used both 2D co-culture and 3D heterospheroid models, consisting of human A549 carcinoma cells co-cultured with wild-type or Ext1-mutated mouse embryonic fibroblasts.

Results and conclusions: Gene expression profiling of differentially expressed genes in fibroblast/A549 heterospheroids identified P311 as a gene substantially down-regulated in A549 cells co-cultured with Ext1-mutated fibroblasts. In addition, we observed that the Ext1 mutants displayed reduced Tgf-β1 mRNA levels and lower levels of secreted active TGF-β protein. Re-introduction of Ext1 in the Ext1 mutant fibroblasts rescued the levels of Tgf-β1 mRNA, increased the amounts of secreted active TGF-β in these cells, as well as P311 mRNA levels in adjacent A549 cells. Accordingly, small interfering RNAs (siRNAs) against fibroblast Tgf-β1 reduced P311 expression in neighboring A549 tumor cells. Our data raises the possibility that fibroblast Ext1 levels play a role in P311 expression in A549/fibroblast co-culture through TGF-β1.

General significance: This study considers a possible novel mechanism of Ext1-regulated heparan sulfate structure in modifying tumor-stroma interactions through altering stromal tgf-ß1 expression.

Keywords: A549 adenocarcinoma cells; EXT1; Fibroblasts; Heparan sulfate; P311; TGF-beta.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • A549 Cells
  • Animals
  • Cell Proliferation / drug effects*
  • Coculture Techniques
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / drug effects
  • Embryo, Mammalian / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation / drug effects*
  • Heparitin Sulfate / pharmacology*
  • Humans
  • Mice
  • N-Acetylglucosaminyltransferases / genetics
  • N-Acetylglucosaminyltransferases / metabolism*
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Transforming Growth Factor beta1 / metabolism

Substances

  • Nerve Tissue Proteins
  • P311 protein, mouse
  • Transforming Growth Factor beta1
  • Heparitin Sulfate
  • N-Acetylglucosaminyltransferases
  • exostosin-1