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

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 Extl 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 hetero-spheroids identified P311 as a gene substantially down-regulated in A549 cells co-cultured with Extl-mutated fibroblasts. In addition, we observed that the Extl mutants displayed reduced Tgf-131 mRNA levels and lower levels of secreted active TGF-13 protein. Re-introduction of Extl in the Extl mutant fibroblasts rescued the levels of Tgf-131 mRNA, increased the amounts of secreted active TGF-13 in these cells, as well as P311 mRNA levels in adjacent A549 cells. Accordingly, small interfering RNAs (siRNAs) against fibroblast Tgf-131 reduced P311 expression in neighboring A549 tumor cells. Our data raises the possibility that fibroblast Extl levels play a role in P311 expression in A549/fibroblast co-culture through TGF-f31. General significance: This study considers a possible novel mechanism of Extl-regulated heparan sulfate structure in modifying tumor-stroma interactions through altering stromal tgf-fil expression.