The effects of gap junction blockage on neuronal differentiation of human NTera2/clone D1 cells

Gap junctions are intercellular channels which provide for the passage of small ions and molecules (MW <1200 D) among adjacent cells. The NTera2/clone D1 (NT2/D1) cells are CNS precursors which differentiate into NT2-N neurons upon treatment with retinoic acid (RA) and antiproliferative agents. In this study, the effects of gap junction blockers 18 alpha-glycyrrhetinic acid (GRA) and carbenoxolone (CBX) have been compared with those of oleanolic acid (OLA) and glycyrrhizic acid (GZA), GRA analogs with no blocking effects. Both control and experimental cultures showed reduction of Cx43 protein after 4 weeks of RA induction. A major reduction was also observed in expression of cytokeratin, vimentin, and nestin in control cells at this time point while the cultures treated with the blockers did not show any significant change. The average number of MAP2-positive NT2-N differentiated neurons per field of view in the cultures treated with the blockers was less than 7% of that of control cultures. NT2-N cells were negative for Cx43, cytokeratin, vimentin, and nestin. The blockers did not appear to be operating through inhibition of RA signaling, as their presence did not affect the expression of retinoic acid receptors (RARalpha and RARgamma) nor did they inhibit RA-mediated gene transcription. These results, together, show that the blockage of gap junctions interferes with neuronal differentiation of NT2/D1 cells.