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 s mall ions and molecules (MW < 1200 D) among adjacent cells. The NTera2/clon e D1 (NTS/D1) cells are CNS precursors which differentiate into NT2-N neuro ns upon treatment with retinoic acid (RA) and antiproliferative agents. In this study, the effects of gap junction blockers 18 alpha-glycyrrhetinic ac id (GRA) and carbenoxolone (CBX) have been compared with those of oleanolic acid (OLA) and glycyrrhizic acid (GZA), GRA analogs with no blocking effec ts. Both control and experimental cultures showed reduction of Cx43 protein after 4 weeks of RA induction. A major reduction was also observed in expr ession of cytokeratin, vimentin, and nestin in control cells at this time p oint while the cultures treated with the blockers did not show any signific ant change. The average number of MAP2-positive NT2-N differentiated neuron s 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, cytoke ratin, vimentin, and nestin. The blockers did not appear to be operating th rough inhibition of RA signaling, as their presence did not affect the expr ession of retinoic acid receptors (RAR alpha and RAR gamma) nor did they in hibit RA-mediated gene transcription, These results, together, show that th e blockage of gap junctions interferes with neuronal differentiation of NT2 /D1 cells, (C) 1999 Academic Press.