The Effects of Gap Junction Blockage on Neuronal Differentiation of Human NTera2/Clone D1 Cells Mahmud Bani-Yaghoub,* John F. Bechberger,* T. Michael Underhill,† and Christian C. G. Naus* *Department of Anatomy and Cell Biology and †Skeletal Biology Group, School of Dentistry, The University of Western Ontario, London, Ontario, N6A 5C1 Canada Received June 4, 1998; accepted September 4, 1998 Gap junctions are intercellular channels which pro- vide for the passage of small ions and molecules (MW F1200 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 -glycyrrhetinic acid (GRA) and carbenoxolone (CBX) have been com- pared with those of oleanolic acid (OLA) and glycyrrhi- zic acid (GZA), GRA analogs with no blocking effects. Both control and experimental cultures showed reduc- tion 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 differenti- ated 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, cytokera- tin, 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 (RAR and RAR) nor did they inhibit RA-mediated gene transcription. These results, to- gether, show that the blockage of gap junctions inter- feres with neuronal differentiation of NT2/D1 cells.  1999 Academic Press Key Words: carbenoxolone; connexin43; dye-cou- pling; embryonal carcinoma; glycyrrhetinic acid; glycyrrhizic acid; intercellular communication; neu- rons; oleanolic acid. INTRODUCTION Differentiation involves a complex series of events that are coordinated among adjacent cells. The organi- zation of this process is mediated, at least partly, by exchange of small ions and molecules through gap junctions (41). These structures are made of connexons, which are hexameric assemblies of the gap junction proteins, connexins. There is accumulating evidence that both cell type and the time of development affect the expression of connexins (25). For example, in rodent skin, the level and type of connexins expressed and the degree of intercellular communication change upon differentiation of keratinocytes (8). In the developing rat brain, Cx26 and Cx43 are expressed prenatally, with a reduction of Cx26 to basal level by postnatal day 6 (P6) while Cx43 is expressed increasingly following P6. Cx32 expression, on the other hand, increases after this time. In adult brain, Cx32 protein is expressed in oligodendrocytes and in some populations of differenti- ated neurons (5, 6, 15, 16). One way to investigate the role of gap junctions in differentiation is chemically inhibiting gap junctional communication (GJC). Several recent reports have described the use of glycyrrhetinic acid (GRA), an aglycone of glycyrrhizic acid (GZA), which is extracted from licorice (glycyrrhiza glabra) roots, to block intercel- lular communication (13, 14, 17, 18, 35, 45). Glycyrrhet- inic acid has shown no detectable effects on viability, cytotoxicity, and total protein synthesis in several cell types (17, 33, 45). Thus, both  and  conformations of glycyrrhetinic acid (-GRA or AGA and -GRA or BGA) as well as their synthetic analog, carbenoxolone (CBX), can be used in examining the role of intercellular communication in differentiation. The effects of these blocking agents can be compared with those of their inactive analogs, oleanolic acid (OLA) and glycyrrhizic acid (GZA) (13, 20, 51). We have used a human teratocarcinoma cell line (NTera-2/clone D1 or NT2/D1) as CNS neuronal precur- sors to study the role of gap junctions in neuronal differentiation. The NT2/D1 cell line removes the com- plicating variables such as reversibility and inconsis- tency of some neuronal cell lines as well as source- related variability of neuronal cultures derived from human fetal brain. NT2/D1 cells have the capacity to differentiate into postmitotic neurons (NT2-N cells) following treatment with retinoic acid (RA) for 4–5 weeks and antiproliferative agents for 3 weeks, respec- tively (43, 44). The addition of RA to these cells induces Experimental Neurology 156, 16–32 (1999) Article ID exnr.1998.6950, available online at http://www.idealibrary.com on 16 0014-4886/99 $30.00 Copyright  1999 by Academic Press All rights of reproduction in any form reserved.