Induction of gap junctional intercellular communication, connexin43 expression, and subsequent differentiation in human fetal neuronal cells by stimulation of the cyclic AMP pathway

Expression of gap junction proteins and cell-cell communication was studied in the human neural-glial cell line, SVG, as a first step in defining whet her the SVG cells could be used as a model system to study the role of gap junctions in neuronal precursor cells. SVG cells were found to express conn exin43 protein that co-migrated with WB-F344 rat liver connexin43 and that reacted with connexin43-specific antibodies on Western blots. However, fluo rescence recovery after photobleaching analysis of 5,6-carboxyfluorescein-l oaded cells failed to show significant dye coupling. Agents that stimulate the adenylyl cyclase/cAMP pathway were used to induce gap junctional interc ellular communication in the SVG cultures. A 24-48 h treatment of SVG cells with 5 mu M forskolin or 5 mu M forskolin + 200 mu M 3-isobutyl-1-methylxa nthine increased the percentage of dye-coupled cells from 5-65%, using the fluorescent recovery after photobleaching method. The increase in dye coupl ing induced by forskolin or forskolin + 3-isobutyl-1-methylxanthine was inh ibited by octanol, which is known to block gap junction-mediated cell commu nication. Western blot analysis of total protein extracts revealed the appe arance of a higher molecular weight connexin43 protein band after treatment of SVG cells with forskolin or forskolin + 3-isobutyl-1-methylxanthine, th at was not observed in vehicle-treated controls. Alkaline phosphatase treat ment of total protein extracts from forskolin or forskolin + 3-isobutyl-1-m ethylxanthine-treated cells reduced the higher molecular weight band to app roximate to 41,000 the same as observed in the control extracts. The alkali ne phosphatase treatment demonstrates that the higher molecular weight band was due to a phosphorylation event stimulated by forskolin or the forskoli n + 3-isobutyl-1-methylxanthine combination. In addition, treatment of the SVG cells with the forskolin or forskolin + 3-isobutyl-1-methylxanthine sti mulated outgrowth of neurite-like processes from the cell body which immuno stained positive for the connexin43 protein as well as protein markers for neurons and oligodendrocytes. We hypothesize that the SVG cells may represent a neuronal progenitor cell population that has the ability to differentiate when exposed to the approp riate signals. (C) 1999 IBRO. Published by Elsevier Science Ltd.