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

Abstract

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 whether 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 connexin43 protein that co-migrated with WB-F344 rat liver connexin43 and that reacted with connexin43-specific antibodies on Western blots. However, fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein-loaded cells failed to show significant dye coupling. Agents that stimulate the adenylyl cyclase/cAMP pathway were used to induce gap junctional intercellular communication in the SVG cultures. A 24–48 h treatment of SVG cells with 5 μM forskolin or 5 μM forskolin+200 μM 3-isobutyl-1-methylxanthine increased the percentage of dye-coupled cells from 5–65%, using the fluorescent recovery after photobleaching method. The increase in dye coupling induced by forskolin or forskolin+3-isobutyl-1-methylxanthine was inhibited by octanol, which is known to block gap junction-mediated cell communication. Western blot analysis of total protein extracts revealed the appearance of a higher molecular weight connexin43 protein band after treatment of SVG cells with forskolin or forskolin+3-isobutyl-1-methylxanthine, that was not observed in vehicle-treated controls. Alkaline phosphatase treatment of total protein extracts from forskolin or forskolin+3-isobutyl-1-methylxanthine-treated cells reduced the higher molecular weight band to ≈41,000 the same as observed in the control extracts. The alkaline phosphatase treatment demonstrates that the higher molecular weight band was due to a phosphorylation event stimulated by forskolin or the forskolin+3-isobutyl-1-methylxanthine combination. In addition, treatment of the SVG cells with the forskolin or forskolin+3-isobutyl-1-methylxanthine stimulated outgrowth of neurite-like processes from the cell body which immunostained 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 appropriate signals.