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

• Published in: Neuroscience 1999-12, Vol.95 (3), p.859-868
• Main Authors: Dowling-Warriner, C.V., Trosko, J.E.
• Format: Article
• Language: English
• Subjects: connexin43; differentiation; gap junctional intercellular communication; neural progenitor cells
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/S0306-4522(99)00411-X

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.