The role of connexin43 in glioma motility

• Main Author: Bates, David Christopher
• Language: English
• Published: 2010
• Online Access: http://hdl.handle.net/2429/17852

Gap junctions, proteinaceous channels which directly link the cytosol of adjacent cells and allow the passage of ions and small molecules, are formed from the hexameric oligomerization of connexin subunits. We are interested in the role of Connexin43 (Cx43), the most abundant isoform expressed in astrocytes, in glioma motility. To achieve this objective, we have isolated a C6 subclone endogenously expressing high levels of Cx43 (C6-H) and have employed in vitro wound healing and transwell assays to evaluate cellular motility. When compared to parental C6 cells in which Cx43 is expressed at low levels (C6-L), the C6-H subclones were more motile. To deduce whether Cx43 was indeed responsible for the observed differences in motility, the C6-H cells were retrovirally infected with Cx43 shRNA to stably knock down Cx43 expression. Coincident with the knockdown of endogenous Cx43, a decrease in motility and invasion was observed. As gap junction intercellular communication (GJIC) was also decreased, motility assays were conducted in the presence of gap junction inhibitors to evaluate the contribution of GJIC to cell motility. Because no significant differences in motility could be detected upon blocking GJIC, C6 cells exogenously expressing full length or truncated Cx43 were subjected to the aforementioned motility assays to expose alternate mechanisms of Cx43-mediated motility. Cells expressing full length Cx43 exhibited increased motility while cells expressing the truncated form of Cx43 did not. Our results indicate that downregulation of Cx43 decreases motility in C6 glioma cells and suggest that the carboxy terminus plays an important role in Cx43-mediated motility. Keywords: Gap junction, Connexin43, C6 cells, Glioma, Motility, Invasion, RNAiGap junctions, proteinaceous channels which directly link the cytosol of adjacent cells and allow the passage of ions and small molecules, are formed from the hexameric oligomerization of connexin subunits. We are interested in the role of Connexin43 (Cx43), the most abundant isoform expressed in astrocytes, in glioma motility. To achieve this objective, we have isolated a C6 subclone endogenously expressing high levels of Cx43 (C6-H) and have employed in vitro wound healing and transwell assays to evaluate cellular motility. When compared to parental C6 cells in which Cx43 is expressed at low levels (C6-L), the C6-H subclones were more motile. To deduce whether Cx43 was indeed responsible for the observed differences in motility, the C6-H cells were retrovirally infected with Cx43 shRNA to stably knock down Cx43 expression. Coincident with the knockdown of endogenous Cx43, a decrease in motility and invasion was observed. As gap junction intercellular communication (GJIC) was also decreased, motility assays were conducted in the presence of gap junction inhibitors to evaluate the contribution of GJIC to cell motility. Because no significant differences in motility could be detected upon blocking GJIC, C6 cells exogenously expressing full length or truncated Cx43 were subjected to the aforementioned motility assays to expose alternate mechanisms of Cx43-mediated motility. Cells expressing full length Cx43 exhibited increased motility while cells expressing the truncated form of Cx43 did not. Our results indicate that downregulation of Cx43 decreases motility in C6 glioma cells and suggest that the carboxy terminus plays an important role in Cx43-mediated motility. === Medicine, Faculty of === Graduate