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Connexin43 (Cx43) is a ubiquitously expressed member of the gap junction protein family that mediates gap junction intercellular communication (GJIC) by allowing exchange of cytosolic materials. Previous studies have used Cx43 truncated at the cytoplasmic tail (C-tail) to demonstrate that the C-tail is essential to regulate cell growth and motility. Therefore, the aim of our study was to delineate the respective role of the truncated Cx43 and the C-tail in mediating Cx43-dependent signaling. A truncated Cx43 expressing the channel part of the protein (TrCx43, amino acid 1-242) and a construct encompassing only the C-tail from amino acid 243 (243Cx43) were transduced into LN18 human glioma cells. Our results showed that the ability of Cx43 to suppress growth was independent of GJIC as assessed by dye transfer, but was dependent on the presence of a rigid extracellular matrix. We further demonstrated that the C-tail alone is sufficient to promote motility. Surprisingly, Cx43 is also able to increase migration in the absence of the C-tail, suggesting the presence of at least two distinct signaling mechanisms utilized by Cx43 to affect motility. Finally, we used time-lapse imaging to examine the behavior of migrating cells and it was apparent that the C-tail was associated with a lamellipodia-based migration not observed in either mock or TrCx43 expressing LN18 cells. Our study shows for the first time that a free C-tail is sufficient to induce Cx43-dependent changes in cell morphology and that Cx43 signaling is linked to the regulation of the actin cytoskeleton., ((c) 2010 Wiley-Liss, Inc.)

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.

Gliomas are particularly difficult to cure owing largely to their invasive nature. The neoplastic changes of astrocytes which give rise to these tumors frequently include a reduction of connexin43 (Cx43), the most abundant connexin isoform expressed in astrocytes. Cx43 is a subunit of gap junctions (GJ), intercellular channels which directly link the cytosol of adjacent cells and allow the regulated passage of ions and small molecules. To examine the role of Cx43 in glioma motility, we identified two variant C6 cell lines which endogenously express high (C6-H) or low (C6-L) levels of Cx43. In wound healing and transwell assays, C6-H cells were more motile than C6-L cells. To deduce whether Cx43 mediated these differences, assays were conducted on C6-H cells retrovirally transduced with Cx43 shRNA. Coincident with the stable knockdown of endogenous Cx43, a decrease in motility and invasion was observed. Gap junctional intercellular communication was also decreased, however motility assays conducted in the presence of GJ inhibitors did not reveal significant differences in cell motility. C6 cells transfected with full length or C-terminal truncated Cx43 (Cx43DeltaCT) 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 Cx43DeltaCT did not. This report, the first in which RNAi has been employed to reduce Cx43 expression in gliomas, indicates that the downregulation of Cx43 decreases motility of C6 cells. Furthermore, it is the first report to suggest that the Cx43 CT plays an important role in glioma motility.