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.

Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of ciliary neurotrophic factor (CNTF) on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble ciliary neurotrophic factor receptor ␣ (CNTFR␣) (200 ng/ml), or CNTF-CNTFR␣. Although CNTF and CNTFR␣ alone had no effect on Cx43 expression, the heterodimer CNTF-CNTFR␣ significantly increased both Cx43 mRNA and protein levels. Cx43 immunostaining correlated with increased intercellular coupling as determined by dye transfer analysis. By using the pharmacological inhibitor ␣-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490), the increase in Cx43 was found to be dependent on the Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Immunocytochemical analysis revealed that CNTF-CNTFR␣ treatment produced nuclear localization of phosphorylated STAT3, whereas CNTF treatment alone did not. Transient transfection of constructs containing various sequences of the Cx43 promoter tagged to a LacZ reporter into ROS 17/2.8 cells confirmed that the promoter region between ؊838 to ؊1693 was deemed necessary for CNTF-CNTFR␣ to induce heightened expression. CNTF-CNTFR␣ did not alter Cx30 mRNA levels, suggesting selectivity of CNTF-CNTFR␣ for connexin signaling. Together in the presence of soluble receptor, CNTF activates the JAK/STAT pathway leading to enhanced Cx43 expression and intercellular coupling. INTRODUCTIONGap junctions are intercellular channels between adjacent cells that permit the passage of various substances Ͻ1.2 kDa in size (Kumar and Gilula, 1996). Such junctions are formed when each cell provides a connexon, which itself is composed of six connexins (Cxs) (Kumar and Gilula, 1996). Cx43, the prominent Cx isoform expressed in the central nervous system (CNS), is highly expressed in astrocytes (Yamamoto et al., 1990;Dermietzel et al., 1991;Giaume et al., 1991), neuronal precursors (Rozental et al., 1998Bittman and LoTurco, 1999), and possibly neurons (Bruzzone and Ressot, 1997;Vaney, 1999;SiuYi et al., 2001;Rouach et al., 2002). Several studies using in vitro and in vivo models have demonstrated that impediment of gap junctional coupling and/or connexin43 expression amplifies cell death and tissue damage in the wake of injuries and diseases (Blanc et al., 1998;Naus et al., 2001;Siushansian et al., 2001;Ozog et al., 2002b;Lin et al., 2003;Nakase et al., 2003). Thus, design of an intervention to up-regulate Cx43 expression and intercellular communication may lead to novel therapies against pathological disturbances.Gap junction expression and activity can be altered on a short-term or long-term basis (reviewed by Giaume and McCarthy, 1996;Rouach and Giaume, 2001). Short-term regulation is the result of posttranslational processing such as phosphorylation or channel blocking. Long-term regulation, however, entails modification of gene transcripti...

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