Signaling pathways involved in connexin43 expression and cellular sub-localization in human astrocytoma

Title: Signaling pathways involved in connexin43 expression and cellular sub-localization in human astrocytoma
Authors: Brunet, Julien
Date: 2009
Abstract: Gap junctions (GJ) are intercellular channels permitting bi-directional communication between adjacent cells. GJ function is associated with homeostasis while GJ deficiency results in uncontrolled proliferation. Most cancer cells are GJ deficient. Astrocytoma is the most common form of brain cancer in young adults and usually causes death within 9-12 months of diagnosis. Connexin 43 (Cx43) expression and sub-localization is usually impaired in human astrocytoma cells. Since phosphorylation by various protein kinases plays a crucial role in Cx43 trafficking, we investigated the signaling pathways involved in Cx43 sub-localization in the human astrocytoma cell lines, U-251 MG and U-87 MG. Cx43 was found to be present in both cell lines and was found to be phosphorylated on specific serine residues. Using antibodies against Ser-255 and Ser-262, Cx43 was shown to be nuclear; this is a novel observation in human astrocytoma. Interestingly Cx43 fragments of less than 43 kDa were shown to be mostly intranuclear when using antibodies against the phosphorylated forms of Cx43, suggesting a role for phosphorylation in the protein's turnover and sub-localization. Intercellular communication assays showed a lack of GJ function in U-251 cells; however, the presence of open hemichannels in U-87 MGs was observed. Protein kinase inhibitors including Bis I (PKC), PKAI (PKA) and PD98059 (ERK-MAPK) were tested on their ability to affect Cx43 expression and/or sub-localization. PD98059 treatment increased Cx43 expression in U-87 MG cells and partially restored normal membrane localization in U-251 MG. All three inhibitors either increased or decreased the presence of Cx43 fragments, depending on the phosphorylation site observed, suggesting a role for these pathways in Cx43 degradation. Morin, a natural flavonoid with anti-tumorigenic properties, was shown to increase Cx43 phosphorylation on Ser-262 but only in U-251 MG. This effect was exerted through the p38 MAPK pathway. Fractionation experiments showed that Cx43 is not present in caveolae, a structure which helps to compartmentalize signaling molecules with their protein targets. Our results suggest that it is worthwhile to continue in our attempts to restore function of Cx43 in human astrocytoma.
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010
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