Proteomic analysis of astroglial connexin43 silencing uncovers a cytoskeletal platform involved in process formation and migration.

Connexin43 (Cx43) is the most abundant gap junction protein of the brain, where it is predominantly expressed in astrocytes. Recent studies imply a role of Cx43 in the regulation of important cellular processes, including migration, proliferation, and shape formation. These processes are assumed to be reflected by the proteome of the Cx43 expressing cells. To analyze the influence of Cx43 on the astrocytic proteome, we used RNA interference to downregulate the expression of this connexin in cultures of mouse astrocytes. We applied difference gel electrophoresis (DIGE) to compare silenced astrocytes with control cells. The differential proteome analysis revealed 15 significantly regulated proteins (between 1.2- and 1.6-fold), of which six are known to belong to a group of cytoskeletal proteins involved in cortical platform formation. Astrocytes treated with Cx43 small interfering (si)RNA showed an increased expression of the cytoskeletal proteins: actin, tropomyosin, microtubule-associated protein RP/EB1, transgelin, and GFAP, and a decreased expression of cofilin-1. Quantitative immunocytochemistry and Western blotting revealed similar results showing an upregulation of actin, tubulin, tropomyosin, EB1, transgelin and GFAP, and a downregulation of Ser-3-phosphorylated cofilin. Furthermore, Cx43 silencing led to phenotypical changes in cell morphology, migratory activity, and cell adhesion. Our results provide mechanistic clues for an understanding of Cx43 interaction with cellular motor activities such as migration and process formation in astrocytes. 2009 Wiley-Liss, Inc.