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

Abstract

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.

Astrocytes promote glioma invasion via the gap junction protein connexin43

“…This conclusion is supported by many studies that have demonstrated the critical role of Cx43 expressed by glioma cells in enhancing cell migration by increasing intercellular adhesion through the extracellular loop 33,34,38,39,90 and/or by interacting with the cytoskeletal signaling molecules, including F-actin, N-cadherin and cortactin via the cytoplasmic tail. 43,88 Reactive astrocytes express pro-inflammatory chemokines 8,10,110 that confer a survival advantage to tumor cells. In this regard, microglia have a major part in cytokine-mediated pathways in glioma progression.…”

Evaluation of Connexin 43 Redistribution and Endocytosis in Astrocytes Subjected to Ischemia/Reperfusion or Oxygen-Glucose Deprivation and Reoxygenation

“…Recent studies revealed that Cx43 modulated cytoskeletal proteins including MAPRE1, which are involved in process formation and migration of astrocytes. Besides, Stephan et al found that MAPRE1 expression depended on the level of Cx43 expression by the method of immunocytochemistry and western blot [42]. In the present study, we showed that there was no difference in protein and gene expression of MAPRE1 after IR/OGDR at time points 0.5, 1, 3, 6, 12, 24, and 72 h. In agreement with this viewpoint, we also found that the total number of Cx43 was unchanged in the normal and IR/OGDR groups.…”

Systems biology and proteomic analysis of cerebral cavernous malformation

“…Expression of actin-stabilizing proteins such as cofilin and transgelin, in our proteomic analysis, is also altered. Binding of cofilin and transgelin to actin stabilizes actin–tubulin complex and therefore regulates cellular process formation and cell migration [39]. Changes in cytoskeletal proteins as a result of CCM dysregulation can further disturb the cell–cell interaction as seen in several dysregulation in cell junction signal (Figure 5A–E).…”