Under pro-inflammatory conditions, astrocytes become reactive and acquire a migratory phenotype. Our results show that hemichannels formed by connexin 43 (Cx43) play an important role in Thy-1-induced astrocyte migration. The neuronal protein Thy-1 binds to αvβ3 integrin in astrocytes, thereby leading to intricate signaling pathways that include calcium (Ca²⁺) release from intracellular stores, opening of Cx43 hemichannels, release of ATP, activation of P2X7 receptor, and Ca²⁺ influx. However, because these Thy-1 effects occur exclusively in reactive astrocytes, we wondered whether by elevating calcium levels and promoting hemichannel opening we could prompt non-reactive astrocytes to respond to Thy-1. Cx43 immunoreactivity increased at juxta-membrane sites, where hemichannels (not gap junctions) participate in astrocyte polarization and migration stimulated by Thy-1. Also, intracellular Ca²⁺ increase, due to ionomycin treatment, induced hemichannel opening, but activated astrocyte migration only partially, and this limitation was overcome by pre-treatment with tumor necrosis factor (TNF) and Thy-1. Finally, αvβ3 integrin formed membrane clusters after TNF stimulation or overexpression of β3 integrin. We suggest that these microclusters are required for cells to respond to Thy-1 stimulation. Therefore, the large increase in intracellular Ca²⁺ and hemichannel opening induced by ionomycin are required, but not sufficient, to permit Thy-1-induced astrocyte migration. Thus, we suggest that proinflammatory stimuli prompt astrocytes to respond to migratory signals of neuronal cells.

Astrogliosis is a process by which astrocytes, when exposed to inflammation, exhibit hypertrophy, motility, and elevated expression of reactivity markers such as Glial Fibrillar Acidic Protein, Vimentin, and Connexin43. Since 1999, our laboratory in Chile has been studying molecular signaling pathways associated with “gliosis” and has reported that reactive astrocytes upregulate Syndecan 4 and α_Vβ₃ Integrin, which are receptors for the neuronal glycoprotein Thy-1. Thy-1 engagement stimulates adhesion and migration of reactive astrocytes and induces neurons to retract neurites, thus hindering neuronal network repair. Reportedly, we have used DITNC1 astrocytes and neuron-like CAD cells to study signaling mechanisms activated by the Syndecan 4–α_Vβ₃ Integrin/Thy-1 interaction. Importantly, the sole overexpression of β₃ Integrin in non-reactive astrocytes turns them into reactive cells. In vitro, extensive passaging is a simile for “aging”, and aged fibroblasts have shown β₃ Integrin upregulation. However, it is not known if astrocytes upregulate β₃ Integrin after successive cell passages. Here, we hypothesized that astrocytes undergoing long-term passaging increase β₃ Integrin expression levels and behave as reactive astrocytes without needing pro-inflammatory stimuli. We used DITNC1 cells with different passage numbers to study reactivity markers using immunoblots, immunofluorescence, and astrocyte adhesion/migration assays. We also evaluated β₃ Integrin levels by immunoblot and flow cytometry, as well as the neurotoxic effects of reactive astrocytes. Serial cell passaging mimicked the effects of inflammatory stimuli, inducing astrocyte reactivity. Indeed, in response to Thy-1, β₃ Integrin levels, as well as cell adhesion and migration, gradually increased with multiple passages. Importantly, these long-lived astrocytes expressed and secreted factors that inhibited neurite outgrowth and caused neuronal death, just like reactive astrocytes in culture. Therefore, we describe two DITNC1 cell types: a non-reactive type that can be activated with Tumor Necrosis Factor (TNF) and another one that exhibits reactive astrocyte features even in the absence of TNF treatment. Our results emphasize the importance of passage numbers in cell behavior. Likewise, we compare the pro-inflammatory stimulus versus long-term in-plate passaging of cell cultures and introduce them as astrocyte models to study the reactivity process.