Abstract
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.
Highlights
Astrocytes are the most abundant cell type in the central nervous system (CNS); their function has always been overshadowed by neurons
Our previously reported evidence indicated that Thy-1 stimulation induces the release of ATP to the extracellular medium through connexin 43 (Cx43) and Px1 hemichannels, thereby activating P2X7 receptor (P2X7R) and Ca2+ entry, which are required for the adhesion, polarization, and migration of astrocyte cell lines, as well as primary rat neonatal astrocytes in culture [4,5]
We assessed Cx43 expression by indirect immunofluorescence in isolated DITNC1 astrocytes. In these cells, which behave as reactive astrocytes, surface staining for Cx43 was detected in the plasma membrane of isolated cells, indicating that Cx43 was present in the form of hemichannels (Figure 1A)
Summary
Astrocytes are the most abundant cell type in the central nervous system (CNS); their function has always been overshadowed by neurons. When bound to Thy-1, the engaged receptors trigger an intracellular signaling cascade involving FAK, PLCγ, inositol 1,4,5-trisphosphate receptor (IP3R) activation, Ca2+ release from the endoplasmic reticulum, connexin (Cx)- and pannexin (Px)-hemichannel opening, ATP release, and P2X7 receptor (P2X7R) activation, which further increases intracellular Ca2+ concentrations ([Ca2+]i) [4,5,6,7,8] In this complex sequence, connexin 43 (Cx43) channels play an important role to allow the release of ATP to the extracellular medium, and to communicate with the surrounding cells by gap junctions [5,9,10]. Ionomycin promotes astrocyte migration; ionomycin and/or Thy-1 require TNF for effective cell migration
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