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Cancer cell adhesion to the vascular endothelium is an important step in tumor metastasis. Thy-1 (CD90), a cell adhesion molecule expressed in activated endothelial cells, has been implicated in melanoma metastasis by binding to integrins present in cancer cells. However, the signaling pathway(s) triggered by this Thy-1-Integrin interaction in cancer cells remains to be defined. Our previously reported data indicate that Ca2+-dependent hemichannel opening, as well as the P2X7 receptor, are key players in Thy-1-αVβ3 Integrin-induced migration of reactive astrocytes. Thus, we investigated whether this signaling pathway is activated in MDA-MB-231 breast cancer cells and in B16F10 melanoma cells when stimulated with Thy-1. In both cancer cell types, Thy-1 induced a rapid increase in intracellular Ca2+, ATP release, as well as cell migration and invasion. Connexin and Pannexin inhibitors decreased cell migration, implicating a requirement for hemichannel opening in Thy-1-induced cell migration. In addition, cell migration and invasion were precluded when the P2X7 receptor was pharmacologically blocked. Moreover, the ability of breast cancer and melanoma cells to transmigrate through an activated endothelial monolayer was significantly decreased when the β3 Integrin was silenced in these cancer cells. Importantly, melanoma cells with silenced β3 Integrin were unable to metastasize to the lung in a preclinical mouse model. Thus, our results suggest that the Ca2+/hemichannel/ATP/P2X7 receptor-signaling axis triggered by the Thy-1-αVβ3 Integrin interaction is important for cancer cell migration, invasion and transvasation. These findings open up the possibility of therapeutically targeting the Thy-1-Integrin signaling pathway to prevent metastasis.

Abstract Background Neuroinflammation involves cytokine release, astrocyte reactivity and migration. Neuronal Thy-1 promotes DITNC1 astrocyte migration by engaging αVβ3 Integrin and Syndecan-4. Primary astrocytes express low levels of these receptors and are unresponsive to Thy-1; thus, inflammation and astrocyte reactivity might be necessary for Thy-1-induced responses. Methods Wild-type rat astrocytes (TNF-activated) or from human SOD1G93A transgenic mice (a neurodegenerative disease model) were used to evaluate cell migration, Thy-1 receptor levels, signaling molecules, and reactivity markers. Results Thy-1 induced astrocyte migration only after TNF priming. Increased expression of αVβ3 Integrin, Syndecan-4, P2X7R, Pannexin-1, Connexin-43, GFAP, and iNOS were observed in TNF-treated astrocytes. Silencing of β3 Integrin prior to TNF treatment prevented Thy-1-induced migration, while β3 Integrin over-expression was sufficient to induce astrocyte reactivity and allow Thy-1-induced migration. Finally, hSOD1G93A astrocytes behave as TNF-treated astrocytes since they were reactive and responsive to Thy-1. Conclusions Therefore, inflammation induces expression of αVβ3 Integrin and other proteins, astrocyte reactivity, and Thy-1 responsiveness. Importantly, ectopic control of β3 Integrin levels modulates these responses regardless of inflammation.

Astrocytes are the most abundant type of glial cell in the central nervous system. Upon injury and inflammation, astrocytes become reactive and undergo morphological and functional changes. Depending on their phenotypic classification as A1 or A2, reactive astrocytes contribute to both neurotoxic and neuroprotective responses, respectively. However, this binary classification does not fully capture the diversity of astrocyte responses observed across different diseases and injuries. Transcriptomic analysis has revealed that reactive astrocytes have a complex landscape of gene expression profiles, which emphasizes the heterogeneous nature of their reactivity. Astrocytes actively participate in regulating central nervous system inflammation by interacting with microglia and other cell types, releasing cytokines, and influencing the immune response. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is a central player in astrocyte reactivity and impacts various aspects of astrocyte behavior, as evidenced by in silico, in vitro, and in vivo results. In astrocytes, inflammatory cues trigger a cascade of molecular events, where nuclear factor-κB serves as a central mediator of the pro-inflammatory responses. Here, we review the heterogeneity of reactive astrocytes and the molecular mechanisms underlying their activation. We highlight the involvement of various signaling pathways that regulate astrocyte reactivity, including the PI3K/AKT/mammalian target of rapamycin (mTOR), αvβ3 integrin/PI3K/AKT/connexin 43, and Notch/PI3K/AKT pathways. While targeting the inactivation of the PI3K/AKT cellular signaling pathway to control reactive astrocytes and prevent central nervous system damage, evidence suggests that activating this pathway could also yield beneficial outcomes. This dual function of the PI3K/AKT pathway underscores its complexity in astrocyte reactivity and brain function modulation. The review emphasizes the importance of employing astrocyte-exclusive models to understand their functions accurately and these models are essential for clarifying astrocyte behavior. The findings should then be validated using in vivo models to ensure real-life relevance. The review also highlights the significance of PI3K/AKT pathway modulation in preventing central nervous system damage, although further studies are required to fully comprehend its role due to varying factors such as different cell types, astrocyte responses to inflammation, and disease contexts. Specific strategies are clearly necessary to address these variables effectively.

Abstract Background In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvβ3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. Methods Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). Results The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. Conclusions Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.