| Summary: | Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y<sub>2</sub> receptor (P2Y<sub>2</sub>R) to modulate its downstream signaling. P2Y<sub>2</sub>R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y<sub>2</sub>R signaling and its pro-survival actions in the 1321N1 astrocytoma cells mechanical injury model system. KD of Cav-1 decreased its expression in 1321N1 cells devoid of or expressing hHAP2Y<sub>2</sub>R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y<sub>2</sub>R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y<sub>2</sub>R) revealed a P2Y2R-dependent temporal increase in both kinases. These temporal increases in pERK1/2 and pAkt were significantly decreased in Cav-1 KD 1321N1 (devoid of or expressing a hHAP2Y<sub>2</sub>R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y<sub>2</sub>R-mediated pERK1/2 and pAkt kinases’ activity, respectively. These early-onset hHAP2Y<sub>2</sub>R-mediated signaling responses in Cav-1-expressing and Cav-1 KD 1321N1 correlated with changes in cell viability (via a resazurin-based method) and apoptosis (via caspase-9 expression). In Cav-1-positive 1321N1 cells, expression of hHAP2Y<sub>2</sub>R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y<sub>2</sub>R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y<sub>2</sub>R. These findings support the importance of Cav-1 in modulating P2Y<sub>2</sub>R signaling during mechanical injury and its protective actions in a human astrocytoma cell line, whilst shedding light on potential new venues for brain injury or trauma interventions.
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