Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma

Abstract

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 P2Y₂ receptor (P2Y₂R) to modulate its downstream signaling. P2Y₂R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y₂R 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 hHAP2Y₂R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y₂R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y₂R) 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 hHAP2Y₂R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y₂R-mediated pERK1/2 and pAkt kinases' activity, respectively. These early-onset hHAP2Y₂R-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 hHAP2Y₂R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y₂R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y₂R. These findings support the importance of Cav-1 in modulating P2Y₂R 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.

Keywords: P2Y2; astrocytoma; brain injury; caveolae; caveolin-1; signaling.

Figure 1

shRNA-mediated knockdown of caveolin-1 expression of 1321N1 astrocytoma cells. (A) Immunoblot analysis of hHAP2Y₂R, caveolin-1 (Cav-1), and GAPDH (control) expression in serum-starved wild-type (WT) 1321N1 cells (lane 1), human 1321N1 cells expressing hHAP2Y₂R (hHAP2Y₂R 1321N1 cells) (lane 3), or cells infected with Cav-1 shRNA lentiviral particles (lanes 2 and 4; Cav-1 knockdown (KD)). (B) Densitometric analysis of immunoblots indicates the level of Cav-1 normalized to GAPDH expression. Results are presented as the means ± S.E.M. (n = 3; *** p < 0.001 as determined by one-way ANOVA).

Figure 2

Cav-1 KD reduced the P2Y₂R-mediated ERK1/2 phosphorylation after mechanical injury. Post-injury ERK1/2 phosphorylation time course of (A–C) Cav-1-expressing and (D–F) Cav-1 KD 1321N1 cell lines. Immunoblots for (A) Cav-1-expressing WT-1321N1 and (B) Cav-1/hHAP2Y₂R-expressing 1321N1 cells. Densitometric analysis of the latter immunoblots is shown in (C), revealing the post-injury hHAP2Y₂R-mediated increased ERK1/2 activity. Immunoblots for (D) Cav-1 KD WT-1321N1 and (E) Cav-1 KD/hHAP2Y₂R-expressing 1321N1 cells. Densitometric analysis of the latter immunoblots is shown in (F), revealing the diminished post-injury hHAP2Y₂R-mediated increased ERK1/2 activity in Cav-1KD 1321N1 cells. Cells were subjected to a severe traumatic injury and immunoblot analysis was done as described in the Materials and Methods section. ERK1/2 phosphorylation and total ERK1/2, Cav-1, and GAPDH (control) expression in equal amounts of protein were determined by Western blot analysis. Immunoblots are representative of at least three independent experiments. In (C) and (F), ERK1/2 phosphorylation was normalized using the formula: phosphorylated ERK1/2/(total ERK1/2 + GAPDH) and expressed as a percentage of untreated controls at 0 min. Values represent the means ± S.E.M. (n = 4), * p < 0.05 and *** p < 0.001 (one-way ANOVA) represent statistically significant differences between P2Y₂R-devoid and P2Y₂R-expressing 1321N1 cells.

Figure 3

Cav-1 KD reduced the P2Y₂R-mediated Akt phosphorylation after mechanical injury. Post-injury Akt phosphorylation time course of (A–C) Cav-1-expressing and (D–F) Cav-1 KD 1321N1 cell lines. Immunoblots for (A) Cav-1-expressing WT-1321N1 and (B) Cav-1/hHAP2Y₂R-expressing 1321N1 cells. Densitometric analysis of the latter immunoblots is shown in (C), revealing the post-injury hHAP2Y₂R-mediated increased Akt activity. Immunoblots for (D) Cav-1 KD WT-1321N1 and (E) Cav-1 KD/hHAP2Y₂R-expressing 1321N1 cells. Densitometric analysis of the latter immunoblots is shown in (F), revealing the diminished post-injury hHAP2Y2R-mediated increased Akt activity in Cav-1 KD 1321N1 cells. Cells were lysed and Akt phosphorylation on Ser473 and total Akt, Cav-1, and GAPDH (control) expression in equal amounts of protein were determined by Western blot analysis. Immunoblots are representative of at least three independent experiments. In panels C and F, Akt phosphorylation on Ser473 was normalized using the formula: phosphorylated Akt/(pan Akt + GAPDH) and expressed as a percentage of untreated controls at 0 min. Values represent the means ± S.E.M. (n = 4), where * p < 0.05, ** p < 0.01, and *** p < 0.001 (one-way ANOVA) represent statistically significant differences between P2Y₂R-devoid and P2Y₂R-expressing 1321N1 cells.

Figure 4

Cav-1 KD inhibits the (A) P2Y₂R-mediated increased cell viability and (B) anti-apoptotic action after mechanical injury. The relative expression of both P2Y₂R and Cav-1 are indicated on the x-axes of panels (A) and (B). After injury, cell cultures were returned to the incubator and further incubated for 24 h. Cell viability and caspase-9 activity were measured using AB and caspase-9 fluorometric kit, as described in the Materials and Methods section. Uninjured cells in wells of Flex Plates served as controls. Values are mean ± S.E.M. (n = 4) expressed as a percentage of responses in non-injured cells (upper panel). *** p < 0.001, * p < 0.05 (one-way ANOVA).

Figure 5

Caveolin-1 is necessary for the P2Y₂R anti-apoptotic action in 1321N1 cells after mechanical injury. A schematic representation describing the Cav-1-dependent P2Y₂R-mediated signaling pathways investigated in this study. (A) Nucleotide agonists’ activation of the P2Y₂R signaling pathways and subsequent antiapoptotic action during mechanical injury. (B) Caveolin-1 knockdown leads to P2Y₂R uncoupling from its signaling pathways.