Abstract
Glioma cells express a specific amiloride‐sensitive cation channel composed of ASIC1, α‐ and γENaC. As these tumors are highly invasive, we hypothesized that channel activity was engaging an intracellular signaling pathway regulating migration and proliferation. We used a truncated construct (ASIC1Y67X) and shRNA to knockdown ASIC1 and integrin β1 in D54MG glioma cells, and scratch assays, FACS, patch‐clamp, and immunoblotting, to determine knockdown effects. Cells were synchronized by removal of FBS for 48h prior to the experiments. ASIC1 knockdown reduced scratch area by 39 ± 16% (n≥4 ±SD P<0.001), and significantly increased the percentage of cells in the cell cycle G0/G1 phase (by 19 ± 5% (SD) n≥3 P<0.001) as compared to controls at 24h. Expression of two cell cycle inhibitors, p21Cip1 and p27Kip1 increased by 2.5 fold (n≤5) and 3 fold (n≤6), respectively. Phosphorylation of ERK1/2, a key mediator of proliferation and migration was also reduced (by 47 ± 20% (SD) n≥6 P<0.001) as compared to control at 24h. Knockdown of δENaC (not part of the glioma channel complex), had no effect on any measured parameter. Knockout of integrin β1 inhibited the amiloride‐sensitive current by 89±24% (n≥9; ±SD P<0.001). Our data reveal novel interactions between Deg/ENaC channels, the MAPK pathway and integrins that may form a mechanism by which Na+ current regulates cell proliferation and migration. Supported by NIH Grant # DK037206.
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