All ETDs from UAB

Advisory Committee Chair

Catherine M Fuller

Advisory Committee Members

James F Collawn

Joanne Murphy-Ullrich

Yancey Gillespie

Mark O Bevensee

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Two of the most striking features of brain tumors are their unusual ability to robustly proliferate and migrate in the brain. Previous results from our laboratory suggest that glioma cells express a non-selective cation channel that is composed of Epithelial Sodium Channel (ENaC) and Acid Sensing Ion Channel (ASIC) subunits. We hypothesize that this glioma specific current regulates glioma cell migration and proliferation by modulating an intracellular signaling pathway. To test our hypothesis, we have attenuated this cationic conductance by either pharmacological blockers (psalmotoxin-1 and benzamil), or by knocking down expression of a particular subunit of this cross-clade ion channel (ASIC-1), or by regulating the extracellular Na+ concentration. We found that these maneuvers resulted in significant inhibition in cell migration and cell cycle progression of D54-MG glioma cells. The expression of p21Cip1 and p27Kip1 were significantly upregulated when the glioma specific conductance was inhibited. The involvement of MAP kinase pathway was determined when we found that phosphorylation of ERK1/2, a key regulator of cell migration and proliferation was also inhibited. We next demonstrated that the glioma specific cation conductance was engaging the MAPK pathway regulating migration and proliferation by interacting with integrin beta 1. We have shown the physical proximity of ASIC1 and integrin beta 1 by co-immunoprecipitation. Stably knocking down integrin beta 1 significantly attenuated the glioma-conductance in D54-MG cells. We have also showed that the proper membrane localization of ASIC1 is dependent on integrin beta 1. The indirect interaction between ASIC-1 and integrin beta 1 was mediated by alpha actinin as we found a physical association between ASIC-1, integrin beta 1 and alpha - actinin in D54-MG glioma cells using co-immunoprecipitation. Mutating the putative alpha-actinin binding site on the C-terminus of ASIC-1 reduced the membrane localization of ASIC-1. Phosphorylation of ERK1/2 was also reduced by knockdown of alpha-actinin-1 and -4 or by mutating the alpha-actinin binding motif in the ASIC-1 C-terminus. These data show that glioma cell migration and proliferation is regulated by a non-selective and amiloride sensitive cationic conductance and a physical association of ASIC1-integrin beta 1-alpha actinins is required to maintain this constitutive activity of this cross-clade channel.

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