Isoform-specific functions of the NA,K-ATPase β1- and β2- subunits in medulloblastoma cells
Date
2018
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
The Na,K-ATPase is a ubiquitously expressed membrane protein that carries out the transport of Na+ and K+ ions across the plasma membranes of most animal cells. The pump consists of a catalytic α-subunit and a regulatory β-subunit, both of which have multiple isoforms. While the α1- and β1-subunits are universally expressed in most cells, suggesting important housekeeping roles of these proteins, other isoforms are expressed in a tissue-specific manner. For example, the β2-isoform is expressed in muscle and brain tissue. Given this tissue-specific expression pattern and its tight regulation during cerebellar maturation, the β2-subunit likely has a unique tissue-specific function. Previous work has shown that the β2-subunit is a cell adhesion molecule on glia, is necessary for astrocyte to neuron adhesion and granule cell migration, and that β2-subunit knockout is lethal in mice, probably due to neuronal degeneration. The β1-subunit has been studied in much more detail, and has been described as a cell signaling scaffold and as a cell adhesion molecule in epithelial and cardiac cells. Molecular pathways describing how the β2-subunit may be involved in cell signaling remain to be identified. This dissertation will focus on determining the isoform-specific functions of the β1- and β2-isoforms in medulloblastoma cells. ☐ Medulloblastoma is the most common malignant form of pediatric brain cancer and is a tumor that originates in the cerebellum. Mutations leading to hyperactive sonic hedgehog (Shh) signaling have been associated with some forms of medulloblastoma. Additionally, the Shh pathway plays an important role in cerebellar development, but molecular targets contributing to Shh-mediated neuronal cell proliferation and transformation are still poorly understood. ☐ Changes in Na,K-ATPase function and subunit expression have been reported in several cancers. Loss of β1-subunit expression has been associated with a poorly differentiated phenotype in carcinoma but its role in medulloblastoma progression is not yet known. Here we show activation of Shh signaling resulted in reduced β1-subunit protein and mRNA expression, both by pharmacologic activation and by overexpression of Gli1, a member of the Shh signaling cascade. Moreover, in human medulloblastoma cells, low β1-subunit levels were associated with increased cell proliferation and in vivo xenograft tumor growth. Thus we established that the Na,K-ATPase β1-subunit is a target of the Shh signaling pathway and that loss of β1-subunit expression may contribute to tumor growth in medulloblastoma, a tumor that originates from cerebellar granule neurons. ☐ The goal of this dissertation was to distinguish between β1- and β2-subunit function. Cerebellar granule cells and medulloblastoma cells express both β1- and β2-isoforms. We found that expression of both the β1- and β2-subunits increased during post-natal cerebellar maturation, and more specifically, during cerebellar granule precursor cell differentiation. However, while we showed that the β1-subunit is a target of Shh signaling and represses its expression, expression of the β2-subunit was unaffected by Shh signaling. This and other data further suggests that the β1- and β2-isoforms have independent functions. ☐ Knockdown studies of the β2-subunit in medulloblastoma cells revealed a role for the β2-subunit in epidermal growth factor-induced reorganization of the actin cytoskeleton. Our studies further showed that β2-subunit, but not β1-subunit, knockdown resulted in increased Merlin/NF2 expression, accompanied by Merlin-mediated changes in the kinetics of epidermal growth factor receptor signaling. Our studies for the first time provide a functional link between the Na,K-ATPase β2-subunit and Merlin/NF2, a member of the Ezrin/Radixin/Moesin protein family, suggesting a role for the β2-subunit in actin-dependent structure organization in medulloblastoma cells. ☐ The work described here distinguished between the molecular functions of the β1- and β2-isoforms in cells that originate from cerebellar granule neurons. We also established a link between the β2-subunit and the actin cytoskeleton and epidermal growth factor receptor signaling. Our work suggests possible unique roles for the β1- and β2-isoforms in cerebellar granule precursor cell differentiation and in cerebellar development.
Description
Keywords
Biological sciences, Cerebellar granule neurons, Medulloblastoma cells, β2-subunit