NKCC1 Regulates Migration Ability of Glioblastoma Cells by Modulation of Actin Dynamics and Interacting with Cofilin

Abstract

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. The mechanisms that confer GBM cells their invasive behavior are poorly understood. The electroneutral Na+-K+-2Cl− co-transporter 1 (NKCC1) is an important cell volume regulator that participates in cell migration. We have shown that inhibition of NKCC1 in GBM cells leads to decreased cell migration, in vitro and in vivo. We now report on the role of NKCC1 on cytoskeletal dynamics. We show that GBM cells display a significant decrease in F-actin content upon NKCC1 knockdown (NKCC1-KD). To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition, we studied NKCC1 protein interactions. We found that NKCC1 interacts with the actin-regulating protein Cofilin-1 and can regulate its membrane localization. Finally, we analyzed whether NKCC1 could regulate the activity of the small Rho-GTPases RhoA and Rac1. We observed that the active forms of RhoA and Rac1 were decreased in NKCC1-KD cells. In summary, we report that NKCC1 regulates GBM cell migration by modulating the cytoskeleton through multiple targets including F-actin regulation through Cofilin-1 and RhoGTPase activity. Due to its essential role in cell migration NKCC1 may serve as a specific therapeutic target to decrease cell invasion in patients with primary brain cancer.

Diallyl disulfide induces downregulation and inactivation of cofilin�1 differentiation via the Rac1/ROCK1/LIMK1 pathway in leukemia cells

“…Two types of cofilin dephosphorylate, Slingshot and chronophin, which can dephosphorylate cofilin, thereby causing the depolymerization and removal of F-actin, promoting actin nucleation through Arp 2/3 and inducing other actin polymerization events and directional movement (14). Cofilin 1 helps regulate glioblastoma cell migration by modulating the cytoskeleton via multiple targets, including F-actin regulation and RhoGTPase activity, such as small Rho-GTPases RhoA and Rac1 activity (15). LIMK1 positively controls the expression and phosphorylation of cofilin 1, leading to rearrangement of the cellular actin cytoskeleton, which serves roles in the growth and motility inhibition of breast cancer cells (12).…”

LTP induction boosts glutamate spillover by driving withdrawal of astroglia

“…What could be the downstream signal of NKCC1? It has recently been proposed that NKCC1 (characteristic of astroglia) serves as a protein scaffold regulating, in a pH-sensitive fashion, the phosphorylation of a small (19 kDa) freely-diffusible protein cofilin-1 (Schiapparelli et al, 2017), and that the ion transporter KCC2 drives a similar cascade in neurons (Llano et al, 2015). Cofilin-1 is a well-established bi-directional regulator of actin filament polymerization, which is directly related to cell protrusion formation and retrieval, such as dendritic spine morphogenesis (Bravo-Cordero et al, 2013;Ethell and Pasquale, 2005).…”

“…In these cells, NKCC1 activity can lead to a (local) accumulation of intracellular chloride of up to 140 mM triggering prominent cellular shrinkage, up to 35% (Habela et al, 2009). It has recently been discovered that in glioblastoma cells NKCC1 regulates phosphorylation of cofilin-1 (Schiapparelli et al, 2017). Whilst NKCC1 typically occurs in astrocytes, the neuron-specific ion transporter KCC2 has been known to also target cofilin-1 activity (Llano et al, 2015).…”

“…What could be the downstream signal of NKCC1? In glioblastoma cells, NKCC1 was found to serve as a protein scaffold regulating the phosphorylation of the small, freelydiffusible protein cofilin-1 (Schiapparelli et al, 2017). In neurons, ion transporter KCC2 plays a similar role in cofilin control (Llano et al, 2015).…”

“…In these cells, NKCC1 activity can accumulate intracellular chloride up to 140 mM, triggering prominent (up to 35%) cellular shrinkage (Habela et al, 2009). In glioblastoma cells, NKCC1 regulates phosphorylation of cofilin-1 (Schiapparelli et al, 2017), which controls, in a pHdependent manner, actin filament polymerization, and thus formation and retrieval of thin cell processes, such as dendritic spines (Bravo-Cordero et al, 2013;Ethell and Pasquale, 2005). Indeed, interfering with cofilin-1 phosphorylation by dialyzing individual astrocytes with peptide S3 (Aizawa et al, 2001;Liu et al, 2016) completely occluded LTP-induced PAP shrinkage, suggesting a shared molecular pathway.…”