WNK1-OSR1 kinase-mediated phospho-activation of Na+-K+-2Cl- cotransporter facilitates glioma migration

Wen Zhu,Kelli Pointer,Gulnaz Begum,Sung-Sen Yang,Dandan Sun,Paul A Clark,John S Kuo,Shih-Hua Lin,Kristopher T Kahle

doi:10.1186/1476-4598-13-31

Abstract

BackgroundThe bumetanide (BMT)-sensitive Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl- content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown.MethodsExpression of NKCC1 and its upstream kinases With-No-K (Lysine) kinase 1 (WNK1) and oxidative stress-responsive kinase-1 (OSR1) in different human glioma cell lines and glioma specimens were detected by western blotting and immunostaining. Live cell imaging and microchemotaxis assay were applied to record glioma cell movements under different treatment conditions. Fluorescence indicators were utilized to measure cell volume, intracellular K+ and Cl- content to reflect the activity of NKCC1 on ion transportation. Small interfering RNA (siRNA)-mediated knockdown of WNK1 or OSR1 was used to explore their roles in regulation of NKCC1 activity in glioma cells. Results of different treatment groups were compared by one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons.ResultsWe show that compared to human neural stem cells and astrocytes, human glioma cells exhibit robust increases in the activation and phosphorylation of NKCC1 and its two upstream regulatory kinases, WNK1 and OSR1. siRNA-mediated knockdown of WNK1 or OSR1 reduces intracellular K+ and Cl- content and RVI in glioma cells by abolishing NKCC1 regulatory phospho-activation. Unexpectedly, TMZ activates the WNK1/OSR1/NKCC1 signaling pathway and enhances glioma migration. Pharmacological inhibition of NKCC1 with its potent inhibitor BMT or siRNA knockdown of WNK1 or OSR1 significantly decreases glioma cell migration after TMZ treatment.ConclusionTogether, our data show a novel role for the WNK1/OSR1/NKCC1 pathway in basal and TMZ-induced glioma migration, and suggest that glioma treatment with TMZ might be improved by drugs that inhibit elements of the WNK1/OSR1/NKCC1 signaling pathway.

Highlights

The bumetanide (BMT)-sensitive Na+-K+-2Cl− cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl− content via regulatory volume increase (RVI)
We investigated whether WNK1SPAK/oxidative stress-responsive kinase-1 (OSR1) signaling pathway regulates NKCC1 activity in glioma cells (GCs) and whether this signaling pathway is involved in regulation of glioma migration, with and without chemotherapeutic treatment
Down-regulation of With-No-K (Lysine) kinase 1 (WNK1)/OSR1 reduces microchemotaxis of GCs Given the important role of WNK1 and OSR1 in regulation of NKCC1 in GCs, we further investigated whether reduced expression of these upstream kinases will affect the migratory behaviors of GCs, especially under TMZ treatment

Summary

Introduction

The bumetanide (BMT)-sensitive Na+-K+-2Cl− cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl− content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. The standard treatment of malignant glioma includes maximal surgical resection followed by concurrent radiation and chemotherapy with temozolomide (TMZ) [1]. Recurrence of the disease is attributed in part to resistance of glioma cells to the standard chemotherapeutic reagent TMZ [3]. It is important to identify new therapeutic targets to hinder the migration of the invasive glioma cells and sensitize glioma cells to chemotherapy

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