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Open Access 01-12-2014 | Research

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

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

Published in: Molecular Cancer | Issue 1/2014

Abstract

Background

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). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown.

Methods

Expression 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.

Results

We 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.

Conclusion

Together, 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.

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Title: WNK1-OSR1 kinase-mediated phospho-activation of Na+-K+-2Cl- cotransporter facilitates glioma migration
Authors: Wen Zhu
Gulnaz Begum
Kelli Pointer
Paul A Clark
Sung-Sen Yang
Shih-Hua Lin
Kristopher T Kahle
John S Kuo
Dandan Sun
Publication date: 01-12-2014
Publisher: BioMed Central
Published in: Molecular Cancer / Issue 1/2014
Electronic ISSN: 1476-4598
DOI: https://doi.org/10.1186/1476-4598-13-31

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