title: WNK1-OSR1 kinase-mediated phospho-activation of Na+-K+-2Cl- cotransporter facilitates glioma migration.
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.
  
  
  CONCLUSIONS
  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.
authors.forename: Wen
authors.initials: W
authors.lastname: Zhu
authors.forename: Gulnaz
authors.initials: G
authors.lastname: Begum
authors.forename: Kelli
authors.initials: K
authors.lastname: Pointer
authors.forename: Paul A
authors.initials: PA
authors.lastname: Clark
authors.forename: Sung-Sen
authors.initials: SS
authors.lastname: Yang
authors.forename: Shih-Hua
authors.initials: SH
authors.lastname: Lin
authors.forename: Kristopher T
authors.initials: KT
authors.lastname: Kahle
authors.forename: John S
authors.initials: JS
authors.lastname: Kuo
authors.affiliation: Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA. sund@upmc.edu.
authors.forename: Dandan
authors.initials: D
authors.lastname: Sun
mesh_terms: D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement; D055785:Gene Knockdown Techniques; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2 / Q000378:metabolism*; D046888:Tissue Array Analysis; D000076204:WNK Lysine-Deficient Protein Kinase 1
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D015778:Minor Histocompatibility Antigens; D064506:Solute Carrier Family 12, Member 2; C118135:OXSR1 protein, human; D017346:Protein Serine-Threonine Kinases; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498743:WNK1 protein, human