The role of SPAK in the potassium chloride cotransporter 3 (KCC3) signaling

Abstract

Ste20-related proline-alanine-rich kinase (SPAK) plays a role in regulating many biological activities and interacts with the K-Cl cotransporter (KCC) 3; however the importance of SPAK for KCC3 function has not been demonstrated Here we investigated the role of SPAK in KCC3-regulated cell invasiveness and tumor formation We show that the induction of KCC3 expression triggers the activation of the NFκB and SPAK signaling cascades leading to the activation of p38 mitogen-activated protein kinase (MAPK) and matrix metalloproteinase-2 (MMP2) and augmented binding of NFκB to its putative SPAK promoter binding site suggesting that the SPAK/MMP2 axis is up-regulated by NFκB A small interference RNA-mediated reduction in SPAK protein levels suppressed the invasive ability and oncogenic potential of cervical cancer cells and decreased tumor formation in mouse xenografts Pharmacological inhibition of NFκB or MMP2 abrogated KCC3-triggered SPAK-dependent cell invasiveness Furthermore p38 MAPK was identified as the upstream regulator of KCC3-dependent MMP2 activation We concluded that SPAK may promote the KCC3-mediated tumor aggressiveness via the NFκB/p38 MAPK /MMP2 axis Our previous data explored KCC3 could enhance cell proliferation and promote epithelial to mesenchymal transition (EMT) Here we indicate the SPAK and EMT have positive correlation human cervical and ovarian cancer lines However the role of SPAK in promoting EMT was unclear We therefore examined whether the role of SPAK involved in KCC3-mediated EMT Our data indicated that SiHa cells exhibited well-organized cell-cell association which is the characteristics of squamous cell carcinoma SiHaKCC3 could dramatically induce the elongation of cell shape and increased scattering similar to the morphology of mesenchymal cells Furthermore a small interference RNA-mediated reduction in SPAK protein levels suppressed the KCC3-mediated cell scattering effects but increase well-organized cell-cell associated and islet-like structure Our data from immunoblotting and confocal immunofluorescent of EMT marker protein indicated KCC3 overexpression could dramatically decrease the expression of the epithelial marker (E-cadherin and β-catenin) and increase the expression of the mesenchymal marker (vimentin) compare to parental SiHa cells In addition KCC3 overexpression was accompanied by the increasing expression of SPAK In contrast a small interference RNA-mediated reduction in SPAK protein levels suppressed mesenchymal marker (vimentin) expression but increased epithelial marker (E-cadherin and β-catenin) We concluded that SPAK may promote the KCC3-mediated EMT

Date of Award	2014 Aug 29
Original language	English
Supervisor	Cheng-Yang Chou (Supervisor)