WWOX Induces MEK2-Mediated Cell Death in a p53 Independent Manner

Abstract

WW domain-containing oxidoreductase WWOX that contains two WW domains in the N-terminus, which are involved in protein-protein interactions, and an alcohol dehydrogenase (ADH) domain is a putative tumor suppressor and a proapoptotic protein. It has been reported that WWOX gene is located at the common fragile site FRA16D in chromosome 16q23.3-24.1 and its expression is downregulated in various cancer types. 1,2 Furthermore, aberrantly spliced WWOX mRNA forms that were not detected in normal cells were detected in cancer cell lines and primary breast tumors. 3 In addition, loss of WWOX expression resulted in resistance to apoptosis induced by tumor necrosis factor, staurosporine, UV, and p53 overexpression whereas overexpressed WWOX promoted apoptosis synergistically with p53. 4 WWOX is known to participate in several physiological functions through interaction with several regulators, such as Dvl proteins for the regulation of Wnt-catenin pathway, 5 JNK and c-Jun for JNK signaling, 4,6 RUNX2 for osteoblast differentiation 7 , and p53/p73 for apoptosis. 8,9 Previous report showed that WWOX is a novel regulator in extracellular signal-regulated kinase (ERK) signaling through interaction with MEK2. 10 ERK signaling is important for the control of cell proliferation, migration, cell division, and differentiation. 11 ERK is phosphorylated and activated by MAP kinase kinases (MAP2Ks) such as MEK1/2. However, ERK activated by either MEK1 or MEK2 might have opposite roles in cell survival. MEK1/ERK signal enhances cell proliferation, whereas the MEK2/ERK induces growth arrest at the G 1 /S boundary. 12 Since the previous study showed that WWOX interacts with MEK2 and activates ERK pathway 10 and WWOX is a putative tumor suppressor, it implicates that the association of WWOX with MEK2 may have a negative role in cell growth.In this study, it was investigated whether WWOX regulates MEK2-mediated cell growth. Since ERK activity is increased by WWOX, 10 cell viability assays were carried out to test whether WWOX regulates MEK2-induced proliferation of HEK 293 cells. HEK 293 cells were transiently co-transfected with FLAG-tagged WWOX wild-type (WT) and HAtagged MEK2 expression plasmids. To enhance MEK2 activity and susceptibility to apoptosis, cells were treated with 0.5 mM H 2 O 2 for 16 h prior to assay. WWOX-induced cell death was significantly increased in the presence of MEK2 while MEK2 alone did not induce cell death (Fig. 1), indicating that WWOX enhances MEK2-mediated cell death.H 2 O 2 decreases the cellular threshold for cell death induction by inducing pro-apoptotic events. The threshold concentration of H 2 O 2 which affects WWOX/MEK2-mediated cell viability was investigated. HEK 293 cells co-transfected with FLAG-WWOX WT and HA-MEK2 expression plasmids were treated with various concentrations of H 2 O 2 for 16 h prior to viability assays. Cells transfected with WWOX or MEK2 alone did not show any change of cell viability in the absence of H 2 O 2 and those treated with 1 mM were severely decreased in cell viabil...