dramatically reduced in MKK7-JNK2-transfected HEK293T cells, even to a greater extent than that in MKK7-JNK1- transfected cells (Figure 1A, lane 3 versus 2), suggesting that both JNK1 and JNK2 activation downregulate b-catenin expression although to a different extend. To determine whether activated JNK2 can inhibit the aberrantly accumulated nuclear b-catenin in cancer cells, a human lung cancer cell line A549 was transfected with MKK7- JNK2. Immunoblotting analysis showed a reduction of endoge- nous b-catenin protein (Figure 1B), which is consistent with the observation made in HEK293T cells. However, the degree of b- catenin suppression in A549 was less than that in HEK293T cell, which might be a result of biological differences between HEK293T cells and the transformed human lung cancer cells. b-catenin interacts with TCF-4 and stimulates gene expression. To determine whether JNK2 activation can affect the b-catenin- driven gene transription, MKK7-JNK2 was co-transfected with b- catenin into HEK293T cells along with TCF-4 reporter plasmid TOPFLASH or the inactive control FOPFLASH. TOPFLASH includes a luciferase reporter driven by three copies of TCF binding elements upstream of the thymidine kinase (TK) minimal promoter, and is specifically regulated by Wnt/b-catenin signaling [18]. TOPFLASH used here is different from LEF1-Luciferase reporter used in a recent report [6]. The latter contains seven LEF1 binding sites, in p301fosCAT plasmid, containing the minimal fos promoter linked to the chloramphenicol accetyltrans- ferase (CAT) gene [19]. Upon transfection of b-catenin, the TOPFLASH reporter activity increased by about 10-fold (Figure 1C), although no change was observed in the cells transfected with the empty pcDNA3-HA control vector. However, MKK7-JNK2 transfection strongly suppressed the b-catenin- induced TOPFLASH reporter activity even to a greater extent than MKK7-JNK1 (Figure 1C). The similarities between the regulations of b-catenin-driven transactivation and b-catenin protein level by JNK1 and JNK2 activation implicates that, the inhibitory effect of activated JNK2 on b-catenin-driven transacti- vation is probably attributed to the downregulation of b-catenin protein. Taken together, these data demonstrate for the first time a negative regulation of Wnt/b-catenin signaling by JNK2 activa- tion. To confirm the suppression of Wnt/b-catenin signaling by JNK2 activation, a constant level of b-catenin was co-transfected with increasing amounts of MKK7-JNK2 into HEK293T cells. The alterations of exogenous b-catenin were assessed by immunoblotting. As shown in figure 2A, a slight decrease in b- catenin protein level was seen with 810 ng of pMKK7-JNK2, and a dramatic reduction in b-catenin protein level was observed with higher amounts of pMKK7-JNK2. The effect of increasing amounts of MKK7-JNK2 on b-catenin-induced transaction was also evaluated using a luciferase activity assay. As shown in figure 2B, co-transfection of 150 ng of pMKK7-JNK2 suppressed the TOPFLASH reporter activity by 50% and 300 ng suppressed it by 65%. Taken together, these data demonstrate a dose- dependent inhibition of Wnt/b-catenin signaling by JNK2 activation. Activated JNK2 Reduces b-catenin Protein Level Through Proteasomal Degradation Cytoplasmic b-catenin protein levels are regulated by ubiqui- tination and subsequent proteasomal degradation. To determine whether the decrease of b-catenin protein induced by JNK2 activation is due to increased proteasomal degradation, a proteasomal inhibitor MG132 [20] was used to treat HEK293T cells after transfection with MKK7-JNK2 and b-catenin. As shown JNK2 Interacts with b-Catenin in figure 3A, MG132 blocked JNK2-induced b-catenin inhibition (lane 4 versus 3), demonstrating that JNK2-caused b-catenin degradation was through proteasomal degradation system. GSK3b Functions Upstream of the Proteasome to Mediate the Reduction of b-catenin by JNK2 Activation Multiple pathways have been reported to mediate b-catenin degradation by the proteasome, such as p53/Siah-1/APC, Wnt/ GSK3b/APC, and retinoid X receptor (RXR)-mediated pathway [21]. To determine the role of the most common GSK3b- regualated pathway in the downregulation of b-catenin by JNK2 activation, we detected the phosphorylation status of GSK3b. GSK3b could be inactivated by phosphorylation at Ser-9 by serine/threonine kinases, such as Akt, protein kinase A (PKA), and protein kinase C (PKC), followed by decreased activities [22–24]. Thus, GSK3b phosphorylation at Ser-9 has been used as an indirect marker of decreased GSK3b activity. As shown in figure 1A, Ser-9 phosphorylation of GSK3b (p-GSK3b) was indeed decreased in MKK7-JNK2-transfected HKE293T cells, which meant that GSK3b activity was increased by activated JNK2. The expression pattern of p-GSK3b is similar to that of b- catenin, leading us to postulate that the b-catenin degradation is attributed to the increased GSK3b activity induced by JNK2 activation. In fact, increased GSK3b activity and corresponding downregulation of b-catenin were also seen in A549 cells (Figure 1B). To further test our hypothesis, GSK3b inhibitor lithium chloride (LiCl) was used to suppress GSK3b activity and the effect of activated JNK2 on b-catenin expression was analyzed. As shown in figure 3B, LiCl inhibited GSK3b activity which was indicated by an increase of GSK3b phosphorylation (lane 2 versus 1; lane 4 versus 3). The ability of activated JNK2 to downregulate b-catenin was almost completely abrogated upon LiCl treatment Figure 4. JNK2 deficiency caused upregulation of b-catenin and its downstream target CDK4, as well as upregulation of GSK3b phosphorylation in JNK2-/- mouse intestinal epithelial cells, compared to those in JNK2+/+ mice. Each lane represents one mouse. b-actin served as loading control. doi:10.1371/journal.pone.0006640.g004 PLoS ONE | www.plosone.org 4 August 2009 | Volume 4 | Issue 8 | e6640