Stimulation with recombinant Wnt5a increased activation of AKT 1

Stimulation with recombinant Wnt5a increased activation of AKT 1.9-fold in SK-N-AS and 6.6-fold in SK-N-DZ cells, whereas Wnt3a significantly increased AKT activation in SK-N-DZ cells only (4-fold) (Figure ?(Figure2).2). 6) co-receptors. This leads to the stabilization of cytoplasmic -catenin and subsequent transcription of Wnt target genes that include (cyclin D1) and others [10, 11]. Importantly, it has been shown that overactivation of the Wnt signaling pathway is due to the overexpression of different FZD receptors in a variety of cancers [12C14]. In high-risk NB without amplification, increased Wnt ligands have been shown together with strongly expressed -catenin [15]. Besides canonical -catenin Wnt signaling, -catenin-independent non-canonical Wnt signaling encompasses those pathways that instead use other modes of downstream signaling [16] and may also affect NB phenotype and growth. In the SCKL -catenin-independent planar cell polarity (PCP) pathway, FZD receptors activate a cascade that involves the small GTPase Rac1 and JUN-N-terminal kinase (JNK), which can lead to changes in cytoskeleton and cell polarity [17]. An important aspect is the crosstalk among canonical and non-canonical signaling pathways. Accordingly, PCP and -catenin-dependent Wnt signaling are well known to antagonize each other, and inhibiting Astragaloside II one will typically upregulate the other. Another -catenin-independent pathway, the Wnt/Ca2+ pathway, can increase the intracellular Ca2+ concentration and activate protein kinase Astragaloside II C (PKC) [17, 18]. In melanoma cells Wnt5a signaling directs migration and invasion of cells in a PKC-dependent manner [19] and can increase phosphorylated AKT via phosphoinositide 3-kinase (PI3K) [20]. FZD2 is one of the most important receptors in non-canonical Wnt pathways and FZD2 expression is strongly correlated with poor prognosis in several types of cancer [12, 21, 22]. The binding of Wnt5a to FZD2 activates the Wnt/Ca2+ pathway in melanoma cell lines [23]. Moreover, Wnt5a/FZD2 signaling has been shown to control cellular migration and invasion in colon cancer [21]. However, in the presence of Wnt3a, FZD2 also activates -catenin-dependent signaling in pulmonary carcinoma [24]. These reports indicate that FZD2 can activate both -catenin-dependent and -catenin-independent signaling. So far no studies have reported the association of FZD2 with NB growth. In this study, we examine the function of FZD2 and its potential ligands Wnt3a and Wnt5a in mRNA in both cell lines was the highest, followed by was low (Supplementary information and supplemental Physique 1). Next, we investigated the mRNA expression of and its potential activators and in both NB cell lines. SK-N-DZ cells expressed significantly higher and levels, whereas was expressed at roughly comparable amounts between SK-N-AS and SK-N-DZ cells (Physique ?(Figure1A).1A). Notably, expression of the canonical Wnt signaling pathway target was significantly higher in was more abundantly expressed in SK-N-DZ cells (Physique ?(Figure1A1A). Open in a separate window Physique 1 Characterization of gene expression in and mRNA expression is different between SK-N-AS and SK-N-DZ cells. B. Representative Western blot images and C. quantitative determination of protein expression in SK-N-AS and SK-N-DZ cells. Untreated cells were harvested to analyze the levels of FZD2, phospho-LRP6 (LRP6 phosphorylated at Ser1490; p-LRP6), total -catenin, active -catenin (non-phospho -catenin; Ser33/37/Thr41), MYC, cyclin D1, pan-phospho-PKC, (II Ser660; p-PKC), total JNK, phospho-JNK (JNK phosphorylated at Thr183/Tyr185; p-JNK), total AKT, phospho-AKT (AKT phosphorylated at Ser473; p-AKT), total ERK and phospho-ERK (ERK1/2 phosphorylated at Thr202/Tyr204; p-ERK) by Western blotting. Comparison of protein expression profiles between SK-N-AS and SK-N-DZ cells revealed differences in basal expression and activation levels of investigated signaling proteins. Graphs represent the mean of 3 impartial experiments SD (* < 0.05). Basal Wnt pathway activity was examined in the cells using several markers. Low-density lipoprotein receptor-related protein 6 (LRP6) is usually a key signaling co-receptor for the -catenin pathway, which is usually phosphorylated following Wnt binding to FZD2 [17]. Thus, LRP6 phosphorylation, total -catenin, stabilized active -catenin, MYC and cyclin D1 were examined by Western blot analysis in both NB cell lines to investigate canonical -catenin Wnt signaling components. In SK-N-AS cells, FZD2 and phosphorylated LRP6 protein levels were lower compared with SK-N-DZ cells. In contrast, levels of total -catenin, active -catenin and MYC were both more abundantly expressed in SK-N-AS cells. SK-N-DZ cells in turn, expressed higher levels of cyclin D1, confirming mRNA findings (Physique ?(Physique1B1B and ?and1C1C). Wnt5a signaling activates the PI3K-AKT pathway in melanoma cells [25] and Wnt3a-induced proliferation involves activation of ERK beside Wnt/-catenin pathway activation in fibroblasts [26]. Therefore, we examined the basal activity of -catenin-independent non-canonical Wnt signaling components by examining phosphorylation of PKC, JNK, AKT and ERK. SK-N-AS NB cells Astragaloside II expressed phosphorylated PKC at Astragaloside II higher levels than SK-N-DZ cells. The levels of total and phosphorylated AKT were comparable in both cell.