Elsevier

Experimental Cell Research

Volume 314, Issue 13, 1 August 2008, Pages 2389-2399
Experimental Cell Research

Research Article
Wnt-11 signaling leads to down-regulation of the Wnt/β-catenin, JNK/AP-1 and NF-κB pathways and promotes viability in the CHO-K1 cells

https://doi.org/10.1016/j.yexcr.2008.04.010Get rights and content

Abstract

The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical β-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical β-catenin mediated Wnt signaling but also JNK/AP-1 and NF-κB signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-κB pathway. Consistent with the central role of Akt, JNK and NF-κB in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways.

Introduction

The Wnt gene family that encodes secreted glycoproteins consists of 19 members in mammals. These growth factors control processes such as ageing, certain diseases, pattern formation and cell proliferation, apoptosis and differentiation in the adult and during embryonic development (for reviews, see [1], [2], [3], see also Wnt home page: http://www.stanford.edu/~rnusse/wntwindow.html). The importance of Wnt signaling is underlined by the fact that several of its signal transduction components are implicated in various cancer types and developmental disorders. Wnt signaling has traditionally been classified as either canonical or noncanonical, based on the capacity of the various Wnts to transform mouse mammary epithelial C57MG cells morphologically and their potential to induce a secondary axis during early embryogenesis in Xenopus [4], [5]. It has become apparent, however, that Wnt signaling is more complex and that the Wnts interact with several receptor types, initiate various signaling pathways and also involve signal transduction that is dependent on endocytosis of the cell surface receptors [6], [7], [8], [9].

The canonical Wnt signaling pathway is the best characterized one so far. Here the binding of Wnts to Frizzled and LRP5/6 receptors stabilizes the cytoplasmic β-catenin that subsequently becomes translocated to the nucleus, where it interacts with the T cell factor/lymphoid enhanced factor (Tcf/Lef) family of transcription factors to regulate the target gene expression. Studies in Drosophila originally suggested the existence of a noncanonical Wnt signaling pathway that controls the process planar cell polarity (PCP) [10], [11], where the cells adopt a specific orientation in the plane of the tissue [12]. Frizzled and Dishevelled are required for this process, but a role for any of Wnt ligands themselves has not been confirmed [13]. The Wnt/β-catenin and PCP signaling are completely separated downstream of Dishevelled. The currently known downstream effectors of the PCP pathway include the small Rho-like GTPases and JNK kinase [10], [14], although the involvement of JNK in Drosophila has been also questioned [15]. The Wnt–Ca2+ pathway comprises another branch of noncanonical signaling. Here the Wnt-Frizzled complex signals through heterotrimeric G and phospholipase C proteins, triggering the release of calcium from intracellular storages and leading to the activation of Ca2+-sensitive factors such as protein kinase C and calmodulin-dependent protein kinase II, CamKII [16], [17], [18]. The precise orchestration of noncanonical Wnt signaling events is only just beginning to be unraveled, however.

Of the mammalian Wnts, Wnt-11 is essential for the development of the heart and kidney [19], [20], [21], [22] but is also implicated in cancer [23], [24], [25], [26]. Wnt-11 signaling is thought to function in part by inhibiting the activity of the β-catenin-dependent Wnt pathway, but the mode of action is still unclear. Inactivation of Tcf by Wnt-11 in the zebrafish involves NLK kinase [27], but neither this pathway nor CamKII mediates Wnt-11 signaling in NIH3T3 or the embryonic carcinoma cell line P19 [28]. Wnt-11 signaling functions in chondrocytes are in turn abolished by the inhibition of protein kinase C [29]. Strikingly, differentiation of the endothelial precursor cells to cardiomyocytes is enhanced by Wnt-11, and the process is blocked by a protein kinase C inhibitor [30]. Consistent with the role of Wnt-11 in cardiomyocyte differentiation, Wnt-11 overexpression activates JNK and expression of the early cardiac marker genes in the Xenopus embryo [19], [31], and JNK kinase mediates Wnt-11 signaling in murine cardiogenesis as well [22].

Besides being involved in noncanonical Wnt signaling and planar cell polarity, JNK has other effects. It is a member of the mitogen-activated protein kinases (MAPKs) and is activated in response to growth factors, inhibition of DNA and protein synthesis, environmental stress and inflammatory cytokines, all of which regulate cell proliferation, differentiation and apoptosis [32], [33]. JNK and other MAPKs are activated by phosphorylation of the threonine and tyrosine residues in the conserved Thr–X–Tyr motif. This phosphorylation is catalyzed by the MAPK kinases (MKKs), which are in turn activated by a serine/threonine phosphorylation catalyzed by the MAPKK kinases (MEKKs). The latter are induced by various upstream activators, including kinases and small GTP-binding proteins. The activated JNK phosphorylates and activates several transcription factors, resulting in activation of a heterodimeric AP-1 factor composed of Jun, Fos and ATF subunits [34].

In view of the critical roles of Wnt-11, elucidation of its signaling mechanism would be of considerable importance. We demonstrate here that the CHO cells respond to Wnt-11 signaling and that Wnt-11 not only inhibits canonical β-catenin signaling but also inhibits the transcriptional activity of AP-1. We show that JNK and its immediate upstream kinase MKK4 are involved in Wnt-11-induced inhibition of the JNK/AP-1 pathway. Wnt-11 signaling phosphorylates the Akt kinase, which probably contributes to the inhibition of JNK/AP-1 activity by catalyzing phosphorylation of MKK at Ser-80, which inhibits the action of MKK4. Moreover, Wnt-11 signaling leads to the inhibition of NF-κB signaling. Consistent with the central role of Akt, JNK and NF-κB in cell survival and stress response, Wnt-11 signaling protected cells from apoptosis, thus promoting their viability.

Section snippets

Antibodies

The affinity-purified rabbit polyclonal antibodies anti-phospho-Akt (Thr-308), anti-phospho-Akt (Ser-473), anti-Akt, anti-phospho-c-Jun (Ser-63), anti-phospho-SAPK/JNK (Thr-183/Tyr-185), anti-phospho-SEK1/MKK4 (Ser-257/Thr261) and anti-phospho-SEK1/MKK4 (Ser-80), together with rabbit monoclonal anti-SAPK/JNK (clone 56G89) antibody, HRP-linked goat anti-rabbit IgG and HRP-linked anti-biotin were all obtained from Cell Signaling Technology, mouse anti-β-catenin (clone 14) was from BD Biosciences,

Wnt-11 inhibits the JNK/AP-1 pathway in the CHO cells as well as the canonical β-catenin pathway

Since CHO cells are responsive to both canonical and noncanonical Wnt signaling [41], [42], we selected this cell line to study the mechanisms of Wnt-11 signaling. The transient cotransfection experiments with the Wnt-11-encoding plasmid pWnt-11 and the SuperTopFlash reporter containing the luciferase gene under the Tcf-responsive promoter revealed that Wnt-11 inhibited the canonical β-catenin signaling pathway in a dose-dependent manner (Fig. 1A). Wnt-11 signaling also inhibited the activity

Discussion

This study unraveled some of the mechanisms involved in noncanonical Wnt-11 signaling. Wnt-11 is an important signaling molecule in gastrulation, where it is expressed in dorsal tissues and regulates the convergent extension movements [43], [44]. Like Wnt-11, another noncanonical Wnt, Wnt-5a, regulates the convergent extension movements in the frog that are perturbed if JNK activity is either inhibited or stimulated, indicating the importance of appropriate JNK activity [58], [59]. Indeed, both

Acknowledgments

We thank Drs Hans Clevers, Tuula Kallunki, Kunihiro Matsumoto and Ugo Moens for generously donating reporter plasmids. Dr. Kallunki is also acknowledged for her helpful advice and discussions. The project was supported financially by grants from the Academy of Finland (107406, 206038), the Sigrid Jusélius Foundation, the European Union (LSHG-CT-2004-005085) and Biocenter Oulu.

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