Research ArticleWnt-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
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.
References (67)
- et al.
Multiplicity of the interactions of wnt proteins and their receptors
Cell. Signal.
(2007) The many ways of wnt in cancer
Curr. Opin. Genet. Dev.
(2007)- et al.
Caveolin is necessary for wnt-3a-dependent internalization of LRP6 and accumulation of beta-catenin
Dev. Cell
(2006) - et al.
Association of dishevelled with the clathrin AP-2 adaptor is required for frizzled endocytosis and planar cell polarity signaling
Dev. Cell
(2007) - et al.
Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling
Cell
(1998) - et al.
Frizzled signaling and the developmental control of cell polarity
Trends Genet.
(1998) - et al.
Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors
J. Biol. Chem.
(2006) - et al.
A second canon. functions and mechanisms of beta-catenin-independent wnt signaling
Dev. Cell.
(2003) - et al.
Asymmetric localization of frizzled and the determination of notch-dependent cell fate in the Drosophila eye
Curr. Biol.
(2002) - et al.
Ca(2+)/calmodulin-dependent protein kinase II is stimulated by wnt and frizzled homologs and promotes ventral cell fates in Xenopus
J. Biol. Chem.
(2000)
Wnt11 signaling promotes proliferation, transformation, and migration of IEC6 intestinal epithelial cells
J. Biol. Chem.
Multiple mechanisms for Wnt11-mediated repression of the canonical wnt signaling pathway
J. Biol. Chem.
Opposing roles of WNT-5A and WNT-11 in interleukin-1beta regulation of type II collagen expression in articular chondrocytes
J. Biol. Chem.
Non-canonical wnt signaling enhances differentiation of human circulating progenitor cells to cardiomyogenic cells
J. Biol. Chem.
Wnt11-R, a protein closely related to mammalian Wnt11, is required for heart morphogenesis in Xenopus
Dev. Biol.
The c-jun kinase/stress-activated pathway: regulation, function and role in human disease
Biochim. Biophys. Acta
Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements
Curr. Biol.
The cAMP signalling pathway activates CREB through PKA, p38 and MSK1 in NIH 3T3 cells
Cell. Signal.
Physiological roles of MKK4 and MKK7: insights from animal models
Biochim. Biophys. Acta
Akt (protein kinase B) negatively regulates SEK1 by means of protein phosphorylation
J. Biol. Chem.
The PI3 kinase-akt pathway mediates Wnt3a-induced proliferation
Cell. Signal.
3-phosphoinositide-dependent protein kinase-1 (PDK1): Structural and functional homology with the Drosophila DSTPK61 kinase
Curr. Biol.
AKT/PKB signaling: navigating downstream
Cell
Elevated cyclic AMP inhibits NF-kappaB-mediated transcription in human monocytic cells and endothelial cells
J. Biol. Chem.
Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway
Dev. Cell.
Regulation of MAP kinases by MAP kinase phosphatases
Biochim. Biophys. Acta
Axin utilizes distinct regions for competitive MEKK1 and MEKK4 binding and JNK activation
J. Biol. Chem.
Differential requirement of MKK4 and MKK7 in JNK activation by distinct scaffold proteins
FEBS Lett.
Increased wnt levels in the neural tube impair the function of adherens junctions during neurulation
Mol. Cell. Neurosci.
Frizzled receptors activate a novel JNK-dependent pathway that may lead to apoptosis
Curr. Biol.
The making of wnt: new insights into wnt maturation, sorting and secretion
Development
Differential transformation of mammary epithelial cells by wnt genes
Mol. Cell. Biol.
Identification of distinct classes and functional domains of wnts through expression of wild-type and chimeric proteins in Xenopus embryos
Mol. Cell. Biol.
Cited by (29)
The many postures of noncanonical Wnt signaling in development and diseases
2017, Biomedicine and PharmacotherapyCitation Excerpt :In an early study, β-catenin has been identified as a negative regulator of NF-κB activity in bacteria-induced epithelial inflammation [119]. Wnt signaling, mediated by Wnt3a, Wnt5a, Wnt7a, Wnt10b and Wnt11, and NF-κB signaling are inter-connected in TNFα induced inflammatory responses in multiple cells and tissues, including but not limited to intestine epithelial cells [120], bronchial epithelial cells [121,122], osteosarcoma cells [123], nucleus pulposus cells [124], smooth muscle cells [125], mammary tissues [126], adipose tissues [127,128], osteoblasts [129], neurons [130], and myofibroblasts [131]. In cancer cells, Wnt/β-catenin signaling regulates cytokine-induced nitric oxide synthase expression by inhibiting NF-κB activation [132].
Wnt/β-catenin signalling in adrenal physiology and tumour development
2012, Molecular and Cellular EndocrinologyCitation Excerpt :Our retrospective analysis of Giordano’s micro-array data (Giordano et al., 2003) shows down-regulation of WNT11 in ACC versus normal adrenal (0.32-fold) and ACA (0.36-fold). Interestingly, WNT11 does not induce β-catenin stabilisation and is thought to inhibit canonical Wnt signalling through mechanisms involving Ca2 + and PKC activation (Maye et al., 2004; Railo et al., 2008). WNT11 expression is downregulated in oral squamous cell carcinoma (Andrade Filho et al., 2011) and in hepatocellular carcinoma (HCC) compared with adjacent uninvolved liver (Toyama et al., 2010).
Secreted Wnt antagonist Dickkopf-1 controls kidney papilla development coordinated by Wnt-7b signalling
2011, Developmental BiologyCitation Excerpt :A MALDI-TOF sequencing of this urine protein component was performed in the protein analysis core facility of Biocenter Oulu (http://www.biocenter.oulu.fi/) using routine methods. A Wnt reporter assay was done according to Railo et al. (2008). The following amounts of plasmids were used; 5 ng of Wnt3a or Wnt-7b expression plasmid, 250 ng of empty pcDNA3 plasmid that served as the carrier DNA, 100 ng of SuperTopFlash canonical Wnt pathway reporter and 5 ng of CMV-β-gal plasmid.
Wnt5a and Wnt11 as acute respiratory distress syndrome biomarkers for severe acute respiratory syndrome coronavirus 2 patients
2020, European Respiratory JournalLong noncoding RNA ABHD11-AS1 promote cells proliferation and invasion of colorectal cancer via regulating the miR-1254-WNT11 pathway
2019, Journal of Cellular Physiology