Wnt-4 activates the canonical β-catenin-mediated Wnt pathway and binds Frizzled-6 CRD: functional implications of Wnt/β-catenin activity in kidney epithelial cells
Introduction
The mammalian kidney is a widely used model organ for studying inductive tissue interactions occurring during organ development. To facilitate the study of kidney organogenesis, some elements of this developmental process have been investigated in vitro. For example, Madin–Darby Canine Kidney (MDCK) cells form tubules with several aspects of normal in vivo polarity and physiology preserved [1], [2], [3], [4], [5], [6], [7], [8], spontaneously forming cysts when grown within three-dimensional collagen matrices and undergoing tubulogenesis in response to hepatocyte growth factor (HGF). Remarkably, induced MDCK tubule structures exhibit the appropriate disposition of cell polarity markers and respond normally to physiological stimuli such as glucocorticoids. MDCK cells have thus provided an in vitro standard for studies of cell polarity and tubulogenesis, with the expectation that this system reflects complex processes such as branching morphogenesis.
Kidney development requires diverse signaling pathways for formation of the transient pronephros and mesonephros to patterning of the final metanephric epithelial tubules [9], [10]. While ureteric bud-derived signals essential for initiating mesenchymal condensation are still being sought and evaluated, it is evident that downstream Wnt signals, likely operating in an autocrine fashion within the mesenchyme, are required to generate epithelial collecting tubules [10], [11], [12], [13], [14], [15], [16], [17]. Of the approximately 20 Wnt genes identified in mouse, five (Wnts-2b, -4, -6, -7b, and -11) are expressed during renal development, with only Wnt-4 having an essential role for the mesenchymal to epithelial transition and morphogenesis required in tubule formation [16], [18]. Importantly, tubule morphology is significantly rescued when explants of Wnt-4−/− kidney mesenchyme are exposed to exogenous Wnt-4 [19], again supporting the key role of Wnt-4 signaling in kidney tubulogenesis and development. In addition to kidney development, Wnt-4 was recently shown to be induced in the collecting ducts of normal adult mouse kidney following renal injury, likely revealing the recapitulation of developmental processes required in kidney repair [20], [21]. In kidney development or repair, the identity of a Frizzled receptor(s) that binds and transduces Wnt-4 signals is unknown. A number of mammalian expression studies have located Frizzled-4 and -6 transcripts in the developing kidney mesenchyme consistent with Wnt-4's temporal and spatial expression [22], [23], [24], [25], [26], [27]; however, kidneys are normal in the Frizzled-4 null mouse [28] while the Frizzled-6 null mouse is currently under investigation (Y. Wang and J. Nathans, J. Hopkins U., Baltimore, MD, personal communication).
Wnts are largely insoluble, secreted proteins thought to diffuse short distances (one or a few cell diameters) before binding transmembrane receptors of the Frizzled family and/or other cell surface proteins or matrix components [15], [27], [29], [30]. Although the cognate pairings of Wnt ligands with Frizzled receptors are unknown in most physiological contexts, the “canonical” intracellular signaling pathway activated upon Wnt–Frizzled interaction and mediated via β-catenin has been well studied and found to be highly conserved across evolution [14], [31], [32], [33]. Regarding the canonical pathway, Wnt–Frizzled interaction ultimately results in the stabilization of the downstream component β-catenin, facilitating its entry into the nucleus and association with transcription factors of the lymphoid enhancer factor (LEF) or T cell factor (TCF) high mobility group (HMG) domain family. Thereupon follows the activation (relief of repression) of largely unidentified gene targets crucial in multiple developmental capacities and cancer progression. Interestingly, β-catenin has an additional role at cell–cell junctions in association with cadherins and indirectly with the actin cytoskeleton [34], [35], [36], [37]. In some contexts, functionally distinct “noncanonical” Wnt–Frizzled pathways are activated, mediated via intracellular components including JNK kinases, calcium, protein kinase C, and small G-proteins [38], [39], [40], [41], [42], [43], [44], [45]. Although some reports have suggested that Wnt-4 mediates its kidney-specific effects via the canonical Wnt pathway [20], [21], [46], [47], [48], essential contributions of the less understood noncanonical Wnt pathways must be considered given their roles in the morphogenesis of other tissues [38], [41], [49], [50], [51], [52], [53], [54].
We report here that the kidney-derived MDCK cell line is responsive to Wnt-4 and two other canonical Wnts as assessed via the transcriptional activation of luciferase reporters sensitive to nuclear β-catenin levels, while considerably less activation is observed following MDCK exposure to Wnt-5A and control conditions. Further, using a PCR-based screening approach, we show that Frizzled-6 is the primary Frizzled receptor expressed in MDCK cells and that Frizzled-6 biochemically associates with Wnt-4. Although functional tests (MDCK cells and Xenopus axis duplication assays) indicate that Frizzled-6 is not likely mediating canonical Wnt-4 signaling, we speculate that Wnt-4/Frizzled-6 may contribute to the activation of one or more noncanonical Wnt pathways and presumably that MDCK cells express an additional unknown Frizzled receptor(s) responsible for transducing the observed canonical Wnt-4 signal. Last, we investigate the contribution of Wnt/β-catenin signaling to in vitro MDCK tubulogenesis and show that β-catenin acts as a survival factor in MDCK cells as it has in mammary gland development and several stem cell lineages [55], [56].
Section snippets
Transfections, luciferase reporter assays, and cell cycle analysis
Transfections were performed using FuGene 6 according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN). MDCK cells grown in D-MEM and 10% fetal bovine serum were seeded at 2 × 105 cells/well in 6-well tissue culture dishes 24 h before transfection. The TOPFLASH and FOPFLASH luciferase reporter constructs were kindly provided by O. Destree and H. Clevers (U. Utrecht) [57], while the Siamois luciferase reporter and control constucts were generous gifts from D.
Wnt-4 transactivates canonical Wnt pathway reporters in MDCK cells
To develop a simplified model of Wnt-4 signal transduction in kidney, we tested if MDCK cells are responsive to Wnt-4. We also tested the MDCK response to Wnts previously characterized as canonical (Wnt-1 and -3A) or as noncanonical (Wnt-5A) transducers. In the presence of stably expressed exogenous mouse Wnts (Wnt-4, -1, -5A), provided by cocultured NIH-3T3 cells [19] or by mouse L cell Wnt-3A conditioned media, we determined that Wnt-4, Wnt-1, and Wnt-3A activated an established
Discussion
The Wnt signaling pathway plays key roles in the embryogenesis of all animals and in cancer progression. During development, the pathway is employed in distinct temporal and spatial capacities; for example, in specifying and patterning the early dorsal axis and limbs, and in forming the brain, heart, and kidney. The canonical Wnt pathway transduces signals via the downstream mediator β-catenin, which enters the nucleus to bind HMG-box transcription factors LEF or TCF, activating largely unknown
Acknowledgements
We are grateful to the following individuals for providing reagents, advice, and/or results before publication: A.P. McMahon (Harvard U.), D. Kimelman (U. Washington), J. Nathans (John's Hopkins U.), Y. Wang (John's Hopkins U.), W.J. Nelson (Stanford U.), A.L. Pollock (U. Arizona), K.E. Mostov (UCSF), M.C. Hung (UT MDACC), S. Dayal (UT MDACC), S.W. Kim (UT MDACC), T.G. Vaught (UT MDACC), and K.M. Ramirez (UT MDACC). This work was supported in part by National Institutes of Health (NIH) RO1
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These authors contributed equally.