PMC:4925210 / 3728-19051 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4925210","sourcedb":"PMC","sourceid":"4925210","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4925210","text":"Results\n\nWNT ligands can bind to the extracellular domain of PKD1\nThe cystic phenotype of Wnt9b-kidney null mice, along with the presence of several CRDs in PKD1 prompted us to test whether WNTs could physically interact with PKD1. Indeed, WNT9B co-immunoprecipitated with PKD1 but not with the structurally similar PKD1L1 or TRPC1 (Fig. 1b). Next, we tested whether WNT9B could associate with various fragments of the extracellular domain unique to PKD1 in the extracellular space. The CRD of FZD8 was used as a reference point, since its affinity to WNT9B is known (KD=482±16 nM24). WNT9B associated with the LRR-WSC-Fc and LDL-A-Fc (Fig. 1c). LRR-WSC-Fc also associated with WNT9B in the extracellular space in trans, whereby cells were singly transfected with Fc constructs or WNT9B, co-cultured for 24 h, and protein-protein interactions were detected in the cultured media (Fig. 1d). PKD1 interacted with WNT9B through a site that included the C-terminal CRD of the LRR and the WSC domain (LRR-CT+WSC-Fc, Fig. 1e). To test whether PKD1 and WNT9B interacted directly, we purified LRR-WSC-Fc from conditioned media of stably transfected HEK293 cells and incubated it with purified WNT9B. 87 ng (or 2.35 pmol) of purified WNT9B was pulled-down with 390 ng (or 5.2 pmol) of purified PKD1-Fc fusion (Fig.1f-h). Under the same conditions, full-length TRPP2-Myc isolated from total cell lysates did not interact with purified WNT9B (Supplementary Fig. 1g).\nWNT5A, WNT4, or WNT3A also bound to LRR-WSC (Supplementary Fig. 1). WNT5A and WNT4 bound also to the LDL-A domain of PKD1 (Supplementary Fig. 1b-e). The affinity of WNT3A to soluble FZD8-CRD is 3.6±1.2 nM24. Thus, using the FZD8-CRD/WNT9B and FZD8-CRD/WNT3A interactions as “affinity rulers”, we deduced that the PKD1/WNT9B or PKD1/WNT3A interactions were of comparable affinity to that of FZD8/WNT3A. Therefore, PKD1 can interact with WNTs that function through β-catenin (WNT3A), independently of β-catenin (WNT5A), or both (WNT9B, WNT4), with an estimated affinity in the low nanomolar range.\n\nWNT9B activates heterologously expressed PKD1/TRPP2 complex\nNext, we tested whether CHO-K1 cells co-transfected with PKD1 and TRPP2 could support an increase in [Ca2+]i in response to WNT9B. CHO-K1 cells do not express endogenous PKD1 or TRPP219. Perfusion of untransfected cells with purified WNT9B did not result in an increase in [Ca2+]i (Fig. 2a). In contrast, WNT9B induced an increase in [Ca2+]i, only in the presence of extracellular Ca2+ in cells co-transfected with PKD1 and TRPP2 (Fig. 2b,c). These data indicated that PKD1 and TRPP2 mediated WNT9B-induced Ca2+ influx rather than Ca2+ release from intracellular stores.\nWNT9B-induced whole cell currents were determined in 50 nM intracellular Ca2+ using the whole cell configuration of the patch clamp technique. Addition of WNT9B (500 ng/ml) induced large currents in cells co-transfected with wild type PKD1 and TRPP2 (Fig. 2d,e). These currents were ~50-100-fold larger than PKD1/TRPP2 currents reported earlier19, 25, 26. Cells co-transfected with the pathogenic PKD1S99I 27 and TRPP2 failed to respond to WNT9B (Fig. 2f,i,l), although PKD1S99I was expressed at comparable levels to wild type PKD1 (Supplementary Figs. 2a and 3a). Cell surface biotinylation revealed that S99I severely compromised surface expression of PKD1S99I (Supplementary Fig. 2b). However, cell surface expression of TRPP2 was unaffected (Supplementary Fig. 2c). Consistently, cell surface expression of TRPP2 was unchanged in wild type and Pkd1-null mouse embryonic fibroblasts (MEFs) (Supplementary Fig. 2d). These data suggested that cell surface expression of PKD1 is required for WNT9B-induced currents.\nBecause at 50 nM of intracellular Ca2+ other Ca2+-activated channels could contribute to whole cell currents, we buffered intracellular Ca2+ with 10 mM EGTA to eliminate contribution of these endogenous channels. However, while the contribution of endogenous channels is suppressed in zero intracellular Ca2+, the activity of TRPP2 is also suppressed by 80-90%28. As expected, in zero intracellular Ca2+, WNT9B-induced PKD1/TRPP2-dependent currents were ~10-fold smaller than currents in 50 nM of intracellular Ca2+ and showed strong outward rectification, which is typical for members of the Polycystin family of ion channels21, 29, 30 (Fig. 3b,h). The reversal potential was near −20 mV, consistent with the much higher K+ permeability of TRPP2 compared to Na+ or Ca2+ 31, 32. Co-expression with PKD1S99I did not result in WNT9B-induced currents (Fig. 3c,h), as shown earlier (Fig. 2f). Co-transfection of PKD1 and the TRPP2 pathogenic mutant, TRPP2D511V, suppressed the WNT9B response by ~75% compared to wild type TRPP2 (Fig. 3d,h). Cells co-transfected with PKD1 and pathogenic TRPP2R872X that does not interact with PKD1 failed to respond (Fig. 3e,h), indicating that the PKD1/TRPP2 interaction is required for the WNT9B response. Replacement of the pore-forming region of TRPP2 with the equivalent region of the K+-permeable Kv1.3 (TRPP2Kv1.3) suppressed inward currents but not outward currents carried by K+ (Fig. 3f,h). The chimeric TRPP2Kv1.3 channel was expressed in comparable levels to wild type TRPP2 and maintained its interactions with wild type TRPP2 or PKD1 (Supplementary Fig. 3c,d). These data suggested that TRPP2 directly contributed to the formation of WNT9B-induced currents and the interaction with PKD1 was required for the effect. PKD1L1 physically interacts with TRPP233, 34. However, PKD1L1 did not interact with WNT9B (Fig. 1b). Consistently, it did not form a WNT9B-inducible channel complex in CHO-K1 cells (Fig. 3g,h). Overall, results from these experiments support the hypothesis that the PKD1/TRPP2 complex was activated by WNT9B, likely through a direct ligand-receptor interaction between WNT9B and PKD1. To test whether activation of the PKD1/TRPP2 channel involves activation of endogenous FZDs, we co-expressed ZNRF3 and tested for an effect on WNT9B-induced currents. ZNRF3 functions as an E3 ubiquitin ligase clearing FZDs from the cell surface35. ZNRF3 robustly suppressed WNT3A-induced activation of the WNT/β-canonical pathway which requires FZDs (Supplementary Fig. 3b), but had no effect on WNT9B-induced currents (Fig. 3h). These data suggested that PKD1/TRPP2 functioned independently of FZDs in CHO-K1 cells.\nTo further strengthen the evidence of FZD independent activation of TRPP2, we co-transfected PKD1 and TRPP2 into Drosophila S2 cells, which lack FZDs36. First, we showed that purified WNT9B bound to the cell surface of S2 cells transiently transfected with PKD1 and TRPP2 (Supplementary Fig. 4a-b). The pattern of cell surface-bound WNT9B was “spotty” suggesting that PKD1/TRPP2 channels are not uniformly distributed at the cell surface, as has been shown for Drosophila Fzd236. Next, we showed that WNT9B (500 ng/ml) induced whole cell currents only in transfected cells (Supplementary Fig. 4c), providing additional evidence for the WNT-induced activation of PKD1/TRPP2 independently of FZDs.\n\nTRPP2 mediates WNT-induced whole cell currents in MEFs\nWild type MEFs express PKD111 and TRPP2 (Fig. 6a and b and Supplementary Fig. 2d) and deletion of Pkd2 is expected to cause an upregulation of the WNT/β-catenin pathway and constitutive activation of p38-MAPK37. Consistently, phospho-β-catenin levels were slightly decreased, whereas phospho-p38MAPK were slightly increased in Pkd2-null MEFs (Supplementary Fig. 5b,c). WNT3A induced activation of the canonical WNT/β-catenin pathway in Pkd2 mutant cells and this increase was 2-3-fold higher compared to wild type cells (Supplementary Fig. 5g). Overexpression of ZNRF3 suppressed this effect (Supplementary Fig. 5h). Expression levels of Fzd1/2/6/7/8, and Ryk mRNAs or LRP6 and ROR2 proteins (LRP5 and ROR1 are not expressed in MEFs) were not different between wild type and Pkd2-null MEFs (Supplementary Fig. 5a,e,f). Expression level of DVL1 or DVL2 was similar between Pkd2+/+ and Pkd2−/− cells, but slightly reduced in Pkd1−/− cells (Supplementary Fig. 5d). In sum, Pkd2-null cells showed higher activation of the canonical WNT/β-catenin pathway compared to wild type cells and no obvious differences in the expression levels of FZDs, ROR2, LRP6, and RYK receptors.\nWhen intracellular Ca2+ was clamped at 50 nM, WNT9B induced large currents in wild type (Fig. 4a, black filled squares, c, red filled squares), but not Pkd2-null cells (Fig. 4a and c, blue open circles). WNT9B-induced currents showed a reversal potential at 0 mV and were reversibly blocked by La3+, as was seen in CHO-K1 cells transfected with PKD1 and TRPP2 (Fig. 2e,h). In zero intracellular Ca2+, WNT9B-induced whole cell currents showed outward rectification and a reversal potential close to −20 mV (Fig. 4d-f), as was seen in CHO-K1 cells transfected with PKD1 and TRPP2 under the same conditions (Fig. 3b). WNT9B did not induce a significant current in Pkd2−/− cells (Fig. 4d and f, blue open circles). Therefore, these experiments demonstrated that WNT9B was able to induce a similar current in native MEFs or CHO-K1 cells transfected only with PKD1/TRPP2, but not in Pkd2-null MEFs. The differential effect of WNT9B in wild type and Pkd2-null cells was specific, since both cell types responded similarly to extracellular ATP (Supplementary Fig. 6a-f). Adding-back wild type TRPP2 in mutant cells restored WNT9B-induced currents (Fig. 4g-i), whereas adding-back TRPP2Kv1.3 restored only outward currents, but not inward currents (Fig. 4j-l), indicating that TRPP2 was directly involved in WNT9B-induced currents. Overexpression of ZNRF3 did not have an effect on WNT9B-induced currents (Fig. 4m-o) indicating that WNT9B-induced TRPP2-mediated currents were independent of FZDs and/or LRP6, as shown in CHO-K1 and Drosophila S2 cells.\nBinding experiments showed that WNT3A was able to interact with the LRR-WSC domain of PKD1 (Supplementary Fig. 1). However, canonical WNT3A was not expected to induce Ca2+ signaling. At zero intracellular Ca2+, WNT3A induced whole cell currents in wild type cells (Fig. 5a-c), but with a noticeable delay (by about a min) compared to WNT9B-induced currents (Fig. 4a,g). Expression of ZNRF3 did not have significant effects on current amplitude, whereas WNT3A failed to induce whole cell currents in Pkd2-null cells (Fig. 5g-i).\n\nPKD1 interacts with DVL1 and DVL2, but DVLs do not affect WNT9B-induced PKD1/TRPP2-mediated currents\nNext, we tested whether PKD1 could physically interact DVL1 and DVL2 (DVL3 is not present in these cells determined by RT-PCR). We found that DVL1 and DVL2 co-immunoprecipitated with PKD1 in wild type or Pkd2-null MEFs (Fig. 6a and b). Deletion analysis showed that DVL2 interacted with PKD1 and the interaction was abrogated by the E499G mutation in DVL238. This mutation is located within the Dishevelled, Egl-10 and Plecstrin (DEP) domain (Fig. 6c), known to mediate the effects of DVL2 on PCP39, 40. It also impairs phosphorylation of DVL238. Nevertheless, depletion of DVL2 or both DVL1 and DVL2 using RNAi (Fig. 6e) did not show a significant effect on WNT9B-induced whole cell currents (Fig. 6d), suggesting that although DVL1/2 physically interact with PKD1, they are not required for the formation of a functional PKD1/TRPP2 channel complex.\n\nWNT9B-induced cell migration is dependent on TRPP2\nWNT5A was shown to promote directional cell migration in a variety of cell types through a process involving both localized Ca2+ release transients originating from the cortical endoplasmic reticulum and Ca2+ influx7. Both types of Ca2+ signaling were required for directional cell migration through the formation of the WNT5A-mediated receptor-actin-myosin polarity structure in the trailing edge of a migrating cell7. Thus, we tested whether WNT9B could induce Ca2+ release. WNT9B did not induce Ca2+ release in wild type or Pkd2-null MEFs, in contrast to ATP which elicited a similar response (Supplementary Fig. 6g,h). Next, we tested whether WNT9B could promote directional cell migration in wild type and Pkd2-null MEFs, through TRPP2 and an increase in intracellular Ca2+ concentration. Wild type or Pkd2-null cells were stimulated with WNT9B for up to 2 h and the number of cells with a typical migratory phenotype (Fig. 7a) was determined in both cultures by double staining for F-actin and Myosin IIB. WNT9B failed to induce this migratory phenotype in Pkd2-null cells, whereas it had a strong effect on wild type cells (Fig. 7a,b). To test whether WNT9B-induced cell polarization was dependent on intracellular Ca2+, Pkd2+/+ MEFs were pretreated with DMSO or 10 μM BAPTA/AM for 45 min before adding WNT9B. BAPTA/AM blocked WNT9B-induced cell polarization (Fig. 7c) to a degree similar to Pkd2-null cells (Fig. 7b). To test whether Pkd2-null MEFs failed to polarize because of TRPP2, we added-back wild type TRPP2 or TRPP2Kv1.3. Wild type, but not TRPP2Kv1.3 rescued cell polarization in Pkd2−/− MEFs (Fig. 7d,e). Consistently, Pkd2-null cells failed to migrate towards a WNT9B gradient using a chemokinetic assay (Fig. 7f,g). In contrast to WNT9B, Pkd2−/− fibroblasts migrated normally in response to PDGF-BB (25 ng/ml) (Fig. 7h), a strong chemokinetic factor acting through Ca2+ flickers41, store-, and/or receptor-activated Ca2+ channels42, indicating that WNT9B-activated PKD1/TRPP2 mediated currents were distinct from the channels activated by PDGF-BB. Overall, these experiments showed that cells lacking TRPP2 exhibited reduced directional migration, suggesting that one of the functions of WNT9B-induced activated TRPP2 in cell culture is to establish polarization during directional cell migration.\n\nPKD1 cooperates with DVL2 to maintain normal kidney tubulogenesis in Xenopus embryos\nNext, we utilized Xenopus as a model organism43, 44 to test whether PKD1 could function within the WNT pathway. We chose DVL2 to experimentally downregulate WNT signaling, because DVL2 is the major isoform expressed in the Xenopus pronephric kidney45. Knockdown of PKD1 using two independent splicing morpholinos (sMOs), one described in Xu et al46 (Pkd1-sMO1) and another one (Pkd1-sMO2) described here (Supplementary Fig. 7), resulted in edema and dysplastic (dilated and shortened) pronephric tubules (Fig. 8i,l). Knockdown of DVL2 resulted in a cystic kidney phenotype grossly similar to PKD1 knockdown (Fig. 8i,l). Importantly, this phenotype could be reversed by co-injection of human DVL2 mRNA, but not DVL2-E499G mRNA, which does not interact with PKD1 (Fig. 8m). Next, we combined a suboptimal dose of the Pkd1-sMO1, which by itself did not cause any cystic phenotype, with decreasing amounts of Dvl2-MO (Fig. 8a-i,l). The combined knockdown resulted in a synergistic effect compared to the individual MOs, suggesting a functional interaction between PKD1 and DVL2. Finally, injection of an MO targeting WNT9A (Wnt9A-MO), one of the WNT ligands specifically expressed in the pronephric kidney43, on its own did not cause edema formation or dysplastic kidneys (Fig. 8j). However, combining Wnt9-MO with a suboptimal dose of Pkd1-sMO1 demonstrated synergism (Fig. 8j-l) similar to the one observed for Dvl2-MO and Pkd1-sMO1. Together these data support the hypothesis that WNT9A, PKD1, and DVL2 function in the same pathway in pronephric 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