![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 19, Issue 6, 2588-2596, June 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Research Department, Nuvelo, San Carlos, CA 94070
Submitted February 21, 2008;
Revised March 24, 2008;
Accepted April 2, 2008
Monitoring Editor: Marianne Bronner-Fraser
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
60% sequence homology and similar domain organization (Kim et al., 2006
). All RSpo proteins contain two furin-like cysteine-rich domains at the N-terminus followed by a thrombospondin domain and a basic charged C-terminal tail. Numerous studies have implicated RSpo proteins in the canonical Wnt signaling pathway. We have previously demonstrated that all four RSpo members induce proliferation of crypt epithelial cells resulting in enlargement of mouse gastrointestinal tract (Kim et al., 2005, 2006). Moreover, RSpo proteins can stabilize the levels of cytosolic β-catenin and dramatically synergize with Wnt3A, suggesting a potential role for RSpo proteins in modulating the Wnt signaling pathway (Kazanskaya et al., 2004; Binnerts et al., 2007; Wei et al., 2007).
The Wnt signaling pathway plays a pivotal role in diverse biological processes during embryonic development, adult homeostasis and disease pathogenesis (Logan and Nusse, 2004; Moon et al., 2004; Nusse, 2005). Wnt ligands are a family of secreted proteins that regulate the β-catenin turnover through interactions with Frizzled receptors and LRP5/6 co-receptors (Davidson et al., 2005; He et al., 2004; Tamai et al., 2004; Zeng et al., 2005). In the absence of Wnt ligand, cytosolic β-catenin is targeted for proteosomal degradation by the APC/Axin/GSK destruction complex (Behrens et al., 1998; Salic et al., 2000). On engagement with the receptors, Wnt ligands trigger a series of events resulting in the stabilization and subsequent translocation of cytosolic β-catenin into the nucleus and transactivation of Wnt target genes through interaction with the transcription factor T-cell factor (TCF; Bejsovec, 2005; Gordon and Nusse, 2006).
Expression of RSpo proteins overlaps with expression of Wnt proteins during mouse development implying a possible link of RSpo proteins with the Wnt signaling pathway (Parr et al., 1993; Echelard et al., 1994; Yoshida et al., 1997; Nam et al., 2006b). Consistent with this, RSpo1 expression was detected in the developing dorsal neural tube and was reduced in Wnt1 and Wnt 3A double knockout mice (Kamata et al., 2004). Likewise, in Xenopus, mRNA expression of RSpo proteins overlapped with expression of Wnt ligands during embryonic development (Kazanskaya et al., 2004). Recently, an important role of RSpo1 in development of the reproductive system was described in humans with a loss of function mutation of RSpo1 (Parma et al., 2006). Similar to phenotypes observed in Wnt-4 knockout mice, a homozygous null mutation of RSpo1 in members of a family with a recessive syndrome was characterized by XX sex reversal. These findings indicate a key role for RSpo1 in Wnt signaling during the development of reproductive organs.
Although RSpo proteins are linked to the Wnt pathway, the precise mechanism by which each member regulates the Wnt pathway is poorly understood. Previously, it was shown that RSpo proteins interact with FZD8 and LRP6 receptors (Nam et al., 2006a; Wei et al., 2007). However, these studies did not demonstrate whether these interactions are required for the activation of the Wnt signaling pathway by RSpo proteins. Recently, we have demonstrated that RSpo1 activity is dependent on the presence of Wnt ligands and that RSpo1 regulates the Wnt signaling by antagonizing Dickkopf (DKK)1-mediated LRP6 internalization (Binnerts et al., 2007). To further explore and compare the mechanism of action of different RSPo proteins, we generated and tested purified recombinant proteins of all RSpo family members. Here we show that all four members regulate the Wnt pathway by a common mechanism. In addition, we provide data that indicates that the furin domain of RSpo protein is sufficient to synergize with Wnt3A and antagonize DKK1 function.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell Culture and Transfection
HEK293 cells were purchased from ATCC (Manassas, VA) and maintained in DMEM containing 10% fetal bovine serum. To generate a HEK293 clone that stably expresses a 16TCF luciferase reporter construct, cells were transfected with a pTA16TCF-Luc construct using Fugene-6 (Roche, Indianapolis, IN) transfection reagent, and stable clones were selected using a culture medium containing 1 mg/ml geneticin (Invitrogen). A HEK 293 stable clone expressing mLRP6-FLAG was generated in a similar way using a pcDNA3.1/Intron mLRP6-FLAG expression construct.
Plasmids and DNA Constructs
A 16TCF luciferase reporter construct was generated as described (Binnerts et al., 2007). Open reading frame (ORF) nucleotide (nt) sequences for hKremen2 (BF312414, ATCC, Manassas, VA) and mLRP6 (BC060704
[GenBank]
, Open Biosystems, Huntsville, AL) were amplified from indicated cDNA clones and inserted into the pcDNA3.1/Intron plasmid. mLRP6
C was generated by deleting the LRP6 cytoplasmic domain as described (Binnerts et al., 2007). LRP6-FLAG was generated by in-frame fusion of the LRP6 ORF sequence with a FLAG epitope tag (DYKDDDDK) and Kremen2-HA was generated with a HA epitope tag (YPYDVPDYA) encoding sequence. The expression constructs of RSpo chimeric molecules in which the furin domain of RSpo1 is replaced by furin domains of RSpo2, 3, or 4 were generated by a stitching PCR method using following primers: a universal forward primer, GTC GTC GAC ACG TGT GAT CAG ATA; RSpo2 reverse primer, TTC ACA TCC TTC CAC ACA TTC CAT; RSpo3 reverse primer, CTC ACA GTG CAC AAT ACT GAC ACA; RSpo4 reverse primer, TTC ACA CTC CCC CTG GCACT, a forward primer for RSpo1 C-terminal with thrombospondin domain, ATG AGC GAG TGG TCT CCG T; a reverse primer for RSpo1 C-terminal with thrombospondin domain, TGG ACA AAC CAC AAC TAG AAT GCA G. PCR amplified furin domains of RSpo2, 3, or 4 were ligated with the thrombospondin domain containing the C-terminal half of the RSpo1 molecule followed by PCR amplification of chimeric molecules using a forward primer, GTC GTC GAC ACG TGT GAT CAG ATA, and a reverse primer, TGG ACA AAC CAC AAC TAG AAT GCA G. The PCR-amplified products of three chimeric molecules were confirmed by sequencing and digested with EcoRI/XbaI to clone into the pcDNA/Intron vector.
Luciferase Reporter Assays Small Interfering RNA Experiment
A 16TCF luciferase reporter construct was generated by cloning 16 repeats of a TCF consensus site (AGATCAAAGG) into the pTA-Luc vector (Clontech, Mountain View, CA) as reported (Binnerts et al., 2007). A stable HEK293 clone, A6 was selected using a geneticin selection marker, and the selected clone demonstrated minimal basal reporter activity. Stable HEK293 cells were seeded in 96-well plates in DMEM containing 1% FBS (Invitrogen) the day before treatment. The stable cells were treated in triplicate for 20 h with indicated concentrations of proteins, and reporter activity was determined using BrightGlo reagent (Promega, Madison, WI) by a Veritas Luminometer (Turner Biosystems, Sunnyvale, CA). Recombinant hDKK1. mWnt3A proteins were purchased from R&D systems (Minneapolis, MN), and recombinant RSpo1, 2, 3, and 4 were purified from conditioned media derived from stable HEK293 cells by standard column chromatography and filtration. Wntless (WLS) gene-specific and nontargeting control small interfering RNA (siRNA) duplexes were purchased from Dharmacon (Chicago, IL); WLS: GCGUCAGUCCAAGUGAA (Banziger et al., 2006). HEK-293 A6 reporter cells were transfected in triplicate in 96-well plates with indicated siRNA duplexes by reverse transfection with Neofect (Ambion, Austin, TX) according to manufacturer's instructions. Cells were treated as indicated 48 h after siRNA transfection and analyzed for reporter activity as described above.
Western Blotting and Coimmunoprecipitation
β-Catenin stabilization was performed as previously described. In brief, the cell lysate was prepared from HEK 293 cells using hypotonic lysis buffer. The cytosolic fraction was prepared by ultracentrifugation of the cell lysates (100,000 x g) for 30 min, resolved by SDS-PAGE, and transferred to nitrocellulose membrane. Immunoblotting was performed using anti- β-catenin antibodies (Cell Signaling). β-Actin was used as a protein-loading control. To measure LRP6 phosphorylation either HEK 293 cells or stable 293 cells expressing FLAG-tagged LRP6 were seeded in six-well dishes and treated with indicated proteins for 4 h. Cells were placed on ice, rinsed with ice-cold PBS, and lysed in cell lysis buffer containing phosphatase inhibitors (Cell Signaling). Lysates were resolved on 3–8% SDS-PAGE gels and LRP6 phosphorylation was analyzed using a phospho-Ser1490–specific anti-LRP6 polyclonal antibody (Cell Signaling). To determine the interaction of Kremen and LRP6, HEK293 cells, stably expressing LRP6-C-FLAG, were transiently transfected with a Kremen2-C-HA expression construct using Fugen-6 transfection reagent (Roche). The total cell lysates were prepared after indicated treatments, and the immunoprecipitation was performed by incubating the cell lysates with anti-HA antibodies (Abcam, Cambridge, MA) overnight at 4°C. The immunoprecipitated molecules were precipitation using protein G Dynabeads (Invitrogen). After stringent washing with lysis buffer, the protein complex was eluted from the beads by boiling in sample buffer with DTT (Pierce) and resolved in 3–8% Tris-acetate gels (Invitrogen) for immunoblotting with anti-FLAG, anti-hemagglutinin (HA; Abcam), or anti-DKK1 (R&D Systems) antibodies.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 2006; Binnerts et al., 2007). Although genetic studies with the various RSpo proteins indicated a wide range of phenotypes, it is unclear whether all RSpo proteins activate the Wnt pathway through a common mechanism. To further understand the biochemical and cellular activities of all RSpo family proteins, we generated recombinant proteins of four RSpo members (Figure 1A) and compared the TCF reporter activity between RSpo1-4 (Figure 1B). As we previously demonstrated, RSpo1 induced TCF reporter activity in a dose-dependent manner. Interestingly, RSpo2 and RSpo3 were more potent than RSpo1, particularly at a lower concentration (<100 ng/ml), whereas RSpo4 was relatively inactive. The relative activities of RSpo family proteins in Wnt/β-catenin signaling were further confirmed by measuring the accumulation of cytosolic β-catenin (Figure 1C). Consistent with the activities observed in the TCF-mediated reporter assay, RSpo2 and 3 induced stabilization of cytosolic β-catenin in HEK293 cells at lower concentrations than RSpo1, whereas RSpo4 had a minimal effect on cytosolic β-catenin levels.
|
; Kim et al., 2005; Binnerts et al., 2007; Wei et al., 2007). To further examine the amplification of Wnt3A activity by RSpo proteins, a HEK293 cell line, stably expressing a TCF reporter gene, was treated with increasing amounts of Wnt3A proteins in the presence of a suboptimal concentration of purified RSpo1-4 (20 ng/ml). Although treating cells with either varying amounts of Wnt3A or RSpo1-4 alone (at 20 ng/ml) was not sufficient to trigger a robust response, a substantial increase of Wnt3A-induced TCF-mediated transcriptional activation (up to 260-fold enhancement) was detected when cells were treated in combination with each of four members of RSpo proteins (Figure 2A). Interestingly, although cells treated with RSpo4 alone were virtually nonresponsive, cotreatment with Wnt3A significantly increased TCF reporter activity (150-fold). As observed in cells treated with RSpo alone (Figure 1), RSpo2 and RSpo3 were slightly more potent than RSpo1 in amplifying the Wnt signaling. Furthermore, RSpo-mediated enhancement of TCF reporter activity was not limited to Wnt3A. A suboptimal dose of RSpo1 and RSpo2 (50 ng/ml) also substantially amplified Wnt1 or Wnt7A activities (Figure 2B), suggesting that RSpo proteins can modulate general canonical Wnt signaling.
|
|
C) reduced the activity of all four RSpo members (Figure 4A). These data imply that all RSpo proteins regulate the Wnt signaling pathway at the level of LRP6.
|
To further explore the link between LRP6 and RSpo proteins, we next examined the effect of LRP6 inhibitor DKK1 on RSpo activity. Previously, we demonstrated that RSpo1 can antagonize DKK1 activity to regulate the cell surface levels of LRP6 (Binnerts et al., 2007). Similar to the observation seen with RSpo1, DKK1 inhibited activity of RSpo2–4 proteins (Figure 4D) but to a lesser extent compared to the inhibition of Wnt3A protein activity. These data further confirm the implication of LRP6 in RSpo-mediated Wnt signaling.
RSpo Proteins Antagonize DKK-1–induced Interaction of Kremen and LRP6
It was previously shown that a direct interaction between DKK1 and LRP6/Kremen is required for the regulation of cell surface levels of LRP6 (Semenov et al., 2001; Mao et al., 2002). DKK1 interacts with Kremen1 or 2 and subsequently triggers the internalization of LRP6 (Mao et al., 2007). We have previously demonstrated that RSpo1 can block DKK1 function and can interact with Kremen expressing cells (Binnerts et al., 2007). We therefore investigated whether all RSpo1-4 can also antagonize DKK1 mediated LRP6 and Kremen interaction. We analyzed the formation of a Kremen2 and LRP6 ternary complex in cells treated either with DKK1 alone or in combination with RSpo proteins. As shown in Figure 5, although LRP6 was coimmunoprecipitated with Kremen2 in cells treated with DKK1, cotreatment with RSpo proteins reduced DKK-1–mediated Kremen2 and LRP6 complex formation. Interaction of Kremen and LRP6 was not altered in cells treated with RSpo proteins alone. Moreover, all RSpo proteins interfered to a similar extent with the association of DKK1 with the Kremen/LRP6 complex. These results suggest that all RSpo proteins can interfere with DKK1 function by disrupting Kremen and LRP6 interaction and subsequent internalization of the receptor complex. Together, these data suggest a common role for RSpo proteins in regulating the Wnt pathway by antagonizing DKK1 activity in LRP6-mediated signaling.
|
60% sequence homology among family members (Figure 6A). The furin-like domains were shown to be sufficient and indispensable for RSpo proteins activity (Kazanskaya et al., 2004). To further map the minimal domain required for RSpo activity, we next generated series of RSpo deletion mutants and tested their activities in the TCF reporter assay. As previously shown (Kazanskaya et al., 2004), two intact furin domains were required and sufficient to activate Wnt signaling (data not shown). Consistent with the TCF reporter activities of full-length proteins, the furin domains of RSpo2 and 3 proteins were more active than RSpo1, whereas RSpo4 was the least active (Figure 6B). Although the furin domain is required and sufficient for the RSpo activity, it retained only 50% of the activity seen with full-length RSpo protein (Figure 6C), suggesting the possible regulatory activity of other domains. Like RSpo wild-type (wt) proteins, the RSpo1 furin domain also amplified Wnt3A activity indicating that the ability of RSpo proteins to amplify Wnt signaling resides in the furin domain (Figure 6D).
|
Furin Domains Are Essential and Sufficient for Inhibition of DKK1 Activity
To address whether the furin domain can also interfere with DKK1 activity, purified RSpo1 furin domain was tested in the TCF reporter assay in the presence of increasing concentrations of DKK1. DKK1 inhibited the TCF reporter activity of purified RSpo1 furin domain in a dose-dependent manner (Figure 7A). However, similar to the activity of full-length RSpo1 protein, the DKK1-mediated inhibition was reversed (20–40%) by increasing concentrations of RSpo1 furin domain (at higher doses, >500 ng/ml; Figure 7B, middle and bottom), indicating that the furin domain is responsible for the ability of RSpo proteins to interfere with DKK1 function. In contrast, the DKK1-mediated inhibition of Wnt3A activity was not reversed by increasing amounts of Wnt3A protein (up to 1000 ng/ml; Figure 7B, top).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). However, expression of the four RSpo genes during mouse development demonstrated striking differences (Nam et al., 2006b), suggesting unique biological activities of individual members during development. Interestingly, however, when injected into adult mice, all purified recombinant RSpo proteins led to a similar dramatic increase of crypt depth and epithelial cell proliferation in the gastrointestinal tract (Kim et al., 2005, 2006), suggesting a common mechanism of action for all RSpo proteins.
Previous studies have suggested a possible implication of the Wnt coreceptor LRP6 in RSpo1-mediated activation of β-catenin signaling (Binnerts et al., 2007; Wei et al., 2007). Consistent with our recent study with RSpo1 (Binnerts et al., 2007), we demonstrated that LRP6 is required for signaling by all RSpo proteins; first, overexpression of full-length LRP6 enhanced TCF activation by all four RSpo proteins, whereas a C-terminal truncated, dominant negative LRP6 (LRP6C) reduced their activity. Second, although RSpo proteins marginally increased LRP6 phosphorylation, they induced a substantial increase of phosphorylation when combined with Wnt3A. These results further suggest that all RSpo proteins regulate Wnt signaling at the level of the LRP6 receptor.
LRP6 is tightly regulated by the soluble inhibitor DKK1 (Mao et al., 2001, 2002; Semenov et al., 2001). DKK1 is a member of a multigene family and is shown to bind to and antagonize LRP5/6. Kremen is a transmembrane protein and a coreceptor for DKK1, which functionally cooperates with DKK1 to induce endocytosis of LRP6 and down-regulate LRP6 from the cell surface. Recently, we have shown that RSpo1 inhibits DKK1-mediated LRP6 internalization and DKK1-dependent association of LRP6 and Kremen1 (Binnerts et al., 2007). In the present study, we compared the activities of all four RSpo family members and demonstrated that all RSpo proteins can antagonize DKK1-dependent inhibition of the canonical Wnt signaling and disrupt DKK1-dependent association of LRP6 and Kremen.
Our data indicate that RSpo proteins modulate the Wnt pathway through a common biochemical mechanism by competing with DKK1 and as shown previously to reduce internalization of LRP6 and subsequently increasing the levels of LRP6 on the cell surface (Binnerts et al., 2007).
Although our data strongly suggest that RSpo proteins amplify the Wnt pathway and antagonize DKK1 function, it is currently unclear whether these two activities are causally linked each other. Therefore, to map the domain responsible for amplification of Wnt signaling and DKK1 inhibition, we analyzed several RSpo deletion mutants. Previously, the functional domain of RSpo proteins required for Wnt activation was mapped to a furin-like cysteine-rich domain (Kazanskaya et al., 2004). Interestingly, the furin domains derived from all four RSpo members were sufficient for induction of TCF reporter activity and their relative activity was consistent with the activity seen with full-length protein. This observation was further validated by analyzing chimeric RSpo proteins in which the furin domain of RSpo1 was replaced with the furin domain of RSpo2–4, which demonstrated that the furin domains dictate the relative potency of RSpo proteins. Moreover, the furin domains were also shown to be essential and sufficient for RSpo1-mediated competition with DKK1 activity and inhibition of DKK1-induced complex formation between LRP6 and Kremen. Although these data suggest that both Wnt amplification and DKK1 interference are mediated by the same functional furin domain, it will remain to be elucidated whether Wnt amplification is a direct consequence of inhibition of DKK1-mediated LRP6 internalization. It is tempting to speculate that RSpo proteins play a pivotal role by uncoupling Kremen from LRP6 and subsequently permitting a favorable interaction with Wnt and the Frizzled receptor complex.
It is conceivable that the in vivo biological activity of RSpo proteins is dictated by expression of Wnt ligands in target tissues. Consistent with this notion, recent findings showed that loss of function mutation of RSpo1 (Parma et al., 2006), 2 (Bell et al., 2003), 3 (Aoki et al., 2006), and 4 (Blaydon et al., 2006) or knockout of RSpo2 gene (Nam et al., 2007) resulted in similar phenotypes as seen for Wnt or Fzd receptor mutations (Johnson and Tabin, 1997; Vainio et al., 1999; Ishikawa et al., 2001). For example, disruption of the RSpo1 gene was shown to cause a recessive XX sex reversal in an affected patient population, which, similar to the phenotype observed in wnt4+ mice, suggests the cooperative interaction of Wnt and RSpo proteins.
Our current data, in conjunction with previous reports, demonstrate a novel mechanism of action for all four RSpo proteins. We have shown that all RSpo proteins activate Wnt signaling through LRP6 by antagonizing DKK1 function. It is most likely that the specific activities of RSpo proteins reported in vivo are achieved by tissue-specific coexpression of different Wnt ligands, as seen during development and in various mouse (Nam et al., 2007) and human genetic studies (Bell et al., 2003; Aoki et al., 2006; Blaydon et al., 2006; Parma et al., 2006). It remains to be elucidated how RSpo proteins trigger Wnt signaling in a given specific microenvironment in which multiple Wnt signaling components contribute to the differential Wnt signaling activity. RSpo proteins may play a pivotal role in setting the threshold of Wnt activation by antagonizing DKK1 and amplifying signaling by Wnt ligands. Furthermore, as a key modulator of the Wnt pathway, RSpo proteins may prove to be valuable therapeutic agents in various disease conditions where Wnt signaling activity is required for tissue repair such as inflammatory bowel disease (Bouma and Strober, 2003; Radtke and Clevers, 2005; Lim and Hanauer, 2004) and in bone diseases characterized by elevated DKK1 levels, such as bone osteolytic lesions (Tian et al., 2003; Colla et al., 2007; Diarra et al., 2007; Yaccoby et al., 2007).
|
Footnotes |
|---|
Address correspondence to: Arie Abo (aabo{at}nuvelo.com).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Banziger, C., Soldini, D., Schutt, C., Zipperlen, P., Hausmann, G., and Basler, K. (2006). Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125, 509–522.[CrossRef][Medline]
Behrens, J., Jerchow, B. A., Wurtele, M., Grimm, J., Asbrand, C., Wirtz, R., Kuhl, M., Wedlich, D., and Birchmeier, W. (1998). Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science 280, (5363), 596–599.
Bejsovec, A. (2005). Wnt pathway activation: new relations and locations. Cell 120, 11–14.[Medline]
Bell, S. M., Schreiner, C. M., Hess, K. A., Anderson, K. P., and Scott, W. J. (2003). Asymmetric limb malformations in a new transgene insertional mutant, footless. Mech. Dev 120, 597–605.[CrossRef][Medline]
Binnerts, M. et al. (2007). R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6. Proc. Natl. Acad. Sci. USA 104, (37), 14700–14705.
Blaydon, D. C. et al. (2006). The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nat. Genet 38, 1245–1247.[CrossRef][Medline]
Bouma, G, and Strober, W. (2003). The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol 3, 521–533.[CrossRef][Medline]
Colla, S., Zhan, F., Xiong, W, Wu, X., Xu, H., Stephens, O., Yaccoby, S., Epstein, J., Barlogie, B., and Shaughnessy, J. D. (2007). The oxidative stress response regulates DKK1 expression through the JNK signaling cascade in multiple myeloma plasma cells. Blood 109, 4470–4477.
Davidson, G., Wu, W., Shen, J., Bilic, J., Fenger, U., Stannek, P., Glinka, A., and Niehrs, C. (2005). Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438, 867–872.[CrossRef][Medline]
Diarra, D. et al. (2007). Dickkopf-1 is a master regulator of joint remodeling. Nat. Med 13, 156–163.[CrossRef][Medline]
Echelard, Y., Vassileva, G., and McMahon, A. P. (1994). Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS. Development 120, 2213–2224.[Abstract]
Gordon, M. D., and Nusse, R. (2006). Wnt signaling: multiple pathways, multiple receptors and multiple transcription factors. J. Biol. Chem 281, 22429–22433.
He, X., Semenov, M., Tamai, K., and Zeng, X. (2004). LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development 131, 1663–1677.
Ishikawa, T., Tamai, Y., Zorn, A. M., Yoshida, H., Seldin, M. F., Nishikawa, S., and Taketo, M. M. (2001). Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis. Development 128, 25–33.[Abstract]
Johnson, R. L., and Tabin, C. J. (1997). Molecular models for vertebrate review limb development. Cell 90, 979–990.[CrossRef][Medline]
Kamata, T., Katsube, K., Michikawa, M., Yamada, M., Takada, S., and Mizusawa, H. (2004). R-spondin, a novel gene with thrombospondin type 1 domain, was expressed in the dorsal neural tube and affected in Wnts mutants. Biochim. Biophys. Acta 1676, 51–62.[Medline]
Kazanskaya, O., Glinka, A., del, B.B.I., Stannek, P., Niehrs, C., and Wu, W. (2004). R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis. Dev. Cell 7, 525–534.[CrossRef][Medline]
Kim, K. A., Zhao, J., Andarmani, S., Kakitani, M., Oshima, T., Binnerts, M. E., Abo, A., Tomizuka, K., and Funk, W. D. (2006). R-Spondin proteins: a novel link to beta-catenin activation. Cell Cycle 5, 23–26.[Medline]
Kim, K. A. et al. (2005). Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256–1259.
Lim, W. C., and Hanauer, S. B. (2004). Emerging biologic therapies in inflammatory bowel disease. Rev. Gastroenterol. Disord 4, 66–85.[Medline]
Logan, C. Y., and Nusse, R. (2004). The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol 20, 781–810.[CrossRef][Medline]
Mao, B. et al. (2002). Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 417, 664–667.[CrossRef][Medline]
Mao, B., Wu, W., Li, Y., Hoppe, D., Stannek, P., Glinka, A., and Niehrs, C. (2001). LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411, 321–325.[CrossRef][Medline]
Moon, R. T., Kohn, A. D., De Ferrari, G. V., and Kaykas, A. (2004). WNT and beta-catenin signalling: diseases and therapies. Nat. Rev. Genet 5, 691–701.[CrossRef][Medline]
Nam, J. S., Turcotte, T. J., Smith, P. F., Choi, S., and Yoon, J. K. (2006a). Mouse Cristin/R-spondin family proteins are novel ligands for the frizzled 8 and LRP6 receptors and activate β-catenin-dependent gene expression. J. Biol. Chem 281, 13247–13257.
Nam, J. S., Turcotte, T. J., and Yoon, J. K. (2006b). Dynamic expression of R-spondin family genes in mouse development Gene Expr. Patterns 7, 306–312.
Nam, J. S., Park, E., Turcotte, T. J, Palencia, S., Zhan, X., Lee, J., Yun, K., Funk, W. D., and Yoon, J. K. (2007). Mouse R-spondin2 is required for apical ectodermal ridge maintenance in the hind limb. Dev. Biol 311, 124–135.[CrossRef][Medline]
Nusse, R. (2005). Wnt signaling in disease and in development. Cell Res 15, (1), 28–32.[CrossRef][Medline]
Parma, P., Radi, O., Vidal, V., Chaboissier, M. C., Dellambra, E., Valentini, S., Guerra, L., Schedl, A., and Camerino, G. (2006). R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat Genet 38, 1233–1244.[CrossRef][Medline]
Parr, B. A., Shea, M. J., Vassileva, G., and McMahon, A. P. (1993). Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds. Development 119, 247–261.[Abstract]
Radtke, F., and Clevers, H. (2005). Self-renewal and cancer of the gut: two sides of a coin. Science 307, 1904–1909.
Salic, A., Lee, E., Mayer, L., and Kirschner, M. W. (2000). Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts. Mol Cell 5, (3), 523–532.[CrossRef][Medline]
Semenov, M. V., Tamai, K., Brott, B. K., Kuhl, M., Sokol, S., and He, X. (2001). Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr. Biol 11, 951–961.[CrossRef][Medline]
Tamai, K., Zeng, X., Liu, C., Zhang, X., Harada, Y., Chang, Z., and He, X. (2004). A mechanism for Wnt coreceptor activation. Mol. Cell 13, 149–156.[CrossRef][Medline]
Tian, E., Zhan, F., Walker, R., Rasmussen, E., Ma, Y., Barlogie, B., and Shaughnessy, J. D. (2003). The role of the Wnt-signaling antagonist DKK1 in the development of osteolytic lesions in multiple myeloma. N. Engl. J. Med 349, 2483–2494.
Vainio, S., Heikkila, M., Kispert, A., Chin, N., and McMahon, A. P. (1999). Female development in mammals is regulated by Wnt-4 signalling. Nature 397, 405–409.[CrossRef][Medline]
Wei, Q., Yokota, C., Semenov, M. V., Doble, B., Woodgett, J., and He, X. (2007). R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and β-catenin signaling. J. Biol. Chem 282, 15903–15911.
Yaccoby, S., Ling, W., Zhan, F., Walker, R., Barlogie, B., and Shaughnessy, J. D. (2007). Antibody-based inhibition of DKK1 suppresses tumor-induced bone resorption and multiple myeloma growth in vivo. Blood 109, 2106–2111.
Yoshida, M., Suda, Y., Matsuo, I., Miyamoto, N., Takeda, N., Kuratani, S., and Aizawa, S. (1997). Emx1 and Emx2 functions in development of dorsal telencephalon. Development 124, 101–111.[Abstract]
Zeng, X., Tamai, K., Doble, B., Li, S., Huang, H., Habas, R., Okamura, H., Woodgett, J., and He, X. (2005). A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438, 873–877.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  O. Kazanskaya, B. Ohkawara, M. Heroult, W. Wu, N. Maltry, H. G. Augustin, and C. Niehrs The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development Development, November 15, 2008; 135(22): 3655 - 3664. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
