Regulator of G Protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning

Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Gα subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling

Wnt expression is not correlated with β-catenin dysregulation in Dupuytren's Disease

BACKGROUND: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in finger flexion contractures. Increased cellular β-catenin levels have been identified as characteristic of this disease. As Wnts are the most widely recognized upstream regulators of cellular β-catenin accumulation, we have examined Wnt gene expression in surgical specimens and in DD-derived primary cell cultures grown in two-dimensional monolayer culture or in three-dimensional FPCL collagen lattice cultures. RESULTS: The Wnt expression profile of patient-matched DD and unaffected control palmar fascia tissue was determined by a variety of complimentary methods; Affymetrix Microarray analysis, specific Wnt and degenerative primer-based Reverse Transcriptase (RT)-PCR, and Real Time PCR. Microarray analysis identified 13 Wnts associated with DD and control tissues. Degenerate Wnt RT-PCR analysis identified Wnts 10b and 11, and to a lesser extent 5a and 9a, as the major Wnt family members expressed in our patient samples. Competitive RT-PCR analysis identified significant differences between the levels of expression of Wnts 9a, 10b and 11 in tissue samples and in primary cell cultures grown as monolayer or in FPCL, where the mRNA levels in tissue > FPCL cultures > monolayer cultures. Real Time PCR data confirmed the down-regulation of Wnt 11 mRNA in DD while Wnt 10b, the most frequently isolated Wnt in DD and control palmar fascia, displayed widely variable expression between the methods of analysis. CONCLUSION: These data indicate that changes in Wnt expression per se are unlikely to be the cause of the observed dysregulation of β-catenin expression in DD

Dkk1 Stabilizes Wnt Co-Receptor LRP6: Implication for Wnt Ligand-Induced LRP6 Down-Regulation

The low density lipoprotein receptor-related protein-6 (LRP6) is an essential co-receptor for canonical Wnt signaling. Dickkopf 1 (Dkk1), a major secreted Wnt signaling antagonist, binds to LRP6 with high affinity and prevents the Frizzled-Wnt-LRP6 complex formation in response to Wnts. Previous studies have demonstrated that Dkk1 promotes LRP6 internalization and degradation when it forms a ternary complex with the cell surface receptor Kremen.In the present study, we found that transfected Dkk1 induces LRP6 accumulation while inhibiting Wnt/LRP6 signaling. Treatment with Dkk1-conditioned medium or recombinant Dkk1 protein stabilized LRP6 with a prolonged half-life and induces LRP6 accumulation both at the cell surface and in endosomes. We also demonstrated that Kremen2 co-expression abrogated the effect of Dkk1 on LRP6 accumulation, indicating that the effect of Kremen2 is dominant over Dkk1 regulation of LRP6. Furthermore, we found that Wnt3A treatment induces LRP6 down-regulation, an effect paralleled with a Wnt/LRP6 signaling decay, and that Dkk1 treatment blocked Wnt3A-induced LRP6 down-regulation. Finally, we found that LRP6 turnover was blocked by an inhibitor of caveolae-mediated endocytosis.Our results reveal a novel role for Dkk1 in preventing Wnt ligand-induced LRP6 down-regulation and contribute significantly to our understanding of Dkk1 function in Wnt/LRP6 signaling

A Novel Role for MAPKAPK2 in Morphogenesis during Zebrafish Development

One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp), which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses

Signaling of Human Frizzled Receptors to the Mating Pathway in Yeast

Frizzled receptors have seven membrane-spanning helices and are considered as atypical G protein-coupled receptors (GPCRs). The mating response of the yeast Saccharomyces cerevisiae is mediated by a GPCR signaling system and this model organism has been used extensively in the past to study mammalian GPCR function. We show here that human Frizzled receptors (Fz1 and Fz2) can be properly targeted to the yeast plasma membrane, and that they stimulate the yeast mating pathway in the absence of added Wnt ligands, as evidenced by cell cycle arrest in G1 and reporter gene expression dependent on the mating pathway-activated FUS1 gene. Introducing intracellular portions of Frizzled receptors into the Ste2p backbone resulted in the generation of constitutively active receptor chimeras that retained mating factor responsiveness. Introducing intracellular portions of Ste2p into the Frizzled receptor backbone was found to strongly enhance mating pathway activation as compared to the native Frizzleds, likely by facilitating interaction with the yeast Gα protein Gpa1p. Furthermore, we show reversibility of the highly penetrant G1-phase arrests exerted by the receptor chimeras by deletion of the mating pathway effector FAR1. Our data demonstrate that Frizzled receptors can functionally replace mating factor receptors in yeast and offer an experimental system to study modulators of Frizzled receptors

A Model of Ischemia-Induced Neuroblast Activation in the Adult Subventricular Zone

We have developed a rat brain organotypic culture model, in which tissue slices contain cortex-subventricular zone-striatum regions, to model neuroblast activity in response to in vitro ischemia. Neuroblast activation has been described in terms of two main parameters, proliferation and migration from the subventricular zone into the injured cortex. We observed distinct phases of neuroblast activation as is known to occur after in vivo ischemia. Thus, immediately after oxygen/glucose deprivation (6–24 hours), neuroblasts reduce their proliferative and migratory activity, whereas, at longer time points after the insult (2 to 5 days), they start to proliferate and migrate into the damaged cortex. Antagonism of ionotropic receptors for extracellular ATP during and after the insult unmasks an early activation of neuroblasts in the subventricular zone, which responded with a rapid and intense migration of neuroblasts into the damaged cortex (within 24 hours). The process is further enhanced by elevating the production of the chemoattractant SDf-1α and may also be boosted by blocking the activation of microglia. This organotypic model which we have developed is an excellent in vitro system to study neurogenesis after ischemia and other neurodegenerative diseases. Its application has revealed a SOS response to oxygen/glucose deprivation, which is inhibited by unfavorable conditions due to the ischemic environment. Finally, experimental quantifications have allowed us to elaborate a mathematical model to describe neuroblast activation and to develop a computer simulation which should have promising applications for the screening of drug candidates for novel therapies of ischemia-related pathologies

Integration of the β-Catenin-Dependent Wnt Pathway with Integrin Signaling through the Adaptor Molecule Grb2

THE COMPLEXITY OF WNT SIGNALING LIKELY STEMS FROM TWO SOURCES: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified.Here we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of beta-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA) LRP6, Dvl2 or CA-beta-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN) Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of beta-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal Adhesion Kinase (FAK), and this is blocked by DN-Grb2.These data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling

Secreted Frizzled-related protein-1 is a negative regulator of androgen receptor activity in prostate cancer

Secreted Frizzled-related protein-1 (sFRP1) associates with Wnt proteins and its loss can lead to activation of Wnt/β-catenin signalling. It is frequently downregulated in cancer, including prostate cancer, but its function in prostate cancer is unclear because it can increase proliferation of prostate epithelial cells. We investigated the function of sFRP1 in androgen-dependent prostate cancer and found that sFRP1 inhibited androgen receptor (AR) transcriptional activity. In addition, sFRP1 inhibited the proliferation of androgen-dependent LNCaP cells but not of an androgen-independent subline LNCaP-r, suggesting a role in androgen-dependent growth. The inhibition of AR by sFRP1 was unaffected by co-expression of Wnt3a, stabilised β-catenin or β-catenin shRNA, suggesting it does not involve Wnt/β-catenin signalling. Wnt5a also inhibited AR and expression of Wnt5a and sFRP1 together did not further inhibit AR, suggesting that Wnt5a and sFRP1 activate the same signal(s) to inhibit AR. However, sFRP1 inhibition of AR was unaffected by inhibitors of kinases involved in Wnt/Ca2+ and Wnt/planar cell polarity non-canonical Wnt signalling. Interestingly, the cysteine-rich domain of sFRP1 interacted with Frizzled receptors expressed in prostate cancer cells, suggesting that sFRP1/Frizzled complexes activate a signal that leads to repression of AR. Taken together, these observations highlight the function of β-catenin-independent Wnt signalling in the control of AR activity and provide one explanation for sFRP1 downregulation in prostate cancer

The Wnt-dependent signaling pathways as target in oncology drug discovery

Our current understanding of the Wnt-dependent signaling pathways is mainly based on studies performed in a number of model organisms including, Xenopus, Drosophila melanogaster, Caenorhabditis elegans and mammals. These studies clearly indicate that the Wnt-dependent signaling pathways are conserved through evolution and control many events during embryonic development. Wnt pathways have been shown to regulate cell proliferation, morphology, motility as well as cell fate. The increasing interest of the scientific community, over the last decade, in the Wnt-dependent signaling pathways is supported by the documented importance of these pathways in a broad range of physiological conditions and disease states. For instance, it has been shown that inappropriate regulation and activation of these pathways is associated with several pathological disorders including cancer, retinopathy, tetra-amelia and bone and cartilage disease such as arthritis. In addition, several components of the Wnt-dependent signaling pathways appear to play important roles in diseases such as Alzheimer’s disease, schizophrenia, bipolar disorder and in the emerging field of stem cell research. In this review, we wish to present a focused overview of the function of the Wnt-dependent signaling pathways and their role in oncogenesis and cancer development. We also want to provide information on a selection of potential drug targets within these pathways for oncology drug discovery, and summarize current data on approaches, including the development of small-molecule inhibitors, that have shown relevant effects on the Wnt-dependent signaling pathways