Regulation of norrin receptor frizzled-4 by Wnt2 in colon-derived cells

Background  

Norrin is a potent Wnt pathway ligand. Aberrant activation of this signaling pathway can result in colon tumors but the role of norrin-based signaling in the genesis of colon cancer, and its relationship to activation of the pathway by traditional Wnt ligands, is not defined.     

A novel SULF1 splice variant inhibits Wnt signalling but enhances angiogenesis by opposing SULF1 activity

The importance of SULF1 in modulating the activities of multiple signalling molecules is now well established. Several studies, however, reported little or no effect of Sulf1 null mutations, questioning the relevance of this gene to in vivo development. The failure of SULF1 deletion to influence development may be predicted if one considers the involvement of a naturally occurring SULF1 antagonist, generated by alternative splicing of the same gene. We demonstrate that while the previously described SULF1 (SULF1A) enhances Wnt signalling, the novel shorter isoform (SULF1B) inhibits Wnt signalling. Our studies show developmental stage specific changes in the proportions of SULF1A and SULF1B isoforms at both the mRNA and protein levels in many developing tissues, with particularly pronounced changes in developing and adult blood vessels. Unlike SULF1A, SULF1B promotes angiogenesis and is highly expressed in endothelial cells during early blood vessel development while SULF1A predominates in mature endothelial cells. We propose that the balance of two naturally occurring SULF1 variants, with opposing functional activities, may regulate the overall net activities of multiple secreted factors and the associated signalling cascades essential for normal development and maintenance of most tissues.     

Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields approximately 3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.     

R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.     

Syndecan-4 regulates non-canonical Wnt signalling and is essential for convergent and extension movements in Xenopus embryos

Early shaping of Xenopus laevis embryos occurs through convergent and extension movements, a process that is driven by intercalation of polarized dorsal mesodermal cells and regulated by non-canonical Wnt signalling. Here, we have identified Xenopus syndecan-4 (xSyn4), a cell-surface transmembrane heparan sulphate proteoglycan. At the gastrula stage, xSyn4 is expressed in the involuting dorsal mesoderm and the anterior neuroectoderm. Later, it is found in the pronephros, branchial arches, brain and tailbud. Both gain- and loss-of-function of xSyn4 impaired convergent extension movements in Xenopus embryos and in activin-treated ectodermal explants. xSyn4 interacts functionally and biochemically with the Wnt receptor Frizzled7 (xFz7) and its signal transducer Dishevelled (xDsh). Furthermore, xSyn4 is necessary and sufficient for translocation of xDsh to the plasma membrane - a landmark in the activation of non-canonical Wnt signalling. Our results suggest that the ability of xSyn4 to translocate xDsh is regulated by fibronectin, a component of the extracellular matrix required for proper convergent extension movements. We propose a model where xSyn4 and fibronectin cooperate with xFz7 and Wnt in the specific activation of the non-canonical Wnt pathway.     

While K-ras is essential for mouse development, expression of the K-ras 4A splice variant is dispensable

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.     

The secreted protein Noggin4 is an activator of the Wnt/PCP-signaling pathway

The protein Noggin4 of the African clawed frog Xenopus laevis has been shown to act as a modulator of the “noncanonical” Wnt/PCP-signaling pathway that plays an important role in the regulation of cell motility. Induction of disturbances in the expression of Noggin4 led to the activation of Wnt/PCP-pathway and the related anomalies of early embryonic development. The Noggin4 protein can bind the Wnt11 protein that normally contributes to the activation of the Wnt/PCP-pathway and of enhancing the activator effect of this protein in luciferase assays. Thus, Noggin4 can be used as a tool for specific experimental regulation of the activity of the Wnt/PCP pathway.     

Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant

Several variants of the serotonin 5-HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5-HT4 gene. Based on sequence analysis seven C-terminal variants (a-g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C-terminal b variant; therefore, we call this receptor 5-HT4(hb). This novel receptor variant was expressed transiently in COS-7 cells, and its pharmacological profile was compared with those of the previously cloned 5-HT4(a) and 5-HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5-HT4 ligands, no significant differences were detected. However, the broadly used 5-HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5-HT4(a) and 5-HT4(b) variant but showed partial agonistic activity on the 5-HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5-HT4 ligands in different model systems.     

CKAP4/p63 is a receptor for the frizzled-8 protein-related antiproliferative factor from interstitial cystitis patients

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.     

A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus.

Anteroposterior (AP) patterning of the vertebrate neural plate is initiated during gastrulation and is regulated by Spemann's organizer and its derivatives. The prevailing model for AP patterning predicts a caudally increasing gradient of a 'transformer' which posteriorizes anteriorly specified neural cells. However, the molecular identity of the transforming gradient has remained elusive. We show that in Xenopus embryos (1) dose-dependent Wnt signalling is both necessary and sufficient for AP patterning of the neuraxis, (2) Wnt/beta-catenin signalling occurs in a direct and long-range fashion within the ectoderm, and (3) that there is an endogenous AP gradient of Wnt/beta-catenin signalling in the presumptive neural plate of the Xenopus gastrula. Our results indicate that an activity gradient of Wnt/beta-catenin signalling acts as transforming morphogen to pattern the Xenopus central nervous system.     

FZD4S, a splicing variant of frizzled-4, encodes a soluble-type positive regulator of the WNT signaling pathway

Frizzled-1 (FZD1)-FZD10 are seven-transmembrane-type WNT receptors, and SFRP1-SFRP5 are soluble-type WNT antagonists. These molecules are encoded by mutually distinct genes. We have previously isolated and characterized the 7.7-kb FZD4 mRNA, encoding a seven-transmembrane receptor with the extracellular cysteine-rich domain (CRD). Here, we have cloned and characterized FZD4S, a splicing variant of the FZD4 gene. FZD4S, corresponding to the 10.0-kb FZD4 mRNA, consisted of exon 1, intron 1, and exon 2 of the FZD4 gene. FZD4S encoded a soluble-type polypeptide with the N-terminal part of CRD, and was expressed in human fetal kidney. Injection of synthetic FZD4S mRNA into the ventral marginal zone of Xenopus embryos at the 4-cell stage did not induce axis duplication by itself, but augmented the axis duplication potential of coinjected Xwnt-8 mRNA. These results indicate that the FZD4 gene gives rise to soluble-type FZD4S as well as seven-transmembrane-type FZD4 due to alternative splicing, and strongly suggest that FZD4S plays a role as a positive regulator of the WNT signaling pathway.     

Modulation of calcium signalling by dominant negative splice variant of ryanodine receptor subtype 3 in native smooth muscle cells

The ryanodine receptor subtype 3 (RYR3) is expressed ubiquitously but its physiological function varies from cell to cell. Here, we investigated the role of a dominant negative RYR3 isoform in Ca2+ signalling in native smooth muscle cells. We used intranuclear injection of antisense oligonucleotides to specifically inhibit endogenous RYR3 isoform expression. In mouse duodenum myocytes expressing RYR2 subtype and both spliced and non-spliced RYR3 isoforms, RYR2 and non-spliced RYR3 were activated by caffeine whereas the spliced RYR3 was not. Only RYR2 was responsible for the Ca2+-induced Ca2+ release mechanism that amplified Ca2+ influx- or inositol 1,4,5-trisphosphate-induced Ca2+ signals. However, the spliced RYR3 negatively regulated RYR2 leading to the decrease of amplitude and upstroke velocity of Ca2+ signals. Immunostaining in injected cells showed that the spliced RYR3 was principally expressed near the plasma membrane whilst the non-spliced isoform was revealed around the nucleus. This study shows for the first time that the short isoform of RYR3 controls Ca2+ release through RYR2 in native smooth muscle cells.     

Isolation of Xenopus frizzled-10A and frizzled-10B genomic clones and their expression in adult tissues and embryos

Frizzled genes, encoding WNT receptors, play key roles in cell fate determination. Here, we isolated two Xenopus frizzled genes (Xfz10A and Xfz10B), probably reflecting pseudotetraploidy in Xenopus. Xfz10A (586 amino acids) and Xfz10B (580 amino acids) both encoded by a single exon, consisted of the N-terminal cysteine-rich domain, seven transmembrane domains, and the C-terminal Ser/Thr-X-Val motif. Xfz10A and Xfz10B were 97.0% identical at the amino acid level, and Xfz10B was 100% identical to previously reported Xfz9, yet Xfz10A was 85.3% and 62.4% identical to FZD10 and FZD9, respectively. Xfz10 mRNA appeared as 3.4 kb in adult tissues and embryos. RT-PCR analyses revealed the expression of more Xfz10A mRNA in stomach, kidney, eye, skeletal muscle, and skin, and more Xfz10B mRNA in heart and ovary, but in embryos, two mRNAs were equally expressed from the blastula stage with their peak expression at the late gastrula stage. The main site of Xfz10 mRNA expression was neural fold at the neurula stage and the dorsal region of midbrain, hindbrain, and spinal cord at the tadpole stage. These results suggest that Xfz10 has important roles in neural tissue formation.