3D structure of DKK1 indicates its involvement in both canonical and non-canonical Wnt pathways

Molecular Biology - Dikkoppf-1 (DKK1) is an antagonist of the canonical Wnt signaling pathway. The importance of DKK1 as a diagnostic and therapeutic agent in a wide range of diseases along with...     

Apigenin supplementation protects the development of dextran sulfate sodium-induced murine experimental colitis by inhibiting canonical and non-canonical inflammasome signaling pathways

The present study was designed to elucidate the protective effects of dietary apigenin (API) enrichment in a chronic colitis model induced by DSS in mice. Inflammatory mediators and the possible role of canonical and non-canonical NLRP3 inflammasome signaling pathways in the beneficial effects of API under chronic inflammatory conditions were also explored. Six-week-old mice were randomized in four dietary groups: sham and control groups received standard diet (SD), and other two groups were fed with API at 0.1%. After 30 days, all groups except sham received 3% DSS in drinking water for 5 days followed by a regime of 21 days of water. Our results revealed that dietary API supplementation decreased the macroscopic and microscopic damage signs of colitis; also, it was capable to down-regulate mPGES, COX-2 and iNOS enzyme colonic expressions and to decrease serum matrix metalloproteinase (MMP-3) levels. Similarly, API diet reduced IL-1β and TNF-α proinflammatory cytokine secretions in primary LPS-stimulated splenocytes. Furthermore, we demonstrated that API anti-inflammatory activity was related with an inhibition of both canonical and non-canonical NLRP3 inflammasome pathways by decreasing proinflammatory IL-1β and IL-18 cytokine levels as a consequence of regulation of cleaved caspase-1 and caspase-11 enzymes. We conclude that API supplement might provide a basis for developing a new dietary strategy for the prevention of chronic ulcerative colitis.     

Zebrafish Naked1 and Naked2 antagonize both canonical and non-canonical Wnt signaling

Wnt signaling controls a wide range of developmental processes and its aberrant regulation can lead to disease. To better understand the regulation of this pathway, we identified zebrafish homologues of Naked Cuticle (Nkd), Nkd1 and Nkd2, which have previously been shown to inhibit canonical Wnt/beta-catenin signaling. Zebrafish nkd1 expression increases substantially after the mid-blastula transition in a pattern mirroring that of activated canonical Wnt/beta-catenin signaling, being expressed in both the ventrolateral blastoderm margin and also in the axial mesendoderm. In contrast, zebrafish nkd2 is maternally and ubiquitously expressed. Overexpression of Nkd1 or Nkd2a suppressed canonical Wnt/beta-catenin signaling at multiple stages of early zebrafish development and also exacerbated the cyclopia and axial mesendoderm convergence and extension (C&E) defect in the non-canonical Wnt/PCP mutant silberblick (slb/wnt11). Thus, Nkds are sufficient to antagonize both canonical and non-canonical Wnt signaling. Reducing Nkd function using antisense morpholino oligonucleotides resulted in increased expression of canonical Wnt/beta-catenin target genes. Finally, reducing Nkd1 function in slb mutants suppressed the axial mesendoderm C&E defect. These data indicate that zebrafish Nkd1 and Nkd2 function to limit both canonical and non-canonical Wnt signaling.     

Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation

Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote beta-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.     

c-Abl promotes osteoblast expansion by differentially regulating canonical and non-canonical BMP pathways and p16INK4a expression

Defects in stem cell renewal or progenitor cell expansion underlie ageing-related diseases such as osteoporosis. Yet much remains unclear about the mechanisms regulating progenitor expansion. Here we show that the tyrosine kinase c-Abl plays an important role in osteoprogenitor expansion. c-Abl interacts with and phosphorylates BMPRIA and the phosphorylation differentially influences the interaction of BMPRIA with BMPRII and the Tab1-Tak1 complex, leading to uneven activation of Smad1/5/8 and Erk1/2, the canonical and non-canonical BMP pathways that direct the expression of p16(INK4a). c-Abl deficiency shunts BMP signalling from Smad1/5/8 to Erk1/2, leading to p16(INK4a) upregulation and osteoblast senescence. Mouse genetic studies revealed that p16(INK4a) controls mesenchymal stem cell maintenance and osteoblast expansion and mediates the effects of c-Abl deficiency on osteoblast expansion and bone formation. These findings identify c-Abl as a regulator of BMP signalling pathways and uncover a role for c-Abl in p16(INK4a) expression and osteoprogenitor expansion.     

Activation of the canonical beta-catenin pathway by histamine

Histamine signaling is a principal regulator in a variety of pathophysiological processes including inflammation, gastric acid secretion, neurotransmission, and tumor growth. We report that histamine stimulation causes transactivation of a T cell factor/beta-catenin-responsive construct in HeLa cells and in the SW-480 colon cell line, whereas histamine did not effect transactivation of a construct containing the mutated response construct FOP. On the protein level, histamine treatment increases phosphorylation of glycogen synthase kinase 3-beta in HeLa cells, murine macrophages, and DLD-1, HT-29, and SW-480 colon cell lines. Furthermore, histamine also decreases the phosphorylated beta-catenin content in HeLa cells and murine macrophages. Finally, pharmacological inhibitors of the histamine H1 receptor counteracted histamine-induced T cell factor/beta-catenin-responsive construct transactivation and the dephosphorylation of beta-catenin in HeLa cells and in macrophages. We conclude that the canonical beta-catenin pathway acts downstream of the histamine receptor H1 in a variety of cell types. The observation that inflammatory molecules, like histamine, activate the beta-catenin pathway may provide a molecular explanation for a possible link between inflammation and cancer.     

Impaired estrogen sensitivity in bone by inhibiting both estrogen receptor alpha and beta pathways

Although it is well established that estrogen deficiency causes osteoporosis among the postmenopausal women, the involvement of estrogen receptor (ER) in its pathogenesis still remains uncertain. In the present study, we have generated rats harboring a dominant negative ERalpha, which inhibits the actions of not only ERalpha but also recently identified ERbeta. Contrary to our expectation, the bone mineral density (BMD) of the resulting transgenic female rats was maintained at the same level with that of the wild-type littermates when sham-operated. In addition, ovariectomy-induced bone loss was observed almost equally in both groups. Strikingly, however, the BMD of the transgenic female rats, after ovariectomized, remained decreased even if 17beta-estradiol (E(2)) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E(2). Moreover, bone histomorphometrical analysis of ovariectomized transgenic rats revealed that the higher rates of bone turnover still remained after treatment with E(2). These results demonstrate that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways and that the maintenance of BMD before ovariectomy might be compensated by other mechanisms distinct from ERalpha and ERbeta pathways.     

Analysis of canonical and non-canonical splice sites in mammalian genomes

A set of 43 337 splice junction pairs was extracted from mammalian GenBank annotated genes. Expressed sequence tag (EST) sequences support 22 489 of them. Of these, 98.71% contain canonical dinucleotides GT and AG for donor and acceptor sites, respectively; 0.56% hold non-canonical GC-AG splice site pairs; and the remaining 0.73% occurs in a lot of small groups (with a maximum size of 0.05%). Studying these groups we observe that many of them contain splicing dinucleotides shifted from the annotated splice junction by one position. After close examination of such cases we present a new classification consisting of only eight observed types of splice site pairs (out of 256 a priori possible combinations). EST alignments allow us to verify the exonic part of the splice sites, but many non-canonical cases may be due to intron sequencing errors. This idea is given substantial support when we compare the sequences of human genes having non-canonical splice sites deposited in GenBank by high throughput genome sequencing projects (HTG). A high proportion (156 out of 171) of the human non-canonical and EST-supported splice site sequences had a clear match in the human HTG. They can be classified after corrections as: 79 GC-AG pairs (of which one was an error that corrected to GC-AG), 61 errors that were corrected to GT-AG canonical pairs, six AT-AC pairs (of which two were errors that corrected to AT-AC), one case was produced from non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two cases left of supported non-canonical splice sites. If we assume that approximately the same situation is true for the whole set of annotated mammalian non-canonical splice sites, then the 99.24% of splice site pairs should be GT-AG, 0.69% GC-AG, 0.05% AT-AC and finally only 0.02% could consist of other types of non-canonical splice sites. We analyze several characteristics of EST-verified splice sites and build weight matrices for the major groups, which can be incorporated into gene prediction programs. We also present a set of EST-verified canonical splice sites larger by two orders of magnitude than the current one (22 199 entries versus approximately 600) and finally, a set of 290 EST-supported non-canonical splice sites. Both sets should be significant for future investigations of the splicing mechanism.     

SpliceDB: database of canonical and non-canonical mammalian splice sites

A database (SpliceDB) of known mammalian splice site sequences has been developed. We extracted 43 337 splice pairs from mammalian divisions of the gene-centered Infogene database, including sites from incomplete or alternatively spliced genes. Known EST sequences supported 22 815 of them. After discarding sequences with putative errors and ambiguous location of splice junctions the verified dataset includes 22 489 entries. Of these, 98.71% contain canonical GT-AG junctions (22 199 entries) and 0.56% have non-canonical GC-AG splice site pairs. The remainder (0.73%) occurs in a lot of small groups (with a maximum size of 0.05%). We especially studied non-canonical splice sites, which comprise 3.73% of GenBank annotated splice pairs. EST alignments allowed us to verify only the exonic part of splice sites. To check the conservative dinucleotides we compared sequences of human non-canonical splice sites with sequences from the high throughput genome sequencing project (HTG). Out of 171 human non-canonical and EST-supported splice pairs, 156 (91.23%) had a clear match in the human HTG. They can be classified after sequence analysis as: 79 GC-AG pairs (of which one was an error that corrected to GC-AG), 61 errors corrected to GT-AG canonical pairs, six AT-AC pairs (of which two were errors corrected to AT-AC), one case was produced from a non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two other cases left of supported non-canonical splice pairs. The information about verified splice site sequences for canonical and non-canonical sites is presented in SpliceDB with the supporting evidence. We also built weight matrices for the major splice groups, which can be incorporated into gene prediction programs. SpliceDB is available at the computational genomic Web server of the Sanger Centre: http://genomic.sanger.ac. uk/spldb/SpliceDB.html and at http://www.softberry. com/spldb/SpliceDB.html.     

Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability

The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system; however, their role in the mammalian vasculature remains ill defined. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165-induced migration, tube formation and permeability in vitro and VEGF-165-stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.     

Targeted degradation of beta-catenin by chimeric F-box fusion proteins

Adenomatous polyposis coli (APC) tumor suppressor protein, together with Axin and glycogen synthase kinase 3beta (GSK-3beta), forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of cytoplasmic beta-catenin by ubiquitin-dependent proteolysis. Degradation of phosphorylated beta-catenin is initiated by interaction through the WD40-repeat of a F-box protein beta-TrCP, a component of SCF ubiquitin ligase complex. Mutations in APC, Axin, and beta-catenin that prevent down-regulation of cytoplasmic beta-catenin are found in various types of cancers. In the search for efficient treatment and prevention of malignancies associated with increased levels of cytoplasmic beta-catenin, we created chimeric F-box fusion proteins by replacing the WD40-repeat of beta-TrCP with the beta-catenin-binding domains of Tcf4 and E-cadherin. Expression of chimeric F-box fusion proteins successfully promotes degradation of beta-catenin independently of GSK-3beta-mediated phosphorylation. More importantly, this degradation does not require intact APC protein (pAPC).     

The roles of canonical and non-canonical Wnt signaling in human de-differentiated articular chondrocytes

Osteoarthritis is the most prevalent form of arthritis in the world and it is becoming a major public health problem. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to de-differentiation. The involvement of signaling pathways, such as the Wnt pathway, during cartilage pathology has been reported. Wnt signaling regulates critical biological processes. Wnt signals are transduced through at least three intracellular signaling pathways including the canonical Wnt/β-catenin pathway, the Wnt/Ca2 + pathway and the Wnt/planar cell polarity pathway. We investigated the involvement of the Wnt canonical and non-canonical pathways in human articular chondrocyte de-differentiation in vitro. Human articular chondrocytes were cultured through four passages with no treatment, or with sFRP3 treatment, an inhibitor of Wnt pathways, or with DKK1 treatment, an inhibitor of the canonical pathway. Chondrocyte-secreted markers and Wnt pathway components were analyzed using western blotting and qPCR. Inhibition of the Wnt pathway showed that the canonical Wnt signaling probably is responsible for inhibition of collagen II expression, activation of metalloproteinase 13 expression and regulation of Wnt7a and c-jun expression during chondrocyte de-differentiation in vitro. Our results also suggest that expressions of eNOS, Wnt5a and cyclinE1 are regulated by non-canonical Wnt signaling.