Inhibition of the Wnt signaling pathway by Idax, a novel Dvl-binding protein.

In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of beta-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax in Xenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not beta-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.

Induction of a mirror-image duplication of anterior wing structures by localized hedgehog expression in the anterior compartment of Drosophila melanogaster wing imaginal discs.

The segment polarity gene hedgehog (hh) encodes a secretory protein involved in cell-cell communication in Drosophila melanogaster. The hh gene is expressed in the posterior compartment and is essential for the establishment and maintenance of the anterior/posterior-compartment boundary of each embryonic parasegment [Ingham, P.W., Nature 366 (1993) 560-562]. To clarify possible hh functions in adult appendage formation, we isolated a fly line (h9D) associated with a wing malformation from among fly lines with an hh transgene whose expression is under the control of trapped enhancers. In h9D flies, the ectopic expression of hh occurred in the anterior edge of wing pouch in the wing disc. This abnormal hh expression resulted in not only a mirror-image duplication and ectopic outgrowth in the anterior wing compartment, but also the ectopic expression of patched and decapentaplegic, strongly suggesting that the hh product serves as a morphogen or an inducer essential for wing development, including the proximal/distal axis formation.

Structure and expression of hedgehog, a Drosophila segment-polarity gene required for cell-cell communication.

The complete nucleotide sequence of the coding region of hedgehog (hh), a segment-polarity gene in Drosophila melanogaster, was determined. The gene was found to include three exons which would encode a 421- (or 471-) amino acid (aa) polypeptide with a long hydrophobic stretch. The hh mRNA was about 2.3 kb long and expressed throughout development. The hh expression in an embryo occurred in stripes, while that in imaginal discs occurred in the posterior compartment. As a whole, the spatial expression pattern of hh mRNA was very similar to that of engrailed (en), a homeobox gene required for the formation of the anterior-posterior compartment boundary. Unlike en, no hh expression was observed in the central nervous system.

Postmortem serum erythropoietin level as a marker of survival time in injury deaths.

Circulating erythropoietin (EPO) is mainly derived from the kidneys, and the serum concentration is rapidly increased in response to anemia and hypoxia. The present study investigated postmortem serum EPO levels in injury death cases (n=185, postmortem time<48 h, survival time <7 days: sharp instrument injury, n=44 and blunt injury, n=141) with regard to survival time, compared with C-reactive protein (CRP) as a marker of inflammation. Serum levels of both markers were independent of postmortem time. A survival time-dependent increase in serum EPO up to about 100 mU/ml was seen within 6h of sharp instrument injury to the heart or a proximal major vessel (thoracic aorta or subclavian/carotid artery) and blunt injury with massive hemorrhages, showing high correlations (r=0.957 and r=0.822, respectively, P<0.0001), whereas the increase was insignificant (P>0.05) for sharp instrument injury to a peripheral vessel or lungs/abdominal viscera and blunt injury with minor hemorrhages over the same survival period. A further increase (>100 mU/ml) was often detected in cases of death about 24h after blunt injury, irrespective of the type of injury. In contrast, a gradual increase in serum CRP level was seen about 12-24h after blunt injury. These findings suggest that serum EPO can be a marker for investigating survival time within 6h of major injury involving acute massive hemorrhaging.

Decreased expression of CXXC4 promotes a malignant phenotype in renal cell carcinoma by activating Wnt signaling.

The Wnt signaling pathway is involved in normal embryonic development and controls the homeostatic self-renewal of stem cells in adult tissues. Constitutive activation of Wnt signaling contributes to cancer development and progression. We identified a CXXC4 homozygous deletion at 4q24 in an aggressive renal cell carcinoma (RCC) using single-nucleotide polymorphism (SNP) arrays. CXXC4 encodes Idax, which negatively regulates Wnt signaling by binding to the PDZ domain of Dishevelled. CXXC4 mRNA levels in tumor samples were significantly lower in patients with metastases compared with those without (P=0.0016). Patients whose tumors had lower CXXC4 expression than normal kidney showed a poorer cause-specific survival outcome than those with higher expression (P=0.0095). Decreased expression of CXXC4 also correlated with cytoplasmic staining of beta-catenin. Knockdown of CXXC4 induced the nuclear translocation of beta-catenin and altered expression of a set of genes involved in cell proliferation, invasion and survival. Furthermore, reduced expression of CXXC4 by small interfering RNAs promoted cell proliferation and inhibited apoptosis after 5-FU and doxorubicin treatment in RCC cells. These data suggest that CXXC4 plays a critical role in tumor progression of RCC through Wnt signaling. Wnt signaling could thus be a potential molecular target in RCC indicating decreased CXXC4 expression.

Postmortem serum erythropoietin levels in establishing the cause of death and survival time at medicolegal autopsy.

Circulating erythropoietin (EPO) is mainly produced in the kidneys, depending on blood oxygen level. The present study investigated the postmortem serum EPO levels with regard to the cause of death and survival time. Serial medicolegal autopsy cases of postmortem time within 48 h (n = 536) were examined. Serum EPO levels were within the clinical reference range in most cases. Uremic patients with medical administration of an EPO agent (n = 11) showed a markedly high level (140-4,850 mU/ml; median, 1,798 mU/ml). Otherwise, an elevation in serum EPO level (>30 mU/ml) was mainly seen in protracted deaths due to blunt injury and fire fatality, depending on the survival time (r = 0.69, p < 0.0001, and r = 0.45, p < 0.0001, respectively), and in subacute deaths from gastrointestinal bleeding and infectious diseases. However, mildly to moderately elevated serum EPO levels were sporadically found in acute deaths due to mechanical asphyxiation, fire fatality, and acute ischemic heart disease, and in fatal hypothermia cases, especially for elderly subjects. Protracted deaths due to mechanical asphyxiation and ischemic heart disease did not show any survival time-dependent increase in serum EPO level (p > 0.05). EPO was immunohistochemically detected in the tubular epithelia and interstitial cells, showing no evident difference among the causes of death, independent of survival time or serum level. These findings suggest that serum EPO can be used as a marker for investigating anemia and/or hypoxia as a consequence of fatal insult in subacute or prolonged deaths, or a predisposition to traumatic deaths or fatal heart attacks in acute deaths.

Mechanism of induction of Bar-like eye malformation by transient overexpression of Bar homeobox genes in Drosophila melanogaster.

The Bar locus of Drosophila is known to be a small complex consisting of two similar homeobox genes, BarH1 and BarH2. Using egr as an ommatidium marker, possible mechanisms of formation of malformed eyes were examined. As in the case of BarH1, overexpression of BarH2 was found to be capable of inducing Bar-like eye malformation. It was suggested that suppression of the anterior progression of the morphogenetic furrow and inhibition of reinitiation of normal ommatidial differentiation were mandatory to formation of the reduced eye morphology in Bar mutants.

Subtype determination of Drosophila embryonic external sensory organs by redundant homeo box genes BarH1 and BarH2.

BarH1 and BarH2 are two closely related homeo box genes that form a small complex at the Bar locus on the X chromosome of Drosophila. By immunostaining, we showed that BarH1 and BarH2 proteins are coexpressed in cells belonging to the central and peripheral nervous systems in embryos. In external sensory (es) organs, their expression was particularly apparent in thecogens (glial cells) and neurons at late development. Although deletion of BarH2 caused no appreciable morphological change in es organs, the simultaneous deletion of BarH1 and BarH2 led to a homeotic change in these organs with consequent conversion from campaniform-like sensilla to trichoid sensilla. In contrast, the overexpression of either BarH1 or BarH2 resulted in opposite morphological change. It would thus follow that BarH1 and BarH2 are a pair of redundant homeo box genes required for the subtype specification of es organs.

Bar homeobox genes are latitudinal prepattern genes in the developing Drosophila notum whose expression is regulated by the concerted functions of decapentaplegic and wingless.

In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. Here, we show that two homeobox genes at the Bar locus (BarH1 and BarH2) may belong to a different class of prepattern genes expressed latitudinally, and suggest that the developing notum consists of checker-square-like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activating achaete-scute. Presutural macrochaetae formation also requires Bar homeobox gene activity. Bar homeobox gene expression is restricted dorsally and posteriorly by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar homeobox genes is determined by wingless whose expression is under the control of Decapentaplegic signaling.

Synergistic activation of the Wnt signaling pathway by Dvl and casein kinase Iepsilon.

Although casein kinase Iepsilon (CKIepsilon) has been shown to regulate the Wnt signaling pathway positively, its mode of action is not clear. In this study we show that CKIepsilon activates the Wnt signaling pathway in co-operation with Dvl. CKIepsilon and Axin associated with different sites of Dvl, and CKIepsilon and Dvl interacted with distinct regions on Axin. Therefore, these three proteins formed a ternary complex. Either low expression of Dvl or CKIepsilon alone did not accumulate beta-catenin, but their co-expression accumulated greatly. Dvl and CKIepsilon activated the transcriptional activity of T cell factor (Tcf) synergistically. Although the Dvl mutant that binds to Axin but not to CKIepsilon activated Tcf, it did not synergize with CKIepsilon. Another Dvl mutant that does not bind to Axin did not activate Tcf irrespective of the presence of CKIepsilon. Furthermore, Dvl and CKIepsilon co-operatively induced axis duplication of Xenopus embryos. These results indicate that Dvl and CKIepsilon synergistically activated the Wnt signaling pathway and that the binding of the complex of Dvl and CKIepsilon to Axin is necessary for their synergistic action.

Inhibition of Wnt signaling pathway by a novel axin-binding protein.

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.

Dual Bar homeo box genes of Drosophila required in two photoreceptor cells, R1 and R6, and primary pigment cells for normal eye development.

In the Bar mutation of Drosophila, ommatidial differentiation is known to be suppressed in the anterior portion of the eye. Our structural analysis shows that the Bar region contains a pair of homeo box genes, BarH1 and BarH2. These genes encode polypeptides similar in size and sequence and share a common homeo domain that is identical in sequence except for putative trans-activator-binding sites. We also show, by mosaic analysis and immunostaining with anti-BarH1/BarH2 antibodies, that BarH1 and BarH2 are not only specifically coexpressed but also functionally required in R1/R6 prephotoreceptors and primary pigment cells in developing ommatidia. In R1/R6, the expression of BarH1 and BarH2 appears to be regulated by rough and glass gene products. BarH1 and BarH2 proteins are essential to normal lens formation, formation of three types of pigment cells, and elimination of excess cells from mature ommatidia. Taken together, our results suggest that Bar homeo domain proteins may play key roles in the fate-determination processes of pigment cells and cone cells.

A novel beta-catenin-binding protein inhibits beta-catenin-dependent Tcf activation and axis formation.

beta-Catenin is efficiently phosphorylated by glycogen synthase kinase-3beta in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, beta-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of beta-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of beta-catenin, thereby inhibiting the binding of Tcf to beta-catenin. It did not affect the stability of beta-catenin but inhibited Wnt- or beta-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and beta-catenin-dependent axis duplication, and prevented the beta-catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits beta-catenin signaling.

Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A.

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3beta-dependent phosphorylation in the complex and the stability of beta-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61beta and -gamma, interact with Axin. PR61beta or -gamma formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61beta on Axin was different from those of GSK-3beta, beta-catenin, APC, and Dvl. Although PR61beta did not affect the stability of beta-catenin, it inhibited Dvl- and beta-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed beta-catenin-induced axis formation and expression of siamois, a Wnt target gene, in Xenopus embryos, suggesting that PR61beta acts either at the level of beta-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of beta-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.