CTLA-4 is a direct target of Wnt/beta-catenin signaling and is expressed in human melanoma tumors.

Our goal was to identify genes regulated by Wnt/beta-catenin signaling in melanoma cells, as this pathway has been implicated in melanocyte development and in melanoma biology. We therefore undertook transcriptional profiling of UACC 1273 human melanoma cells following treatment with recombinant Wnt-3a and found that cytotoxic T-lymphocyte antigen-4 (CTLA-4) was the most highly induced gene. We observed CTLA-4 expression in human epidermal melanocytes and in patient-derived primary melanoma tumors and found that Wnt/beta-catenin signaling elevates CTLA-4 expression in two cultured melanoma cell lines. CTLA-4 is likely a direct target of Wnt/beta-catenin signaling, as the beta-catenin responsiveness of a 1.7 kb region of the CTLA-4 promoter requires a T-cell factor-1/lymphoid enhancing factor-1 consensus site present at -114 to -119 bp from the transcriptional start site. These findings are the initial demonstration that CTLA-4 is a direct target of Wnt/beta-catenin signaling and the first report of its expression in primary melanoma tumors and melanocytes. Given the described role of CTLA-4 in inhibiting the immune response, these findings may shed light on the role of Wnt/beta catenin signaling in melanoma and on the mechanism of action of human anti-CTLA-4 antibody, currently in phase III clinical trials for the treatment of melanoma.

The Wnt5A/protein kinase C pathway mediates motility in melanoma cells via the inhibition of metastasis suppressors and initiation of an epithelial to mesenchymal transition.

We have shown that Wnt5A increases the motility of melanoma cells. To explore cellular pathways involving Wnt5A, we compared gain-of-function (WNT5A stable transfectants) versus loss-of-function (siRNA knockdown) of WNT5A by microarray analysis. Increasing WNT5A suppressed the expression of several genes, which were re-expressed after small interference RNA-mediated knockdown of WNT5A. Genes affected by WNT5A include KISS-1, a metastasis suppressor, and CD44, involved in tumor cell homing during metastasis. This could be validated at the protein level using both small interference RNA and recombinant Wnt5A (rWnt5A). Among the genes up-regulated by WNT5A was the gene vimentin, associated with an epithelial to mesenchymal transition (EMT), which involves decreases in E-cadherin, due to up-regulation of the transcriptional repressor, Snail. rWnt5A treatment increases Snail and vimentin expression, and decreases E-cadherin, even in the presence of dominant-negativeTCF4, suggesting that this activation is independent of Wnt/beta-catenin signaling. Because Wnt5A can signal via protein kinase C (PKC), the role of PKC in Wnt5A-mediated motility and EMT was also assessed using PKC inhibition and activation studies. Treating cells expressing low levels of Wnt5A with phorbol ester increased Snail expression inhibiting PKC in cells expressing high levels of Wnt5A decreased Snail. Furthermore, inhibition of PKC before Wnt5A treatment blocked Snail expression, implying that Wnt5A can potentiate melanoma metastasis via the induction of EMT in a PKC-dependent manner.

Mutant Frizzled 4 associated with vitreoretinopathy traps wild-type Frizzled in the endoplasmic reticulum by oligomerization.

nt signalling pathways regulate cell proliferation, cell fate and morphogenetic movements. Here, we demonstrate that the Frizzled (Fz) family of Wnt receptors, similarly to G-protein-coupled receptors (GPCRs), form specific homo- and hetero-oligomers. Two lines of evidence suggest that oligomerization occurs in the endoplasmic reticulum: first, a mutant allele of Fz4, encoding a truncated protein that is retained in the endoplasmic reticulum, is linked to the autosomal-dominant retinal degenerative disease, familial exudative vitreoretinopathy (FEVR). We show that this mutant form of Fz4 oligomerizes with wild-type Fz4, retains it in the endoplasmic reticulum and inhibits its signalling. Second, a derivative of Fz1 targeted to the endoplasmic reticulum traps wild-type Fz1 in the endoplasmic reticulum and blocks its signalling. These data support the hypothesis that oligomerization of mutant and wild-type Fz proteins occurs in the endoplasmic reticulum and may explain the genetic dominance of this FEVR allele.

Genetics of behaviour: phenotypic and molecular study of rats derived from high- and low-alcohol consuming lines.

The aim of this study was to analyse the genetic basis of excessive ethanol consumption and its relationship with emotional reactivity. The high-ethanol preferring line of rats used is characterized by a: (i) high voluntary consumption of alcohol; (ii) high sensitivity to taste reinforcement (saccharine, quinine); (iii) high locomotor activity in a novel environment; and (iv) low emotional reactivity, these features being opposite in the Wistar-Kyoto (WKY) rat strain. The F2 population demonstrated a very large variability in these behavioural traits, and factor analysis revealed that these characteristics appear to be largely unrelated to each other. The molecular bases for these differences were investigated by quantitative trait loci (QTL) analysis. For this purpose, the 196 F2 rats were genotyped with regularly distributed markers on the whole genome, and genetic linkage maps were generated for all subsequent QTL analyses. A locus with a maximum LOD score of 7.6 and accounting for approximately 61% of the genetic variance of the trait in the F2 population was detected on chromosome 4 for alcohol drinking. In the same region, we found a QTL related to the reinforcement properties of saccharin, with a significant LOD score of 4.9 and explaining 46% of the variance of the trait. Other significant QTL were found for plus maze behaviour and open field activity on chromosome 1. Current research aims to identify the gene(s) involved.

Wnt-5A augments repopulating capacity and primitive hematopoietic development of human blood stem cells in vivo.

Human hematopoietic stem cells are defined by their ability to repopulate multiple hematopoietic lineages in the bone marrow of transplanted recipients and therefore are functionally distinct from hematopoietic progenitors detected in vitro. Although factors capable of regulating progenitors are well established, in vivo regulators of hematopoietic repopulating function are unknown. By using a member of the vertebrate Wnt family, Wnt-5A, the proliferation and differentiation of progenitors cocultured on stromal cells transduced with Wnt-5A or treated with Wnt-5A conditioned medium (CM) was unaffected. However, i.p. injection of Wnt-5A CM into mice engrafted with human repopulating cells increased multilineage reconstitution by >3-fold compared with controls. Furthermore, in vivo treatment of human repopulating cells with Wnt-5A CM produced a greater proportion of phenotypically primitive hematopoietic progeny that could be isolated and shown to possess enhanced progenitor function independent of continued Wnt-5A treatment. Our study demonstrates that Wnt-5A augments primitive hematopoietic development in vivo and represents an in vivo regulator of hematopoietic stem cell function in the human. Based on these findings, we suggest a potential role for activation of Wnt signaling in managing patients exhibiting poor hematopoietic recovery shortly after stem cell transplantation.