Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled (Fz)/PCP factors and, although their molecular interactions are beginning to be understood, the upstream input providing the directional bias and polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with its extracellular domain acting as a ligand for Van Gogh (Vang). We demonstrate in Drosophila melanogaster wings that Wg (Wingless) and dWnt4 (Drosophila Wnt homologue) provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg and dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process.

Significance: The establishment and maintenance of epithelial polarity relies on the tight regulation of vesicular trafficking, which ensures that apical and basolateral membrane proteins reach their designated plasma membrane domains. Here, we report the direct visualization of biosynthetic trans -endosomal trafficking of apically targeted rhodopsin in polarized epithelial cells. Our work provides novel insights into the crosstalk between biosynthetic and endocytic pathways. We demonstrate that the small GTPase Rab11a regulates sorting at apical recycling endosomes (AREs) and is also implicated in carrier vesicle docking at the apical plasma membrane. We further unveil a surprising role for dynamin-2 in the release of apical carriers from AREs. Our data indicate that trans -endosomal trafficking is indispensable for accurate and high-fidelity apical delivery.

The transcription factor aryl hydrocarbon receptor (AhR) has relevant functions in cell proliferation. Interestingly, the AhR can either promote or inhibit proliferation depending on the cell phenotype. Although recent data reveal potential pathways for AhR signaling in cell proliferation, the mechanisms that regulate its activity in tumor cells remain unknown. Here, we have analyzed promoter hypermethylation as a potential mechanism controlling AhR expression in human tumor cells. AhR promoter CpG methylation was sporadic in a panel of 19 tumor cell lines except for the chronic myeloid leukemia (CML) K562 and the acute lymphoblastic leukemia (ALL) REH. When compared with normal lymphocytes, REH had very low constitutive AhR expression that could be attributed to promoter hypermethylation since treatment with the DNA demethylating agent 5-aza-2′-deoxycitidine (AZA) significantly increased AhR mRNA and protein. These results in leukemia-derived cell lines were further confirmed in primary ALL, where 33% of the patients (7/21) had AhR promoter hypermethylation. Chromatin immunoprecipitation (ChIP) showed that methylation impaired binding of the transcription factor Sp1 to the AhR promoter, thus providing a mechanism for AhR downregulation in REH cells. Therefore, promoter hypermethylation represents a novel epigenetic mechanism downregulating AhR activity in hematological malignancies such as ALL.

Alterations in tissue-specific gene expression greatly affect cell function. Transcription factors (TFs) interact with cis -acting binding sites in noncoding enhancer promoter regions. Transposable elements (TEs) are abundant and similarly represented among mammalian genomes. TEs are important in gene regulation, but their function is not well understood. We have characterized a TE containing functional TF-binding sites for the carcinogen-activated dioxin receptor xenobiotic responsive element (XRE) and the epithelial–mesenchymal transition regulator Slug (Slug site). A Mus promoter database was scanned for XREs to predict coregulation with other TFs. We identified an overrepresented (1,398 genes) B1 retrotransposon containing XRE and Slug sites within 35 bp of each other (designated as B1-X35S). This B1-X35S retrotransposon differed from classic B1s by the presence of the Slug site and by its differential nucleotide conservation outside the X35S region. Phylogenetically, B1-X35S appeared recently in evolution, close to the B1-B subfamily. Comparative gene expression in 61 mouse tissues revealed that B1-X35S-containing genes had lower median expression levels than those with canonical B1 TEs, suggesting a repressive role for X35S. Indeed, X35S was functional and able to bind aryl hydrocarbon (dioxin) receptor (AhR) and Slug and, importantly, to repress cis -reporter genes. Moreover, AhR and Slug were recruited to X35S in vivo and repressed the endogenous expression of X35S-containing genes. Our results demonstrate the existence of a widely present B1 subfamily in the mouse. Because AhR and Slug are relevant in tumor development and differentiation, X35S may represent a genome-wide regulatory mechanism and a tool to modulate gene expression.

Angiogenesis has key roles in development and in the progression of human diseases such as cancer. Consequently, identifying the novel markers and regulators of angiogenesis is a critical task. The dioxin receptor (AhR) contributes to vascular homeostasis and to the endothelial response to toxins, although the mechanisms involved are largely uncharacterized. Here, we show that AhR-null mice (AhR(-/-)) have impaired angiogenesis in vivo that compromises tumor xenograft growth. Aortic rings emigration experiments and RNA interference indicated that AhR(-/-) endothelial cells failed to branch and to form tube-like structures. Such a phenotype was found to be vascular endothelial growth factor (VEGF)-dependent, as AhR(-/-) aortic endothelial cells (MAECs) secreted lower amounts of active VEGF-A and their treatment with VEGF-A rescued angiogenesis in culture and in vivo. Further, the addition of anti-VEGF antibody to AhR(+/+) MAECs reduced angiogenesis. Treatment under hypoxic conditions with 2-methoxyestradiol suggested that HIF-1alpha modulates endothelial VEGF expression in an AhR-dependent manner. Importantly, AhR-null stromal myofibroblasts produced increased transforming growth factor-beta (TGFbeta) activity, which inhibited angiogenesis in human endothelial cells (HMECs) and AhR(-/-) mice, whereas the co-culture of HMECs with AhR(-/-) myofibroblasts or with their conditioned medium inhibited branching, which was restored by an anti-TGFbeta antibody. Moreover, VEGF and TGFbeta activities cooperated in modulating angiogenesis, as the addition of TGFbeta to AhR(-/-) MAECs further reduced their low basal VEGF-A activity. Thus, AhR modulates angiogenesis through a mechanism requiring VEGF activation in the endothelium and TGFbeta inactivation in the stroma. These data highlight the role of AhR in cardiovascular homeostasis and suggest that this receptor can be a novel regulator of angiogenesis during tumor development.