Eva M. Rico-Leo

Dioxin Receptor Deficiency Impairs Angiogenesis by a Mechanism Involving VEGF-A Depletion in the Endothelium and Transforming Growth Factor-β Overexpression in the Stroma

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-1α modulates endothelial VEGF expression in an AhR-dependent manner. Importantly, AhR-null stromal myofibroblasts produced increased transforming growth factor-β (TGFβ) 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-TGFβ antibody. Moreover, VEGF and TGFβ activities cooperated in modulating angiogenesis, as the addition of TGFβ 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 TGFβ 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.

Dioxin receptor deficiency impairs angiogenesis by a mechanism involving VEGF-a depletion in the endothelium and TGFβ over-expression in the stroma

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-1α modulates endothelial VEGF expression in an AhR-dependent manner. Importantly, AhR-null stromal myofibroblasts produced increased transforming growth factor-β (TGFβ) 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-TGFβ antibody. Moreover, VEGF and TGFβ activities cooperated in modulating angiogenesis, as the addition of TGFβ 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 TGFβ 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.

Loss of dioxin-receptor expression accelerates wound healing in vivo by a mechanism involving TGFβ

Delayed wound healing caused by inefficient re-epithelialization underlines chronic skin lesions such as those found in diabetes. The dioxin receptor (AhR) modulates cell plasticity and migration and its activation by occupational polycyclic aromatic hydrocarbons (PAHs) results in severe skin lesions such as contact hypersensitivity, dermatitis and chloracne. Using wild-type (Ahr+/+) and AhR-null (Ahr–/–) mouse primary keratinocyte cultures and tissue explants, we show that lack of AhR increases keratinocyte migration and accelerates skin re-epithelialization without affecting cell proliferation or recruitment of inflammatory cells. Wounds in Ahr–/– animals had elevated numbers of fibroblasts and increased collagen content in their granulation tissue. Importantly, Ahr–/– dermal fibroblasts secreted higher levels of active TGFβ that increased keratinocyte migration in culture and that could account for over-activation of the TGFβ pathway and for faster wound healing in the AhR-null neo-epithelium. Consistently, a TGFβ neutralizing antibody decreased keratinocyte migration in culture and halted re-epithelialization in Ahr–/– mice. Moreover, in vivo treatment with an antisense oligonucleotide for AhR increased TGFβ signaling and improved re-epithelialization in wounds of wild-type mice. These data indicate that AhR is relevant for wound repair and suggest that AhR downmodulation might be a potential new tool for the treatment of chronic, surgical or accidental wounds.

The dioxin receptor has tumor suppressor activity in melanoma growth and metastasis

Melanoma is a highly metastatic and malignant skin cancer having poor rates of patient survival. Since the incidence of melanoma is steadily increasing in the population, finding prognostic and therapeutic targets are crucial tasks in cancer. The dioxin receptor (AhR) is required for xenobiotic-induced toxicity and carcinogenesis and for cell physiology and organ homeostasis. Yet, the mechanisms by which AhR affects tumor growth and dissemination are largely uncharacterized. We report here that AhR contributes to the tumor-stroma interaction, blocking melanoma growth and metastasis when expressed in the tumor cell but supporting melanoma when expressed in the stroma. B16F10 cells engineered to lack AhR (small hairpin RNA for AhR) exacerbated melanoma primary tumorigenesis and lung metastasis when injected in AhR+/+ recipient mice but not when injected in AhR- /- mice or when co-injected with AhR-/- fibroblasts in an AhR+/+ stroma. Contrary, B16F10 cells expressing a constitutively active AhR had reduced tumorigenicity and invasiveness in either AhR genetic background. The tumor suppressor role of AhR in melanoma cells correlated with reduced migration and invasion, with lower numbers of cancer stem-like cells and with altered levels of β1-integrin and caveolin1. Human melanoma cell lines with highest AHR expression also had lowest migration and invasion. Moreover, AHR expression was reduced in human melanomas with respect to nevi lesions. We conclude that AhR knockdown in melanoma cells requires stromal AhR for maximal tumor progression and metastasis. Thus, AhR can be a molecular marker in melanoma and its activity in both tumor and stromal compartments should be considered.

Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition

Background: The dioxin receptor (AhR) regulates cell migration and has a role in TGFβ activation. Results: AhR expression inhibits basal and TGFβ-induced epithelial-to-mesenchymal transition (EMT). Conclusion: AhR has an intrinsic role in EMT and cross talks with TGFβ. Significance: The involvement of AhR in EMT can help explain its functions in organ homeostasis and tumor progression. Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR+/+ and AhR−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells.

Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor

Abstract Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.

The Dioxin receptor modulates Caveolin-1 mobilization during directional migration: role of cholesterol

BackgroundAdhesion and migration are relevant physiological functions that must be regulated by the cell under both normal and pathological conditions. The dioxin receptor (AhR) has emerged as a transcription factor regulating both processes in mesenchymal, epithelial and endothelial cells. Indirect results suggest that AhR could cooperate not only with additional transcription factors but also with membrane-associated proteins to drive such processes.ResultsIn this study, we have used immortalized and primary dermal fibroblasts from wild type (AhR+/+) and AhR-null (AhR?/?) mice to show that AhR modulates membrane distribution and mobilization of caveolin-1 (Cav-1) during directional cell migration. AhR co-immunoprecipitated with Cav-1 and a fraction of both proteins co-localized to detergent-resistant membrane microdomains (DRM). Consistent with a role of AhR in the process, AhR?/? cells had a significant reduction in Cav-1 in DRMs. Moreover, high cell density reduced AhR nuclear levels and moved Cav-1 from DRMs to the soluble membrane in AhR+/+ but not in AhR?/? cells. Tyrosine-14 phosphorylation had a complex role in the mechanism since its upregulation reduced Cav-1 in DRMs in both AhR+/+ and AhR?/? cells, despite the lower basal levels of Y14-Cav-1 in the null cells. Fluorescence recovery after photobleaching revealed that AhR knock-down blocked Cav-1 transport to the plasma membrane, a deficit possibly influencing its depleted levels in DRMs. Membrane distribution of Cav-1 in AhR-null fibroblasts correlated with higher levels of cholesterol and with disrupted membrane microdomains, whereas addition of exogenous cholesterol changed the Cav-1 distribution of AhR+/+ cells to the null phenotype. Consistently, higher cholesterol levels enhanced caveolae-dependent endocytosis in AhR-null cells.ConclusionsThese results suggest that AhR modulates Cav-1 distribution in migrating cells through the control of cholesterol-enriched membrane microdomains. Our study also supports the likely possibility of membrane-related, transcription factor independent, functions of AhR.

piRNA-associated proteins and retrotransposons are differentially expressed in murine testis and ovary of aryl hydrocarbon receptor deficient mice

Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR+/+ and AhR−/− mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR−/− than in AhR+/+ testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Unexpectedly, AhR−/− males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR+/+ mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR−/− ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR+/+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements.

A dioxin-like compound induces hyperplasia and branching morphogenesis in mouse mammary gland, through alterations in TGF-β1 and aryl hydrocarbon receptor signaling

&NA; Hexachlorobenzene (HCB) is a widespread environmental pollutant and a dioxin‐like compound that binds weakly to the aryl hydrocarbon receptor (AhR). Because AhR and transforming growth factor &bgr;1 (TGF‐&bgr;1) converge to regulate common signaling pathways, alterations in this crosstalk might contribute to developing preneoplastic lesions. The aim of this study was to evaluate HCB action on TGF‐&bgr;1 and AhR signaling in mouse mammary gland, through AhR +/+ and AhR −/− models. Results showed a differential effect in mouse mammary epithelial cells (NMuMG), depending on the dose: 0.05 &mgr;M HCB induced cell migration and TGF‐&bgr;1 signaling, whereas 5 &mgr;M HCB reduced cell migration, promoted cell cycle arrest and stimulated the dioxin response element (DRE) ‐dependent pathway. HCB (5 &mgr;M) enhanced &agr;‐smooth muscle actin expression and decreased TGF‐&bgr; receptor II mRNA levels in immortalized mouse mammary fibroblasts AhR +/+, resembling the phenotype of transformed cells. Accordingly, their conditioned medium was able to enhance NMuMG cell migration. Assays in C57/Bl6 mice showed HCB (3 mg/kg body weight) to enhance ductal hyperplasia, cell proliferation, estrogen receptor &agr; nuclear localization, branch density, and the number of terminal end buds in mammary gland from AhR +/+ mice. Primary culture of mammary epithelial cells from AhR +/+ mice showed reduced AhR mRNA levels after HCB exposure (0.05 and 5 &mgr;M). Interestingly, AhR −/− mice exhibited an increase in ductal hyperplasia and mammary growth in the absence of HCB treatment, thus revealing the importance of AhR in mammary development. Our findings show that environmental HCB concentrations modulate AhR and TGF‐&bgr;1 signaling, which could contribute to altered mammary branching morphogenesis, likely leading to preneoplastic lesions and retaining terminal end buds. Highlights0.05 &mgr;M HCB induces migration and TGF‐&bgr;1 signaling in mammary epithelial cells NMuMG.5 &mgr;M HCB reduces migration, and promotes cell cycle arrest and AhR nuclear pathway in NMuMG.HCB enhances &agr;‐SMA and decreases TGF‐&bgr; receptor II expression in fibroblasts AhR +/+.Conditioned medium from fibroblasts AhR +/+ exposed to HCB enhances NMuMG migration.HCB increases hyperplasia and branching morphogenesis in AhR +/+ mice mammary gland.