Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu 648 and Glu 651 rendered minor effects on VR1 pore attributes, while Glu 636 slightly modulated pore blockade. In contrast, replacement of Asp 646 by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg 2؉ with respect to Na ؉ without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp 646 is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.The molecular mechanism underlying chemical and thermal nociception is starting to be understood, thanks to the cloning of a capsaicin-operated neuronal receptor referred to as the vanilloid receptor subunit 1 (VR1) 1 (1). VR1 is a nonselective cation channel with high Ca 2ϩ permeability that integrates both types of pain-producing stimuli (1-5). These channels are activated by vanilloids such as capsaicin, the pungent ingredient of hot red peppers, and by temperatures higher than 40°C (1, 2, 4). Recently, the lipid-based anandanamide was shown to be a potential endogenous VR1 agonist (6). Activation of the VR1 channel raises intracellular Ca 2ϩ and excites a subset of dorsal root and trigeminal ganglion primary neurons (5). These neurons transmit noxious information to the central nervous system and release proinflammatory neuropeptides at peripheral terminals (5, 7). In addition to playing a role in nociception, the high Ca 2ϩ permeability exhibited by VR1 strongly desensitizes capsaicin-operated responses (5, 7). This property partially accounts for the antinociceptive activity exhibited by vanilloids (5,8,9).VR1 subunits are membrane proteins with a predicted relative molecular mass of 95 kDa that show similarity to the family of putative store-operated calcium channels (1,3,10). Although the molecular composition and stoichiometry of neuronal VR1 channels is undetermined, heterologous expression of VR1 subunits gives rise to homomeric receptors that recapitulate most of the reported physiological properties (1, 2, 4, 5, 7). Nonetheles...

Resveratrol (RES), a chemopreventive molecule, inhibits the proliferation of tumor cells of different etiologies. We previously showed that RES alters the cell cycle and induces apoptosis in MCF-7 breast tumor cells by interfering with the estrogen receptor (ERa)-dependent phosphoinositide 3-kinase (PI3K) pathway. Here, we analyzed signaling downstream of PI3K, to understand the mechanisms of RES-induced apoptosis. Apoptotic death by RES in MCF-7 was mediated by Bcl-2 downregulation since overexpression of this protein abolished apoptosis. Decreased Bcl-2 levels were not related to cytochrome c release, activation of caspases 3/8 or poly(ADP-ribose) polymerase proteolysis. However, RES decreased mitochondrial membrane potential and increased reactive oxygen species and nitric oxide production. NF-kB, a regulator of Bcl-2 expression, and calpain protease activity, a regulator of NF-kB, were both inhibited by RES. The patterns for NFkB and calpain activities followed that of PI3K and were inhibited by LY294002. NF-kB inhibition coincided with diminished MMP-9 activity and cell migration. These data suggest that RES-induced apoptosis in MCF-7 could involve an oxidative, caspase-independent mechanism, whereby inhibition of PI3K signaling converges to Bcl-2 through NF-kB and calpain protease activity. Therefore, Bcl-2 and NF-kB could be considered potential targets for the chemopreventive activity of RES in estrogen-responsive tumor cells. ' 2005 Wiley-Liss, Inc.Key words: resveratrol; caspase; Bcl-2; NF-kB; apoptosis Among natural compounds with beneficial effects on human health, RES (3,4 0 ,5-trihydroxystilbene) has attracted considerable interest. This molecule, present at relevant concentrations in red wine, 1 has been associated with a lower incidence of cardiovascular disease. Different studies have also suggested a beneficial effect of RES in cancer since it inhibits proliferation and promotes death in tumor cell lines of different origins, 2-4 and, in vivo, suppresses the formation of skin 5 and mammary gland 6 tumors in rodent models of carcinogenesis.We, as well as other laboratories, have found that the ability of RES to inhibit cell viability and proliferation in the human breast cancer cell lines MCF-7 and MDA-MB-231 was unrelated to ERa status. 7,8 However, apoptotic cell death was present only in ERapositive MCF-7 and involved cell-specific regulation of the G 1 /S and G 2 /M transitions of the cell cycle. 2,8 RES properties are related to ERa since this compound has estrogenic or antiestrogenic activities depending on its concentration and the phenotype of the target cell. 9,10 ERa, in addition to its nuclear role as a transcription factor, is involved in regulating the PI3K pathway, which controls cell growth, proliferation and apoptosis. [11][12][13] In MCF-7 cells, RES induced a biphasic pattern of PI3K activity that increased at low concentrations and decreased at high concentrations. Activation of downstream PI3K effectors PKB/AKT and GSK-3 closely followed the pattern of PI3K activity. 14 The ...

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 (AhR) modulates cell plasticity and migration, although the signaling involved remains unknown. Here, we report a mechanism that integrates AhR into these cytoskeleton-related functions. Immortalized and mouse embryonic fibroblasts lacking AhR (AhR؊/؊) had increased cell area due to spread cytoplasms that reverted to wild-type morphology upon AhR re-expression. The AhR-null phenotype included increased F-actin stress fibers, depolarized focal adhesions, and enhanced spreading and adhesion. The cytoskeleton alterations of AhR؊/؊ cells were due to downregulation of constitutive Vav3 expression, a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases and a novel transcriptional target of AhR. AhR was recruited to the vav3 promoter and maintained constitutive mRNA expression in a ligand-independent manner. Consistently, AhR؊/؊ fibroblasts had reduced Rac1 activity and increased activation of the RhoA/Rho kinase (Rock) pathway. Pharmacological inhibition of Rac1 shifted AhR؉/؉ fibroblasts to the null phenotype, whereas Rock inhibition changed AhR-null cells to the AhR؉/؉ morphology. Knockdown of vav3 transcripts by small interfering RNA induced cytoskeleton defects and changes in adhesion and spreading mimicking those of AhR-null cells. Moreover, vav3؊/؊ MEFs, as AhR؊/؊ mouse embryonic fibroblasts, had increased cell area and enhanced stress fibers. By modulating Vav3-dependent signaling, AhR could regulate cell shape, adhesion, and migration under physiological conditions and, perhaps, in certain pathological states.

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SummaryAryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR−/−) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR−/− mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/β-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR−/− liver. Consistently, PI3K, ERK, and Wnt/β-catenin inhibition partially rescued polyploidy in AhR−/− mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver.

Vanilloid receptors (VRs) play a fundamental role in the transduction of peripheral tissue injury and/or inflammation responses. Molecules that antagonize VR channel activity may act as selective and potent analgesics. We report that synthetic arginine-rich hexapeptides block heterologously expressed VR-1 channels with submicromolar efficacy in a weak voltagedependent manner, consistent with a binding site located near/ at the entryway of the aqueous pore. Dynorphins, natural arginine-rich peptides, also blocked VR-1 activity with micromolar affinity. Notably, synthetic and natural arginine-rich peptides attenuated the ocular irritation produced by topical capsaicin application onto the eyes of experimental animals. Taken together, our results imply that arginine-rich peptides are VR-1 channel blockers with analgesic activity. These findings may expand the development of novel analgesics by targeting receptor sites distinct from the capsaicin binding site. ß

The aryl hydrocarbon receptor (AhR) has roles in cell proliferation, differentiation and organ homeostasis, including the liver. AhR depletion induces undifferentiation and pluripotency in normal and transformed cells. Here, AhR-null mice (AhR−/−) were used to explore whether AhR controls liver regeneration and carcinogenesis by restricting the expansion of stem-like cells and the expression of pluripotency genes. Short-term CCl4 liver damage was earlier and more efficiently repaired in AhR−/− than in AhR+/+ mice. Stem-like CK14 + and TBX3 + and pluripotency-expressing OCT4 + and NANOG + cells expanded sooner in AhR−/− than in AhR+/+ regenerating livers. Stem-like side population cells (SP) isolated from AhR−/− livers had increased β-catenin (β-Cat) signaling with overexpression of Axin2, Dkk1 and Cyclin D1. Interestingly, β-Cat, Axin2 and Dkk1 also increased during regeneration but more notably in AhR-null livers. Liver carcinogenesis induced by diethylnitrosamine (DEN) produced large carcinomas in all AhR−/− mice but mostly premalignant adenomas in less than half of AhR+/+ mice. AhR-null tumoral tissue, but not their surrounding non-tumoral parenchyma, had nuclear β-Cat and Axin2 overexpression. OCT4 and NANOG were nevertheless similarly expressed in AhR+/+ and AhR−/− lesions. We suggest that AhR may serve to adjust liver repair and to block tumorigenesis by modulating stem-like cells and β-Cat signaling.