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Binding of an extracellular signaling molecule to a G protein-coupled receptor (GPCR) results in G protein activation, which in turn triggers the production of second messengers. Although many advances have been achieved in understanding GPCR activation, the early steps of GPCR of this process are still poorly understood. In the case of rhodopsin, the paradigmatic member of class A GPCRs, the endogenous ligand is retinal which is covalently bound to K296 through a Schiff base. The activation of rhodopsin is triggered by the absorption of a photon and the isomerization of 11-cis-retinal to all-trans-retinal. When rhodopsin in ground state (λmax = 500 nm) is photoactivated, it progresses through short-lived intermediates leading to the Meta-I state (λmax = ~478 nm), which then establishes an equilibrium with Meta-II (λmax = 380 nm), the G-protein binding state. We have developed a llama antibody (nanobody Nb2) that binds to bovine rhodopsin and stabilizes its early activation intermediate Meta-I. Here we present the crystal structures of Nb2 in complex with ground-state bovine rhodopsin, as well as Nb2 in complex with apo-rhodopsin (opsin). The binding site of Nb2 includes rhodopsin's N-terminus and extracellular loops ECL2 and ECL3. Whereas rhodopsin's structure in the ground-state rhodopsin/Nb2 complex is virtually identical to that of rhodopsin alone, Nb2 binding induces a dramatic structural change in opsin, trapping it in a conformation similar to that of ground-state rhodopsin. In a similar way, when Nb2 binds in solution to photoactivated, deprotonated rhodopsin (Meta-II) it shifts the equilibrium towards a protonated Schiff base state corresponding to the Meta-I conformation. Finally, co-expression of Nb2 and P23H mutant rhodopsin in HEK293 cell lines is able to rescue the phenotype of misfolding and degradation in vitro, suggesting a therapeutic potential of this nanobody. In summary, we have developed a nanobody that traps photoactivated rhodopsin in the elusive Meta-I state; in addition, Nb2 stabilizes amisfolding-prone mutant of rhodopsin. Nb2 might also be a useful tool to facilitate the crystallization of wild type and mutants of rhodopsin in different conformational states bound to different ligands. Current efforts include solving the crystal structure of the Meta-I rhodopsin/nanobody complex.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.

Lactate can be used by neurons as an energy substrate to support their activity. Evidence suggests that lactate also acts on a metabotropic receptor called HCAR1, first described in the adipose tissue. Whether HCAR1 also modulates neuronal circuits remains unclear. In this study, using qRT-PCR, we show that HCAR1 is present in the human brain of epileptic patients who underwent resective surgery. In brain slices from these patients, pharmacological HCAR1 activation using a non-metabolized agonist decreased the frequency of both spontaneous neuronal Ca2+ spiking and excitatory post-synaptic currents (sEPSCs). In mouse brains, we found HCAR1 expression in different regions using a fluorescent reporter mouse line and in situ hybridization. In the dentate gyrus, HCAR1 is mainly present in mossy cells, key players in the hippocampal excitatory circuitry and known to be involved in temporal lobe epilepsy. By using whole-cell patch clamp recordings in mouse and rat slices, we found that HCAR1 activation causes a decrease in excitability, sEPSCs, and miniature EPSCs frequency of granule cells, the main output of mossy cells. Overall, we propose that lactate can be considered a neuromodulator decreasing synaptic activity in human and rodent brains, which makes HCAR1 an attractive target for the treatment of epilepsy.

Compound 21 (C21), a selective agonist of angiotensin II type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of the present study was to assess the effect of C21 on thoracic aorta endothelial function in a model of diet-induced obesity (DIO) and to elucidate the potential cross-talk among AT2R, Mas receptor (MasR) and/or bradykinin type 2 receptor (B2R) in this response. Five-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1 mg/kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (HECs; EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels. Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favored the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine (ACh) due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways. In conclusion, C21 favors the interaction among AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.

G protein-coupled receptors (GPCRs) constitute a large family of receptors that activate intracellular signaling pathways upon detecting specific extracellular ligands. While many aspects of GPCR signaling have been uncovered through decades of studies, some fundamental properties, like its channel capacity—a measure of how much information a given transmission system can reliably transduce—are still debated. Previous studies concluded that GPCRs in individual cells could transmit around one bit of information about the concentration of the ligands, allowing only for a reliable on or off response. Using muscarinic receptor-induced calcium response measured in individual cells upon repeated stimulation, we show that GPCR signaling systems possess a significantly higher capacity. We estimate the channel capacity of this system to be above two, implying that at least four concentration levels of the agonist can be distinguished reliably. These findings shed light on the basic principles of GPCR signaling., G protein-coupled receptors (GPCRs) activate intracellular signalling pathways upon extracellular stimulation. Here authors record single cell responses of GPCR signalling which allows the direct estimation of its channel capacity for each cell along with the reproducibility of its response.

The interaction between oxysterols and the G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2) fine-tunes immune cell migration, a mechanism efficiently targeted by several disease-modifying treatments developed to treat multiple sclerosis (MS), such as natalizumab. We previously showed that memory CD4(+) T lymphocytes migrate specifically in response to 7α,25-dihydroxycholesterol (7α,25-OHC) via EBI2 in the MS murine model experimental autoimmune encephalomyelitis. However, the EBI2 expression profile in human lymphocytes in both healthy and MS donors is unknown. Here, we characterize EBI2 biology in human lymphocytes. We observed that EBI2 is functionally expressed on memory CD4(+) T cells and is enhanced under natalizumab treatment. These data suggest a significant role for EBI2 in human CD4(+) T cell migration, notably in patients with MS. Better knowledge of EBI2 involvement in autoimmunity may therefore lead to an improved understanding of the physiopathology of MS.

Adult intestinal stem cells are located at the bottom of crypts of Lieberkuhn, where they express markers such as LGR51,2 and fuel the constant replenishment of the intestinal epithelium1. Although fetal LGR5-expressing cells can give rise to adult intestinal stem cells3,4, it remains unclear whether this population in the patterned epithelium represents unique intestinal stem-cell precursors. Here we show, using unbiased quantitative lineage-tracing approaches, biophysical modelling and intestinal transplantation, that all cells of the mouse intestinal epithelium—irrespective of their location and pattern of LGR5 expression in the fetal gut tube—contribute actively to the adult intestinal stem cell pool. Using 3D imaging, we find that during fetal development the villus undergoes gross remodelling and fission. This brings epithelial cells from the non-proliferative villus into the proliferative intervillus region, which enables them to contribute to the adult stem-cell niche. Our results demonstrate that large-scale remodelling of the intestinal wall and cell-fate specification are closely linked. Moreover, these findings provide a direct link between the observed plasticity and cellular reprogramming of differentiating cells in adult tissues following damage5–9, revealing that stem-cell identity is an induced rather than a hardwired property. Lineage tracing, biophysical modelling and intestinal transplantation approaches are used to demonstrate that, in the mouse fetal intestinal epithelium, cells are highly plastic with respect to cellular identity and, independent of LGR5 expression and cell position, can contribute to the adult stem cell compartment.

Thirty years ago, a novel axis of the renin-angiotensin system (RAS) was unveiled by the discovery of angiotensin-(1−7) [ANG-(1−7)] generation in vivo. Later, angiotensin-converting enzyme 2 (ACE2) was shown to be the main mediator of this reaction, and Mas was found to be the receptor for the heptapeptide. The functional analysis of this novel axis of the RAS that followed its discovery revealed numerous protective actions in particular for cardiovascular diseases. In parallel, similar protective actions were also described for one of the two receptors of ANG II, the ANG II type 2 receptor (AT2R), in contrast to the other, the ANG II type 1 receptor (AT1R), which mediates deleterious actions of this peptide, e.g., in the setting of cardiovascular disease. Very recently, another branch of the RAS was discovered, based on angiotensin peptides in which the amino-terminal aspartate was replaced by alanine, the alatensins. Ala-ANG-(1−7) or alamandine was shown to interact with Mas-related G protein-coupled receptor D, and the first functional data indicated that this peptide also exerts protective effects in the cardiovascular system. This review summarizes the presentations given at the International Union of Physiological Sciences Congress in Rio de Janeiro, Brazil, in 2017, during the symposium entitled “The Renin-Angiotensin System: Going Beyond the Classical Paradigms,” in which the signaling and physiological actions of ANG-(1−7), ACE2, AT2R, and alatensins were reported (with a focus on noncentral nervous system-related tissues) and the therapeutic opportunities based on these findings were discussed.

The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we show that Lgr6 marks rare populations of cells in both basal and luminal mammary gland compartments in mice. Lineage tracing analysis showed that Lgr6+ cells are unipotent progenitors, which expand clonally during puberty but diminish in adulthood. In pregnancy or following stimulation with ovarian hormones, adult Lgr6+ cells regained proliferative potency and their progeny formed alveoli over repeated pregnancies. Oncogenic mutations in Lgr6+ cells resulted in expansion of luminal cells, culminating in mammary gland tumours. Conversely, depletion of Lgr6+ cells in the MMTV-PyMT model of mammary tumorigenesis significantly impaired tumour growth. Thus, Lgr6 marks mammary gland progenitor cells that can initiate tumours, and cells of luminal breast tumours required for efficient tumour maintenance.

XCR1 is selectively expressed on a conventional dendritic cell subset, the cDC1 subset, through phylogenetically distant species. The outcome of antigen-targeting to XCR1 may therefore be similar across species, permitting the translation of results from experimental models to human and veterinary applications. Here we evaluated in pigs the immunogenicity of bivalent protein structures made of XCL1 fused to the external portion of the influenza virus M2 proton pump, which is conserved through strains and a candidate for universal influenza vaccines. Pigs represent a relevant target of such universal vaccines as pigs can be infected by swine, human and avian strains. We found that cDC1 were the only cell type labeled by XCR1-targeted mCherry upon intradermal injection in pig skin. XCR1-targeted M2e induced higher IgG responses in seronegative and seropositive pigs as compared to non-targeted M2e. The IgG response was less significantly enhanced by CpG than by XCR1 targeting, and CpG did not further increase the response elicited by XCR1 targeting. Monophosphoryl lipid A with neutral liposomes did not have significant effect. Thus altogether M2e-targeting to XCR1 shows promises for a trans-species universal influenza vaccine strategy, possibly avoiding the use of classical adjuvants.

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.

Introduction The renin angiotensin system plays a crucial role in erectile function. It has been shown that elevated angiotensin‐II levels contribute to the development of erectile dysfunction (ED). Oppositely, angiotensin‐(1‐7) (Ang‐[1‐7]) mediates penile erection by activation of receptor Mas. Recently, we have developed a formulation based on Ang‐(1‐7) inclusion in cyclodextrin (CyD) [Ang‐(1‐7)‐CyD], which allows for the oral administration of Ang‐(1‐7). Aim In the present study, we evaluated the effects of chronic treatment with Ang‐(1‐7)‐CyD on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemic mice. Methods Apolipoprotein(Apo)E −/− mice fed a Western‐type diet for 11 weeks received Ang‐(1‐7)‐CyD or vehicle during the final 3 weeks. Collagen content and reactive oxygen species (ROS) production within the corpus cavernosum were evaluated by Sirius red and dihydroethidium staining, respectively. Protein expression of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH) subunits (p67‐phox and p22‐phox), and AT1 and Mas receptors in the penis was assessed by Western blotting. Nitric oxide (NO) production was measured by Griess assay in the mice serum. Cavernosal strips were mounted in an isometric organ bath to evaluate the endothelial function. Main Outcome Measures The effect of Ang‐(1‐7)‐CyD treatment on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemia‐induced ED. Results Ang‐(1‐7)‐CyD treatment reduced collagen content in the corpus cavernosum of ApoE −/− mice. This effect was associated with an attenuation of ROS production and a diminished expression of NADPH. Furthermore, Ang‐(1‐7)‐CyD treatment augmented the expression of nNOS and eNOS in the penis and elevated vascular NO production. Importantly, these effects were accompanied by an improvement in cavernosal endothelial function. Conclusion Long‐term treatment with Ang‐(1‐7)‐CyD reduces penile fibrosis associated with attenuation of oxidative stress. Additionally, cavernosal endothelial function in hypercholesterolemic mice was markedly improved. These results suggest that Ang‐(1‐7)‐CyD might have significant therapeutic benefits for the treatment of erectile dysfunction. Fraga‐Silva RA, Costa‐Fraga FP, Savergnini SQ, De Sousa FB, Montecucco F, da Silva D, Sinisterra RD, Mach F, Stergiopulos N, da Silva RF, and Santos RAS. An oral formulation of angiotensin‐(1–7) reverses corpus cavernosum damages induced by hypercholesterolemia. J Sex Med 2013;10:2430–2442.

CC chemokine receptor 5 (CCR5), the major HIV coreceptor, is a G protein-coupled receptor (GPCR) involved in cell activation and migration in response to chemokines. Blockade of CCR5 is an effective anti-HIV strategy, and potent anti-HIV chemokine analogs such as PSC-RANTES have been developed. These inhibitors act by interfering with receptor trafficking, thereby inducing prolonged intracellular sequestration of CCR5. Like many GPCRs, CCR5 is desensitized following agonist activation. The initial steps in this process are well understood, but later stages, including where CCR5 is sequestered during desensitization, and how anti-HIV chemokine analogs intervene to achieve prolonged sequestration, have yet to be elucidated in detail. In this study we demonstrate that CCR5 cycles to and from the cell surface via the endosome recycling compartment and the trans-Golgi network during desensitization, accumulating in the trans-Golgi network following internalization by both PSC-RANTES and CCL5, the native ligand from which it was derived. In addition, we show that unlike CCR5 sequestered by CCL5, CCR5 sequestered by PSC-RANTES cannot be induced to return to the cell surface by addition of the small molecule CCR5 inhibitor, TAK-779, and that association of PSC-RANTES with CCR5 is more durable than that of native CCL5 during desensitization. Our findings reconcile the previously conflicting descriptions of the location of sequestered CCR5 during desensitization, as well as providing more general insights into potential trafficking routes for endocytosed GPCRs and further elucidation of the unusual inhibitory mechanism of chemokine analogs with potent anti-HIV activity.

CXCR3 ligands were secreted by tissue fibroblasts and peripheral blood–derived mononuclear leukocytes in response to interferon-γ (IFN-γ) and Toll-like receptor (TLR) ligands. Subsequent purification and identification revealed the presence of truncated CXCL11 variants missing up to 6 amino acids. In combination with CD26/dipeptidyl peptidase IV, the metalloprotease aminopeptidase N (APN), identical to the myeloid cell marker CD13, rapidly processed CXCL11, but not CXCL8, to generate truncated CXCL11 forms. Truncated CXCL11 had reduced binding, signaling, and chemotactic properties for lymphocytes and CXCR3- or CXCR7-transfected cells. CD13/APN-truncated CXCL11 failed to induce an intracellular calcium increase but was still able to bind and desensitize CXCR3 for intact CXCL11 signaling. CXCL11 efficiently bound to CXCR7, but CXCL11 was not able to induce calcium signaling or ERK1/2 or Akt phosphorylation through CXCR7. CD26-truncated CXCL11 failed to attract lymphocytes but still inhibited microvascular endothelial cell (HMVEC) migration. However, further processing of CXCL11 by CD13 resulted in significant reduction of inhibition of HMVEC migration. Taken together, during inflammation or cancer, CXCL11 processing by CD13 may lead to a reduced number of tumor-infiltrating lymphocytes and in a more angiogenic environment.

Tachykinins are comprised of the family of related peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). NKB has emerged as regulator of kisspeptin release in the arcuate nucleus (ARC), whereas the roles of SP and NKA in reproduction remain unknown. This work explores the roles of SP and NKA in the central regulation of GnRH release. First, central infusion of specific agonists for the receptors of SP (neurokinin receptor 1, NK1R), NKA (NK2R) and NKB (NK3R) each induced gonadotropin release in adult male and ovariectomized, estradiol-replaced female mice, which was absent in Kiss1r(-/-) mice, indicating a kisspeptin-dependent action. The NK2R agonist, however, decreased LH release in ovariectomized-sham replaced females, as documented for NK3R agonists but in contrast to the NK1R agonist, which further increased LH release. Second, Tac1 (encoding SP and NKA) expression in the ARC and ventromedial nucleus was inhibited by circulating estradiol but did not colocalize with Kiss1 mRNA. Third, about half of isolated ARC Kiss1 neurons expressed Tacr1 (NK1R) and 100% Tacr3 (NK3R); for anteroventral-periventricular Kiss1 neurons and GnRH neurons, approximately one-fourth expressed Tacr1 and one-tenth Tacr3; Tacr2 (NK2R) expression was absent in all cases. Overall, these results identify a potent regulation of gonadotropin release by the SP/NK1R and NKA/NK2R systems in the presence of kisspeptin-Kiss1r signaling, indicating that they may, along with NKB/NK3R, control GnRH release, at least in part through actions on Kiss1 neurons.

Aberrant regulation of the Wnt/β-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or β-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or β-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind β-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or β-catenin depend on Wnt ligands and their secretion for a sufficient level of β-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls., Activating mutations in the Wnt signalling pathway are associated with colon cancer. Here the authors show that tumour cells carrying mutations in APC and β-catenin are still regulated by Wnt ligands, suggesting that Wnt secretion and receptor signalling remains important to control downstream signalling.

Pancreatic β-cells, located within the islets of Langerhans, are key players in the control of blood glucose homeostasis. The amount of insulin secreted by these cells is precisely adjusted to avoi...

Pattern recognition receptors (PRRs) are commonly known as sensor proteins crucial for the early detection of microbial or host-derived stress signals by innate immune cells. Interestingly, some PRRs are also expressed and functional in cells of the adaptive immune system. These receptors provide lymphocytes with innate sensing abilities; for example, B cells express Toll-like receptors, which are important for the humoral response. Strikingly, certain other NOD-like receptors are not only highly expressed in adaptive immune cells, but also exert functions related specifically to adaptive immune system pathways, such as regulating antigen presentation. In this review, we will focus particularly on the current understanding of PRR functions intrinsic to B and T lymphocytes; a developing aspect of PRR biology.

Angiotensin (Ang)-(1–7) is now recognized as a biologically active component of the renin–angiotensin system (RAS). Ang-(1–7) appears to play a central role in the RAS because it exerts a vast array of actions, many of them opposite to those attributed to the main effector peptide of the RAS, Ang II. The discovery of the Ang-converting enzyme (ACE) homolog ACE2 brought to light an important metabolic pathway responsible for Ang-(1–7) synthesis. This enzyme can form Ang-(1–7) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(1–9) with subsequent Ang-(1–7) formation by ACE. In addition, it is now well established that the G protein-coupled receptor Mas is a functional binding site for Ang-(1–7). Thus, the axis formed by ACE2/Ang-(1–7)/Mas appears to represent an endogenous counterregulatory pathway within the RAS, the actions of which are in opposition to the vasoconstrictor/proliferative arm of the RAS consisting of ACE, Ang II, and AT1receptor. In this brief review, we will discuss recent findings related to the biological role of the ACE2/Ang-(1–7)/Mas arm in the cardiovascular and renal systems, as well as in metabolism. In addition, we will highlight the potential interactions of Ang-(1–7) and Mas with AT1and AT2receptors.

Nicotinic acid (at a daily dose of grams) has been shown to induce potent anti-atherosclerotic effects in human and animal models. Evidence from clinical studies performed in the 1950s has shown that nicotinic acid treatment remarkably improves the plasma lipid profile. Large clinical studies showed that nicotinic acid improves clinical cardiovascular outcomes. Given the protective effects of niacin, basic research studies were designed to explore additional anti-atherosclerotic pathways, such as those involved in cardiovascular inflammation. After the discovery of the nicotinic acid receptor GPR109A on adipocytes and immune cells, novel direct immunomodulatory properties of nicotinic acid have been identified. Importantly, the regulation of the release of inflammatory mediators from adipose tissue was observed, independent of lipid level amelioration. Less is known about the possible direct anti-inflammatory activities of nicotinic acid in other cells (such as hepatocytes, endothelial and vascular cells) previously indicated as key players in atherogenesis. Thus, further studies are needed to clarify this promising topic. Emerging evidence from clinical and basic research studies indicates that novel direct anti-atherosclerotic properties might mediate nicotinic acid-induced cardiovascular protection. Despite some limitations in its clinical use (mainly due to the incidence of adverse events, such as cutaneous flushing and hepatotoxicity), nicotinic acid should be considered as a very potent therapeutic approach to reduce atherosclerosis. Promising research developments are warranted in the near future.

The authors review recent research on the molecular mechanisms of addiction and propose a new classification for addictive drugs.

The higher incidence of thyroid carcinoma (TC) in women during reproductive years compared with men and the increased risk associated with the therapeutic use of estrogens have suggested a pathogenetic role exerted by these steroids in the development of TC. In the present study, we evaluated the potential of 17beta-estradiol (E2), genistein (G), and 4-hydroxyta-moxifen (OHT) to regulate the expression of diverse estrogen target genes and the proliferation of human WRO, FRO, and ARO thyroid carcinoma cells, which were used as a model system. We have ascertained that ARO cells are devoid of estrogen receptors (ERs), whereas both WRO and FRO cells express a single variant of ERalpha that was neither transactivated, modulated, nor translocated into the nucleus upon treatment with ligands. However, E2, G, and OHT were able either to induce the transcriptional activity of c-fos promoter constructs, including those lacking the estrogen-responsive elements, or to increase c-fos, cyclin A, and D1 expression. It is noteworthy that we have demonstrated that the G protein-coupled receptor 30 (GPR30) and the mitogen-activated protein kinase (MAPK) pathway mediate both the up-regulation of c-fos and the growth response to E2, G, and OHT in TC cells studied, because these stimulatory effects were prevented by silencing GPR30 and using the MEK inhibitor 2'-amino-3'-methoxyflavone (PD 98059). Our findings provide new insight into the molecular mechanisms through which estrogens may induce the progression of TC.