Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Cancer Cells

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics

Analysis of HPV-16 Late Gene Expression

Human papillomaviruses (HPVs) are present in 99.7% of all cervical cancers and HPV type 16 (HPV-16) is the major cause of cervical cancer. Expression of the viral capsid gene L1 and L2 can be detected only in the terminally epithelial cells and we speculate that inhibition of HPV-16 late gene expression in the early stage of the life cycle is probably a prerequisite for persistence of infection. The products of the late genes, L1 and L2, are highly immunogenic and expression of these proteins in the lower layers of the cervical epithelium could lead to clearance of the virus. Therefore, it is of interest to understand how HPV late gene expression is regulated. The goal of this thesis was to examine the regulation of late genes in HPV-16. To this end we wished to generate reporter plasmids based on the HPV-16 genome with the L1 gene replaced by an easily measurable reporter gene, such as chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), secreted alkaline phosphatase (SEAP) or luciferase, and to establish reporter stable cell lines useful for large scale screening of small molecules or cellular factors that influence RNA processing events during late gene expression. CAT and GFP proved to be functional surrogate markers of late gene expression and their expression was dependent on the levels of known inducers of HPV-16 late gene expression such as adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB), arginine/serine-rich SRp30c protein (SRp30c) or alternative splicing factor/splicing factor 2 (ASF/SF2). Functional stable cell lines with CAT reporter plasmids, separately integrated into the HeLa cellular genome, were also generated allowing the identification of a number of small molecules capable of modulating CAT expression. Phorbol 12-myristate 13-acetate (TPA), valproic acid and tannic acid were identified as inducers of HPV-16 late gene expression. Further experiments identified the TPA inducible, hnRNP A2/B1 protein as a novel regulator of HPV-16 late gene expression. Immunohistochemical analysis of this protein in cervical epithelium at the different stages of the development of cervical cancer demonstrated that hnRNP A2/B1 is highly expressed in normal cervical epithelium and low-grade squamous intraepithelial lesion (LSIL) and decreased in highgrade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC). In conclusion, the HPV-16 reporter plasmids and reporter cell lines described herein are functional and can be used for the investigations of HPV-16 late gene expression

CaV channels and cancer: canonical functions indicate benefits of repurposed drugs as cancer therapeutics

The importance of ion channels in the hallmarks of many cancers is increasingly recognised. This article reviews current knowledge of the expression of members of the voltage-gated calcium channel family (Ca(V)) in cancer at the gene and protein level and discusses their potential functional roles. The ten members of the Ca(V) channel family are classified according to expression of their pore-forming α-subunit; moreover, co-expression of accessory α2δ, β and γ confers a spectrum of biophysical characteristics including voltage dependence of activation and inactivation, current amplitude and activation/inactivation kinetics. Ca(V) channels have traditionally been studied in excitable cells including neurones, smooth muscle, skeletal muscle and cardiac cells, and drugs targeting the channels are used in the treatment of hypertension and epilepsy. There is emerging evidence that several Ca(V) channels are differentially expressed in cancer cells compared to their normal counterparts. Interestingly, a number of Ca(V) channels also have non-canonical functions and are involved in transcriptional regulation of the expression of other proteins including potassium channels. Pharmacological studies show that Ca(V) canonical function contributes to the fundamental biology of proliferation, cell-cycle progression and apoptosis. This raises the intriguing possibility that calcium channel blockers, approved for the treatment of other conditions, could be repurposed to treat particular cancers. Further research will reveal the full extent of both the canonical and non-canonical functions of Ca(V) channels in cancer and whether calcium channel blockers are beneficial in cancer treatment

REGULATION OF NCX3 EXPRESSION BY HISTONE DEACETYLASES (HDACS) IN CORTICAL NEURONS AND IN BRAIN ISCHEMIA

Na+- Ca2+ exchanger isoform 3 (NCX3) plays a fundamental role in the pathogenesis of stroke damage. Indeed its ablation worsens the experimentally-induced ischemic damage. Interestingly it has been found that NCX3 mRNA and protein are both reduced after stroke. However, the mechanism by which stroke-induced ncx3 gene reduction is still unclear. Notably, in the last decades it has been found that histone deacetylases (HDACs) inhibition by regulating specific neuroprotective genes ameliorates the neurodegeneration that occurs in brain ischemia. Interestingly, we found that neurons treated with Trichostatin A (TSA), a pan HDACs inhibitor (HDACi), and MC1568, a class II HDACs inhibitor, significantly increased ncx3 promoter activity, whereas MS-275 (class I HDACs inhibitor) had no significant effect. Notably, among the HDACs class II A, we found that when the HDAC4 and HDAC5 isoforms were overexpressed by construct transfection or knocked-down by small interfering RNA (siRNA) transfection, NCX3 mRNA and protein levels were downregulated or increased, respectively. Moreover, experiments of site direct mutagenesis of DREAM (downstream regulatory element antagonist modulator) consensus sequence on ncx3 promoter in MC1568 treated neurons, corroborated that NCX3 downregulation induced by HDACs is achieved by DREAM. Notably, Chromatin Immunoprecipitation (ChIP) assay demonstrated that HDAC4 and HDAC5 binding on ncx3 promoter was significantly increased after transient middle cerebral artery occlusion (tMCAO). Our findings identify a new epigenetic regulatory mechanism that controls NCX3 gene transcription and demonstrated that HDAC class II A inhibition, by blocking HDAC4 and HDAC5 and modulating the acetylation of ncx3 gene promoter sequence, could be a new therapeutic strategy in stroke treatment

Investigación del splicing del pre-ARNm en el mieloma múltiple: desde su función en la patogenia a su abordaje terapeútico

Tesis por compendio de publicaciones[ES]El origen del cáncer se ha relacionado tradicionalmente con modificaciones que se producen en la secuencia del ADN de los genomas de las células cancerosas. Sin embargo, la mayoría de los cambios en la expresión de los genes no está aparentemente precedida por una alteración del ADN en forma de mutaciones, anomalías cromosómicas o incluso eventos epigenéticos. En los últimos 30 años el estudio de la expresión génica a escala global se ha utilizado extensamente para caracterizar todo tipo de neoplasias. El desarrollo de los microarrays de expresión y más recientemente de la RNA-Seq ha sido clave para disponer de una información detallada de los patrones de expresión génica de los diferentes tumores. En algunas neoplasias, el estudio del perfil de expresión génica ha permitido la identificación de subtipos, ha ayudado a mejorar la estratificación de los pacientes e incluso ha contribuido a llevar a cabo tratamientos más personalizados o dirigidos. No obstante, la mayoría de estos estudios se han centrado en investigar la expresión génica del transcrito canónico, sin tener en cuenta la expresión del resto de los transcritos o isoformas que pueden obtenerse como resultado del splicing o procesamiento del pre-ARNm a partir de un mismo gen. En este sentido, actualmente es bien conocido que las diferentes isoformas pueden expresarse de forma desigual a la isoforma canónica y desempeñar funciones distintas, incluso divergentes, interviniendo de forma muy diferente en la patogenia tumoral. De hecho, el splicing del pre-ARNm es uno de los mecanismos celulares de regulación postranscripcional más importantes. Puesto que la mayoría de los genes pueden dar lugar a múltiples transcritos una consecuencia de este procesamiento es a veces, incluso, la producción de proteínas con funciones opuestas. Este fenómeno está bien ilustrado en el hecho de que varios de los genes que codifican proteínas implicadas en rutas apoptóticas son capaces de dar lugar a isoformas pro y anti apoptóticas mediante este proceso. La expresión génica se ha analizado minuciosamente en el MM. Los primeros estudios ya mostraron que los perfiles de expresión génica de las CPs tumorales eran claramente diferentes a los de las CPs normales de donantes sanos, y que dentro del grupo de los mielomas se podían encontrar subgrupos moleculares más parecidos a las GMSI, y otros similares a las líneas celulares de MM302. Posteriormente, se elaboró una clasificación molecular del MM basada en los perfiles de expresión génica que identificaba siete subgrupos distintos52. Estas subentidades moleculares con algunas modificaciones se han confirmado en numerosos estudios posteriores, no solo utilizando microarrays de expresión sino también mediante RNA-Seq. En las neoplasias de CPs, al igual que sucede en otros cánceres, la investigación del splicing del pre-ARNm y de la variedad de isoformas generadas mediante este mecanismo, ha sido muy limitada, tanto a nivel del transcriptoma completo como enfocándose en genes particulares. Por este motivo, hemos pensado que resultaría interesante avanzar en el estudio de cómo este mecanismo de regulación postranscripcional puede influir en la patogenia del MM y otras discrasias de células plasmáticas, e incluso averiguar si la modulación farmacológica del spliceosoma podría servir como abordaje terapéutico. Hasta ahora no se ha descrito ninguna alteración cromosómica ni mutación o desregulación significativa de un gen o ruta molecular particular que esté presente en la LCPp, la forma más agresiva de las neoplasias de CPs, y no lo esté en el MM. Esto indica que otros procesos biológicos pueden ser los responsables de la trasformación de la célula plasmática en una célula tumoral mucho más agresiva e incontrolable que la del MM. La búsqueda de diferencias, tanto en el patrón de splicing como en la expresión de las distintas isoformas, entre el MM y la LCPp podría ayudarnos a identificar posibles mecanismos moleculares involucrados en el desarrollo de la LCPp, y que no sean dependientes de la presencia de las alteraciones genéticas ya conocidas. TP53 es un conocido gen supresor tumoral que ha sido ampliamente estudiado. Concretamente el splicing del pre-ARNm es uno de los mecanismos postranscripcionales que ha emergido en los últimos años como uno de los procesos más relevantes en la regulación de p53. Se han descrito al menos 12 isoformas proteicas codificadas a partir de 9 ARNm (TAp53α, TAp53β, TAp53γ, Δ40p53α, Δ40p53β, Δ40p53γ, Δ133p53α, Δ133p53β, Δ133p53γ, Δ160p53α, Δ160p53β y Δ160p53γ). Algunas de las isoformas difieren entre sí en los dominios de transactivación y en el dominio C-terminal, lo que les permite regular diferencialmente la expresión de diferentes genes dianas y ser reguladas también de forma variable por los reguladores de p53. Varios trabajos han demostrado que la alteración de los niveles de expresión de las isoformas de p53 puede inhibir o potenciar su propia actividad como supresor tumoral en diferentes neoplasias y por tanto, influir en su respuesta al tratamiento y pronóstico. La expresión de las isoformas de p53 no se ha investigado aún en el MM. Por este motivo, el análisis de las isoformas de p53, tanto a nivel de proteína como de ARNm, en muestras de pacientes con MM tratados homogéneamente en el marco de un ensayo clínico, podría proporcionar información novedosa de su valor pronóstico. Actualmente se están desarrollando estrategias terapéuticas dirigidas a modular la maquinaria del splicing con resultados esperanzadores. De hecho, existen una serie de fármacos que modifican los patrones de splicing del pre-ARNm, cuyo mecanismo de acción es bastante desconocido. Algunos de ellos se están investigando como drogas antitumorales en ensayos clínicos. En este sentido, se cree que la investigación del efecto de estos agentes en las células mielomatosas sería útil para ampliar el espectro de fármacos con potencial utilidad en el MM y además, para descifrar los mecanismos moleculares del proceso de splicing y de su regulación en el MM. Los objetivos que se plantean a la hora de abordar este trabajo son: Objetivo 1: Analizar el transcriptoma de casos de LCPp y de MM que comparten un fondo genético similar y compararlos entre sí. • Analizar el perfil de expresión génica de muestras de LCPp y compararlo con el de las muestras de MM. • Explorar los eventos de splicing alternativo en las LCPp en relación a los observados en el MM. • Identificar las isoformas de ARNm diferencialmente expresadas entre la LCPp y el MM. • Analizar la existencia de sitios reguladores de splicing alternativo en ambas neoplasias de CPs mediante análisis bioinformáticos. Objetivo 2: Evaluar la expresión de las isoformas de p53 en pacientes con MM. • Identificar y cuantificar las isoformas proteicas de p53 en pacientes con MM. • Analizar el efecto de la expresión de las isoformas proteicas de p53 en el pronóstico de los pacientes. • Examinar los patrones de expresión de las isoformas de p53 a nivel de ARNm y compararlo con lo observado a nivel de proteínas. Objetivo 3: Investigar si la modulación farmacológica del spliceosoma, mediante la amilorida, puede servir como abordaje terapéutico en el MM. • Analizar el efecto citotóxico in vitro e in vivo de la amilorida en el MM. • Estudiar el mecanismo de acción de la amilorida en las células del MM

Characterizaton of the role played by NCX isoform 3 in a transgenic model of Alzheimer's disease by electrophysiological and biochemical studies

Alzheimer’s disease (AD), the most common neurodegenerative disorder is characterized by progressive memory loss and impairment of cognitive ability. Aβ1-42 deposition, the principal hallmark of AD, triggers several mechanisms, including dysregulation of ionic homeostasis, contributing to neuronal dysfunction and death. In particular, the dysregulation of intracellular calcium concentrations ([Ca2+]i) triggers a series of events including oxidative damage and activation of apoptotic machinery. Furthermore, the dysregulation of intracellular sodium concentrations ([Na+]i) affects neuronal excitability and contributes to epileptogenesis in AD. The Na+/Ca2+ exchanger (NCX) couples in a bidirectional manner the exchange of 3Na+ for 1Ca2+, thereby playing a relevant role in maintaining intracellular Na+ and Ca2+ homeostasis. For this reason, we investigated the role of NCX3 in Aβ1-42-induced ionic dysregulation in primary hippocampal neurons from Tg2576 mice, a transgenic animal model of AD. First, we validated primary hippocampal neurons from Tg2576 mice, here set up for the first time, as an in vitro model of AD by confirming the presence of Aβ1-42 oligomers through western blot experiments. In particular, we observed Aβ1-42 trimers, detectable as a ~ 12 kDa band, in Tg2576 primary hippocampal neurons, whereas they were absent in Wild Type (WT) neurons. Importantly, the same band has been detected in the hippocampus of 3-month-old Tg2576 mice. Patch clamp experiments revealed that NCX activity was progressively up-regulated in the reverse mode of operation in Tg2576 hippocampal neurons compared to WT, at 8 and 12 DIV, whereas no modulation occurred in the forward mode. Furthermore, silencing experiments with a specific siRNA directed against NCX3, revealed that this increase of NCX currents was mediated by only NCX isoform 3. However, as revealed by western blot analyses, the up-regulation of NCX3 activity was not accompanied by a significant increase of NCX3 protein expression. Interestingly, [Na+] detection with SBFI probe showed a significant increase of [Na+]i in Tg2576 hippocampal neurons at 12 DIV compared to WT, thus indicating that the up-regulation of NCX activity was Na+-dependent. Moreover, electrophysiological experiments revealed that NaV currents were progressively up-regulated in Tg2576 hippocampal neurons compared to WT at 8 and 12 DIV. To determine whether the up-regulation of NCX activity results in increased [Ca2+]i or rather in Ca2+ refilling into ER, we performed Fura-2 AM measurements to determine both [Ca2+]i and ER Ca2+ content. In particular, we found a significant reduction of [Ca2+]i in Tg2576 hippocampal neurons at 8 DIV compared to WT and a significant increase in ER Ca2+ content at 12 DIV. Western blot on Tg2576 mouse brain, revealed that NCX3 protein expression was significantly increased in the hippocampus of 3-month-old Tg2576 mice compared to WT. Importantly, immunohistochemical analyses confirmed western blot results. In fact, in both CA1 and CA3 hippocampal regions as well as within the corpus callosum of 3-month-old Tg2576 mice, the anti-NCX3 antibody revealed an increased NCX3 immunoreactivity signal, which was mainly confined along the processes of cells and dendrites of pyramidal cells. By contrast, we observed a significant reduction of NCX3 protein expression in the hippocampus of 8-month-old Tg2576 compared to WT, although an increase in the hippocampus of 8-month-old WT mice has been observed in comparison with 3-month-old WT mice. Importantly, immunohistochemical analyses confirmed western blot results. In fact, in both CA1 and CA3 hippocampal regions as well as within the corpus callosum of 8-month-old Tg2576 mice, NCX3 immunostaining appeared robustly decreased. Moreover, western blot experiments did not detect any modulation of NCX3 protein expression in the cerebral cortex of both 3 and 8-month-old Tg2576 mice compared to WT. On the other hand, a clear loss of intensity of immunoreactivity signal has been observed in cortical sections from both 3 and 8-month-old Tg2576 mice. All together, these results suggest that NCX3 up-regulation could represent a protective mechanism against Na+ disruption occurring in Tg2576 hippocampal neurons. Notably, this evidence points to a possible role of NCX3 in neuronal survival against hyperexcitability and subsequent epileptiform activity observed in AD. Moreover, the role of NCX3 in Ca2+ refilling into ER further supports its positive implication

Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis

Tese de doutoramento, Bioquímica (Genética Molecular), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic Fibrosis (CF) is the most common autosomal disease in Caucasians, with an estimated incidence of 1:6000 births in Portugal. The most relevant clinical aspect of its classic manifestation is chronic lung disease, which is the main cause of morbidity and mortality. Other symptoms include, pancreatic dysfunction, male infertility and high concentrations of chloride (Cl-) in sweat. However, even though the classic form of the disease is well defined, its pathophysiology is not completely understood with this pleiotropic disease having highly variable manifestations of clinical phenotypes. Novel therapies aim to correct the basic defect, specifically focusing on the rescue of Cystic fibrosis transmembrane conductance regulator (CFTR) function in CF airways. Most of these CFTR modulator strategies target the F508del, the most common mutation. Nevertheless, widespread evidence has demonstrated that a significant number of CFcausing mutations affect splicing efficiency and the stability of mRNA molecules. Here, we propose to elucidate the regulatory mechanisms underlying these CF-associated mutations. To this end, our aims are: 1) to identify CFTR gene mutations in individuals with non-CF chronic lung diseases, namely chronic obstructive pulmonary disease (COPD), asthma and disseminated bronchiectasis (DB); 2) to identify CFTR gene mutations in individuals with a suspicion of CF, followed by the analysis of CFTR expression in their native tissues to characterize the impact of CFTR splicing or premature termination codon (PTC) mutations in the structure and levels of mRNA; 3) to identify key factors in the nonsense/mediated decay (NMD) pathway by automated microscopy screens using a cell model expressing a novel CFTR NMD-PTC/read-through mini-gene reporter; and 4) to screen for novel compounds suppressing PTC mutations by automated microscopy screens using the previous cell-based model, as potential corrective therapies for CF. The expected results will provide knowledge on RNA-processing dysfunction and on the efficacy of novel RNA modulator compounds towards a "personalised medicine" approach. Regarding the first objective, our data show that 7 (out of 136) patients with non-CF respiratory diseases presented CFTR mutations in one allele, in contrast with the control group, in which no mutations were detected. We analysed the association of CFTR gene mutations with each of the three respiratory diseases considered. For asthma our data did not show an increase in mutation frequency when compared to the control group. For DB, we found an increase in the frequency of CFTR gene mutations, albeit with no statistical significance, which is in agreement with previous reports. For COPD however, we found a statistically significant increase in CFTR gene mutation incidence, relative to the control group. Our data reinforce the importance of characterizing CFTR gene mutations on non-CF respiratory diseases in Portuguese patients, to gain a better understanding of the epidemiology and etiology of these diseases. The results also lead to the identification of groups of patients who may benefit from the new therapeutic compounds currently under development to correct the basic CFTR protein defect in CF. Concerning the second objective, we have developed a novel RNA-based approach to detect unknown CFTR mutations [Felício et al (2017) Clin Genet 91: 476-481]. We are currently using this protocol for patients with a suspicion of CF and none or just one CFTR mutation identified. With this method we identified one mutation (711+3A>T) which had been previously reported but had not been characterized. We can conclude that this is a rapid, robust and inexpensive method to detect rare mutations, and therefore a method that can be easily used after a first screen. Regarding the third objective, we used this CFTR-NMD reporter to identify novel NMD factors by screening a previously validated shRNA library – a subset of The RNAi Consortium (TRC) – which is enriched in shRNAs targeting genes with a known or predicted involvement in transcript processing (425 genes), using HT microscopy. We selected the 24 top hits for the confirmation: 11 genes with NMD score ≥ 2 and more than 2 shRNAs with the same phenotype; 2 genes from the screen that showed read-through activity; and 11 other genes resulting from a high-throughput screen (HTS) aimed at the identification of CFTR splicing regulators (unpublished data). We chose these genes related to splicing because this is a process known to be required for NMD to occur and thus the knock-down of such genes can lead to NMD inhibition. The confirmation screen was performed using a library of siRNAs targeting the previously selected genes, however the results were inconclusive due to the low NMD score obtained. We have identified 4 genes with higher values but NMD score ≤ 1, three (eIF4A3, SREK1 and RPS19) are related to splicing, elF4A3 is directly and RPS19 is indirectly related to NMD, SREK1 is related only to splicing, with the fourth gene, ADIRF, having unknown functional properties. Some of the hits identified within this screen may be potential drug targets by their effects in inhibiting NMD, when knocked-down. With the results obtained in the confirmation screen we decided to follow the 24 genes identified in the primary screen for validation studies using two different techniques: WB and qRT-PCR (study in progress). Lastly, the fourth objective was to restore functional protein production to PTC mutations using read-through compounds, with the ultimate aim of CF patient treatment. The CFTRNMD construct used has the G542X nonsense mutation, mCherry at the N terminus and eGFP fused at the CFTR C-terminus. Through the screen of the library, we identified new small-molecule compounds that induced PTC read-through. To confirm the read-through efficiency future experiments are needed using additional techniques, such as, WB and transcript analysis by semi-quantitative PCR and qRT-PCR. Finally, to validate the top hit compounds, it is necessary to test them in patient’s materials, including nasal primary cells for functional activity and intestinal organoids to determine a dose response and to test these compounds in combination with potentiators and correctors. The studies presented in this dissertation had the overall aims of advancing the current knowledge on RNA-processing dysfunction and of identifying novel RNA modulator compounds towards a "personalised medicine" approach. The results obtained have indeed provided new insights into: 1) the relationship of other respiratory diseases with the presence of CFTR mutations; 2) new approaches to detect CFTR mutations in DNA and RNA; and 3) our understanding of key factors in NMD and read-through activity in relation to CFTR nonsense mutations

The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology

The last decade has witnessed tremendous progress in the understanding of the mineralocorticoid receptor (MR), its molecular mechanism of action, and its implications for physiology and pathophysiology. After the initial cloning of MR, and identification of its gene structure and promoters, it now appears as a major actor in protein-protein interaction networks. The role of transcriptional coregulators and the determinants of mineralocorticoid selectivity have been elucidated. Targeted oncogenesis and transgenic mouse models have identified unexpected sites of MR expression and novel roles for MR in non-epithelial tissues. These experimental approaches have contributed to the generation of new cell lines for the characterization of aldosterone signaling pathways, and have also facilitated a better understanding of MR physiology in the heart, vasculature, brain and adipose tissues. This review describes the structure, molecular mechanism of action and transcriptional regulation mediated by MR, emphasizing the most recent developments at the cellular and molecular level. Finally, through insights obtained from mouse models and human disease, its role in physiology and pathophysiology will be reviewed. Future investigations of MR biology should lead to new therapeutic strategies, modulating cell-specific actions in the management of cardiovascular disease, neuroprotection, mineralocorticoid resistance, and metabolic disorders

Defining the organizational structure of dopamine and muscarninic acetylcholine receptors

No abstract available