RESUMEN
Wnt signalling has been implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Here, using a unique intravital imaging platform and fluorescent reporters, we visualised ß-catenin/TCF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double-reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings might inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis.
Asunto(s)
Neoplasias , beta Catenina , Animales , Embrión de Pollo , Humanos , beta Catenina/metabolismo , Vía de Señalización Wnt , Neoplasias/genética , Línea Celular Tumoral , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismoRESUMEN
CD14 is an innate immune receptor that senses pathogen-associated molecular patterns, such as lipopolysaccharide, to activate the innate immune response. Although CD14 is known to be glycosylated, detailed understanding about the structural and functional significance of this modification is still missing. Herein, an NMR and MS-based study, assisted by MD simulations, has provided a 3D-structural model of glycosylated CD14. Our results reveal the existence of a key N-glycosylation site at Asn282 that exclusively contains unprocessed oligomannnose N-glycans that perfectly fit the concave cavity of the bent-solenoid shaped protein. This site is not accessible to glycosidases and is fundamental for protein folding and secretion. A second N-site at Asn151 displays mostly complex N-glycans, with the typical terminal epitopes of the host cell-line expression system (i.e. ßGal, α2,3 and α2,6 sialylated ßGal, here), but also particularities, such as the lack of core fucosylation. The glycan at this site points outside the protein surface, resulting in N-glycoforms fully exposed and available for interactions with lectins. In fact, NMR experiments show that galectin-4, proposed as a binder of CD14 on monocytes to induce their differentiation into macrophages-like cells, interacts in vitro with CD14 through the recognition of the terminal glycoepitopes on Asn151. This work provides key information about CD14 glycosylation, which helps to better understand its functional roles and significance. Although protein glycosylation is known to be dynamic and influenced by many factors, some of the features found herein (presence of unprocessed N-glycans and lack of core Fuc) are likely to be protein specific.
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Lectinas , Polisacáridos , Glicosilación , Polisacáridos/química , Lectinas/metabolismo , Línea Celular , Lipopolisacáridos/metabolismoRESUMEN
A significant number of breast cancers develop resistance to hormone therapy. This progression, while posing a major clinical challenge, is difficult to predict. Despite important contributions made by cell models and clinical studies to tackle this problem, both present limitations when taken individually. Experiments with cell models are highly reproducible but do not reflect the indubitable heterogenous landscape of breast cancer. On the other hand, clinical studies account for this complexity but introduce uncontrolled noise due to external factors. Here, we propose a new approach for biomarker discovery that is based on a combined analysis of sequencing data from controlled MCF7 cell experiments and heterogenous clinical samples that include clinical and sequencing information from The Cancer Genome Atlas. Using data from differential gene expression analysis and a Bayesian logistic regression model coupled with an original simulated annealing-type algorithm, we discovered a novel 6-gene signature for stratifying patient response to hormone therapy. The experimental observations and computational analysis built on independent cohorts indicated the superior predictive performance of this gene set over previously known signatures of similar scope. Together, these findings revealed a new gene signature to identify patients with breast cancer with an increased risk of developing resistance to endocrine therapy.
Asunto(s)
Neoplasias de la Mama , Perfilación de la Expresión Génica , Humanos , Femenino , Teorema de Bayes , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Hormonas/uso terapéutico , PronósticoRESUMEN
Tissue culture has evolved considerably over the last few years, including cell culture in three dimensions, organoids, cocultures of different cell types and the use of diverse types of matrices in an attempt to mimic conditions that more closely resemble those found in the original tissue or organ. In this chapter, we describe how patient-derived breast tissue can be cultured on sponges for several days, maintaining their original architecture and with the capacity to respond to treatments. This protocol facilitates the study of the tissue responses without the need for extensive tissue manipulation, cell digestion or use of a biomaterial as scaffold, while maintaining the stroma and extracellular matrix organization. This method has the potential to improve preclinical testing by contributing to provide more accurate data reflecting cell-cell and cell-matrix interactions, tumor microenvironment, drug effects or stem cell function in normal- and pathophysiology of the breast.
Asunto(s)
Neoplasias de la Mama , Organoides , Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula/métodos , Femenino , Humanos , Organoides/metabolismo , Células Madre , Microambiente TumoralRESUMEN
The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at lysine 4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus of the five ING proteins. ING5 facilitates histone H3 acetylation by the HBO1 complex, and also H4 acetylation by the MOZ/MORF complex. We show that ING5 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING5, which contains the nuclear localization sequence, is flexible and disordered, but it binds dsDNA with micromolar affinity. NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule, and they bind H3K4me3 in the same way as the isolated PHD. We have observed that ING5 can form heterodimers with the highly homologous ING4, and that two of three primary tumor-associated mutants in the N-terminal domain strongly destabilize the coiled-coil structure. They also affect cell proliferation and cell cycle phase distribution, suggesting a driver role in cancer progression.
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Histonas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Histonas/química , Humanos , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Alineación de Secuencia , Factores de Transcripción/química , Proteínas Supresoras de Tumor/químicaRESUMEN
Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Mama/metabolismo , Resistencia a Antineoplásicos , Células Madre Neoplásicas/metabolismo , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción SOXB1/metabolismo , Mama/citología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular , Proliferación Celular , Células Epiteliales/citología , Estrógenos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Factor de Transcripción SOX9/genética , Transducción de Señal , Tamoxifeno/farmacología , Regulación hacia ArribaRESUMEN
Aberrant transforming growth factor-ß (TGF-ß) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-ß signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-ß signaling activity and that stromal cell-conditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-ß-induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment.
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Proteínas de la Matriz Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias de la Próstata/patología , Factor de Crecimiento Transformador beta1/metabolismo , Microambiente Tumoral/fisiología , Proteínas Adaptadoras Transductoras de Señales , Quimiocinas , Humanos , Masculino , Neoplasias de la Próstata/metabolismoRESUMEN
Fluorescent-activated cell sorting (FACS) represents one of the key techniques that have been used to isolate and characterize stem cells, including cells from the mammary gland. A combination of approaches, including recognition of cell surface antigens and different cellular activities, has facilitated the identification of stem cells from the healthy mammary gland and from breast tumors. In this chapter we describe the protocol to use FACS to separate breast cancer stem cells, but most of the general principles discussed could be applied to sort other types of cells.
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Neoplasias de la Mama/metabolismo , Citometría de Flujo , Células Madre Neoplásicas/metabolismo , Animales , Antígenos de Superficie/metabolismo , Neoplasias de la Mama/patología , Separación Celular/métodos , Citometría de Flujo/métodos , Humanos , Inmunofenotipificación/métodosRESUMEN
The heterogeneous nature of breast cancer is a result of intrinsic tumor complexity and also of the tumor microenvironment, which is known to be hypoxic. We found that hypoxia expands different breast stem/progenitor cell populations (cells with increased aldehyde dehydrogenase activity (Aldefluor+), high mammosphere formation capacity and CD44+CD24-/low cells) both in primary normal epithelial and tumor cells. The presence of the estrogen receptor (ER) limits hypoxia-dependent CD44+CD24-/low cell expansion.We further show that the hypoxia-driven cancer stem-like cell enrichment results from a dedifferentiation process. The enhanced mammosphere formation and Aldefluor+ cell content observed in breast cancer cells relies on hypoxia-inducible factor 1α (HIF1α). In contrast, the CD44+CD24-/low population expansion is HIF1α independent and requires prolyl hydroxylase 3 (PHD3) downregulation, which mimics hypoxic conditions, leading to reduced CD24 expression through activation of NFkB signaling. These studies show that hypoxic conditions expand CSC populations through distinct molecular mechanisms. Thus, potential therapies that combine current treatments for breast cancer with drugs that target CSC should take into account the heterogeneity of the CSC subpopulations.
Asunto(s)
Neoplasias de la Mama/patología , Diferenciación Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Hipoxia/fisiopatología , Células Madre Neoplásicas/patología , Adulto , Apoptosis , Mama/citología , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Antígeno CD24/genética , Antígeno CD24/metabolismo , Proliferación Celular , Células Cultivadas , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Células Madre Neoplásicas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Adulto JovenRESUMEN
We show here for the first time that sphingosine-1-phosphate (Sph-1-P) stimulates cortisol secretion in zona fasciculata cells of bovine adrenal glands. This effect was dependent upon protein kinase C (PKC) and extracellular Ca2+, and was inhibited by pertussis toxin. Sph-1-P activated phospholipase D (PLD) through a pertussis toxin-sensitive mechanism, also involving extracellular Ca2+ and PKC. Primary alcohols, which attenuate formation of phosphatidic acid (the product of PLD), and cell-permeable ceramides, which inhibit PLD, blocked Sph-1-P-induced cortisol secretion. In conclusion, Sph-1-P stimulates cortisol secretion through a mechanism involving Gi/o protein-coupled receptors, extracellular Ca2+, PKC and PLD.
Asunto(s)
Hidrocortisona/metabolismo , Lisofosfolípidos , Esfingosina/análogos & derivados , Esfingosina/farmacología , Zona Fascicular/efectos de los fármacos , Zona Fascicular/metabolismo , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Ceramidas/farmacología , Quelantes/farmacología , Activación Enzimática/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Lípidos/farmacología , Toxina del Pertussis/farmacología , Ácidos Fosfatidicos/biosíntesis , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Zona Fascicular/citologíaRESUMEN
Angiotensin II (Ang-II) regulates a variety of cellular functions including cortisol secretion. In the present report, we demonstrate that Ang-II activates phospholipase D (PLD) in zona fasciculata (ZF) cells of bovine adrenal glands, and that this effect is associated to the stimulation of cortisol secretion by this hormone. PLD activation was dependent upon extracellular Ca2+, and was blocked by inhibition of protein kinase C (PKC). Using the reverse transcription-polymerase chain reaction technique, we demonstrated that ZF cells express both PLD-1 and PLD-2 isozymes. Primary alcohols, which attenuate the formation of phosphatidate (the product of PLD), and cell-permeable ceramides, which inhibit PLD potently, blocked Ang-II-stimulated cortisol secretion. Furthermore, propranolol or chlorpromazine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked cortisol secretion. These data suggest that the PLD/PAP pathway plays an important role in the regulation of cortisol secretion by Ang-II in ZF cells.
Asunto(s)
Angiotensina II/farmacología , Hidrocortisona/metabolismo , Fosfolipasa D/metabolismo , Vasoconstrictores/farmacología , Zona Fascicular/efectos de los fármacos , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/metabolismo , Animales , Calcio/metabolismo , Bovinos , Ceramidas/metabolismo , Inhibidores Enzimáticos/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfatidato Fosfatasa/antagonistas & inhibidores , Fosfolipasa D/genética , Proteína Quinasa C/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Zona Fascicular/citología , Zona Fascicular/enzimologíaRESUMEN
Dickkopf-3 (Dkk-3) and Dkkl-1 (Soggy) are secreted proteins of poorly understood function that are highly expressed in subsets of neurons in the brain. To explore their potential roles during neuronal development, we examined their expression in Ntera-2 (NT2) human embryonal carcinoma cells, which differentiate into neurons upon treatment with retinoic acid (RA). RA treatment increased the mRNA and protein levels of Dkk-3 but not of Dkkl-1. Ectopic expression of both Dkk-3 and Dkkl-1 induced apoptosis in NT2 cells. Gene silencing of Dkk-3 did not affect NT2 cell growth or differentiation but altered their response to RA in suspension cultures. RA treatment of NT2 cells cultured in suspension resulted in morphological changes that led to cell attachment and flattening out of cell aggregates. Although there were no significant differences in the expression levels of cell adhesion molecules in control and Dkk-3-silenced cells, this morphological response was not observed in Dkk-3-silenced cells. These findings suggest that Dkk-3 plays a role in the regulation of cell interactions during RA-induced neuronal differentiation.
Asunto(s)
Células Madre de Carcinoma Embrionario/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Moduladores de la Mitosis/farmacología , Neurogénesis/fisiología , Tretinoina/farmacología , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/fisiología , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular Tumoral , Quimiocinas , Células Madre de Carcinoma Embrionario/citología , Células Madre de Carcinoma Embrionario/efectos de los fármacos , Silenciador del Gen , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Neurogénesis/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , TransfecciónRESUMEN
Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo and radiotherapy. Here, we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifenresistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely, ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2expressing cells, and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure, and also in the primary tumours of these patients, compared to those of responders. Together, these results suggest that development of tamoxifen resistance is driven by Sox2dependent activation of Wnt signalling in cancer stem/progenitor cells.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Factores de Transcripción SOXB1/metabolismo , Tamoxifeno/uso terapéutico , Animales , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Retículo Endoplásmico/metabolismo , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Interferencia de ARN , Recurrencia , Factores de Transcripción SOXB1/antagonistas & inhibidores , Factores de Transcripción SOXB1/genética , Análisis de Supervivencia , Tamoxifeno/farmacología , Trasplante Heterólogo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by selective motoneurons degeneration. There is today no clear-cut pathogenesis sequence nor any treatment. However growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. To better characterize the time course of pathological events in an animal model that recapitulates human ALS symptoms, we investigated functional and cellular characteristics of hSOD1(G93A) mice. METHODS AND FINDINGS: We have evaluated locomotor function of hSOD1(G93A) mice through dynamic walking patterns and spontaneous motor activity analysis. We detected early functional deficits that redefine symptoms onset at 60 days of age, i.e. 20 days earlier than previously described. Moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. To tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. We show that (1) decrease in neuromuscular junction's number correlates with motor impairment, (2) astrocytes number is not altered at pre- and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and (3) microglia modifications precede disease onset. At pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge. CONCLUSIONS: In conclusion, precise motor analysis updates the onset of the disease in hSOD1(G93A) mice and allows locomotor monitoring until the end stage of the disease. Early functional deficits coincide with alterations of neuromuscular junctions. Importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. This finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets.
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Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Microglía/patología , Animales , Astrocitos/patología , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Ratones , Ratones Transgénicos , Actividad Motora/fisiología , Mutación/genética , Unión Neuromuscular/patología , Unión Neuromuscular/fisiopatología , Médula Espinal/patología , Médula Espinal/fisiopatología , Superóxido Dismutasa/genética , Factores de TiempoRESUMEN
We reported recently that sphingosine-1-phosphate (S1P) is a novel regulator of aldosterone secretion in zona glomerulosa cells of adrenal glands and that phospholipase D (PLD) is implicated in this process. We now show that S1P causes the phosphorylation of protein kinase B (PKB) and extracellularly regulated kinases 1/2 (ERK 1/2), which is an indication of their activation, in these cells. These effects are probably mediated through the interaction of S1P with the Gi protein-coupled receptors S1P1/3, as pretreatment with pertussis toxin or with the S1P1/3 antagonist VPC 23019 completely abolished the phosphorylation of these kinases. Inhibitors of phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) blocked S1P-stimulated aldosterone secretion. This inhibition was only partial when the cells were incubated independently with inhibitors of each pathway. However, aldosterone output was completely blocked when the cells were pretreated with LY 294002 and PD 98059 simultaneously. These inhibitors also blocked PLD activation, which indicates that this enzyme is downstream of PI3K and MEK in this system. We propose a working model for S1P in which stimulation of the PI3K/PKB and MEK/ERK pathways leads to the stimulation of PLD and aldosterone secretion.
Asunto(s)
Aldosterona/metabolismo , Lisofosfolípidos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingosina/análogos & derivados , Animales , Bovinos , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Modelos Biológicos , Toxina del Pertussis/farmacología , Fosforilación , Esfingosina/metabolismo , Zona Glomerular/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid capable of regulating critical physiological and pathological functions. Here, we report for the first time that S1P stimulates aldosterone secretion in cells of the zona glomerulosa of the adrenal gland. Regulation of aldosterone secretion is important because this hormone controls electrolyte and fluid balance and is implicated in cardiovascular homeostasis. S1P-stimulated aldosterone secretion was dependent upon the protein kinase C (PKC) isoforms alpha and delta and extracellular Ca2+, and it was inhibited by pertussis toxin (PTX). S1P activated phospholipase D (PLD) through a PTX-sensitive mechanism, also involving PKC alpha and delta and extracellular Ca2+. Primary alcohols, which attenuate the formation of phosphatidic acid (the product of PLD), and cell-permeable ceramides, which inhibit PLD activity, blocked S1P-stimulated aldosterone secretion. Furthermore, propranolol, chlorpromazine, and sphingosine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked aldosterone secretion. These data suggest that the PLD/PAP pathway plays a crucial role in the regulation of aldosterone secretion by S1P and that Gi protein-coupled receptors, extracellular Ca2+, and the PKC isoforms alpha and delta are all important components in the cascade of events controlling this process.