RESUMEN

In cardiac myocytes, the type 2a sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) plays a key role in intracellular Ca regulation. Due to its critical role in heart function, SERCA2a activity is tightly regulated by different mechanisms, including micropeptides. While phospholamban (PLB) is a well-known SERCA2a inhibitor, dwarf open reading frame (DWORF) is a recently identified SERCA2a activator. Since PLB phosphorylation is the most recognized mechanism of SERCA2a activation during adrenergic stress, we studied whether PLB phosphorylation also affects SERCA2a regulation by DWORF. By using confocal Ca imaging in a HEK293 expressing cell system, we analyzed the effect of the co-expression of PLB and DWORF using a bicistronic construct on SERCA2a-mediated Ca uptake. Under these conditions of matched expression of PLB and DWORF, we found that SERCA2a inhibition by non-phosphorylated PLB prevails over DWORF activating effect. However, when PLB is phosphorylated at PKA and CaMKII sites, not only PLB's inhibitory effect was relieved, but SERCA2a was effectively activated by DWORF. Förster resonance energy transfer (FRET) analysis between SERCA2a and DWORF showed that DWORF has a higher relative affinity for SERCA2a when PLB is phosphorylated. Thus, SERCA2a regulation by DWORF responds to the PLB phosphorylation status, suggesting that DWORF might contribute to SERCA2a activation during conditions of adrenergic stress.

Asunto(s)

Proteínas de Unión al Calcio , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Humanos , Fosforilación , Células HEK293 , Sistemas de Lectura Abierta/genética , Calcio/metabolismo , Activación Enzimática , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo

RESUMEN

Plant Elicitor Peptides (Peps) induce plant immune responses and inhibit root growth through their receptors PEPR1 and PEPR2, two receptor-like kinases. In our study, we found a previously unknown function of Peps that enhance root hair growth in a PEPRs-independent manner. When we characterized the expression patterns of PROPEP genes, we found several gene promoters of PROPEP gene family were particularly active in root hairs. Furthermore, we observed that PROPEP2 is vital for root hair development, as disruption of PROPEP2 gene led to a significant reduction in root hair density and length. We also discovered that PROPEP2 regulates root hair formation via the modulation of CPC and GL2 expression, thereby influencing the cell-fate determination of root hairs. Additionally, calcium signaling appeared to be involved in PROPEP2/Pep2-induced root hair growth. These findings shed light on the function of Peps in root hair development.

RESUMEN

2-aminoethoxydiphenyl borate (2-APB) is commonly used as a tool to modulate calcium signaling in physiological studies. 2-APB has a complex pharmacology and acts as activator or inhibitor of a variety of Ca2+ channels and transporters. While unspecific, 2-APB is one of the most-used agents to modulate store-operated calcium entry (SOCE) mediated by the STIM-gated Orai channels. Due to its boron core structure, 2-APB tends to readily hydrolyze in aqueous environment, a property that results in a complex physicochemical behavior. Here, we quantified the degree of hydrolysis in physiological conditions and identified the hydrolysis products diphenylborinic acid and 2-aminoethanol by NMR. Notably, we detected a high sensitivity of 2-APB/diphenylborinic acid towards decomposition by hydrogen peroxide to compounds such as phenylboronic acid, phenol, and boric acid, which were, in contrast to 2-APB itself and diphenylborinic acid, insufficient to affect SOCE in physiological experiments. Consequently, the efficacy of 2-APB as a Ca2+ signal modulator strongly depends on the reactive oxygen species (ROS) production within the experimental system. The antioxidant behavior of 2-APB towards ROS and its resulting decomposition are inversely correlated to its potency to modulate Ca2+ signaling as shown by electron spin resonance spectroscopy (ESR) and Ca2+ imaging. Finally, we observed a strong inhibitory effect of 2-APB, i.e., its hydrolysis product diphenylborinic acid, on NADPH oxidase (NOX2) activity in human monocytes. These new 2-APB properties are highly relevant for Ca2+ and redox signaling studies and for pharmacological application of 2-APB and related boron compounds.

Asunto(s)

Canales de Calcio , Señalización del Calcio , Humanos , Canales de Calcio/metabolismo , NADPH Oxidasa 2 , Especies Reactivas de Oxígeno/farmacología , Calcio/metabolismo

RESUMEN

BACKGROUND: Agents targeting the PI3K pathway in triple negative breast cancer did not show any significant efficacy so far mostly because of the complex nature of these targeted inhibitors. Targeting the cancer cells with the combination of inhibitors may help in decelerating the regulatory pathways further achieving optimum clinical benefit. In this study, we investigated the effect of PIK3CA and mTOR inhibition in-vitro in triple-negative breast cancer (TNBC) cell lines. OBJECTIVE AND METHODS: Three TNBC cell lines; MDA MB231, MDA MB468, and MDA MB453 were subtyped using immunohistochemistry and were screened for hotspot mutations in PIK3CA and AKT1. All cell lines were treated with different concentrations of inhibitors; PI3K inhibitor (BKM 120), mTOR inhibitor (AZD 8055), and dual PI3K/mTOR inhibitor (BEZ 235), and cell viability was assessed by MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide), Trypan blue and Annexin-V/PI Assays. RESULTS: Using immunohistochemistry, TNBC cell lines were subtyped as; mesenchymal subtype-specific cell line (MDA MB231), basal subtype-specific cell line (MDA MD468), and Luminal androgen receptor (LAR) subtype-specific cell line (MDA MB453). PIK3CA hot spot mutation (p.H1047R) in exon 20 was identified in the Luminal androgen receptor subtype (MDA MB453 cells) cell line. Cell viability assays showed that the Mesenchymal subtype-specific cell line (MDA MB231) was the most resistant to all inhibitors and the Luminal Androgen subtype (MDA MB453 cells) cell line was more sensitive to BKM120 (PI3K inhibitor) inhibition compared to other subtypes. CONCLUSIONS: This study identified that the Luminal androgen receptor subtype of triple-negative breast cancer with PIK3CA mutation may be targeted with PIK3CA inhibitors with a favorable outcome.

Asunto(s)

Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/uso terapéutico , Femenino , Humanos , Inhibidores mTOR , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/farmacología , Receptores Androgénicos/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética

RESUMEN

PURPOSE: The endocrine secretion of TSH is a finely orchestrated process controlled by the thyrotropin-releasing hormone (TRH). Its homeostasis and signaling rely on many calcium-binding proteins belonging to the "EF-hand" protein family. The Ca2+/calmodulin (CaM) complex is associated with Ca2+/CaM-dependent kinases (Ca2+/CaMK). We have investigated Ca2+/CaMK expression and regulation in the rat pituitary. METHODS: The expression of CaMKII and CaMKIV in rat anterior pituitary cells was shown by immunohistochemistry. Cultured anterior pituitary cells were stimulated by TRH in the presence and absence of KN93, the pharmacological inhibitor of CaMKII and CaMKIV. Western blotting was then used to measure the expression of these kinases and of the cAMP response element-binding protein (CREB). TSH production was measured by RIA after time-dependent stimulation with TRH. Cells were infected with a lentiviral construct coding for CaMKIV followed by measurement of CREB phosphorylation and TSH. RESULTS: Our study shows that two CaM kinases, CaMKII and CaMKII, are expressed in rat pituitary cells and their phosphorylation in response to TRH occurs at different time points, with CaMKIV being activated earlier than CaMKII. TRH induces CREB phosphorylation through the activity of both CaMKII and CaMKIV. The activation of CREB increases TSH gene expression. CaMKIV induces CREB phosphorylation while its dominant negative and KN93 exert the opposite effects. CONCLUSION: Our data indicate that the expression of Ca2+/CaMK in rat anterior pituitary are correlated to the role of CREB in the genetic regulation of TSH, and that TRH stimulation activates CaMKIV, which in turn phosphorylates CREB. This phosphorylation is linked to the production of thyrotropin.

Asunto(s)

Señalización del Calcio/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Hipófisis/metabolismo , Hormona Liberadora de Tirotropina/metabolismo , Tirotropina , Animales , Bencilaminas/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunoquímica , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Transducción de Señal , Sulfonamidas/farmacología , Tirotropina/análisis , Tirotropina/genética , Tirotropina/metabolismo

RESUMEN

The Wnt signaling pathway is a crucial regulator of the intestinal epithelium homeostasis and is altered in most colon cancers. While the role of aberrant canonical, ß-catenin-dependent Wnt signaling has been well established in colon cancer promotion, much less is known about the role played by noncanonical, ß-catenin-independent Wnt signaling in this type of cancer. This work aimed to characterize the noncanonical signal transduction pathway in colon cancer cells. To this end, we used the prototype noncanonical ligand, Wnt5a, in comparison with Wnt3a, the prototype of a canonical ß-catenin activating ligand. The analysis of the expression profile of Wnt receptors in colon cancer cell lines showed a clear increase in both level expression and variety of Frizzled receptor types expressed in colon cancer cells compared with non-malignant cells. We found that Wnt5a activates a typical Wnt/Ca++ - noncanonical signaling pathway in colon malignant cells, inducing the hyperphosphorylation of Dvl1, Dvl2 and Dvl3, promoting Ca++ mobilization as a result of phospholipase C (PLC) activation via pertussis toxin-sensitive G-protein, and inducing PLC-dependent cell migration. We also found that while the co-receptor Ror2 tyrosine kinase activity is not required for Ca++ mobilization-induced by Wnt5a, it is required for the inhibitory effects of Wnt5a on the ß-catenin-dependent transcriptional activity. Unexpectedly, we found that although the prototype canonical Wnt3a ligand was unique in stimulating the ß-catenin-dependent transcriptional activity, it also simultaneously activated PLC, promoted Ca++ mobilization, and induced Rho kinase and PLC-dependent cell migration. Our data indicate, therefore, that a Wnt ligand can activate at the same time the so-called Wnt canonical and noncanonical pathways inducing the formation of complex signaling networks to integrate both pathways in colon cancer cells.

Asunto(s)

Neoplasias del Colon/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Calcio/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Neoplasias del Colon/patología , Proteínas de Unión al GTP/metabolismo , Humanos , Ligandos , Ratones , Modelos Biológicos , Toxina del Pertussis/farmacología , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Estabilidad Proteica/efectos de los fármacos , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Receptores Wnt/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismo

RESUMEN

The leucine-rich repeat kinase 2 (LRRK2) is expressed in various immune cells and involved in regulating inflammatory processes. LRRK2 facilitates calcium extrusion exchanger and sodium-calcium exchanger activity and hence influences intracellular Ca2+ concentration in dendritic cells (DCs). DC maturation and migration are governed by the intracellular Ca2+ concentration, but the related mechanisms whereby LRRK2 regulates DC function and involved Ca2+ channels are still under investigation. In the previous study, we found that LRRK2-/- DCs exhibited higher store-operated Ca2+ entry (SOCE) activity than LRRK2+/+ DCs. Herein, we ascertained the exact SOCE components by using genetic, pharmacological, and fluorescent approaches. Ca2+ imaging showed that LRRK2 kinase activity negatively modulated SOCE activity. Moreover, LRRK2 deficiency resulted in an enhanced migration capacity of DCs but had little effect on the maturation process. SOCE is widely known to regulate DC functions; we wanted to dissect the reason why LRRK2 specifically influenced DC migration and therefore silenced ORAI1, ORAI2, and ORAI3, respectively. Transwell assays showed that both ORAI1 and ORAI2 silencing markedly decreased the migration of DCs, but only ORAI1 deficiency influenced the expression of maturation markers CD11c, CD86, and major histocompatibility complex class II. Of note, LRRK2 deficiency increased ORAI2 expression but not that of ORAI1 and ORAI3. Thus, we suggest that LRRK2 modulates DC migration by interfering with ORAI2.-Yan, J., Zhao, W., Gao, C., Liu, X., Zhao, X., Wei, T., Gao, Z. Leucine-rich repeat kinase 2 regulates mouse dendritic cell migration by ORAI2.

Asunto(s)

Movimiento Celular/fisiología , Células Dendríticas/fisiología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína ORAI2/metabolismo , Animales , Compuestos de Boro/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Silenciador del Gen , Imidazoles/farmacología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Proteína ORAI2/genética , Molécula de Interacción Estromal 2/genética , Molécula de Interacción Estromal 2/metabolismo

RESUMEN

The type 2a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a key role in Ca2+ regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca2+ sensor R-CEPIA1er was used to measure the concentration of Ca2+ in the lumen of the endoplasmic reticulum (ER) ([Ca2+]ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+ release channel ryanodine receptor (RyR2) created a Ca2+ release/reuptake system that mimicked aspects of cardiac myocyte Ca2+ handling. SERCA2a function was quantified from the rate of [Ca2+]ER refilling after ER Ca2+ depletion; then, ER Ca2+ leak was measured after SERCA inhibition. ER Ca2+ uptake and leak were analyzed as a function of [Ca2+]ER to determine maximum ER Ca2+ uptake rate and maximum ER Ca2+ load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function in the cellular environment. The newly developed Ca2+ sensor R-CEPIA1er was used to successfully analyze Ca2+ uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca2+ pump regulation.

Asunto(s)

Calcio/metabolismo , Retículo Endoplásmico/enzimología , Miocitos Cardíacos/enzimología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Retículo Sarcoplasmático/enzimología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico , Técnicas Biosensibles , Proteínas de Unión al Calcio/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Mutación , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo , Canal Liberador de Calcio Receptor de Rianodina/genética , Retículo Sarcoplasmático/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Factores de Tiempo

RESUMEN

We have shown that calcium (Ca2+) oscillations in human pulmonary fibroblasts (HPFs) contribute to profibrotic effects of transforming growth factor-ß (TGF-ß) and that disruption of these oscillations blunts features of pulmonary fibrosis. Prostaglandin E2 (PGE2) exerts antifibrotic effects in the lung, but the mechanisms for this action are not well defined. We thus sought to explore interactions between PGE2 and the profibrotic agent TGF-ß in pulmonary fibroblasts (PFs) isolated from patients with or without idiopathic pulmonary fibrosis (IPF). PGE2 inhibited TGF-ß-promoted [Ca2+] oscillations and prevented the activation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) but did not prevent activation of Smad-2 or ERK. PGE2 also eliminated TGF-ß-stimulated expression of collagen A1, fibronectin, and α-smooth muscle actin and reduced stress fiber formation in the HPFs. RNA sequencing revealed that HPFs preferentially express EP2 receptors relative to other prostanoid receptor subtypes: EP2 expression is ~10-fold higher than that of EP4 receptors; EP1 and EP3 receptors are barely detectable; and EP2-receptor expression is ~3.5-fold lower in PFs from IPF patients than in normal HPFs. The inhibitory effects of PGE2 on synthetic function and stress fiber formation were blocked by selective EP2 or EP4 antagonists and mimicked by selective EP2 or EP4 agonists, the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin, all of which elevate cellular cAMP concentrations. We conclude that PGE2, likely predominantly via EP2 receptors, interferes with Ca2+ signaling, CaMK-II activation, and Akt activation in IPF-HPFs and HPFs treated with TGF-ß. Moreover, a decreased expression of EP2 receptors in pulmonary fibroblasts from IPF patients may contribute to the pathophysiology of this disease.

Asunto(s)

Señalización del Calcio/efectos de los fármacos , Dinoprostona/farmacología , Fibroblastos/metabolismo , Pulmón/metabolismo , Fibrosis Pulmonar/metabolismo , Anciano , Anciano de 80 o más Años , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/biosíntesis , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Femenino , Fibroblastos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Pulmón/patología , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Fibrosis Pulmonar/patología , Subtipo EP2 de Receptores de Prostaglandina E/biosíntesis , Proteína Smad2/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis

RESUMEN

Tributyltin (TBT) is an organotin environmental pollutant widely used as an agricultural and wood biocide and in antifouling paints. Countries began restricting TBT use in the 2000s, but their use continues in some agroindustrial processes. We studied the acute effect of TBT on cardiac function by analyzing myocardial contractility and Ca2+ handling. Cardiac contractility was evaluated in isolated papillary muscle and whole heart upon TBT exposure. Isolated ventricular myocytes were used to measure calcium (Ca2+) transients, sarcoplasmic reticulum (SR) Ca2+ content and SR Ca2+ leak (as Ca2+ sparks). Reactive oxygen species (ROS), as superoxide anion (O2•-) was detected at intracellular and mitochondrial myocardium. TBT depressed cardiac contractility and relaxation in papillary muscle and intact whole heart. TBT increased cytosolic, mitochondrial ROS production and decreased mitochondrial membrane potential. In isolated cardiomyocytes TBT decreased both Ca2+ transients and SR Ca2+ content and increased diastolic SR Ca2+ leak. Decay of twitch and caffeine-induced Ca2+ transients were slowed by the presence of TBT. Dantrolene prevented and Tiron limited the reduction in SR Ca2+ content and transients. The environmental contaminant TBT causes cardiotoxicity within minutes, and may be considered hazardous to the mammalian heart. TBT acutely induced a negative inotropic effect in isolated papillary muscle and whole heart, increased arrhythmogenic SR Ca2+ leak leading to reduced SR Ca2+ content and reduced Ca2+ transients. TBT-induced myocardial ROS production, may destabilize the SR Ca2+ release channel RyR2 and reduce SR Ca2+ pump activity as key factors in the TBT-induced negative inotropic and lusitropic effects.

Asunto(s)

Cardiotoxicidad/metabolismo , Compuestos de Trialquiltina/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Calcio/metabolismo , Mitocondrias/metabolismo , Contracción Miocárdica , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático/metabolismo

RESUMEN

Transmembrane member 16A (TMEM16A) is the Ca2+-activated chloride channel in airways and intestine. It has been associated with goblet cell metaplasia, as expression of TMEM16A is strongly up-regulated in cystic fibrosis and asthma during mucus hypersecretion. However, the possible role of TMEM16A for mucus production or mucus secretion remains obscure, and whether TMEM16A controls the function of intestinal goblet cells is entirely unknown. Basal mucus secretion in lungs occurs through low levels of ATP in the airway surface liquid. Here, we report for the first time that TMEM16A is essential for basal secretion of mucus in airways and intestine. Airway-ciliated and intestinal epithelial-specific knockout of TMEM16A ( TMEM16Aflox/floxFoxJ1, TMEM16Aflox/floxVil1) leads to accumulation of mucus in airway club (Clara) cells and intestinal goblet cells, respectively. Acute ATP-induced mucus secretion by airway club cells is inhibited when TMEM16A is knocked out in ciliated cells, possibly as a result of compromised release of prosecretory cytokines. Knockdown or inhibition of TMEM16A in human Calu3 airway epithelial cells indicates compromised IL-8 release. In intestinal goblet cells lacking expression of TMEM16A, mucus accumulates as a result of compromised ATP-induced secretion. In contrast, cholinergic mucus secretion by compound exocytosis is independent of TMEM16A. The data demonstrate a previously unrecognized role of TMEM16A for membrane exocytosis and describe a novel, ATP-driven pathway for intestinal mucus secretion. We conclude that ATP-dependent mucus secretion in both airways and intestine requires TMEM16A. The present results may form the basis for a novel, therapeutic approach for the treatment of mucus hypersecretion in inflammatory airway and intestinal disease.-Benedetto, R., Cabrita, I., Schreiber, R., Kunzelmann, K. TMEM16A is indispensable for basal mucus secretion in airways and intestine.

Asunto(s)

Anoctamina-1/fisiología , Mucosa Intestinal/metabolismo , Moco/metabolismo , Proteínas de Neoplasias/fisiología , Mucosa Respiratoria/metabolismo , Adenosina Trifosfato/metabolismo , Alérgenos/toxicidad , Animales , Anoctamina-1/antagonistas & inhibidores , Anoctamina-1/genética , Señalización del Calcio , Línea Celular , Cilios , Cruzamientos Genéticos , Exocitosis/efectos de los fármacos , Técnicas de Inactivación de Genes , Células Caliciformes/metabolismo , Células HEK293 , Humanos , Interleucina-8/metabolismo , Ratones , Ratones Noqueados , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Especificidad de Órganos , Ovalbúmina/toxicidad , Técnicas de Placa-Clamp , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/metabolismo , Toxina Tetánica/farmacología , Tiofenos/farmacología

RESUMEN

Increased levels of the calcium-binding protein neuronal calcium sensor 1 (NCS1) predict an unfavorable patient outcome in several aggressive cancers, including breast and liver tumors. Previous studies suggest that NCS1 overexpression facilitates metastatic spread of these cancers. To investigate this hypothesis, we explored the effects of NCS1 overexpression on cell proliferation, survival, and migration patterns in vitro in 2- and 3-dimensional (2/3-D). Furthermore, we translated our results into an in vivo mouse xenograft model. Cell-based proliferation assays were used to demonstrate the effects of overexpression of NCS1 on growth rates. In vitro colony formation and wound healing experiments were performed and 3-D migration dynamics were studied using collagen gels. Nude mice were injected with breast cancer cells to monitor NCS1-dependent metastasis formation over time. We observed that increased NCS1 levels do not change cellular growth rates, but do significantly increase 2- and 3-D migration dynamics in vitro. Likewise, NCS1-overexpressing cells have an increased capacity to form distant metastases and demonstrate better survival and less necrosis in vivo. We found that NCS1 preferentially localizes to the leading edge of cells and overexpression increases the motility of cancer cells. Furthermore, this phenotype is correlated with an increased number of metastases in a xenograft model. These results lay the foundation for exploring the relevance of an NCS1-mediated pathway as a metastatic biomarker and as a target for pharmacologic interventions.-Apasu, J. E., Schuette, D., LaRanger, R., Steinle, J. A., Nguyen, L. D., Grosshans, H. K., Zhang, M., Cai, W. L., Yan, Q., Robert, M. E., Mak, M., Ehrlich, B. E. Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.

Asunto(s)

Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Femenino , Humanos , Ratones , Ratones Desnudos , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Ca2+ is an essential trigger for most forms of synaptic plasticity. Ca2+ signaling occurs not only by Ca2+ entry via plasma membrane channels but also via Ca2+ signals generated by intracellular organelles. These organelles, by dynamically regulating the spatial and temporal extent of Ca2+ elevations within neurons, play a pivotal role in determining the downstream consequences of neural signaling on synaptic function. Here, we review the role of three major intracellular stores: the endoplasmic reticulum, mitochondria, and acidic Ca2+ stores, such as lysosomes, in neuronal Ca2+ signaling and plasticity. We provide a comprehensive account of how Ca2+ release from these stores regulates short- and long-term plasticity at the pre- and postsynaptic terminals of central synapses.

Asunto(s)

Señalización del Calcio , Plasticidad Neuronal , Neuronas/metabolismo , Sinapsis/metabolismo , Animales , Espinas Dendríticas/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Lisosomas/metabolismo , Mitocondrias/metabolismo , Terminales Presinápticos/metabolismo

RESUMEN

Heart contraction vitally depends on tightly controlled intracellular Ca regulation. Because contraction is mainly driven by Ca released from the sarcoplasmic reticulum (SR), this organelle plays a particularly important role in Ca regulation. The type two ryanodine receptor (RyR2) is the major SR Ca release channel in ventricular myocytes. Several cardiac pathologies, including myocardial infarction and heart failure, are associated with increased RyR2 activity and diastolic SR Ca leak. It has been suggested that the increased RyR2 activity plays an important role in arrhythmias and contractile dysfunction. Several studies have linked increased SR Ca leak during myocardial infarction and heart failure to the activation of RyR2 in response to oxidative stress. This activation might include direct oxidation of RyR2 as well as indirect activation via phosphorylation or altered interactions with regulatory proteins. Out of ninety cysteine residues per RyR2 subunit, twenty one were reported to be in reduced state that could be potential targets for redox modifications that include S-nitrosylation, S-glutathionylation, and disulfide cross-linking. Despite its clinical significance, molecular mechanisms of RyR dysfunction during oxidative stress are not fully understood. Herein we review the most recent insights into redox-dependent modulation of RyR2 during oxidative stress and heart diseases.

RESUMEN

[Ca2+] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca2+ release of extrinsic Ca2+ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca2+ buffer is added to the myoplasm, part of the released Ca2+ binds to it, reducing the Ca2+ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca2+ released. Monitoring release by measuring [Ca2+] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca2+] in the SR ([Ca2+]SR) continuously from a starting value close to 400 µM reaching the range of 100 µM in about half an hour. The effect of reducing resting [Ca2+]SR on the Ca2+ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca2+] ([Ca2+]myo) transients were simultaneously recorded with Rhod2. The Ca2+ release flux was calculated from [Ca2+]myo and divided by [Ca2+]SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026 ± 0.005 ms-1 at resting [Ca2+]SR = 372 ± 5 µM to 0.021 ± 0.004 ms-1 at resting [Ca2+]SR = 120 ± 16 µM (n = 4, p = 0.03). The time averaged permeability was not significantly changed (0.009 ± 0.003 and 0.010 ± 0.003 ms-1, at the higher and lower [Ca2+]SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca2+]SR (Δ[Ca2+]SR) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca2+]SR = 30.2 ± 3.5 µM from resting [Ca2+]SR = 88.8 ± 13.6 µM, n = 5) than in 40 mM EGTA (Δ[Ca2+]SR = 72.2 ± 10.4 µM from resting [Ca2+]SR = 98.2 ± 15.6 µM, n = 4) suggesting that a Ca2+ activated component of release was suppressed by BAPTA.

Asunto(s)

Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Fibras Musculares Esqueléticas/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Ranidae

RESUMEN

Cystic fibrosis (CF) is the most common genetic disorder that causes a significant damage in secretory epithelial cells due to the defective ion flux across the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Pancreas is one of the organs most frequently damaged by the disease leading to pancreatic insufficiency, abdominal pain and an increased risk of acute pancreatitis in CF patients causing a significant decrease in the quality of life. CFTR plays a central role in the pancreatic ductal secretory functions by carrying Cl- and HCO3 - ions across the apical membrane. Therefore pathophysiological studies in CF mostly focused on the effects of impaired ion secretion by pancreatic ductal epithelial cells leading to exocrine pancreatic damage. However, several studies indicated that CFTR has a central role in the regulation of intracellular signaling processes and is now more widely considered as a signaling hub in epithelial cells. In contrast, elevated intracellular Ca2+ level was observed in the lack of functional CFTR in different cell types including airway epithelial cells. In addition, impaired CFTR expression has been correlated with damaged mitochondrial function in epithelial cells. These alterations of intracellular signaling in CF are not well characterized in the exocrine pancreas yet. Therefore in this review we would like to summarize the complex role of CFTR in the exocrine pancreas with a special focus on the intracellular signaling and mitochondrial function.

RESUMEN

BACKGROUND: Allergic and non-allergic childhood asthma has been characterized by distinct immune mechanisms. While interferon regulating factor 1 (IRF-1) polymorphisms (SNPs) influence atopy risk, the effect of SNPs on asthma phenotype-specific immune mechanisms is unclear. We assessed whether IRF-1 SNPs modify distinct immune-regulatory pathways in allergic and non-allergic childhood asthma (AA/NA). METHODS: In the CLARA study, asthma was characterized by doctor's diagnosis and AA vs NA by positive or negative specific IgE. Children were genotyped for four tagging SNPs within IRF-1 (n = 172). mRNA expression was measured with qRT-PCR. Gene expression was analyzed depending on genetic variants within IRF-1 and phenotype including haplotype estimation and an allelic risk score. RESULTS: Carrying the risk alleles of IRF-1 in rs10035166, rs2706384, or rs2070721 was associated with increased risk for AA. Carrying the non-risk allele in rs17622656 was associated with lower risk for AA but not NA. In AA carrying the risk alleles, an increased pro-inflammatory expression of ICAM3, IRF-8, XBP-1, IFN-γ, RGS13, RORC, and TSC2 was observed. NOD2 expression was decreased in AA with risk alleles in rs2706384 and rs10035166 and with risk haplotype. Further, AA with risk haplotype showed increased IL-13 secretion. NA with risk allele in rs2070721 compared to non-risk allele in rs17622656 showed significantly upregulated calcium, innate, mTOR, neutrophil, and inflammatory-associated genes. CONCLUSION: IRF-1 polymorphisms influence the risk for childhood allergic asthma being associated with increased pro-inflammatory gene regulation. Thus, it is critical to implement IRF-1 genetics in immune assessment for childhood asthma phenotypes.

Asunto(s)

Asma/genética , Factor 1 Regulador del Interferón/genética , Adolescente , Niño , Preescolar , Citocinas/metabolismo , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Inmunoglobulina E/sangre , Polimorfismo de Nucleótido Simple , Reacción en Cadena en Tiempo Real de la Polimerasa , Pruebas de Función Respiratoria/métodos , Riesgo

RESUMEN

It is now clear that in addition to activating several complex kinase pathways (Smad, MAP kinase, PI3 kinase), TGFß also acts by elevating cytosolic Ca2+ concentration within human pulmonary fibroblasts. Ca2+/calmodulin-dependent protein kinase II (CamK II) is also known to regulate gene expression in fibroblasts. In this study, we examined the interactions between calcium signaling, activation of CamK and other kinases, and extracellular matrix (ECM) gene expression. Human pulmonary fibroblasts were cultured and stimulated with artificially generated Ca2+ pulses in the absence of TGFß, or with TGFß (1 nM) or vehicle in the presence of various blockers of Ca2+ signaling. PCR and Western blotting were used to measure gene expression and protein levels, respectively. We found that Ca2+ pulses in the absence of TGFß increased ECM gene expression in a pulse frequency-dependent manner, and that blocking Ca2+ signaling and the CamK II pathway significantly reduced TGFß-mediated ECM gene expression, without having any effects on other kinase pathways (Smad, PI3 kinase, or MAP kinase). We also found that TGFß elevated the expression of CamK IIß and CamK IIδ, while siRNA silencing of those two subtypes significantly reduced TGFß-mediated expression of collagen A1 and fibronectin 1. Our data suggest that TGFß induces the expression of CamK IIß and CamK IIδ, which in turn are activated by TGFß-evoked Ca2+ waves in a frequency-dependent manner, leading to increased expression of ECM proteins.

Asunto(s)

Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Pulmón/citología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Anciano , Anciano de 80 o más Años , Western Blotting , Señalización del Calcio , Cadena alfa 1 del Colágeno Tipo I , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Indoles/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Rianodina/farmacología , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

By serendipity and good fortune, as a postdoctoral fellow in 1967, I landed at the right place at the right time, as I was allowed to investigate the mechanism by which hormones activate the enzyme adenylyl cyclase (then adenyl cyclase) in Martin Rodbell's Laboratory at the NIH in Bethesda, Maryland. The work uncovered first, the existence of receptors separate from the enzyme and then, the existence of transduction mechanisms requiring guanosine-5'-triphosphate (GTP) and Mg(2+). With my laboratory colleagues first and postdoctoral fellows after leaving NIH, I participated in the development of the field "signal transduction by G proteins," uncovered by molecular cloning several G-protein-coupled receptors (GPCRs) and became interested in both the molecular makeup of voltage-gated Ca channels and Ca2+ homeostasis downstream of activation of phospholipase C (PLC) by the Gq/11 signaling pathway. We were able to confirm the hypothesis that there would be mammalian homologues of the Drosophila "transient receptor potential" channel and discovered the existence of six of the seven mammalian genes, now called transient receptor potential canonical (TRPC) channels. In the present article, I summarize from a bird's eye view of what I feel were key findings along this path, not only from my laboratory but also from many others, that allowed for the present knowledge of cell signaling involving G proteins to evolve. Towards the end, I summarize roles of TRPC channels in health and disease.

Asunto(s)

Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Guanosina Trifosfato/genética , Humanos , Transducción de Señal/genética , Transducción de Señal/fisiología , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/genética

RESUMEN

Despite key advances in the clinical management of life-threatening ventricular arrhythmias, culminating with the development of implantable cardioverter-defibrillators and catheter ablation techniques, pharmacologic/biologic therapeutics have lagged behind. The fundamental issue is that biological targets are molecular factors. Diseases, however, represent emergent properties at the scale of the organism that result from dynamic interactions between multiple constantly changing molecular factors. For a pharmacologic/biologic therapy to be effective, it must target the dynamic processes that underlie the disease. Here we propose a classification of ventricular arrhythmias that is based on our current understanding of the dynamics occurring at the subcellular, cellular, tissue and organism scales, which cause arrhythmias by simultaneously generating arrhythmia triggers and exacerbating tissue vulnerability. The goal is to create a framework that systematically links these key dynamic factors together with fixed factors (structural and electrophysiological heterogeneity) synergistically promoting electrical dispersion and increased arrhythmia risk to molecular factors that can serve as biological targets. We classify ventricular arrhythmias into three primary dynamic categories related generally to unstable Ca cycling, reduced repolarization, and excess repolarization, respectively. The clinical syndromes, arrhythmia mechanisms, dynamic factors and what is known about their molecular counterparts are discussed. Based on this framework, we propose a computational-experimental strategy for exploring the links between molecular factors, fixed factors and dynamic factors that underlie life-threatening ventricular arrhythmias. The ultimate objective is to facilitate drug development by creating an in silico platform to evaluate and predict comprehensively how molecular interventions affect not only a single targeted arrhythmia, but all primary arrhythmia dynamics categories as well as normal cardiac excitation-contraction coupling.

Asunto(s)

Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/etiología , Disfunción Ventricular/diagnóstico , Disfunción Ventricular/etiología , Animales , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/fisiopatología , Descubrimiento de Drogas , Humanos , Disfunción Ventricular/tratamiento farmacológico , Disfunción Ventricular/fisiopatología