RESUMEN
Lineage plasticity is a recognized hallmark of cancer progression that can shape therapy outcomes. The underlying cellular and molecular mechanisms mediating lineage plasticity remain poorly understood. Here, we describe a versatile in vivo platform to identify and interrogate the molecular determinants of neuroendocrine lineage transformation at different stages of prostate cancer progression. Adenocarcinomas reliably develop following orthotopic transplantation of primary mouse prostate organoids acutely engineered with human-relevant driver alterations (e.g., Rb1-/-; Trp53-/-; cMyc+ or Pten-/-; Trp53-/-; cMyc+), but only those with Rb1 deletion progress to ASCL1+ neuroendocrine prostate cancer (NEPC), a highly aggressive, androgen receptor signaling inhibitor (ARSI)-resistant tumor. Importantly, we show this lineage transition requires a native in vivo microenvironment not replicated by conventional organoid culture. By integrating multiplexed immunofluorescence, spatial transcriptomics and PrismSpot to identify cell type-specific spatial gene modules, we reveal that ASCL1+ cells arise from KRT8+ luminal epithelial cells that progressively acquire transcriptional heterogeneity, producing large ASCL1+;KRT8- NEPC clusters. Ascl1 loss in established NEPC results in transient tumor regression followed by recurrence; however, Ascl1 deletion prior to transplantation completely abrogates lineage plasticity, yielding adenocarcinomas with elevated AR expression and marked sensitivity to castration. The dynamic feature of this model reveals the importance of timing of therapies focused on lineage plasticity and offers a platform for identification of additional lineage plasticity drivers.
RESUMEN
Presenilin 1 (PS1) is an intramembrane protease, the active subunit of the γ-secretase complex. Its well-studied function is the amyloidogenic cleavage of the C-terminal fragment of the amyloid precursor protein, also known as C99, to produce the Abeta peptide. Recent findings from the Greengard laboratory suggest that PS1 also have anti-amyloidogenic activities, which reduce Abeta levels. First, it redirects APP-C99 toward autophagic degradation, lowering the amount that can be converted into Abeta. The protein kinase CK1γ2 phosphorylates PS1 at Ser367. Phosphorylated PS1 at this position interacts with Annexin A2, which, in turn, interacts with the lysosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Vamp8. Annexin A2 facilitates the binding of Vamp8 to the autophagosomal SNARE Syntaxin 17 to modulate the fusion of autophagosomes with lysosomes. Thus, PS1 phosphorylated at Ser367 has an anti-amyloidogenic function, promoting autophagosome-lysosome fusion and increasing C99 degradation. Second, it enhances the ability of microglia to phagocyte and degrade extracellular Abeta oligomer, through regulating the expression of the lysosomal master regulator TFEB. Thus, PS1 has a role in both the production and the clearance of Abeta. Drugs designed to activate CK1γ2 and increase the level of PS1 phosphorylated at Ser367 should be useful in the treatment of Alzheimer's disease.
Asunto(s)
Amiloide/metabolismo , Presenilina-1/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Autofagia , Humanos , Degeneración Nerviosa/patologíaRESUMEN
INTRODUCTION: The levels and distribution of amyloid deposits in the brain does not correlate well with Alzheimer's disease (AD) progression. Therefore, it is likely that amyloid precursor protein and its proteolytic fragments other than amyloid b (Ab) contribute to the onset of AD. METHODS: We developed a sensitive assay adapted to the detection of C99, the direct precursor of b-amyloid. Three postmortem groups were studied: control with normal and stable cognition; patients with moderate AD, and individuals with severe AD. The amount of C99 and Aß was quantified and correlated with the severity of AD. RESULTS: C99 accumulates in vulnerable neurons, and its levels correlate with the degree of cognitive impairment in patients suffering from AD. In contrast, Aß levels are increased in both vulnerable and resistant brain areas. DISCUSSION: These results raise the possibility that C99, rather than Aß plaques, is responsible for the death of nerve cells in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Progresión de la Enfermedad , Neuronas/metabolismo , Fragmentos de Péptidos/efectos adversos , Precursor de Proteína beta-Amiloide/efectos adversos , Animales , Autopsia , Encéfalo/metabolismo , HumanosRESUMEN
The visceral endoderm is a polarized epithelial monolayer necessary for early embryonic development in rodents. A key feature of this epithelium is an active endocytosis and degradation of maternal nutrients, in addition to being the source of various signaling molecules or inhibitors required for the differentiation and patterning of adjacent embryonic tissues. Endocytosis across the visceral endoderm epithelium involves specific cell surface receptors and an extensive sub-membrane vesicular system with numerous apical vacuoles/lysosomes. We previously reported that Cubilin, the endocytic receptor for intrinsic factor-vitamin B12, albumin and apolipoproteinA-I/HDL allows maternal nutrient uptake by the visceral endoderm. In the present study, we show that the germline ablation of Cubilin impairs endodermal and mesodermal patterning, and results in developmental arrest at gastrulation. Notably, visceral endoderm dispersal is impeded in Cubilin null embryos. We further confirm the essential role of Cubilin in nutrient internalization by the early visceral endoderm and highlight its involvement in the formation of apical vacuoles. Our results reveal essential roles for Cubilin in early embryonic development, and suggest that in addition to its nutritive function, Cubilin sustains signaling pathways involved in embryonic differentiation and patterning.
Asunto(s)
Endocitosis/fisiología , Endodermo/citología , Receptores de Superficie Celular/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Desarrollo Embrionario/fisiología , Endodermo/metabolismo , Femenino , Gastrulación/fisiología , Factor Intrinseco/metabolismo , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Transporte de Proteínas , Receptores de Superficie Celular/fisiología , Vitamina B 12/metabolismoRESUMEN
Alzheimer's disease (AD) is characterized by accumulation of the ß-amyloid peptide (Aß), which is generated through sequential proteolysis of the amyloid precursor protein (APP), first by the action of ß-secretase, generating the ß-C-terminal fragment (ßCTF), and then by the Presenilin 1 (PS1) enzyme in the γ-secretase complex, generating Aß. γ-Secretase is an intramembranous protein complex composed of Aph1, Pen2, Nicastrin, and Presenilin 1. Although it has a central role in the pathogenesis of AD, knowledge of the mechanisms that regulate PS1 function is limited. Here, we show that phosphorylation of PS1 at Ser367 does not affect γ-secretase activity, but has a dramatic effect on Aß levels in vivo. We identified CK1γ2 as the endogenous kinase responsible for the phosphorylation of PS1 at Ser367. Inhibition of CK1γ leads to a decrease in PS1 Ser367 phosphorylation and an increase in Aß levels in cultured cells. Transgenic mice in which Ser367 of PS1 was mutated to Ala, show dramatic increases in Aß peptide and in ßCTF levels in vivo. Finally, we show that this mutation impairs the autophagic degradation of ßCTF, resulting in its accumulation and increased levels of Aß peptide and plaque load in the brain. Our results demonstrate that PS1 regulates Aß levels by a unique bifunctional mechanism. In addition to its known role as the catalytic subunit of the γ-secretase complex, selective phosphorylation of PS1 on Ser367 also decreases Aß levels by increasing ßCTF degradation through autophagy. Elucidation of the mechanism by which PS1 regulates ßCTF degradation may aid in the development of potential therapies for Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Presenilina-1/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Autofagia , Encéfalo/metabolismo , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Fosforilación , Presenilina-1/metabolismo , Dominios Proteicos , Serina/química , Resultado del TratamientoRESUMEN
Presenilin 1 (PS1), the catalytic subunit of the γ-secretase complex, cleaves ßCTF to produce Aß. We have shown that PS1 regulates Aß levels by a unique bifunctional mechanism. In addition to its known role as the catalytic subunit of the γ-secretase complex, selective phosphorylation of PS1 on Ser367 decreases Aß levels by increasing ßCTF degradation through autophagy. Here, we report the molecular mechanism by which PS1 modulates ßCTF degradation. We show that PS1 phosphorylated at Ser367, but not nonphosphorylated PS1, interacts with Annexin A2, which, in turn, interacts with the lysosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Vamp8. Annexin A2 facilitates the binding of Vamp8 to the autophagosomal SNARE Syntaxin 17 to modulate the fusion of autophagosomes with lysosomes. Thus, PS1 phosphorylated at Ser367 has an antiamyloidogenic function, promoting autophagosome-lysosome fusion and increasing ßCTF degradation. Drugs designed to increase the level of PS1 phosphorylated at Ser367 should be useful in the treatment of Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/genética , Autofagosomas/metabolismo , Lisosomas/metabolismo , Presenilina-1/genética , Animales , Anexina A2/metabolismo , Autofagia/fisiología , Encéfalo/metabolismo , Línea Celular Tumoral , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Neuroblastoma/metabolismo , Neuronas/metabolismo , Fagosomas/metabolismo , Fosforilación , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Transducción de SeñalRESUMEN
Mixl1 is the only member of the Mix/Bix homeobox gene family identified in mammals. During mouse embryogenesis, Mixl1 is first expressed at embryonic day (E)5.5 in cells of the visceral endoderm (VE). At the time of gastrulation, Mixl1 expression is detected in the vicinity of the primitive streak. Mixl1 is expressed in cells located within the primitive streak, in nascent mesoderm cells exiting the primitive streak, and in posterior VE overlying the primitive streak. Genetic ablation of Mixl1 in mice has revealed its crucial role in mesoderm and endoderm cell specification and tissue morphogenesis during early embryonic development. However, the early lethality of the constitutive Mixl1(-/-) mutant precludes the study of its role at later stages of embryogenesis and in adult mice. To circumvent this limitation, we have generated a conditional Mixl1 allele (Mixl1(cKO) that permits temporal as well as spatial control of gene ablation. Animals homozygous for the Mixl1(cKO) conditional allele were viable and fertile. Mixl1(KO/KO) embryos generated by crossing of Mixl1(cKO/cKO) mice with Sox2-Cre or EIIa-Cre transgenic mice were embryonic lethal at early somite stages. By contrast to wild-type embryos, Mixl1(KO/KO) embryos contained no detectable Mixl1, validating the Mixl1(cKO) as a protein null after Cre-mediated excision. Mixl1(KO/KO) embryos resembled the previously reported Mixl1(-/-) mutant phenotype. Therefore, the Mixl1 cKO allele provides a tool for investigating the temporal and tissue-specific requirements for Mixl1 in the mouse.
Asunto(s)
Endodermo/embriología , Técnicas de Inactivación de Genes/métodos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mesodermo/embriología , Animales , Cruzamientos Genéticos , Endodermo/metabolismo , Gastrulación , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Genes Letales , Mesodermo/metabolismo , RatonesRESUMEN
Arterial smooth muscle (ASM) Na(+)/Ca(2+) exchanger type 1 (NCX1) and TRPC/Orai-containing receptor/store-operated cation channels (ROC/SOC) are clustered with α2 Na(+) pumps in plasma membrane microdomains adjacent to the underlying junctional sarcoplasmic reticulum. This arrangement enables these transport proteins to function as integrated units to help regulate local Na(+) metabolism, Ca(2+) signaling, and arterial tone. They thus influence vascular resistance and blood pressure (BP). For instance, upregulation of NCX1 and TRPC6 has been implicated in the pathogenesis of high BP in several models of essential hypertension. The models include ouabain-induced hypertensive rats, Milan hypertensive rats, and Dahl salt-sensitive hypertensive rats, all of which exhibit elevated plasma ouabain levels. We suggest that these molecular mechanisms are key contributors to the increased vascular resistance ("whole body autoregulation") that elevates BP in essential hypertension. Enhanced expression and function of ASM NCX1 and TRPC/Orai1-containing channels in hypertension implies that these proteins are potential targets for pharmacological intervention.
Asunto(s)
Señalización del Calcio , Hipertensión/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Humanos , Hipertensión/genética , Hipertensión/patología , Proteínas Musculares/genética , Músculo Liso Vascular/patología , Ratas , Ratas Endogámicas Dahl , Sodio/metabolismo , Intercambiador de Sodio-Calcio/genética , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6RESUMEN
Cardiotonic steroids (CTS) of the strophanthus and digitalis families have opposing effects on long-term blood pressure (BP). This implies hitherto unrecognized divergent signaling pathways for these CTS. Prolonged ouabain treatment upregulates Ca(2+) entry via Na(+)/Ca(2+) exchanger-1 (NCX1) and TRPC6 gene-encoded receptor-operated channels in mesenteric artery smooth muscle cells (ASMCs) in vivo and in vitro. Here, we test the effects of digoxin on Ca(2+) entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 µg·kg(-1)·day(-1) for 3 wk) did not raise BP and had no effect on resting cytolic free Ca(2+) concentration ([Ca(2+)](cyt)) or phenylephrine-induced Ca(2+) signals in isolated ASMCs. Expression of transporters in the α2 Na(+) pump-NCX1-TRPC6 Ca(2+) signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated α2 Na(+) pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ~4.5-fold) were observed in ASMCs from rats treated with ouabain but not digoxin. Moreover, in primary cultured ASMCs from normal rats, treatment with digoxin (100 nM, 72 h) did not upregulate NCX1 and TRPC6 but blocked the ouabain-induced upregulation of these transporters. Pretreatment of ASMCs with the c-Src inhibitor PP2 (1 µM; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analog eliminated the effect of ouabain on NCX1 and TRPC6 expression and ATP-induced Ca(2+) entry. Thus, in contrast to ouabain, the interaction of digoxin with α2 Na(+) pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca(2+) signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Cardiotónicos/farmacología , Digoxina/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Ouabaína/farmacología , Familia-src Quinasas/metabolismo , Animales , Aorta/citología , Canales de Calcio/metabolismo , Cardiotónicos/administración & dosificación , Células Cultivadas , Digoxina/administración & dosificación , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Arterias Mesentéricas/citología , Miocitos del Músculo Liso/efectos de los fármacos , Nifedipino/farmacología , Ouabaína/administración & dosificación , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Canales Catiónicos TRPC/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Endogenous cardiotonic steroids (CTS) raise blood pressure (BP) via vascular sodium calcium exchange (NCX1.3) and transient receptor-operated channels (TRPCs). Circulating CTS are superelevated in pregnancy-induced hypertension and preeclampsia. However, their significance in normal pregnancy, where BP is low, is paradoxical. Here we test the hypothesis that vascular resistance to endogenous ouabain (EO) develops in normal pregnancy and is mediated by reduced expression of NCX1.3 and TRPCs. We determined plasma and adrenal levels of EO and the impact of exogenous ouabain in pregnancy on arterial expression of Na(+) pumps, NCX1.3, TRPC3, and TRPC6 and BP. Pregnant (embryonic day 4) and nonpregnant rats received infusions of ouabain or vehicle. At 14-16 days, tissues and plasma were collected for blotting and EO assay by radioimmunoassay (RIA), liquid chromatography (LC)-RIA, and LC-multidimensional mass spectrometry (MS3). BP (-8 mmHg; P < 0.05) and NCX1.3 expression fell (aorta -60% and mesenteric artery -30%; P < 0.001) in pregnancy while TRPC expression was unchanged. Circulating EO increased (1.14 ± 0.13 nM) vs. nonpregnant (0.6 ± 0.08 nM; P < 0.05) and was confirmed by LC-MS3 and LC-RIA. LC-MS3 revealed two previously unknown isomers of EO; one increased â¼90-fold in pregnancy. Adrenal EO but not isomers were increased in pregnancy. In nonpregnant rats, similar infusions of ouabain raised BP (+24 ± 3 mmHg; P < 0.001). In ouabain-infused rats, impaired fetal and placental growth occurred with no BP increase. In summary, normal pregnancy is an ouabain-resistant state associated with low BP, elevated circulating levels of EO, two novel steroidal EO isomers, and increased adrenal mass and EO content. Ouabain raises BP only in nonpregnant animals. Vascular resistance to the chronic pressor activity of endogenous and exogenous ouabain is mediated by suppressed NCX1.3 and reduced sensitivity of events downstream of Ca(2+) entry. The mechanisms of EO resistance and the impaired fetal and placental growth due to elevated ouabain may be important in pregnancy-induced hypertension (PIH) and preeclampsia (PE).
Asunto(s)
Arterias/efectos de los fármacos , Arterias/metabolismo , Presión Sanguínea/efectos de los fármacos , Cardiotónicos/administración & dosificación , Resistencia a Medicamentos , Ouabaína/administración & dosificación , Intercambiador de Sodio-Calcio/metabolismo , Glándulas Suprarrenales/metabolismo , Animales , Calcio/metabolismo , Cardenólidos/sangre , Cardenólidos/metabolismo , Cardiotónicos/toxicidad , Cromatografía Liquida , Regulación hacia Abajo , Femenino , Retardo del Crecimiento Fetal/inducido químicamente , Homeostasis , Infusiones Subcutáneas , Espectrometría de Masas , Ouabaína/toxicidad , Péptidos Cíclicos , Placenta/efectos de los fármacos , Placentación , Embarazo , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Saponinas/sangre , Saponinas/metabolismo , Canales Catiónicos TRPC/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Studies in Xenopus laevis suggested that cell-extracellular matrix (ECM) interactions regulate the development of the left-right axis of asymmetry; however, the identities of ECM components and their receptors important for this process have remained unknown. We discovered that FN is required for the establishment of the asymmetric gene expression pattern in early mouse embryos by regulating morphogenesis of the node, while cellular fates of the nodal cells, canonical Wnt and Shh signaling within the node were not perturbed by the absence of FN. FN is also required for the expression of Lefty 1/2 and activation of SMADs 2 and 3 at the floor plate, while cell fate specification of the notochord and the floor plate, as well as signaling within and between these two embryonic organizing centers remained intact in FN-null mutants. Furthermore, our experiments indicate that a major cell surface receptor for FN, integrin α5ß1, is also required for the development of the left-right asymmetry, and that this requirement is evolutionarily conserved in fish and mice. Taken together, our studies demonstrate the requisite role for a structural ECM protein and its integrin receptor in the development of the left-right axis of asymmetry in vertebrates.
Asunto(s)
Tipificación del Cuerpo , Matriz Extracelular/metabolismo , Fibronectinas/fisiología , Integrina alfa5beta1/metabolismo , Animales , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/genética , Peces/embriología , Peces/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Factores de Determinación Derecha-Izquierda/metabolismo , Ratones , Notocorda/embriología , Notocorda/crecimiento & desarrollo , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Prolonged ouabain administration (25 microg kg(-1) day(-1) for 5 wk) induces "ouabain hypertension" (OH) in rats, but the molecular mechanisms by which ouabain elevates blood pressure are unknown. Here, we compared Ca(2+) signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free Ca(2+) concentration ([Ca(2+)](cyt); measured with fura-2) and phenylephrine-induced Ca(2+) transients were augmented in freshly dissociated OH ASMCs. Immunoblots revealed that the expression of the ouabain-sensitive alpha(2)-subunit of Na(+) pumps, but not the predominant, ouabain-resistant alpha(1)-subunit, was increased (2.5-fold vs. NT ASMCs) as was Na(+)/Ca(2+) exchanger-1 (NCX1; 6-fold vs. NT) in OH arteries. Ca(2+) entry, activated by sarcoplasmic reticulum (SR) Ca(2+) store depletion with cyclopiazonic acid (SR Ca(2+)-ATPase inhibitor) or caffeine, was augmented in OH ASMCs. This reflected an augmented expression of 2.5-fold in OH ASMCs of C-type transient receptor potential TRPC1, an essential component of store-operated channels (SOCs); two other components of some SOCs were not expressed (TRPC4) or were not upregulated (TRPC5). Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol [a measure of receptor-operated channel (ROC) activity] was much greater in OH than NT ASMCs. This correlated with a sixfold upregulation of TRPC6 protein, a ROC family member. Importantly, in primary cultured mesenteric ASMCs from normal rats, 72-h treatment with 100 nM ouabain significantly augmented NCX1 and TRPC6 protein expression and increased resting [Ca(2+)](cyt) and ROC activity. SOC activity was also increased. Silencer RNA knockdown of NCX1 markedly downregulated TRPC6 and eliminated the ouabain-induced augmentation; silencer RNA knockdown of TRPC6 did not affect NCX1 expression but greatly attenuated its upregulation by ouabain. Clearly, NCX1 and TRPC6 expression are interrelated. Thus, prolonged ouabain treatment upregulates the Na(+) pump alpha(2)-subunit-NCX1-TRPC6 (ROC) Ca(2+) signaling pathway in arterial myocytes in vitro as well as in vivo. This may explain the augmented myogenic responses and enhanced phenylephrine-induced vasoconstriction in OH arteries (83) as well as the high blood pressure in OH rats.
Asunto(s)
Cardiotónicos , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Músculo Liso Vascular/metabolismo , Ouabaína , Intercambiador de Sodio-Calcio/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Western Blotting , Canales de Calcio/metabolismo , Colorantes Fluorescentes , Fura-2 , Homeostasis/fisiología , Procesamiento de Imagen Asistido por Computador , Masculino , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/fisiología , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Canales Catiónicos TRPC/biosíntesis , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6 , Regulación hacia ArribaRESUMEN
Ca(2+) entry through store-operated channels (SOCs) in the plasma membrane plays an important role in regulation of vascular smooth muscle contraction, tone, and cell proliferation. The C-type transient receptor potential (TRPC) channels have been proposed as major candidates for SOCs in vascular smooth muscle. Recently, two families of transmembrane proteins, Orai [also known as Ca(2+) release-activated Ca(2+) channel modulator (CRACM)] and stromal interacting molecule 1 (STIM1), were shown to be essential for the activation of SOCs mainly in nonexcitable cells. Here, using small interfering RNA, we show that Orai1 plays an essential role in activating store-operated Ca(2+) entry (SOCE) in primary cultured proliferating human aortic smooth muscle cells (hASMCs), whereas Orai2 and Orai3 do not contribute to SOCE. Knockdown of Orai1 protein expression significantly attenuated SOCE. Moreover, inhibition of Orai1 downregulated expression of Na(+)/Ca(2+) exchanger type 1 (NCX1) and plasma membrane Ca(2+) pump isoform 1 (PMCA1). The rate of cytosolic free Ca(2+) concentration decay after Ca(2+) transients in Ca(2+)-free medium was also greatly decreased under these conditions. This reduction of Ca(2+) extrusion, presumably via NCX1 and PMCA1, may be a compensation for the reduced SOCE. Immunocytochemical observations indicate that Orai1 and NCX1 are clustered in plasma membrane microdomains. Cell proliferation was attenuated in hASMCs with disrupted Orai1 expression and reduced SOCE. Thus Orai1 appears to be a critical component of SOCE in proliferating vascular smooth muscle cells, and may therefore be a key player during vascular growth and remodeling.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Miocitos del Músculo Liso/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Aorta/metabolismo , Western Blotting , Canales de Calcio/genética , Señalización del Calcio/fisiología , Proliferación Celular , Células Cultivadas , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Músculo Liso Vascular/metabolismo , Proteína ORAI1 , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , ARN Interferente Pequeño , Intercambiador de Sodio-Calcio/genéticaRESUMEN
Phenotypic modulation of vascular myocytes is important for vascular development and adaptation. A characteristic feature of this process is alteration in intracellular Ca(2+) handling, which is not completely understood. We studied mechanisms involved in functional changes of inositol 1,4,5-trisphosphate (IP(3))- and ryanodine (Ry)-sensitive Ca(2+) stores, store-operated Ca(2+) entry (SOCE), and receptor-operated Ca(2+) entry (ROCE) associated with arterial myocyte modulation from a contractile to a proliferative phenotype in culture. Proliferating, cultured myocytes from rat mesenteric artery have elevated resting cytosolic Ca(2+) levels and increased IP(3)-sensitive Ca(2+) store content. ATP- and cyclopiazonic acid [CPA; a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium are significantly larger in proliferating arterial smooth muscle cells (ASMCs) than in freshly dissociated myocytes, whereas caffeine (Caf)-induced Ca(2+) release is much smaller. Moreover, the Caf/Ry-sensitive store gradually loses sensitivity to Caf activation during cell culture. These changes can be explained by increased expression of all three IP(3) receptors and a switch from Ry receptor type II to type III expression during proliferation. SOCE, activated by depletion of the IP(3)/CPA-sensitive store, is greatly increased in proliferating ASMCs. Augmented SOCE and ROCE (activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol) in proliferating myocytes can be attributed to upregulated expression of, respectively, transient receptor potential proteins TRPC1/4/5 and TRPC3/6. Moreover, stromal interacting molecule 1 (STIM1) and Orai proteins are upregulated in proliferating cells. Increased expression of IP(3) receptors, SERCA2b, TRPCs, Orai(s), and STIM1 in proliferating ASMCs suggests that these proteins play a critical role in an altered Ca(2+) handling that occurs during vascular growth and remodeling.
Asunto(s)
Señalización del Calcio , Proliferación Celular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Vasoconstricción , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Cafeína/farmacología , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes , Fura-2 , Indoles/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Arteria Mesentérica Superior/metabolismo , Microscopía Fluorescente , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Fenotipo , Ratas , Ratas Sprague-Dawley , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Molécula de Interacción Estromal 1 , Canales Catiónicos TRPC/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Many neurodegenerative disorders are accompanied by chronic glial activation, which is characterized by the abundant production of proinflammatory cytokines, such as IL-1beta. IL-1beta disrupts Ca(2+) homeostasis and stimulates astrocyte reactivity. The mechanisms by which IL-1beta induces Ca(2+) dysregulation are not completely defined. Here, we examined how acute and chronic (24-48 h) treatment with IL-1beta affect Ca(2+) homeostasis in freshly dissociated and primary cultured mouse cortical astrocytes. Cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) was measured with fura-2 using digital imaging. An acute application of 10 ng/ml IL-1beta induced Ca(2+) mobilization from intracellular stores and activated store-operated Ca(2+) entry (SOCE) and receptor-operated Ca(2+) entry (ROCE) in both freshly dissociated and cultured actrocytes. Treatment of cultured astrocytes with IL-1beta for 24 and 48 h elevated resting [Ca(2+)](cyt), decreased Ca(2+) store content [associated with sarco(endo)plasmic reticulum Ca(2+)-ATPase 2b downregulation], and augmented ROCE. Based on evidence that receptor-operated, but not store-operated Ca(2+) channels are Ba(2+) permeable, Ba(2+) entry was used to distinguish receptor-operated Ca(2+) channels from store-operated Ca(2+) channels. ROCE was activated by the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In the presence of extracellular Ba(2+), OAG-induced elevations of cytosolic Ba(2+) (fura-2 340-to-380-nm ratio) were significantly larger in astrocytes treated with IL-1beta. These changes in IL-1beta-treated astrocytes correlate with augmented expression of transient receptor potential cation channel (TRPC)6 protein, which likely mediates ROCE. Knockdown of the TRPC6 gene markedly reduced ROCE. The data suggest that IL-1beta-induced dysregulation of Ca(2+) homeostasis is the result of enhanced ROCE and TRPC6 expression. The disruption of Ca(2+) homeostasis appears to be an upstream component in the cascade of IL-1beta-activated pathways leading to neurodegeneration.
Asunto(s)
Astrocitos/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Interleucina-1beta/metabolismo , Animales , Astrocitos/citología , Calcio/farmacocinética , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Diglicéridos/farmacología , Femenino , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Interleucina-1beta/farmacología , Ratones , Ratones Endogámicos C57BL , Embarazo , Receptores de Interleucina-1/metabolismo , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6 , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the epsilon and zeta isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca2+ was investigated by co-expressing NCX2 and 14-3-3epsilon in HeLa cells together with the recombinant Ca2+ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3epsilon to dispose of a Ca2+ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3epsilon protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity.