RESUMEN
Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.
Asunto(s)
Glucagón/farmacología , Gluconeogénesis/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Hígado/efectos de los fármacos , Acetilcoenzima A/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Diabetes Mellitus Tipo 2/fisiopatología , Activación Enzimática/efectos de los fármacos , Glucagón/sangre , Receptores de Inositol 1,4,5-Trifosfato/genética , Lipasa/metabolismo , Lipólisis/efectos de los fármacos , Lipólisis/genética , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Oxidación-Reducción/efectos de los fármacosRESUMEN
BACKGROUND & OBJECTIVES: Alcoholic hepatitis (AH) is a common but life-threatening disease with limited treatment options. It is thought to result from hepatocellular damage, but the presence of cholestasis worsens prognosis, so we examined whether bile ducts participate in the pathogenesis of this disease. DESIGN: Cholangiocytes derived from human bile ducts were co-cultured with neutrophils from patients with AH or controls. Loss of type 3 inositol 1,4,5-trisphosphate receptor (ITPR3), an apical intracellular calcium channel necessary for cholangiocyte secretion, was used to reflect cholestatic changes. Neutrophils in contact with bile ducts were quantified in liver biopsies from patients with AH and controls and correlated with clinical and pathological findings. RESULTS: Liver biopsies from patients with AH revealed neutrophils in contact with bile ducts, which correlated with biochemical and histological parameters of cholestasis. Cholangiocytes co-cultured with neutrophils lost ITPR3, and neutrophils from patients with AH were more potent than control neutrophils. Biochemical and histological findings were recapitulated in an AH animal model. Loss of ITPR3 was attenuated by neutrophils in which surface membrane proteins were removed. RNA-seq analysis implicated integrin ß1 (ITGB1) in neutrophil-cholangiocyte interactions and interference with ITGB1 on cholangiocytes blocked the ability of neutrophils to reduce cholangiocyte ITPR3 expression. Cell adhesion molecules on neutrophils interacted with ITGB1 to trigger RAC1-induced JNK activation, causing a c-Jun-mediated decrease in ITPR3 in cholangiocytes. CONCLUSIONS: Neutrophils bind to ITGB1 on cholangiocytes to contribute to cholestasis in AH. This previously unrecognised role for cholangiocytes in this disease alters our understanding of its pathogenesis and identifies new therapeutic targets.
Asunto(s)
Conductos Biliares/citología , Colestasis/complicaciones , Hepatitis Alcohólica/etiología , Neutrófilos/fisiología , Adulto , Animales , Conductos Biliares/patología , Colestasis/patología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Hepatitis Alcohólica/patología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana EdadRESUMEN
Cholangiocarcinoma (CCA) is the second most common malignancy arising in the liver. It carries a poor prognosis, in part because its pathogenesis is not well understood. The type 3 inositol 1,4,5-trisphosphate receptor (ITPR3) is the principal intracellular calcium ion (Ca2+ ) release channel in cholangiocytes, and its increased expression has been related to the pathogenesis of malignancies in other types of tissues, so we investigated its role in CCA. ITPR3 expression was increased in both hilar and intrahepatic CCA samples as well as in CCA cell lines. Deletion of ITPR3 from CCA cells impaired proliferation and cell migration. A bioinformatic analysis suggested that overexpression of ITPR3 in CCA would have a mitochondrial phenotype, so this was also examined. ITPR3 normally is concentrated in a subapical region of endoplasmic reticulum (ER) in cholangiocytes, but both immunogold electron microscopy and super-resolution microscopy showed that ITPR3 in CCA cells was also in regions of ER in close association with mitochondria. Deletion of ITPR3 from these cells impaired mitochondrial Ca2+ signaling and led to cell death. Conclusion: ITPR3 expression in cholangiocytes becomes enhanced in CCA. This contributes to malignant features, including cell proliferation and migration and enhanced mitochondrial Ca2+ signaling.
Asunto(s)
Neoplasias de los Conductos Biliares/etiología , Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/etiología , Colangiocarcinoma/patología , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Células Cultivadas , HumanosRESUMEN
Clinical conditions that result in endotoxemia, such as sepsis and alcoholic hepatitis (AH), often are accompanied by cholestasis. Although hepatocellular changes in response to lipopolysaccharide (LPS) have been well characterized, less is known about whether and how cholangiocytes contribute to this form of cholestasis. We examined effects of endotoxin on expression and function of the type 3 inositol trisphosphate receptor (ITPR3), because this is the main intracellular Ca2+ release channel in cholangiocytes, and loss of it impairs ductular bicarbonate secretion. Bile duct cells expressed the LPS receptor, Toll-like receptor 4 (TLR4), which links to activation of nuclear factor-κB (NF-κB). Analysis of the human ITPR3 promoter revealed five putative response elements to NF-κB, and promoter activity was inhibited by p65/p50. Nested 0.5- and 1.0-kilobase (kb) deletion fragments of the ITPR3 promoter were inhibited by NF-κB subunits. Chromatin immunoprecipitation (ChIP) assay showed that NF-κB interacts with the ITPR3 promoter, with an associated increase in H3K9 methylation. LPS decreased ITPR3 mRNA and protein expression and also decreased sensitivity of bile duct cells to calcium agonist stimuli. This reduction was reversed by inhibition of TLR4. ITPR3 expression was decreased or absent in cholangiocytes from patients with cholestasis of sepsis and from those with severe AH. Conclusion: Stimulation of TLR4 by LPS activates NF-κB to down-regulate ITPR3 expression in human cholangiocytes. This may contribute to the cholestasis that can be observed in conditions such as sepsis or AH.
Asunto(s)
Conductos Biliares/efectos de los fármacos , Conductos Biliares/metabolismo , Endotoxemia/metabolismo , Endotoxinas/toxicidad , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Adulto , Señalización del Calcio/efectos de los fármacos , Colestasis/etiología , Colestasis/metabolismo , Endotoxemia/complicaciones , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hepatitis Alcohólica/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismoRESUMEN
BACKGROUND & OBJECTIVES: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+ signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+ release channels in HCC. DESIGN: Expression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects of de novo ITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice. RESULTS: ITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. The ITPR3 gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+ signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion of ITPR3 from human HCC cells enhanced apoptosis. CONCLUSIONS: These results provide evidence that de novo expression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias Hepáticas/metabolismo , Adulto , Animales , Apoptosis/fisiología , Señalización del Calcio/fisiología , Carcinogénesis/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proliferación Celular/fisiología , Células Cultivadas , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Hepatocitos/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/deficiencia , Receptores de Inositol 1,4,5-Trifosfato/genética , Hígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Regeneración Hepática/fisiología , Masculino , Ratones Noqueados , Persona de Mediana Edad , Análisis de SupervivenciaRESUMEN
The type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) is the principal intracellular Ca2+ release channel in hepatocytes, and so is important for bile secretion and other functions. IP3R2 activity is regulated in part by post-translational modifications but little is known about transcriptional regulation of its expression. We found that both IP3R2 mRNA and protein levels in liver were increased during fasting. Treatment of hepatocytes with forskolin or 8-CPT-cAMP also increased IP3R2, and this was reduced by actinomycin D. Analysis of the IP3R2 promoter revealed five CREs, and CREB potently increased promoter activity. Mutation of CRE4 or CRE5 decreased induction by CREB, and ChIP assay showed recruitment of CREB to these sites. Adenylyl cyclase (AC) 6 and 9 were the principal AC isoforms detected in rat hepatocytes, and silencing either one decreased organic anion secretion, which depends on IP3R2. Secretion furthermore was increased by overnight but not acute treatment with forskolin or 8-CPT-cAMP. These findings provide evidence that IP3R2 expression is transcriptionally regulated by cAMP via CREB binding to CRE elements in its promoter. The findings furthermore suggest that this mechanism is relevant for hormonal regulation of bile secretion.
Asunto(s)
Proteína de Unión a CREB/genética , AMP Cíclico/metabolismo , Hepatocitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , ARN Mensajero/genética , Elementos de Respuesta , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Sitios de Unión , Proteína de Unión a CREB/metabolismo , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Dactinomicina/farmacología , Ayuno , Regulación de la Expresión Génica , Células Hep G2 , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Mutación , Cultivo Primario de Células , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Tionucleótidos/farmacologíaRESUMEN
The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP3R3) is apically localized and triggers Ca(2+) waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP3R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP3R3 in cholangiocyte Ca(2+) signaling and secretion is well established and because loss of InsP3R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3'-UTR of human InsP3R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP3R3-3'UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP3R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP3R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP3-mediated Ca(2+) signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP3R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP3R3 expression and InsP3R3-mediated Ca(2+) signaling and secretion.
Asunto(s)
Calcio/metabolismo , Epigénesis Genética , Células Epiteliales/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Cirrosis Hepática Biliar/genética , MicroARNs/genética , Regiones no Traducidas 3' , Secuencia de Bases , Conductos Biliares/metabolismo , Conductos Biliares/patología , Sitios de Unión , Señalización del Calcio , Línea Celular , Células Epiteliales/patología , Genes Reporteros , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Biliar/metabolismo , Cirrosis Hepática Biliar/patología , Luciferasas/genética , Luciferasas/metabolismo , MicroARNs/metabolismo , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Alcoholic hepatitis affects up to one-third of individuals who abuse alcohol and can be associated with high mortality. Although this disorder is characterized by hepatocellular damage, steatosis and neutrophil infiltration, recent evidence suggests that cholestasis or impaired bile secretion may be a frequent occurrence as well. Bile secretion results from the concerted activity of hepatocytes and cholangiocytes, the epithelial cells that line the bile ducts. Hepatocytes secrete bile acids and conjugated products into the bile canaliculi, which then are modified by cholangiocytes through secretion of bicarbonate and water to give rise to the final secreted bile. Here the molecular mechanisms regulating bile secretion in cholangiocytes are reviewed. Moreover, we discuss how the expression of intracellular Ca(2+) channels might be regulated in cholangiocytes, plus evidence that components of the Ca(2+) signaling machinery are altered in a range of cholestatic diseases of the bile ducts.
Asunto(s)
Conductos Biliares/patología , Señalización del Calcio , Células Epiteliales/patología , Animales , Bicarbonatos/metabolismo , Conductos Biliares/citología , Conductos Biliares/metabolismo , Colestasis/patología , Hepatitis Alcohólica/metabolismo , Hepatitis Alcohólica/patología , HumanosRESUMEN
Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, nondestructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here, we report a completely different mechanism by which neutrophils act nondestructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of Serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy individuals. This nondestructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.
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Señalización del Calcio , Hepatitis Alcohólica , Hepatocitos , Neutrófilos , Elastasa Pancreática , Humanos , Neutrófilos/metabolismo , Neutrófilos/patología , Animales , Ratones , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatitis Alcohólica/metabolismo , Hepatitis Alcohólica/patología , Hepatitis Alcohólica/genética , Elastasa Pancreática/metabolismo , Masculino , Proliferación Celular , Canales de Calcio/metabolismo , Canales de Calcio/genética , FemeninoRESUMEN
Mitochondria-associated membranes (MAMs) are signaling domains formed at points of contact between the endoplasmic reticulum and mitochondria that are essential for mitochondrial Ca2+ signaling, energy metabolism and cell survival. Thoudam et al. now show that MAMs are dynamically regulated by pyruvate dehydrogenase kinase 4 in alcohol-associate liver disease, adding one more piece to the ever more complex puzzle of ER-mitochondria interactions in health and disease.
Asunto(s)
Señalización del Calcio , Membranas Mitocondriales , Membranas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismoAsunto(s)
Regeneración Hepática , Mitocondrias , Hepatectomía , Humanos , Hígado , Mitocondrias HepáticasRESUMEN
UNLABELLED: Bile salt secretion is mediated primarily by the bile salt export pump (Bsep), a transporter on the canalicular membrane of the hepatocyte. However, little is known about the short-term regulation of Bsep activity. Ca(2+) regulates targeting and insertion of transporters in many cell systems, and Ca(2+) release near the canalicular membrane is mediated by the type II inositol 1,4,5-trisphosphate receptor (InsP3R2), so we investigated the possible role of InsP3R2 in modulating Bsep activity. The kinetics of Bsep activity were monitored by following secretion of the fluorescent Bsep substrate cholylglycylamido-fluorescein (CGamF) in rat hepatocytes in collagen sandwich culture, an isolated cell system in which structural and functional polarity is preserved. CGamF secretion was nearly eliminated in cells treated with Bsep small interfering RNA (siRNA), demonstrating specificity of this substrate for Bsep. Secretion was also reduced after chelating intracellular calcium, inducing redistribution of InsP3R2 by depleting the cell membrane of cholesterol, or reducing InsP3R function by either knocking down InsP3R2 expression using siRNA or pharmacologic inhibition using xestospongin C. Confocal immunofluorescence showed that InsP3R2 and Bsep are in close proximity in the canalicular region, both in rat liver and in hepatocytes in sandwich culture. However, after knocking down InsP3R2 or inducing its dysfunction with cholesterol depletion, Bsep redistributed intracellularly. Finally, InsP3R2 was lost from the pericanalicular region in animal models of estrogen- and endotoxin-induced cholestasis. CONCLUSION: These data provide evidence that pericanalicular calcium signaling mediated by InsP3R2 plays an important role in maintaining bile salt secretion through posttranslational regulation of Bsep, and suggest that loss or redistribution of InsP3R2 may contribute to the pathophysiology of intrahepatic cholestasis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ácidos y Sales Biliares/metabolismo , Señalización del Calcio/fisiología , Colestasis/metabolismo , Hepatocitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Animales , Canalículos Biliares/metabolismo , Calcio/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Colágeno , Modelos Animales de Enfermedad , Hepatocitos/citología , Lipopolisacáridos/farmacología , Masculino , Procesamiento Proteico-Postraduccional/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
UNLABELLED: Subcellular Ca(2+) signals control a variety of responses in the liver. For example, mitochondrial Ca(2+) (Ca(mit)(2+)) regulates apoptosis, whereas Ca(2+) in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca(mit)(2+) also affects liver regeneration. The Ca(2+)-buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin-mitochondrial targeting sequence-green fluorescent protein (PV-MITO-GFP). This construct properly localized to and effectively buffered Ca(2+) signals in the mitochondrial matrix. Additionally, the expression of PV-MITO-GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl-2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2-associated X protein (bax), apoptotic peptidase activating factor 1, and caspase-6]. PV-MITO-GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca(mit)(2+) buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl-2 and the decreased expression of bax. CONCLUSION: Together, these results reveal an essential role for Ca(mit)(2+) in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis.
Asunto(s)
Apoptosis/fisiología , Calcio/metabolismo , Regeneración Hepática/fisiología , Mitocondrias Hepáticas/metabolismo , Animales , Señalización del Calcio/fisiología , Proliferación Celular , Masculino , Modelos Animales , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Fluid and bicarbonate secretion is a principal function of cholangiocytes, and impaired secretion results in cholestasis. Cholangiocyte secretion depends on peri-apical expression of the type 3 inositol trisphosphate receptor (ITPR3), and loss of this intracellular Ca2+ release channel is a final common event in most cholangiopathies. Here we investigated the mechanism by which ITPR3 localizes to the apical region to regulate secretion. Isolated bile duct units, primary mouse cholangiocytes, and polarized Madin-Darby canine kidney (MDCK) cells were examined using a combination of biochemical and fluorescence microscopy techniques to investigate the mechanism of ITPR3 targeting to the apical region. Apical localization of ITPR3 depended on the presence of intact lipid rafts as well as interactions with both caveolin 1 (CAV1) and myosin heavy chain 9 (MYH9). Chemical disruption of lipid rafts or knockdown of CAV1 or MYH9 redistributed ITPR3 away from the apical region. MYH9 interacted with the five c-terminal amino acids of the ITPR3 peptide. Disruption of lipid rafts impaired Ca2+ signaling, and absence of CAV1 impaired both Ca2+ signaling and fluid secretion. Conclusion: A cooperative mechanism involving MYH9, CAV1, and apical lipid rafts localize ITPR3 to the apical region to regulate Ca2+ signaling and secretion in cholangiocytes.
Asunto(s)
Señalización del Calcio , Caveolina 1 , Aminoácidos/metabolismo , Animales , Bicarbonatos/metabolismo , Señalización del Calcio/fisiología , Caveolina 1/genética , Perros , Inositol , Receptores de Inositol 1,4,5-Trifosfato/genética , Ratones , Cadenas Pesadas de Miosina/genéticaRESUMEN
Liver test abnormalities are frequently observed in patients with coronavirus disease 2019 (COVID-19) and are associated with worse prognosis. However, information is limited about pathological changes in the liver in this infection, so the mechanism of liver injury is unclear. Here we describe liver histopathology and clinical correlates of 27 patients who died of COVID-19 in Manaus, Brazil. There was a high prevalence of liver injury (elevated alanine aminotransferase and aspartate aminotransferase in 44% and 48% of patients, respectively) in these patients. Histological analysis showed sinusoidal congestion and ischemic necrosis in more than 85% of the cases, but these appeared to be secondary to systemic rather than intrahepatic thrombotic events, as only 14% and 22% of samples were positive for CD61 (marker of platelet activation) and C4d (activated complement factor), respectively. Furthermore, the extent of these vascular findings did not correlate with the extent of transaminase elevations. Steatosis was present in 63% of patients, and portal inflammation was present in 52%. In most cases, hepatocytes expressed angiotensin-converting enzyme 2 (ACE2), which is responsible for binding and entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), even though this ectoenzyme was minimally expressed on hepatocytes in normal controls. However, SARS-CoV-2 staining was not observed. Most hepatocytes also expressed inositol 1,4,5-triphosphate receptor 3 (ITPR3), a calcium channel that becomes expressed in acute liver injury. Conclusion: The hepatocellular injury that commonly occurs in patients with severe COVID-19 is not due to the vascular events that contribute to pulmonary or cardiac damage. However, new expression of ACE2 and ITPR3 with concomitant inflammation and steatosis suggests that liver injury may result from inflammation, metabolic abnormalities, and perhaps direct viral injury.
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COVID-19/complicaciones , Hepatopatías/patología , Hepatopatías/virología , Hígado/patología , Hígado/virología , Adulto , Anciano , Anciano de 80 o más Años , Brasil , COVID-19/mortalidad , COVID-19/patología , COVID-19/fisiopatología , Femenino , Humanos , Hígado/fisiopatología , Hepatopatías/diagnóstico , Hepatopatías/fisiopatología , Pruebas de Función Hepática , Masculino , Persona de Mediana EdadRESUMEN
UNLABELLED: Multidrug resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. CONCLUSION: InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Adenosina Trifosfato/farmacología , Animales , Bilis/metabolismo , Bilirrubina/sangre , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ácido Tauroquenodesoxicólico/farmacologíaRESUMEN
Calcium is a second messenger in virtually all cells and tissues. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nuclear component of InsP3-mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus.
Asunto(s)
Señalización del Calcio/genética , Calcio/metabolismo , Estructuras del Núcleo Celular/metabolismo , Células Eucariotas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Canales de Calcio/metabolismo , Membrana Celular/metabolismo , Estructuras del Núcleo Celular/ultraestructura , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Células Eucariotas/citología , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Receptores de Inositol 1,4,5-Trifosfato , Microscopía Confocal , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Fotoquímica , Proteína Quinasa C/metabolismo , Transporte de Proteínas/fisiología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Células Tumorales CultivadasRESUMEN
Inositol 1,4,5 trisphosphate receptors (ITPRs) are a family of endoplasmic reticulum Ca2+ channels essential for the control of intracellular Ca2+ levels in virtually every mammalian cell type. The three isoforms (ITPR1, ITPR2 and ITPR3) are highly homologous in amino acid sequence, but they differ considerably in terms of biophysical properties, subcellular localization, and tissue distribution. Such differences underscore the variety of cellular responses triggered by each isoform and suggest that the expression/activity of specific isoforms might be linked to particular pathophysiological states. Indeed, recent findings demonstrate that changes in expression of ITPR isoforms are associated with a number of human diseases ranging from fatty liver disease to cancer. ITPR3 is emerging as the isoform that is particularly important in the pathogenesis of various human diseases. Here we review the physiological and pathophysiological roles of ITPR3 in various tissues and the mechanisms by which the expression of this isoform is modulated in health and disease.
Asunto(s)
Canales de Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Animales , Fenómenos Biofísicos , Enfermedad , Retículo Endoplásmico/metabolismo , Humanos , Mitocondrias/metabolismo , Modelos BiológicosRESUMEN
Hepatic ischemia-reperfusion injury is seen in a variety of clinical conditions, including hepatic thrombosis, systemic hypotension, and liver transplantation. Calcium (Ca2+) signaling mediates several pathophysiological processes in the liver, but it is not known whether and how intracellular Ca2+ channels are involved in the hepatocellular events secondary to ischemia-reperfusion. Using an animal model of hepatic ischemia-reperfusion injury, we observed a progressive increase in expression of the type 3 isoform of the inositol trisphosphate receptor (ITPR3), an intracellular Ca2+ channel that is not normally expressed in healthy hepatocytes. ITPR3 expression was upregulated, at least in part, by a combination of demethylation of the ITPR3 promoter region and the increased transcriptional activity of the nuclear factor of activated T-cells (NFAT). Additionally, expression of pro-inflammatory interleukins and necrotic surface area were less pronounced in livers of control animals compared to liver-specific ITPR3 KO mice subjected to hepatic damage. Corroborating these findings, ITPR3 expression and activation of NFAT were observed in hepatocytes of liver biopsies from patients who underwent liver ischemia caused by thrombosis after organ transplant. Together, these results are consistent with the idea that ITPR3 expression in hepatocytes plays a protective role during hepatic injury induced by ischemia-reperfusion.
Asunto(s)
Hepatocitos/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Hígado/metabolismo , Hígado/patología , Sustancias Protectoras/metabolismo , Daño por Reperfusión/metabolismo , Animales , Señalización del Calcio , Desmetilación del ADN , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción NFATC/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
In the heart, cytosolic Ca(2+) signals are well-characterized events that participate in the activation of cell contraction. In contrast, nuclear Ca(2+) contribution to cardiomyocyte function remains elusive. Here, we examined functional consequences of buffering nuclear Ca(2+) in neonatal cardiomyocytes. We report that cardiomyocytes contain a nucleoplasmic reticulum, which expresses both ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (InsP(3)R), providing a possible way for active regulation of nuclear Ca(2+). Adenovirus constructs encoding the Ca(2+) buffer protein parvalbumin were targeted to the nucleus with a nuclear localization signal (Ad-PV-NLS) or to the cytoplasm with a nuclear exclusion signal (Ad-PV-NES). A decrease in the amplitude of global Ca(2+) transients and RyR-II expression, as well as an increase in cell beating rate were observed in Ad-PV-NES and Ad-PV-NLS cells. When nuclear Ca(2+) buffering was imposed nuclear enlargement, increased calcineurin expression, NFAT translocation to the nucleus and subcellular redistribution of atrial natriuretic peptide were observed. Furthermore, prolongation of action potential duration occurred in adult ventricular myocytes. These results suggest that nuclear Ca(2+) levels underlie the regulation of specific protein targets and thereby modulate cardiomyocyte function. The local nuclear Ca(2+) signaling and the structures that control it constitute a novel regulatory motif in the heart.