RESUMEN
Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, cross talk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools' specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent nuclear factor of activated T cells nuclear translocation, suggesting that it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA-ROCK-myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.
Asunto(s)
Señalización del Calcio/genética , Calcio/metabolismo , Fototransducción/genética , Optogenética/métodos , Proteína de Unión al GTP rhoA/genética , Animales , Técnicas Biosensibles/métodos , Diferenciación Celular , Proliferación Celular , Perros , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Luz , Células de Riñón Canino Madin Darby , Especificidad de Órganos , Fosfoinositido Fosfolipasa C/genética , Fosfoinositido Fosfolipasa C/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismo , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). Given that FAs are the components of membranes, FA metabolic enzymes might be associated with regulation of ER membrane functions. However, it remains unclear whether there is the interplay between FA metabolic enzymes and ER membrane proteins. Trans-2-enoyl-CoA reductase (TER) is an FA reductase present in the ER membrane and catalyzes the last step in the FA elongation cycle and sphingosine degradation pathway. Here we identify sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), an ER Ca2+ pump responsible for Ca2+ accumulation in the ER, as a TER-binding protein by affinity purification from HEK293 cell lysates. We show that TER directly binds to SERCA2b by in vitro assays using recombinant proteins. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes of HEK293 cells. Depletion of TER increases Ca2+ storage in the ER and accelerates SERCA2b-dependent Ca2+ uptake to the ER after ligand-induced Ca2+ release. Moreover, depletion of TER reduces the Ca2+-dependent nuclear translocation of nuclear factor of activated T cells 4. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca2+ accumulation in the ER.
Asunto(s)
Retículo Endoplásmico/metabolismo , Ácidos Grasos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Transporte Activo de Núcleo Celular/genética , Calcio/metabolismo , Señalización del Calcio/genética , Retículo Endoplásmico/genética , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Ligandos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Unión Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/químicaRESUMEN
Dilated cardiomyopathy (DCM) is clinically characterized by dilated ventricular cavities and reduced ejection fraction, leading to heart failure and increased thromboembolic risk. Mutations in thin-filament regulatory proteins can cause DCM and have been shown in vitro to reduce contractility and myofilament Ca2+-affinity. In this work we have studied the functional consequences of mutations in cardiac troponin T (R131W), cardiac troponin I (K36Q) and α-tropomyosin (E40K) using adenovirally transduced isolated guinea pig left ventricular cardiomyocytes. We find significantly reduced fractional shortening with reduced systolic Ca2+. Contraction and Ca2+ reuptake times were slowed, which contrast with some findings in murine models of myofilament Ca2+ desensitization. We also observe increased sarcoplasmic reticulum (SR) Ca2+ load and smaller fractional SR Ca2+ release. This corresponds to a reduction in SR Ca2+-ATPase activity and increase in sodium-calcium exchanger activity. We also observe dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT), with concordant RAC-α-serine/threonine protein kinase (Akt) phosphorylation but no change to extracellular signal-regulated kinase activation in chronically paced cardiomyocytes expressing DCM mutations. These changes in Ca2+ handling and signaling are common to all three mutations, indicating an analogous pathway of disease pathogenesis in thin-filament sarcomeric DCM. Previous work has shown that changes to myofilament Ca2+ sensitivity caused by DCM mutations are qualitatively opposite from hypertrophic cardiomyopathy (HCM) mutations in the same genes. However, we find several common pathways such as increased relaxation times and NFAT activation that are also hallmarks of HCM. This suggests more complex intracellular signaling underpinning DCM, driven by the primary mutation.NEW & NOTEWORTHY Dilated cardiomyopathy (DCM) is a frequently occurring cardiac disorder with a degree of genetic inheritance. We have found that DCM mutations in proteins that regulate the contractile machinery cause alterations to contraction, calcium-handling, and some new signaling pathways that provide stimuli for disease development. We have used guinea pig cells that recapitulate human calcium-handling and introduced the mutations using adenovirus gene transduction to look at the initial triggers of disease before remodeling.
Asunto(s)
Señalización del Calcio , Cardiomiopatía Dilatada/genética , Proteínas de Microfilamentos/genética , Mutación , Contracción Miocárdica , Miocitos Cardíacos/enzimología , Factores de Transcripción NFATC/metabolismo , Proteína Oncogénica v-akt/metabolismo , Función Ventricular Izquierda , Animales , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/fisiopatología , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/fisiopatología , Células Cultivadas , Predisposición Genética a la Enfermedad , Cobayas , Masculino , Proteínas de Microfilamentos/metabolismo , Fenotipo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismo , Troponina I/genética , Troponina I/metabolismo , Troponina T/genética , Troponina T/metabolismoRESUMEN
Calreticulin is a highly conserved, ubiquitous Ca2+-buffering protein in the endoplasmic reticulum that controls transcriptional activity of various developmental programs and also of embryonic stem cell (ESC) differentiation. Calreticulin activates calcineurin, which dephosphorylates and induces the nuclear import of the osteogenic transcription regulator nuclear factor of activated T cells 1 (NFATC1). We investigated whether calreticulin controls a switch between osteogenesis and chondrogenesis in mouse ESCs through NFATC1. We found that in the absence of calreticulin, intranuclear transport of NFATC1 is blocked and that differentiation switches from osteogenic to chondrogenic, a process that could be mimicked by chemical inhibition of NFAT translocation. Glycogen synthase kinase 3ß (GSK3ß) deactivation and nuclear localization of ß-catenin critical to osteogenesis were abrogated by calreticulin deficiency or NFAT blockade. Chemically induced GSK3ß inhibition bypassed the calreticulin/calcineurin axis and increased osteoblast output from both control and calreticulin-deficient ESCs, while suppressing chondrogenesis. Calreticulin-deficient ESCs or cells treated with an NFAT blocker had enhanced expression of dickkopf WNT-signaling pathway inhibitor 1 (Dkk1), a canonical Wnt pathway antagonist that blocks GSK3ß deactivation. The addition of recombinant mDKK1 switched osteogenic ESC differentiation toward chondrogenic differentiation. The results of our study indicate a role for endoplasmic reticulum calcium signaling via calreticulin in the differentiation of ESCs to closely associated osteoblast or chondrocyte lineages.
Asunto(s)
Señalización del Calcio , Calreticulina/metabolismo , Diferenciación Celular , Condrocitos/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Osteoblastos/metabolismo , Animales , Calreticulina/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Noqueados , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismoRESUMEN
The calcineurin/nuclear factor of activated T cell (CN/NFAT) signaling pathway plays a critical role in the immune response. Therefore, inhibition of the CN/NFAT pathway is an important target for inflammatory disease. The conserved PXIXIT and LXVP motifs of CN substrates and targeting proteins have been recognized. Based on the affinity ability and inhibitory effect of these docking sequences on CN, we designed a bioactive peptide (named pep3) against the CN/NFAT interaction, which has two binding sites derived from the RCAN1-PXIXIT motif and the NFATc1-LXVP motif. The shortest linker between the two binding sites in pep3 is derived from A238L, a physiological binding partner of CN. Microscale thermophoresis revealed that pep3 has two docking sites on CN. Pep3 also has the most potent inhibitory effect on CN. It is suggested that pep3 contains an NFATc1-LXVP-substrate recognition motif and RCAN1-PXIXIT-mediated anchoring to CN. Expression of this peptide significantly suppresses CN/NFAT signaling. Cell-permeable 11-arginine-modified pep3 (11R-pep3) blocks the NFAT downstream signaling pathway. Intranasal administration of the 11R-pep3 peptide inhibits airway inflammation in an ovalbumin-induced asthma model. Our results suggest that pep3 is promising as an immunosuppressive agent and can be used in topical remedies.
Asunto(s)
Calcineurina/metabolismo , Terapia de Inmunosupresión/métodos , Factores de Transcripción NFATC/metabolismo , Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Secuencias de Aminoácidos , Animales , Sitios de Unión , Humanos , Inmunosupresores/farmacología , Inflamación/tratamiento farmacológico , Inflamación/prevención & control , Transducción de Señal/efectos de los fármacosRESUMEN
The transcription factor signal transducer and activator of transcription 3 (STAT3) plays a central role in cell survival and function. STAT3 has been demonstrated to participate in the maintenance of bone homeostasis in osteoblasts, but its role in osteoclasts in vivo remains poorly defined. Here, we generated a conditional knockout mouse model in which Stat3 was deleted in osteoclasts using a cathepsin K-Cre (Ctsk-Cre) driver. We observed that osteoclast-specific Stat3 deficiency caused increased bone mass in mice, which we attributed to impaired bone catabolism by osteoclasts. Stat3-deficient bone marrow macrophages (BMMs) showed decreased expression of nuclear factor of activated T cells, cytoplasm 1 (NFATc1), and reduced osteoclast differentiation determined by decreases in osteoclast number, tartrate-resistant acid phosphatase activity, and expression of osteoclast marker genes. Enforced expression of NFATc1 in Stat3-deficient BMMs rescued the impaired osteoclast differentiation. Mechanistically, we revealed that STAT3 could drive the transcription of NFATc1 by binding to its promoter. Furthermore, preventing STAT3 activation by using an inhibitor of upstream phosphorylases, AG490, also impaired osteoclast differentiation and formation in a similar way as gene deletion of Stat3 In summary, our data provide the first evidence that STAT3 is significant in osteoclast differentiation and bone homeostasis in vivo, and it may be identified as a potential pharmacological target for the treatment of bone metabolic diseases through regulation of osteoclast activity.
Asunto(s)
Huesos/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Factor de Transcripción STAT3/metabolismo , Animales , Femenino , Regulación de la Expresión Génica , Homeostasis , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Factores de Transcripción NFATC/genética , Osteoclastos/citología , Factor de Transcripción STAT3/genética , Transducción de SeñalRESUMEN
We previously reported that among the 37 RING finger protein (RNF) family members, RNF183 mRNA is specifically expressed in the kidney under normal conditions. However, the mechanism supporting its kidney-specific expression pattern remains unclear. In this study, we elucidated the mechanism of the transcriptional activation of murine Rnf183 in inner-medullary collecting duct cells. Experiments with anti-RNF183 antibody revealed that RNF183 is predominantly expressed in the renal medulla. Among the 37 RNF family members, Rnf183 mRNA expression was specifically increased in hypertonic conditions, a hallmark of the renal medulla. RNF183 up-regulation was consistent with the activation of nuclear factor of activated T cells 5 (NFAT5), a transcription factor essential for adaptation to hypertonic conditions. Accordingly, siRNA-mediated knockdown of NFAT5 down-regulated RNF183 expression. Furthermore, the -3,466 to -3,136-bp region upstream of the mouse Rnf183 promoter containing the NFAT5-binding motif is conserved among mammals. A luciferase-based reporter vector containing the NFAT5-binding site was activated in response to hypertonic stress, but was inhibited by a mutation at the NFAT5-binding site. ChIP assays revealed that the binding of NFAT5 to this DNA site is enhanced by hypertonic stress. Of note, siRNA-mediated RNF183 knockdown increased hypertonicity-induced caspase-3 activation and decreased viability of mIMCD-3 cells. These results indicate that (i) RNF183 is predominantly expressed in the normal renal medulla, (ii) NFAT5 stimulates transcriptional activation of Rnf183 by binding to its cognate binding motif in the Rnf183 promoter, and (iii) RNF183 protects renal medullary cells from hypertonicity-induced apoptosis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Túbulos Renales Colectores/metabolismo , Presión Osmótica , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/biosíntesis , Regulación hacia Arriba , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Células HEK293 , Células HeLa , Humanos , Túbulos Renales Colectores/citología , Ratones , Elementos de Respuesta , Factores de Transcripción/genética , Transcripción Genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Pancreatic ß-cell dysfunction and death contribute to the onset of diabetes, and novel strategies of ß-cell function and survival under diabetogenic conditions need to be explored. We previously demonstrated that Isx9, a small molecule based on the isoxazole scaffold, drives neuroendocrine phenotypes by increasing the expression of genes required for ß-cell function and improves glycemia in a model of ß cell regeneration. We further investigated the role of Isx9 in ß-cell survival. We find that Isx9 drives the expression of Calbindin-D28K (D28K), a key regulator of calcium homeostasis, and plays a cytoprotective role through its calcium buffering capacity in ß cells. Isx9 increased the activity of the calcineurin (CN)/cytoplasmic nuclear factor of the activated T-cells (NFAT) transcription factor, a key regulator of D28K, and improved the recruitment of NFATc1, cAMP response element-binding protein (CREB), and p300 to the D28K promoter. We found that nutrient stimulation increased D28K plasma membrane enrichment and modulated calcium channel activity in order to regulate glucose-induced insulin secretion. Isx9-mediated expression of D28K protected ß cells against chronic stress induced by serum withdrawal or chronic inflammation by reducing caspase 3 activity. Consequently, Isx9 improved human islet function after transplantation in NOD-SCID mice in a streptozotocin-induced diabetes model. In summary, Isx9 significantly regulates expression of genes relevant to ß cell survival and function, and may be an attractive therapy to treat diabetes and improve islet function post-transplantation.
Asunto(s)
Calbindinas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Isoxazoles/farmacología , Tiofenos/farmacología , Animales , Calbindinas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Diabetes Mellitus Experimental/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Trasplante de Islotes Pancreáticos , Ratones Endogámicos NOD , Ratones SCID , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , RatasRESUMEN
Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca2+ sensitivity. Mouse models exhibit increased Ca2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca2+ buffering and altered Ca2+ handling contribute to HCM pathogenesis via activation of Ca2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca2+ handling and Ca2+-dependent signaling in a model system possessing Ca2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised â¼50% of the total protein. HCM mutations significantly lowered the Kd of Ca2+ binding, resulting in higher Ca2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca2+] and slowed Ca2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal-regulated kinase pathways. Altered myofilament Ca2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca2+ sensitivity provides an attractive therapeutic approach in HCM.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Cardiomiopatía Hipertrófica/patología , Mutación , Tropomiosina/genética , Troponina I/genética , Troponina T/genética , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Células Cultivadas , Cobayas , Humanos , Miofibrillas/metabolismo , Miofibrillas/patología , Fosforilación , Sarcómeros/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tropomiosina/metabolismo , Troponina I/metabolismo , Troponina T/metabolismoRESUMEN
The nucleus pulposus (NP) of intervertebral discs experiences dynamic changes in tissue osmolarity because of diurnal loading of the spine. TonEBP/NFAT5 is a transcription factor that is critical in osmoregulation as well as survival of NP cells in the hyperosmotic milieu. The goal of this study was to investigate whether cyclooxygenase-2 (COX-2) expression is osmoresponsive and dependent on TonEBP, and whether it serves an osmoprotective role. NP cells up-regulated COX-2 expression in hyperosmotic media. The induction of COX-2 depended on elevation of intracellular calcium levels and p38 MAPK pathway, but independent of calcineurin signaling as well as MEK/ERK and JNK pathways. Under hyperosmotic conditions, both COX-2 mRNA stability and its proximal promoter activity were increased. The proximal COX-2 promoter (-1840/+123 bp) contained predicted binding sites for TonEBP, AP-1, NF-κB, and C/EBP-ß. While COX-2 promoter activity was positively regulated by both AP-1 and NF-κB, AP-1 had no effect and NF-κB negatively regulated COX-2 protein levels under hyperosmotic conditions. On the other hand, TonEBP was necessary for both COX-2 promoter activity and protein up-regulation in response to hyperosmotic stimuli. Ex vivo disc organ culture studies using hypomorphic TonEBP+/- mice confirmed that TonEBP is required for hyperosmotic induction of COX-2. Importantly, the inhibition of COX-2 activity under hyperosmotic conditions resulted in decreased cell viability, suggesting that COX-2 plays a cytoprotective and homeostatic role in NP cells for their adaptation to dynamically loaded hyperosmotic niches.
Asunto(s)
Calcio/metabolismo , Ciclooxigenasa 2/metabolismo , Factores de Transcripción NFATC/metabolismo , Núcleo Pulposo/citología , Presión Osmótica , Transducción de Señal , Animales , Señalización del Calcio , Células Cultivadas , Ciclooxigenasa 2/genética , Femenino , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/genética , Núcleo Pulposo/metabolismo , Osmorregulación , Regiones Promotoras Genéticas , Ratas , Regulación hacia ArribaRESUMEN
Heart failure is an aging-associated disease that is the leading cause of death worldwide. Sirtuin family members have been largely studied in the context of aging and aging-associated diseases. Sirtuin 2 (SIRT2) is a cytoplasmic protein in the family of sirtuins that are NAD+-dependent class III histone deacetylases. In this work, we studied the role of SIRT2 in regulating nuclear factor of activated T-cells (NFAT) transcription factor and the development of cardiac hypertrophy. Confocal microscopy analysis indicated that SIRT2 is localized in the cytoplasm of cardiomyocytes and SIRT2 levels are reduced during pathological hypertrophy of the heart. SIRT2-deficient mice develop spontaneous pathological cardiac hypertrophy, remodeling, fibrosis, and dysfunction in an age-dependent manner. Moreover, young SIRT2-deficient mice develop exacerbated agonist-induced hypertrophy. In contrast, SIRT2 overexpression attenuated agonist-induced cardiac hypertrophy in cardiomyocytes in a cell-autonomous manner. Mechanistically, SIRT2 binds to and deacetylates NFATc2 transcription factor. SIRT2 deficiency stabilizes NFATc2 and enhances nuclear localization of NFATc2, resulting in increased transcription activity. Our results suggest that inhibition of NFAT rescues the cardiac dysfunction in SIRT2-deficient mice. Thus, our study establishes SIRT2 as a novel endogenous negative regulator of NFAT transcription factor.
Asunto(s)
Cardiomegalia/metabolismo , Factores de Transcripción NFATC/metabolismo , Sirtuina 2/metabolismo , Acetilación , Animales , Regulación de la Expresión Génica/genética , Histona Desacetilasas del Grupo III/metabolismo , Insuficiencia Cardíaca/metabolismo , Homeostasis , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Sirtuina 2/fisiologíaRESUMEN
Binding of receptor activator of NF-κB ligand (RANKL) to its receptor RANK on osteoclast (OC) precursors up-regulates c-Fos and CCAAT/enhancer-binding protein-α (C/EBPα), two critical OC transcription factors. However, the effects of c-Fos and C/EBPα on osteoclastogenesis have not been compared. Herein, we demonstrate that overexpression of c-Fos or C/EBPα in OC precursors up-regulates OC genes and initiates osteoclastogenesis independently of RANKL. However, although C/EBPα up-regulated c-Fos, c-Fos failed to up-regulate C/EBPα in OC precursors. Consistently, C/EBPα overexpression more strongly promoted OC differentiation than did c-Fos overexpression. RANK has a cytoplasmic 535IVVY538 (IVVY) motif that is essential for osteoclastogenesis, and we found that mutation of the IVVY motif blocked OC differentiation by partly inhibiting expression of C/EBPα but not expression of c-Fos. We therefore hypothesized that C/EBPα overexpression might rescue osteoclastogenesis in cells expressing the mutated IVVY motif. However, overexpression of C/EBPα or c-Fos failed to stimulate osteoclastogenesis in the mutant cells. Notably, the IVVY motif mutation abrogated OC gene expression compared with a vector control, suggesting that the IVVY motif might counteract OC inhibitors during osteoclastogenesis. Consistently, the IVVY motif mutant triggered up-regulation of recombinant recognition sequence-binding protein at the Jκ site (RBP-J) protein, a potent OC inhibitor. Mechanistically, C/EBPα or c-Fos overexpression in the mutant cells failed to control the up-regulated RBP-J expression, leading to suppression of OC genes. Accordingly, RBP-J silencing in the mutant cells rescued osteoclastogenesis with C/EBPα or c-Fos overexpression with C/EBPα exhibiting a stronger osteoclastogenic effect. Collectively, our findings indicate that C/EBPα is a stronger inducer of OC differentiation than c-Fos, partly via C/EBPα regulation by the RANK 535IVVY538 motif.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular , Mutación , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Regulación hacia Arriba , Secuencias de Aminoácidos , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Ratones , Osteoclastos/citología , Proteínas Proto-Oncogénicas c-fos/genética , Receptor Activador del Factor Nuclear kappa-B/genéticaRESUMEN
Intervertebral disc degeneration (IDD) causes chronic back pain and is linked to production of proinflammatory molecules by nucleus pulposus (NP) and other disc cells. Activation of tonicity-responsive enhancer-binding protein (TonEBP)/NFAT5 by non-osmotic stimuli, including proinflammatory molecules, occurs in cells involved in immune response. However, whether inflammatory stimuli activate TonEBP in NP cells and whether TonEBP controls inflammation during IDD is unknown. We show that TNF-α, but not IL-1ß or LPS, promoted nuclear enrichment of TonEBP protein. However, TNF-α-mediated activation of TonEBP did not cause induction of osmoregulatory genes. RNA sequencing showed that 8.5% of TNF-α transcriptional responses were TonEBP-dependent and identified genes regulated by both TNF-α and TonEBP. These genes were over-enriched in pathways and diseases related to inflammatory response and inhibition of matrix metalloproteases. Based on RNA-sequencing results, we further investigated regulation of novel TonEBP targets CXCL1, CXCL2, and CXCL3 TonEBP acted synergistically with TNF-α and LPS to induce CXCL1-proximal promoter activity. Interestingly, this regulation required a highly conserved NF-κB-binding site but not a predicted TonE, suggesting cross-talk between these two members of the Rel family. Finally, analysis of human NP tissue showed that TonEBP expression correlated with canonical osmoregulatory targets TauT/SLC6A6, SMIT/SLC5A3, and AR/AKR1B1, supporting in vitro findings that the inflammatory milieu during IDD does not interfere with TonEBP osmoregulation. In summary, whereas TonEBP participates in the proinflammatory response to TNF-α, therapeutic strategies targeting this transcription factor for treatment of disc disease must spare osmoprotective, prosurvival, and matrix homeostatic activities.
Asunto(s)
Disco Intervertebral/metabolismo , Osmorregulación , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Anciano , Aldehído Reductasa/biosíntesis , Aldehído Reductasa/genética , Animales , Línea Celular , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Niño , Preescolar , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Humanos , Lactante , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/patología , Lipopolisacáridos/toxicidad , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Ratas , Simportadores/biosíntesis , Simportadores/genética , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Although the involvement of Rho proteins in the pathogenesis of vascular diseases is well studied, little is known about the role of their upstream regulators, the Rho guanine nucleotide exchange factors (RhoGEFs). Here, we sought to identify the RhoGEFs involved in monocyte chemotactic protein 1 (MCP1)-induced vascular wall remodeling. We found that, among the RhoGEFs tested, MCP1 induced tyrosine phosphorylation of p115 RhoGEF but not of PDZ RhoGEF or leukemia-associated RhoGEF in human aortic smooth muscle cells (HASMCs). Moreover, p115 RhoGEF inhibition suppressed MCP1-induced HASMC migration and proliferation. Consistent with these observations, balloon injury (BI) induced p115 RhoGEF tyrosine phosphorylation in rat common carotid arteries, and siRNA-mediated down-regulation of its levels substantially attenuated BI-induced smooth muscle cell migration and proliferation, resulting in reduced neointima formation. Furthermore, depletion of p115 RhoGEF levels also abrogated MCP1- or BI-induced Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling, which, as we reported previously, is involved in vascular wall remodeling. Our findings also show that protein kinase N1 (PKN1) downstream of Rac1-cyclin D1/CDK6 and upstream of CDK4-PAK1 in the p115 RhoGEF-Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling axis is involved in the modulation of vascular wall remodeling. Of note, we also observed that CCR2-Gi/o-Fyn signaling mediates MCP1-induced p115 RhoGEF and Rac1 GTPase activation. These findings suggest that p115 RhoGEF is critical for MCP1-induced HASMC migration and proliferation in vitro and for injury-induced neointima formation in vivo by modulating Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling.
Asunto(s)
Quimiocina CCL2/agonistas , Modelos Biológicos , Músculo Liso Vascular/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Proteína de Unión al GTP rac1/agonistas , Animales , Aorta , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Común , Movimiento Celular , Proliferación Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Activación Enzimática , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/patología , Neointima/metabolismo , Neointima/patología , Fosforilación , Interferencia de ARN , Ratas , Factores de Intercambio de Guanina Nucleótido Rho/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Especificidad por Sustrato , Remodelación Vascular , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Phospholipase C-related, but catalytically inactive protein (PRIP) was previously identified as a novel inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ but lacking phospholipase activity. We recently showed that PRIP gene knock-out (KO) in mice increases bone formation and concomitantly decreases bone resorption, resulting in increased bone mineral density and trabecular bone volume. However, the role of PRIP in osteoclastogenesis has not yet been fully elucidated. Here, we investigated the effects of PRIP on bone remodeling by investigating dynamic tooth movement in mice fitted with orthodontic devices. Morphological analysis indicated that the extent of tooth movement was smaller in the PRIP-KO mice than in wild-type mice. Histological analysis revealed fewer osteoclasts on the bone-resorption side in maxillary bones of PRIP-KO mice, and osteoclast formation assays and flow cytometry indicated lower osteoclast differentiation in bone marrow cells isolated from these mice. The expression of genes implicated in bone resorption was lower in differentiated PRIP-KO cells, and genes involved in osteoclast differentiation, such as the transcription factor NFATc1, exhibited lower expression in immature PRIP-KO cells initiated by M-CSF. Moreover, calcineurin expression and activity were also lower in the PRIP-KO cells. The PRIP-KO cells also displayed fewer M-CSF-induced changes in intracellular Ca2+ and exhibited reduced nuclear localization of NFATc1. Up-regulation of intracellular Ca2+ restored osteoclastogenesis of the PRIP-KO cells. These results indicate that PRIP deficiency impairs osteoclast differentiation, particularly at the early stages, and that PRIP stimulates osteoclast differentiation through calcium-calcineurin-NFATc1 signaling via regulating intracellular Ca2.
Asunto(s)
Calcineurina/metabolismo , Calcio/metabolismo , Factores de Transcripción NFATC/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Osteoclastos/citología , Fosfolipasas de Tipo C/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Resorción Ósea , Catálisis , Diferenciación Celular , Técnicas de Cocultivo , Femenino , Citometría de Flujo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Maxilar/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ortodoncia , Osteoclastos/metabolismo , Transducción de Señal , Microtomografía por Rayos XRESUMEN
Members of the adhesion G protein-coupled receptor (aGPCR) family carry an agonistic sequence within their large ectodomains. Peptides derived from this region, called the Stachel sequence, can activate the respective receptor. As the conserved core region of the Stachel sequence is highly similar between aGPCRs, the agonist specificity of Stachel sequence-derived peptides was tested between family members using cell culture-based second messenger assays. Stachel peptides derived from aGPCRs of subfamily VI (GPR110/ADGRF1, GPR116/ADGRF5) and subfamily VIII (GPR64/ADGRG2, GPR126/ADGRG6) are able to activate more than one member of the respective subfamily supporting their evolutionary relationship and defining them as pharmacological receptor subtypes. Extended functional analyses of the Stachel sequences and derived peptides revealed agonist promiscuity, not only within, but also between aGPCR subfamilies. For example, the Stachel-derived peptide of GPR110 (subfamily VI) can activate GPR64 and GPR126 (both subfamily VIII). Our results indicate that key residues in the Stachel sequence are very similar between aGPCRs allowing for agonist promiscuity of several Stachel-derived peptides. Therefore, aGPCRs appear to be pharmacologically more closely related than previously thought. Our findings have direct implications for many aGPCR studies, as potential functional overlap has to be considered for in vitro and in vivo studies. However, it also offers the possibility of a broader use of more potent peptides when the original Stachel sequence is less effective.
Asunto(s)
Péptidos/química , Péptidos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Pollos , Chlorocebus aethiops , Células HEK293 , Humanos , Ratones , Mutación , Filogenia , Dominios Proteicos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Transcription factor tonicity-responsive enhancer-binding protein (TonEBP/NFAT5) is critical for osmo-adaptation and extracellular matrix homeostasis of nucleus pulposus (NP) cells in their hypertonic tissue niche. Recent studies implicate TonEBP signaling in inflammatory disease and rheumatoid arthritis pathogenesis. However, broader functions of TonEBP in the disc remain unknown. RNA sequencing was performed on NP cells with TonEBP knockdown under hypertonic conditions. 1140 TonEBP-dependent genes were identified and categorized using Ingenuity Pathway Analysis. Bioinformatic analysis showed enrichment of matrix homeostasis and cytokine/chemokine signaling pathways. C-C motif chemokine ligand 2 (CCL2), interleukin 6 (IL6), tumor necrosis factor (TNF), and nitric oxide synthase 2 (NOS2) were studied further. Knockdown experiments showed that TonEBP was necessary to maintain expression levels of these genes. Gain- and loss-of-function experiments and site-directed mutagenesis demonstrated that TonEBP binding to a specific site in the CCL2 promoter is required for hypertonic inducibility. Despite inhibition by dominant-negative TonEBP, IL6 and NOS2 promoters were not hypertonicity-inducible. Whole-disc response to hypertonicity was studied in an ex vivo organ culture model, using wild-type and haploinsufficient TonEBP mice. Pro-inflammatory targets were induced by hypertonicity in discs from wild-type but not TonEBP-haploinsufficient mice. Mechanistically, NF-κB activity increased with hypertonicity and was necessary for hypertonic induction of target genes IL6, TNF, and NOS2 but not CCL2 Although TonEBP maintains transcription of genes traditionally considered pro-inflammatory, it is important to note that some of these genes also serve anabolic and pro-survival roles. Therefore, in NP cells, this phenomenon may reflect a physiological adaptation to diurnal osmotic loading of the intervertebral disc.
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Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Homeostasis , Mediadores de Inflamación/metabolismo , Factores de Transcripción NFATC/metabolismo , Núcleo Pulposo/metabolismo , Ósmosis/fisiología , Animales , Disco Intervertebral , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , FN-kappa B/genética , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Técnicas de Cultivo de Órganos , Regiones Promotoras Genéticas/genética , Ratas , Transducción de SeñalRESUMEN
Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/ßC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.
Asunto(s)
Factores de Transcripción NFATC/metabolismo , Proteínas Nucleares/metabolismo , Linfocitos T/metabolismo , Humanos , Células Jurkat , Espectrometría de Masas , Factores de Transcripción NFATC/genética , Proteínas Nucleares/genéticaRESUMEN
The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Complejos Multiproteicos/metabolismo , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Secuencias de Aminoácidos , Línea Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Complejos Multiproteicos/genética , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/citología , Proteínas Proto-Oncogénicas c-vav/genética , Receptor Activador del Factor Nuclear kappa-B/genética , Factor 6 Asociado a Receptor de TNF/genética , Ubiquitinación/fisiologíaRESUMEN
The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.