RESUMO
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
Assuntos
Carcinogênese/metabolismo , Carcinoma Hepatocelular/genética , Compartimento Celular/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas de Choque Térmico HSP40/genética , Neoplasias Hepáticas/genética , Transdução de Sinais , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinoma Hepatocelular/metabolismo , Compartimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , AMP Cíclico/farmacologia , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Oncogenes/genética , Domínios Proteicos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão , Espectroscopia de Infravermelho com Transformada de Fourier , Imagem com Lapso de Tempo/métodosRESUMO
Today, the majority of patients with pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL) survive their disease, but many of the survivors suffer from life-limiting late effects of the treatment. ALL develops in the bone marrow, where the cells are exposed to cAMP-generating prostaglandin E2. We have previously identified the cAMP signaling pathway as a putative target for improved efficacy of ALL treatment, based on the ability of cAMP signaling to reduce apoptosis induced by DNA damaging agents. In the present study, we have identified the antioxidant N-acetyl cysteine (NAC) as a powerful modifier of critical events downstream of the cell-permeable cAMP analog 8-(4-chlorophenylthio) adenosine-3', 5'- cyclic monophosphate (8-CPT). Accordingly, we found NAC to turn 8-CPT into a potent killer of ALL cells in vitro both in the presence and absence of DNA damaging treatment. Furthermore, we revealed that NAC in combination with 8-CPT is able to delay the progression of ALL in a xenograft model in NOD-scid IL2Rγnull mice. NAC was shown to rely on the ability of 8-CPT to activate the guanine-nucleotide exchange factor EPAC, and we demonstrated that the ALL cells are killed by apoptosis involving sustained elevated levels of calcium imposed by the combination of the two drugs. Taken together, we propose that 8-CPT in the presence of NAC might be utilized as a novel strategy for treating pediatric ALL patients, and that this powerful combination might be exploited to enhance the therapeutic index of current ALL targeting therapies.
Assuntos
Acetilcisteína , AMP Cíclico , Fatores de Troca do Nucleotídeo Guanina , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Tionucleotídeos , Animais , Criança , Humanos , Camundongos , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , AMP Cíclico/uso terapêutico , DNA/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/agonistas , Camundongos Endogâmicos NOD , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Masculino , Feminino , Pré-Escolar , Tionucleotídeos/farmacologia , Tionucleotídeos/uso terapêutico , Dano ao DNA , Quimioterapia CombinadaRESUMO
Hepatitis B virus (HBV) is a major risk factor for serious liver diseases. The liver plays a unique role in controlling carbohydrate metabolism to maintain the glucose level within the normal range. Chronic HBV infection has been reported to associate with a high prevalence of diabetes. However, the detailed molecular mechanism underlying the potential association remains largely unknown. Here, we report that liver-targeted delivery of small HBV surface antigen (SHBs), the most abundant viral protein of HBV, could elevate blood glucose levels and impair glucose and insulin tolerance in mice by promoting hepatic gluconeogenesis. Hepatocytes with SHB expression also exhibited increased glucose production and expression of gluconeogenic genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (PEPCK) in response to glucagon stimulation. Mechanistically, SHBs increased cellular levels of cyclic AMP (cAMP) and consequently activated protein kinase A (PKA) and its downstream effector cAMP-responsive element binding protein (CREB). SHBs-induced activation of CREB enhanced transcripts of gluconeogenic genes, thus promoting hepatic gluconeogenesis. The elevated cAMP level resulted from increased transcription activity and expression of adenylyl cyclase 1 (AC1) by SHBs through a binary E-box factor binding site (BEF). Taken together, we unveiled a novel pathogenic role and mechanism of SHBs in hepatic gluconeogenesis, and these results might highlight a potential target for preventive and therapeutic intervention in the development and progression of HBV-associated diabetes. IMPORTANCE Chronic HBV infection causes progressive liver damage and is found to be a risk factor for diabetes. However, the mechanism in the regulation of glucose metabolism by HBV remains to be established. In the current study, we demonstrate for the first time that the small hepatitis B virus surface antigen (SHBs) of HBV elevates AC1 transcription and expression to activate cAMP/PKA/CREB signaling and subsequently induces the expression of gluconeogenic genes and promotes hepatic gluconeogenesis both in vivo and in vitro. This study provides a direct link between HBV infection and diabetes and implicates that SHBs may represent a potential target for the treatment of HBV-induced metabolic disorders.
Assuntos
Gluconeogênese , Antígenos de Superfície da Hepatite B , Hepatite B Crônica , Animais , Camundongos , Antígenos de Superfície/metabolismo , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glucagon/metabolismo , Glucagon/farmacologia , Gluconeogênese/genética , Glucose/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.
Assuntos
AMP Cíclico/fisiologia , Dexametasona/farmacologia , Dinoprostona/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Sistemas do Segundo Mensageiro/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Criança , Cromograninas/antagonistas & inibidores , Colforsina/farmacologia , AMP Cíclico/farmacologia , Dexametasona/administração & dosagem , Dinoprostona/administração & dosagem , Dinoprostona/antagonistas & inibidores , Dinoprostona/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/deficiência , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Modelos Animais , Terapia de Alvo Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Quimera por Radiação , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiologia , Receptores de Prostaglandina E Subtipo EP4/biossíntese , Receptores de Prostaglandina E Subtipo EP4/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.
Assuntos
Artrite Experimental , Sirtuínas , Ratos , Camundongos , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/farmacologia , Osteoblastos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/farmacologia , Hipóxia , Artrite Experimental/genética , Artrite Experimental/metabolismo , Fosforilação , Oxigênio/metabolismo , Oxigênio/farmacologia , Sirtuínas/metabolismo , Sirtuínas/farmacologia , AMP Cíclico/metabolismo , AMP Cíclico/farmacologiaRESUMO
Neural response properties that typify primary sensory afferents are critical to fully appreciate because they establish and, ultimately represent, the fundamental coding design used for higher-level processing. Studies illuminating the center-surround receptive fields of retinal ganglion cells, for example, were ground-breaking because they determined the foundation of visual form detection. For the auditory system, a basic organizing principle of the spiral ganglion afferents is their extensive electrophysiological heterogeneity establishing diverse intrinsic firing properties in neurons throughout the spiral ganglion. Moreover, these neurons display an impressively large array of neurotransmitter receptor types that are responsive to efferent feedback. Thus, electrophysiological diversity and its neuromodulation are a fundamental encoding mechanism contributed by the primary afferents in the auditory system. To place these features into context, we evaluated the effects of hyperpolarization and cAMP on threshold level as indicators of overall afferent responsiveness in CBA/CaJ mice of either sex. Hyperpolarization modified threshold gradients such that distinct voltage protocols could shift the relationship between sensitivity and stimulus input to reshape resolution. This resulted in an "accordion effect" that appeared to stretch, compress, or maintain responsivity across the gradient of afferent thresholds. cAMP targeted threshold and kinetic shifts to rapidly adapting neurons, thus revealing multiple cochleotopic properties that could potentially be independently regulated. These examples of dynamic heterogeneity in primary auditory afferents not only have the capacity to shift the range, sensitivity, and resolution, but to do so in a coordinated manner that appears to orchestrate changes with a seemingly unlimited repertoire.SIGNIFICANCE STATEMENT How do we discriminate the more nuanced qualities of the sound around us? Beyond the basics of pitch and loudness, aspects, such as pattern, distance, velocity, and location, are all attributes that must be used to encode acoustic sensations effectively. While higher-level processing is required for perception, it would not be unexpected if the primary auditory afferents optimized receptor input to expedite neural encoding. The findings reported herein are consistent with this design. Neuromodulation compressed, expanded, shifted, or realigned intrinsic electrophysiological heterogeneity to alter neuronal responses selectively and dynamically. This suggests that diverse spiral ganglion phenotypes provide a rich substrate to support an almost limitless array of coding strategies within the first neural element of the auditory pathway.
Assuntos
Potenciais de Ação/fisiologia , Gânglio Espiral da Cóclea/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , AMP Cíclico/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos CBA , Técnicas de Cultura de Órgãos , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/efeitos dos fármacosRESUMO
Traditionally, lipolysis has been regarded as an enzymatic activity that liberates fatty acids as metabolic fuel. However, recent work has shown that novel substrates, including a variety of lipid compounds such as fatty acids and their derivatives, release lipolysis products that act as signaling molecules and transcriptional modulators. While these studies have expanded the role of lipolysis, the mechanisms underpinning lipolysis signaling are not fully defined. Here, we uncover a new mechanism regulating glucose uptake, whereby activation of lipolysis, in response to elevated cAMP, leads to the stimulation of thioredoxin-interacting protein (TXNIP) degradation. This, in turn, selectively induces glucose transporter 1 surface localization and glucose uptake in 3T3-L1 adipocytes and increases lactate production. Interestingly, cAMP-induced glucose uptake via degradation of TXNIP is largely dependent upon adipose triglyceride lipase (ATGL) and not hormone-sensitive lipase or monoacylglycerol lipase. Pharmacological inhibition or knockdown of ATGL alone prevents cAMP-dependent TXNIP degradation and thus significantly decreases glucose uptake and lactate secretion. Conversely, overexpression of ATGL amplifies the cAMP response, yielding increased glucose uptake and lactate production. Similarly, knockdown of TXNIP elicits enhanced basal glucose uptake and lactate secretion, and increased cAMP further amplifies this phenotype. Overexpression of TXNIP reduces basal and cAMP-stimulated glucose uptake and lactate secretion. As a proof of concept, we replicated these findings in human primary adipocytes and observed TXNIP degradation and increased glucose uptake and lactate secretion upon elevated cAMP signaling. Taken together, our results suggest a crosstalk between ATGL-mediated lipolysis and glucose uptake.
Assuntos
Proteínas de Transporte/genética , Transportador de Glucose Tipo 1/genética , Glucose/metabolismo , Lipase/genética , Lipólise/genética , Tiorredoxinas/genética , Células 3T3-L1 , Adipócitos/enzimologia , Adipócitos/metabolismo , Animais , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Glucose/genética , Humanos , Ácido Láctico/biossíntese , Ácido Láctico/metabolismo , Camundongos , Proteólise/efeitos dos fármacos , Esterol Esterase/genéticaRESUMO
Cilostamide, a phosphodiesterase 3A (Pde3A) inhibitor, is known to increase intraoocyte cyclic adenosine monophosphate (cAMP) level which is involved in sustaining meiotic arrest of the oocytes. To explore the mechanisms involved in the cilostamide-mediated meiotic arrest of the oocytes, the present study describes the effects of cilostamide on cAMP level and related factors involved in maturation of the oocytes at its different meiotic stages; diplotene, metaphase I (MI) and metaphase II (MII). The oocytes from these three stages were collected from rat ovary and incubated with 10 µM cilostamide for 3 h in CO2 incubator. The levels of cAMP, cyclic guanosine monophosphate (cGMP) and the key players of maintaining meiotic arrest during oocyte maturation; Emi2, Apc, Cyclin B1, and Cdk1, were analyzed in diplotene, MI and MII stages. Pde3A was found to be expressed at all three stages but with the lowest level in MI oocyte. As compared to the control sets, the cAMP concentration was found to be highest in MII whereas cGMP was highest in the diplotene stage of cilostamide-treated group. The treated group showed declined reactive oxygen species level as compared with the control counterparts. Relatively increased levels of the Emi2, Cyclin B1, and phosphorylated thr161 of Cdk1 versus declined levels of phosphorylated thr14/tyr15 of Cdk1 in diplotene and MII stage oocytes are known to be involved in maintaining meiotic arrest and all these factors were found to undergo similar pattern of change due to the treatment with cilostamide. The findings thus suggest that cilostamide treatment promotes meiotic arrest by Pde3A inhibition led increase of both cAMP and cGMP level vis-a-vis modulation of the related regulatory factors such as Emi2, CyclinB1, and phosphorylated status of Cdk1 in diplotene and MII stage oocytes. Such a mechanism of meiotic arrest could allow the oocyte to prepare itself for meiotic maturation and thereby to improve oocyte quality.
Assuntos
Fator Promotor de Maturação , Inibidores de Fosfodiesterase , Feminino , Ratos , Animais , Ciclina B1 , Inibidores de Fosfodiesterase/farmacologia , Meiose , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Oócitos , AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Monofosfato de Adenosina/farmacologiaRESUMO
The decidua is a hallmark of reproduction in many placental mammals. Differentiation of decidual stromal cells is known to be induced by progesterone and the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Several candidates have been identified as the physiological stimulus for adenylyl cyclase activation, but their relative importance remains unclear. To bypass this uncertainty, the standard approach for in vitro experiments uses membrane-permeable cAMP and progestin. We phylogenetically infer that prostaglandin E2 (PGE2) likely was the signal that ancestrally induced decidualization in conjunction with progesterone. This suggests that PGE2 and progestin should be able to activate the core gene regulatory network of decidual cells. To test this prediction, we performed a genome-wide study of gene expression in human endometrial fibroblasts decidualized with PGE2 and progestin. Comparison to a cAMP-based protocol revealed shared activation of core decidual genes and decreased induction of senescence-associated genes. Single-cell transcriptomics of PGE2-mediated decidualization revealed a distinct, early-activated state transitioning to a differentiated decidual state. PGE2-mediated decidualization was found to depend upon progestin-dependent induction of PGE2 receptor 2 (PTGER2) which in turn leads to PKA activation upon PGE2 stimulation. Progesterone-dependent induction of PTGER2 is absent in opossum, an outgroup taxon of placental mammals which is incapable of decidualization. Together, these findings suggest that the origin of decidualization involved the evolution of progesterone-dependent activation of the PGE2/PTGER2/PKA axis, facilitating entry into a PKA-dominant rather than AKT-dominant cellular state. We propose the use of PGE2 for in vitro decidualization as an alternative to 8-Br-cAMP.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Decídua/citologia , Dinoprostona/farmacologia , Linhagem Celular Transformada , Células Cultivadas , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Decídua/fisiologia , Endométrio/citologia , Endométrio/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Acetato de Medroxiprogesterona/farmacologia , Gravidez , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
Asiaticoside, the major bioactive constituent purified from Centella asiatica, is a pentacyclic triterpene saponin with sugar moieties (glucose-glucose-rhamnose). Its biological activities including anti-inflammation and antioxidant have been widely reported. This study aimed to investigate the role of asiaticoside in diabetic retinopathy (DR). Human retinal pigment epithelium (RPE) cells ARPE-19 were induced by high glucose. Then, cell survival rate, expression of inflammatory factors, oxidative stress, and apoptosis were measured by MTT method, western blot, oxidative stress detection kits and TUNEL respectively. To uncover the underlying mechanism, the levels of cyclic AMP (cAMP) and protein kinase A (PKA) were measured by Enzyme linked immunosorbent assay (ELISA) and PKA activities were detected by the Kemptide phosphorylation assay. Furthermore, cAMP inhibitor SQ22536 was also used to validate the mechanism. Asiaticoside suppressed the inflammation and apoptosis of ARPE-19 cells, and the activities of cAMP and PKA were inhibited upon HG induction while again released after further administration of asiaticoside. However, these effects were all abolished by SQ22536. In conclusion, we have demonstrated in this paper that asiaticoside ameliorates high glucose-induced inflammation and apoptosis of RPE cells by activating cAMP/PKA signaling pathway. asiaticoside-mediated activation of cAMP/PKA signaling pathway may serve as a potential target for the management of DR.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , AMP Cíclico , Apoptose , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Células Epiteliais/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Pigmentos da Retina/metabolismo , Pigmentos da Retina/farmacologia , TriterpenosRESUMO
Tubulointerstitial inflammation plays an important role in the progression of diabetic nephropathy (DN), and tubular epithelial cells (TECs) are crucial promoters of the inflammatory cascade. Exchange protein activated by cAMP (Epac) has been shown to suppress the angiotensin II (Ang-II)-induced release of inflammatory cytokines in tubular cells. However, the role of Epac in TEC-mediated tubulointerstitial inflammation in DN remains unknown. We found that administering the Epac agonist 8-pCPT-2'-O-Me-cAMP (8-O-cAMP) to db/db mice inhibited tubulointerstitial inflammation characterized by macrophage infiltration and increased inflammatory cytokine release and consequently alleviated tubulointerstitial fibrosis in the kidney. Furthermore, 8-O-cAMP administration restored CCAAT/enhancer binding protein ß (C/EBP-ß) expression and further upregulated the expression of Suppressor of cytokine signaling 3 (SOCS3), while inhibiting p-STAT3, MCP-1, IL-6, and TNF-α expression in the kidney cortex in db/db mice. And in vitro study showed that macrophage migration and MCP-1 expression induced by high glucose (HG, 30 mM) were notably reduced by 8-O-cAMP in human renal proximal tubule epithelial (HK-2) cells. In addition, 8-O-cAMP treatment restored C/EBP-ß expression in HK-2 cells and promoted C/EBP-ß translocation to the nucleus, where it transcriptionally upregulated SOCS3 expression, subsequently inhibiting STAT3 phosphorylation. Under HG conditions, siRNA-mediated knockdown of C/EBP-ß or SOCS3 in HK-2 cells partially blocked the inhibitory effect of Epac activation on the release of MCP-1. In contrast, SOCS3 overexpression inhibited HG-induced activation of STAT3 and MCP-1 expression in HK-2 cells. These findings indicate that Epac activation via 8-O-cAMP ameliorates tubulointerstitial inflammation in DN through the C/EBP-ß/SOCS3/STAT3 pathway.
Assuntos
Nefropatias Diabéticas/patologia , Fatores de Troca do Nucleotídeo Guanina/agonistas , Inflamação/patologia , Túbulos Renais/efeitos dos fármacos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/efeitos dos fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Citocinas/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Fator de Transcrição STAT3/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Supressora da Sinalização de Citocinas/efeitos dos fármacos , Regulação para CimaRESUMO
The chemotaxis of Dictysotelium discoideum cells in response to a chemical gradient of cyclic adenosine 3',5'-monophosphate (cAMP) was studied using a newly designed microfluidic device. The device consists of 800 cell-sized channels in parallel, each 4 µm wide, 5 µm high, and 100 µm long, allowing us to prepare the same chemical gradient in all channels and observe the motility of 500-1000 individual cells simultaneously. The percentage of cells that exhibited directed migration was determined for various cAMP concentrations ranging from 0.1 pM to 10 µM. The results show that chemotaxis was highest at 100 nM cAMP, consistent with previous observations. At concentrations as low as 10 pM, about 16% of cells still exhibited chemotaxis, suggesting that the receptor occupancy of only 6 cAMP molecules/cell can induce chemotaxis in very sensitive cells. At 100 pM cAMP, chemotaxis was suppressed due to the self-production and secretion of intracellular cAMP induced by extracellular cAMP. Overall, systematic observations of a large number of individual cells under the same chemical gradients revealed the heterogeneity of chemotaxis responses in a genetically homogeneous cell population, especially the existence of a sub-population with extremely high sensitivity for chemotaxis.
Assuntos
Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , AMP Cíclico/farmacologia , Dictyostelium/fisiologia , Dictyostelium/efeitos dos fármacos , Microfluídica/métodos , Análise de Célula Única/métodosRESUMO
Bacterial persisters are phenotypic variants that form from the action of stress response pathways triggering toxin-mediated antibiotic tolerance. Although persisters form during normal growth from native stresses, the pathways responsible for this phenomenon remain elusive. Here we have discovered that carbon source transitions stimulate the formation of fluoroquinolone persisters in Escherichia coli. Further, through a combination of genetic, biochemical, and flow cytometric assays in conjunction with a mathematical model, we have reconstructed a molecular-level persister formation pathway from initial stress (glucose exhaustion) to the activation of a metabolic toxin-antitoxin (TA) module (the ppGpp biochemical network) resulting in inhibition of DNA gyrase activity, the primary target of fluoroquinolones. This pathway spans from initial stress to antibiotic target and demonstrates that TA behavior can be exhibited by a metabolite-enzyme interaction (ppGpp-SpoT), in contrast to classical TA systems that involve only protein and/or RNA.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Antibacterianos/farmacologia , Carbono/metabolismo , Escherichia coli/metabolismo , Adaptação Fisiológica/genética , Ampicilina/farmacologia , Antitoxinas/genética , Antitoxinas/metabolismo , Carga Bacteriana , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , AMP Cíclico/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoroquinolonas/farmacologia , Glucose/metabolismo , Guanosina Tetrafosfato/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Ofloxacino/farmacologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de TempoRESUMO
In mammalian olfactory transduction, odorants activate a cAMP-mediated signaling pathway that leads to the opening of cyclic nucleotide-gated (CNG), nonselective cation channels and depolarization. The Ca2+ influx through open CNG channels triggers an inward current through Ca2+-activated Cl channels (ANO2), which is expected to produce signal amplification. However, a study on an Ano2-/- mouse line reported no elevation in the behavioral threshold of odorant detection compared with wild type (WT). Subsequent studies by others on the same Ano2-/- line, nonetheless, found subtle defects in olfactory behavior and some abnormal axonal projections from the olfactory receptor neurons (ORNs) to the olfactory bulb. As such, the question regarding signal amplification by the Cl current in WT mouse remains unsettled. Recently, with suction-pipette recording, we have successfully separated in frog ORNs the CNG and Cl currents during olfactory transduction and found the Cl current to predominate in the response down to the threshold of action-potential signaling to the brain. For better comparison with the mouse data by others, we have now carried out similar current-separation experiments on mouse ORNs. We found that the Cl current clearly also predominated in the mouse olfactory response at signaling threshold, accounting for â¼80% of the response. In the absence of the Cl current, we expect the threshold stimulus to increase by approximately sevenfold.
Assuntos
Anoctaminas/fisiologia , Encéfalo/fisiologia , Cálcio/farmacologia , Cloretos/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Animais , Encéfalo/citologia , AMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/efeitos dos fármacos , Técnicas de Patch-Clamp , Transdução de Sinais/efeitos dos fármacos , Olfato/efeitos dos fármacosRESUMO
Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM-ER-mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling.
Assuntos
Tumor de Células de Leydig/metabolismo , Lipídeos de Membrana/metabolismo , Esteroides/biossíntese , Neoplasias Testiculares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Bucladesina/farmacologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colesterol/metabolismo , AMP Cíclico/farmacologia , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Tumor de Células de Leydig/ultraestrutura , Lipidômica , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Neoplasias Testiculares/ultraestruturaRESUMO
Arrestins are key adaptor proteins that control the fate of cell-surface membrane proteins and modulate downstream signaling cascades. The Dictyostelium discoideum genome encodes six arrestin-related proteins, harboring additional modules besides the arrestin domain. Here, we studied AdcB and AdcC, two homologs that contain C2 and SAM domains. We showed that AdcC - in contrast to AdcB - responds to various stimuli (such as the chemoattractants cAMP and folate) known to induce an increase in cytosolic calcium by transiently translocating to the plasma membrane, and that calcium is a direct regulator of AdcC localization. This response requires the calcium-dependent membrane-targeting C2 domain and the double SAM domain involved in AdcC oligomerization, revealing a mode of membrane targeting and regulation unique among members of the arrestin clan. AdcB shares several biochemical properties with AdcC, including in vitro binding to anionic lipids in a calcium-dependent manner and auto-assembly as large homo-oligomers. AdcB can interact with AdcC; however, its intracellular localization is insensitive to calcium. Therefore, despite their high degree of homology and common characteristics, AdcB and AdcC are likely to fulfill distinct functions in amoebae.
Assuntos
Arrestinas/metabolismo , Cálcio/metabolismo , Fatores Quimiotáticos/farmacologia , Dictyostelium/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Arrestinas/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , AMP Cíclico/farmacologia , Dictyostelium/efeitos dos fármacos , Ácido Fólico/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Espaço Intracelular/metabolismo , Lipossomos , Fosfolipídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Multimerização Proteica , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/química , Proteínas Recombinantes/metabolismoRESUMO
Ras GTPases act as molecular switches to control various cellular processes by coupling integrated signals in eukaryotes. Activities of Ras GTPases are triggered by Ras GTPase guanine nucleotide exchange factors (RasGEFs) in general, whereas the role of RasGEF in plant pathogenic fungi is largely unknown. In this study, we characterized the only RasGEF protein in Fusarium graminearum, FgCdc25, by combining genetic, cytological and phenotypic strategies. FgCdc25 directly interacted with RasGTPase FgRas2, but not FgRas1, to regulate growth and sexual reproduction. Mutation of the FgCDC25 gene resulted in decreased toxisome formation and deoxynivalenol (DON) production, which was largely depended on cAMP signalling. In addition, FgCdc25 indirectly interacted with FgSte11 in FgSte11-Ste7-Gpmk1 cascade, and the ΔFgcdc25 strain totally abolished the formation of infection structures and was nonpathogenic in planta, which was partially recovered by addition of exogenous cAMP. In contrast, FgCdc25 directly interplayed with FgBck1 in FgBck1-MKK1-Mgv1 cascade to negatively control cell wall integrity. Collectively, these results suggest that FgCdc25 modulates cAMP and MAPK signalling pathways and further regulates fungal development, DON production and plant infection in F. graminearum.
Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Fusarium/patogenicidade , Transdução de Sinais , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , Parede Celular/metabolismo , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Fusarium/metabolismo , Doenças das Plantas/microbiologia , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Tricotecenos/metabolismo , Virulência/genética , Proteínas ras/metabolismoRESUMO
High-affinity fluorescent derivatives of cyclic adenosine and guanosine monophosphate are powerful tools for investigating their natural targets. Cyclic nucleotide-regulated ion channels belong to these targets and are vital for many signal transduction processes, such as vision and olfaction. The relation of ligand binding to activation gating is still challenging, and there is a need for fluorescent probes that enable the process to be broken down to the single-molecule level. This inspired us to prepare fluorophore-labeled cyclic nucleotides, which are composed of a bright dye and a nucleotide derivative with a thiophenol motif at position 8 that has already been shown to enable superior binding affinity. These bioconjugates were prepared by a novel cross-linking strategy that involves substitution of the nucleobase with a modified thiophenolate in good yield. Both fluorescent nucleotides are potent activators of different cyclic nucleotide-regulated ion channels with respect to the natural ligand and previously reported substances. Molecular docking of the probes excluding the fluorophore reveals that the high potency can be attributed to additional hydrophobic and cation-π interactions between the ligand and the protein. Moreover, the introduced substances have the potential to investigate related target proteins, such as cAMP- and cGMP-dependent protein kinases, exchange proteins directly activated by cAMP or phosphodiesterases.
Assuntos
AMP Cíclico/química , AMP Cíclico/farmacologia , GMP Cíclico/química , GMP Cíclico/farmacologia , Corantes Fluorescentes/química , Canais Iônicos/agonistas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Conformação ProteicaRESUMO
Nonmuscle myosin II has been implicated in regulation of von Willebrand factor (VWF) release from endothelial Weibel-Palade bodies (WPBs), but the specific role of myosin IIa isoform is poorly defined. Here, we report that myosin IIa is expressed both in primary human endothelial cells and intact mouse vessels, essential for cyclic adenosine monophosphate (cAMP)-mediated endothelial VWF secretion. Downregulation of myosin IIa by shRNAs significantly suppressed both forskolin- and epinephrine-induced VWF secretion. Endothelium-specific myosin IIa knockout mice exhibited impaired epinephrine-stimulated VWF release, prolonged bleeding time, and thrombosis. Further study showed that in resting cells, myosin IIa deficiency disrupted the peripheral localization of Rab27-positive WPBs along stress fibers; on stimulation by cAMP agonists, myosin IIa in synergy with zyxin promotes the formation of a functional actin framework, which is derived from preexisting cortical actin filaments, around WPBs, facilitating fusion and subsequent exocytosis. In summary, our findings not only identify new functions of myosin IIa in regulation of WPB positioning and the interaction between preexisting cortical actin filaments and exocytosing vesicles before fusion but also reveal myosin IIa as a physiological regulator of endothelial VWF secretion in stress-induced hemostasis and thrombosis.
Assuntos
AMP Cíclico/farmacologia , Células Endoteliais/efeitos dos fármacos , Miosina não Muscular Tipo IIA/fisiologia , Fator de von Willebrand/metabolismo , Animais , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosina não Muscular Tipo IIA/genética , Via Secretória/efeitos dos fármacos , Via Secretória/genéticaRESUMO
The human HEPC-CB.1 cell line with many characteristics of endothelial progenitor cells (EPC) was tested for its proangiogenic properties as a potentially therapeutic compound. HEPC-CB.1 cells' potential to differentiate into endothelial cells was revealed after treating the cells with a mixture of ATRA, cAMP and VEGF, as shown by the reduced expression levels of CD133, CD271 and CD90 antigens, augmentation of CD146 and CD31, and a decrease in cell clonogenicity. The cooperation of HEPC-CB.1 with the endothelial cell line HSkMEC.2 resulted in the formation of a common network. Tube formation was significantly more effective when resulting from HEPC-CB.1 and HSkMEC.2 cell co-culture as compared to a monoculture of each cell line. The exocrine mechanism of HEPC-CB.1 and HSkMEC.2 cross talk by secreted factors was evidenced using the HEPC-CB.1 supernatant to increase the efficacy of HSkMEC.2 tube formation. The proangiogenic factors produced by HEPC-CB.1 were identified using cytokine antibody array. Out of 120 examined factors, the HEPC-CB.1 cell line produced 63, some with known angiogenic activity. As in vivo the angiogenic process occurs at low oxygen tension, it was observed that in hypoxia, the production of defined factors was augmented. The presented results demonstrate that HEPC-CB.1 cells are able to both cooperate and integrate in a newly formed network and produce factors that help the network formation. The results suggest that HEPC-CB.1 cells are indeed endothelial progenitors and may prove to be an effective tool in regenerative medicine.