RESUMEN
LDL-C lowering is the main goal of atherosclerotic cardiovascular disease prevention, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is now a validated therapeutic strategy that lowers serum LDL-C and reduces coronary events. Ironically, the most widely used medicine to lower cholesterol, statins, has been shown to increase circulating PCSK9 levels, which limits their efficacy. Here, we show that geranylgeranyl isoprenoids and hepatic Rap1a regulate both basal and statin-induced expression of PCSK9 and contribute to LDL-C homeostasis. Rap1a prenylation and activity is inhibited upon statin treatment, and statin-mediated PCSK9 induction is dependent on geranylgeranyl synthesis and hepatic Rap1a. Accordingly, treatment of mice with a small-molecule activator of Rap1a lowered PCSK9 protein and plasma cholesterol and inhibited statin-mediated PCSK9 induction in hepatocytes. The mechanism involves inhibition of the downstream RhoA-ROCK pathway and regulation of PCSK9 at the post-transcriptional level. These data further identify Rap1a as a novel regulator of PCSK9 protein and show that blocking Rap1a prenylation through lowering geranylgeranyl levels contributes to statin-mediated induction of PCSK9.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas , Proproteína Convertasa 9 , Ratones , Animales , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , LDL-Colesterol , Anticuerpos Monoclonales/farmacología , ColesterolRESUMEN
Ras-related Rap1A GTPase is implicated in pancreas ß-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein ß (Ero1lß), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lß and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15-30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function.
Asunto(s)
Insulina , Ratones Endogámicos C57BL , Páncreas , Proteómica , Transducción de Señal , Proteínas de Unión al GTP rap1 , Animales , Proteínas de Unión al GTP rap1/metabolismo , Proteínas de Unión al GTP rap1/genética , Ratones , Proteómica/métodos , Insulina/metabolismo , Páncreas/metabolismo , Células Secretoras de Insulina/metabolismo , Ratones Noqueados , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Secreción de Insulina , Masculino , Glucosa/metabolismoRESUMEN
The integrity of the structure and function of the endometrium is essential for the maintenance of fertility. However, the repair mechanisms of uterine injury remain largely unknown. Here, we showed that the disturbance of mechanical cue homeostasis occurs after uterine injury. Applying a multimodal approach, we identified YAP as a sensor of biophysical forces that drives endometrial regeneration. Through protein activation level analysis of the combinatorial space of mechanical force strength and of the presence of particular kinase inhibitors and gene silencing reagents, we demonstrated that mechanical cues related to extracellular matrix rigidity can turn off the Rap1a switch, leading to the inactivation of ARHGAP35and then induced activation of RhoA, which in turn depends on the polymerization of the agonist protein F-actin to activate YAP. Further study confirmed that mechanotransduction significantly accelerates remodeling of the uterus by promoting the proliferation of endometrial stromal cells in vitro and in vivo. These studies provide new insights into the dynamic regulatory mechanisms behind uterine remodeling and the function of mechanotransduction. Video Abstract.
Asunto(s)
Actinas , Proteínas Adaptadoras Transductoras de Señales , Femenino , Humanos , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transducción de Señal/genética , Proteínas Señalizadoras YAP , Mecanotransducción Celular/fisiología , Matriz Extracelular/metabolismo , Útero/metabolismoRESUMEN
Statins inhibit the mevalonate pathway by impairing protein prenylation via depletion of lipid geranylgeranyl diphosphate (GGPP). Rab27b and Rap1a are small GTPase proteins involved in dense granule secretion, platelet activation, and regulation. We analyzed the impact of statins on prenylation of Rab27b and Rap1a in platelets and the downstream effects on fibrin clot properties. Whole blood thromboelastography revealed that atorvastatin (ATV) delayed clot formation time (P < .005) and attenuated clot firmness (P < .005). ATV pre-treatment inhibited platelet aggregation and clot retraction. Binding of fibrinogen and P-selectin exposure on stimulated platelets was significantly lower following pre-treatment with ATV (P < .05). Confocal microscopy revealed that ATV significantly altered the structure of platelet-rich plasma clots, consistent with the reduced fibrinogen binding. ATV enhanced lysis of Chandler model thrombi 1.4-fold versus control (P < .05). Western blotting revealed that ATV induced a dose-dependent accumulation of unprenylated Rab27b and Rap1a in the platelet membrane. ATV dose-dependently inhibited ADP release from activated platelets. Exogenous GGPP rescued the prenylation of Rab27b and Rap1a, and partially restored the ADP release defect, suggesting these changes arise from reduced prenylation of Rab27b. These data demonstrate that statins attenuate platelet aggregation, degranulation, and binding of fibrinogen thereby having a significant impact on clot contraction and structure.
What is the context? Statins such as Atorvastatin (ATV) are 3-hydroxy, 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which block the cholesterol biosynthetic pathway to lower total serum levels and LDL-cholesterol.The cholesterol pathway also provides a supply of isoprenoids (farnesyl and geranylgeranyl) for the prenylation of signaling molecules, which include the families of Ras and Rho small GTPases.Prenyl groups provide a membrane anchor that is essential for the correct membrane localization and function of these proteins.Statins deplete cells of lipid geranylgeranyl diphosphate (GGPP) thereby inhibiting progression of the mevalonate pathway and prenylation of proteins.Rab27b and Rap1 are small GTPase proteins in platelets that are involved in the secretion of platelet granules and integrin activation.What is new?In this study, we found that ATV impairs prenylation of Rab27b and Rap1a and attenuates platelet function.These effects were partially rescued by GGPP, indicating the involvement of the mevalonate pathway.Platelet aggregation and degranulation was significantly attenuated by ATV.The impact of statins on platelet function altered clot formation, structure and contraction generating a clot that was more susceptible to degradation.What is the impact?This study demonstrates a novel mechanism whereby statins alter platelet responses and ultimately clot structure and stability.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas , Trombosis , Humanos , Adenosina Difosfato/metabolismo , Atorvastatina/farmacología , Plaquetas/metabolismo , Fibrinógeno/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Prenilación , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Trombosis/tratamiento farmacológico , Trombosis/metabolismoRESUMEN
The G protein-coupled α2-adrenoceptor subtype C (abbreviated α2C-AR) has been implicated in peripheral vascular conditions and diseases such as cold feet-hands, Raynaud's phenomenon, and scleroderma, contributing to morbidity and mortality. Microvascular α2C-adrenoceptors are expressed in specialized smooth muscle cells and mediate constriction under physiological conditions and the occlusion of blood supply involving vasospastic episodes and tissue damage under pathological conditions. A crucial step for receptor biological activity is the cell surface trafficking of intracellular receptors, triggered by cAMP-Epac-Rap1A GTPase signaling, which involves protein-protein association with the actin-binding protein filamin-2, mediated by critical amino acid residues in the last 14 amino acids of the receptor carboxyl (C)-terminus. This study assessed the role of the C-terminus in Rap1A GTPase coupled receptor trafficking by domain-swapping studies using recombinant tagged receptors in transient co-transfections and compared with wild-type receptors using immunofluorescence microscopy. We further tested the biological relevance of the α2C-AR C-terminus, when introduced as competitor peptides, to selectively inhibit intracellular α2C-AR surface translocation in transfected as well as in microvascular smooth muscle cells expressing endogenous receptors. These studies contribute to establishing proof of principle to target intracellular α2C-adrenoceptors to reduce biological activity, which in clinical conditions can be a target for therapy.
Asunto(s)
Miocitos del Músculo Liso , Péptidos , Receptores Adrenérgicos alfa 2 , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Receptores Adrenérgicos/metabolismo , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Diabetics have an increased risk for heart failure due to cardiac fibroblast functional changes occurring as a result of AGE/RAGE signaling. Advanced glycation end products (AGEs) levels are higher in diabetics and stimulate elevated RAGE (receptor for AGE) signaling. AGE/RAGE signaling can alter the expression of proteins linked to extracellular matrix (ECM) remodeling and oxidative stressors. Our lab has identified a small GTPase, Rap1a, that may overlap the AGE/RAGE signaling pathway. We sought to determine the role Rap1a plays in mediating AGE/RAGE changes and to assess the impact of isolated collagen on further altering these changes. Primary cardiac fibroblasts from non-diabetic and diabetic mice with and without RAGE expression and from mice lacking Rap1a were cultured on tail collagen extracted from non-diabetic or diabetic mice, and in addition, cells were treated with Rap1a activator, EPAC. Protein analyses were performed for changes in RAGE-associated signaling proteins (RAGE, PKC-ζ, ERK1/2) and downstream RAGE signaling outcomes (α-SMA, NF-κB, SOD-2). Increased levels of endogenous AGEs within the diabetic collagen and increased Rap1a activity promoted myofibroblast transition and oxidative stress, suggesting Rap1a activity elevated the impact of AGEs in the diabetic ECM to stimulate myofibroblast transition and oxidative stress.
Asunto(s)
Diabetes Mellitus Experimental , Miofibroblastos , Animales , Colágeno/metabolismo , Diabetes Mellitus Experimental/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Ratones , Miofibroblastos/metabolismo , Estrés Oxidativo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Proteínas de Unión al GTP rap1RESUMEN
MicroRNA-501-3p (miR-501-3p) has been reported to play tumor-suppressive roles in different cancers; however, its expression pattern and biological function in non-small cell lung cancer (NSCLC) remain unknown. In this study, we noted downregulation of miR-501-3p in NSCLC tissues and cell lines. Functional assays showed that overexpression of miR-501-3p suppressed NSCLC cell proliferation, clonogenicity, migration, and invasion. Moreover, miR-501-3p overexpression attenuated in vivo tumor growth in a nude mouse model. In terms of the mechanism, RAP1A was identified as a novel target of miR-501-3p. Overexpression of RAP1A strongly attenuated the inhibitory effects of miR-501-3p on the capacity of NSCLC cells for proliferation and motility. In the clinical samples of NSCLC, miR-501-3p levels negatively correlated with RAP1A expression, which was upregulated in NSCLC. Collectively, these results indicate that miR-501-3p acts as a tumor suppressor in NSCLC by directly targeting RAP1A mRNA and may serve as a theranostic biomarker for patients with NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación hacia Abajo/genética , Neoplasias Pulmonares/genética , MicroARNs/genética , Proteínas de Unión al GTP rap1/genética , Animales , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor/fisiología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Regulación hacia Arriba/genéticaRESUMEN
Neuropathic pain (NP) after spinal cord injury (SCI) leads to compromised physical and cognitive functions in a majority of patients. Aberrant miRNA expression plays vital roles in the pathogenesis of SCI. This study aims to investigate the effect of miR-331-3p in rats following SCI. Microarray assay was performed in SCI- and sham-operated rats to evaluate the expression of miR-331-3p. Assigned SCI rats were treated with miR-331-3p agomiR alone or miR-331-3p agomiR plus RAP1A-expressing lentivirus or control agomiR. Rat locomotor performance was evaluated by BBB locomotor rating scale. Neuronal tissue damage and apoptosis were detected by histological analyses and Western blot. Inflammation in spinal cord was determined by detection of the expression of inflammatory genes with qRT-PCR, and ELISA. Downstream expression of RAP1A was measured by Western blot. The results showed that SCI induced the downregulation of miR-331-3p in the spinal cord of SCI rats. Overexpression of miR-331-3p improved the locomotor performance, reduced tissue damage, neuronal apoptosis and inflammation in rat SCI model. Rap1a (Ras-related protein Rap-1A) was predicted as a downstream target for miR-331-3p, and upregulation of RAP1A impaired the beneficial effect of miR-331-3p post- SCI, which was shown as worse locomotor activity, more severe tissue damage, as well as promoting apoptosis and inflammation in SCI rats. Furthermore, miR-331-3p reduced the activation of RAP1A downstream genes via inhibiting RAP1A expression. These findings indicate a protective role of miR- 331-3p in the development of SCI via the modulation of RAP1A, and may help to develop novel therapy against SCI-induced complications.
Asunto(s)
MicroARNs/genética , Neuralgia/genética , Traumatismos de la Médula Espinal/patología , Proteínas de Unión al GTP rap1/genética , Animales , Ratas , Ratas Sprague-Dawley , Médula EspinalRESUMEN
Neuropathic pain is a somatosensory disorder which is caused by disease or nerve injury that affects the nervous system. microRNAs (miRNAs) are proved to play crucial roles in the development of neuropathic pain. However, the role of miR-202 in neuropathic pain is still unknown. Sprague-Dawley rats were used for constructing the neuropathic pain model. The expression of miR-202 was determined by quantitative real-time polymerase chain reaction. Potential target gene for miR-202 was measured using bioinformatics methods and Western blot analysis. In this study, we used rats to establish a neuropathic pain model and measured the effect of miR-202 in neuropathic pain. We demonstrated that miR-202 expression was downregulated in the spinal dorsal horn of bilateral sciatic nerve chronic constriction injury (bCCI) rat. However, miR-202 expression was not changed in the dorsal root ganglion, hippocampus, and anterior cingulated cortex of bCCI rat. We identified that RAP1A was a direct target gene of miR-202 in the PC12 cell. RAP1A expression was upregulated in the spinal dorsal horn of bCCI rat. Overexpression of miR-202 could improve the pain threshold for bCCI rats in both hindpaws, indicating that miR-202 overexpression could lighten the pain threshold for model rats. Moreover, RAP1A overexpression increased the pain threshold effect of miR-202 overexpression treated bCCI rats, indicating that miR-202 could lighten the pain threshold through inhibiting RAP1A expression. These data suggested that miR-202 acted pivotal roles in the development of neuropathic pain partly through targeting RAP1A gene.
Asunto(s)
MicroARNs/genética , Neuralgia/genética , Traumatismos de los Nervios Periféricos/genética , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Regiones no Traducidas 3' , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica , Masculino , Neuralgia/etiología , Neuralgia/metabolismo , Células PC12 , Traumatismos de los Nervios Periféricos/etiología , Traumatismos de los Nervios Periféricos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H-Ras, N-Ras, and K-Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best-known member and has gained significant scientific as well as public attention as an endocrine disrupting chemical, a fact that eventually led to its replacement. However, compounds used to replace BPA still contain the molecular scaffold of bisphenols. BPA, BPAF, BPB, BPE, BPF, and an amine-substituted BPAF-derivate all interact with all GDP-bound Ras-Isoforms through binding to a common site on these proteins. NMR-, SOScat-, and GDI- assay-based data revealed a new bisphenol-induced, allosterically activated GDP-bound Ras conformation that define these plasticisers as Ras allosteric agonists.
Asunto(s)
Sitio Alostérico , Compuestos de Bencidrilo/química , Disruptores Endocrinos/química , Fenoles/química , Proteínas ras/química , Regulación Alostérica , Compuestos de Bencidrilo/farmacología , Disruptores Endocrinos/farmacología , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Fenoles/farmacología , Unión Proteica , Proteínas ras/agonistas , Proteínas ras/metabolismoRESUMEN
Hepatitis B virus (HBV) causes acute and chronic hepatitis, and is one of the major causes of cirrhosis and hepatocellular carcinoma. Accumulating evidence suggests that inflammation is the key factor for liver cirrhosis and hepatocellular carcinoma. MicroRNAs play important roles in many biological processes. Here, we aim to explore the function of microRNAs in the HBX-induced inflammation. First, microarray experiment showed that HBV+ liver samples expressed higher level of miR-203a compared to HBV- liver samples. To verify these alterations, HBx-coding plasmid was transfected into HepG2 cells to overexpress HBx protein. The real-time PCR results suggested that over-expression of HBx could induce up-regulation of miR-203a. To define how up-regulation of miR-203a can induce liver cells inflammation, we over-expressed miR-203a in HepG2 cells. Annexin V staining and BrdU staining suggested that overexpression of miR-203a significantly increased the cell apoptosis and proliferation, meanwhile, over-expression of miR-203a could lead to a decrease in G0/G1 phase cells and an increase in G2/M phase cells. Some cytokines production including IL-6 and IL-8 were significantly increased, but TGFß and IFNγ were decreased in miR-203a over-expressed HepG2 cells. Luciferase reporter assay experiments, protein mass-spectrum assay and real-time PCR all together demonstrated that Rap1a was the target gene of miR-203a. Further experiments showed that these alterations were modulated through PI3K/ERK/p38/NFκB pathways. These data suggested that HBV-infection could up-regulate the expression of miR-203a, thus down regulated the expression of Rap1a and affected the PI3K/ERK/p38/NFκB pathways, finally induced the hepatitis inflammation.
Asunto(s)
Inflamación/genética , MicroARNs/metabolismo , Transactivadores/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Apoptosis/genética , Ciclo Celular , Proliferación Celular , Citocinas/genética , Citocinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células Hep G2 , Virus de la Hepatitis B/fisiología , Humanos , Inflamación/patología , Hígado/metabolismo , Hígado/patología , Hígado/virología , Sistema de Señalización de MAP Quinasas/genética , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Reguladoras y Accesorias ViralesRESUMEN
MicroRNAs are small, non-coding RNAs which regulate post-transcriptionally hundreds of target mRNAs. Given that their expression is deregulated in several cancer types, they represent potential diagnostic, prognostic, and predictive biomarkers, as well as next-generation therapeutic targets. Nevertheless, the involvement of miRNAs in non-melanoma skin cancer, a cancer type with increasing prevalence, is not extensively studied, and their comprehensive characterization as regard to the initiation, promotion, and progression stages is missing. To this end, we exploited a well-established multistage mouse skin carcinogenesis model in order to identify miRNAs consistently implicated in different stages of skin carcinogenesis. The cell lines comprising this model were subjected to miRNA expression profiling using microarrays, followed by bioinformatics analysis and validation with Q-PCR, as well as treatment with miRNA modulators. We showed that among all deregulated miRNAs in our system, only a functionally coherent group consisting of the miR-200 family members and miR-205-5p displays a pattern of progressive co-downregulation from the early toward the most aggressive stages of carcinogenesis. Their overlapping, co-regulated putative targets are potentially inter-associated and, of these, the EMT-related Rap1a is overexpressed toward aggressive stages. Ectopic expression of miR-205-5p in spindle cancer cells reduces Rap1a, mitigates cell invasiveness, decreases proliferation, and delays tumor onset. We conclude that deregulation of this miRNA group is primarily associated with aggressive phenotypes of skin cancer cells. Restoration of the miR-205-5p member of this group in spindle cells reduces the expression of critical, co-regulated targets that favor cancer progression, thus reversing the EMT characteristics. © 2015 Wiley Periodicals, Inc.
Asunto(s)
Perfilación de la Expresión Génica/métodos , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias Cutáneas/patología , Proteínas de Unión al GTP rap1/genética , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias Cutáneas/genéticaRESUMEN
BACKGROUND: MicroRNAs (miRNAs) are a class of small, well-conserved, non-coding RNAs that regulate the translation of RNAs. They have a role in biological and pathological process including cell differentiation, apoptosis, proliferation and metabolism. Since their discovery, they have been shown to have a potential role in cancer pathogenesis through their function as oncogenes or tumor suppressors. A substantial number of miRNAs show differential expression in esophageal cancer tissues, and so have been investigated for possible use in diagnosis. Furthermore, there is increasing interest in their use as prognostic markers and determining treatment response, as well as identifying their downstream targets and understanding their mode of action. METHODS: We analyzed the most recent studies on miRNAs in esophageal cancer and/or Barrett's esophagus (BE). The publications were identified by searching in PuBMed for the following terms: Barrett's esophagus and microRNA; esophageal cancer and microRNA. RESULTS: Four miRNAs (mi-R-25, -99a, -133a and -133b) showed good potential as diagnostic markers and interestingly five (mi-R-21, -27b, -126, - 143 and -145) appeared to be useful both as diagnostic and prognostic/predictive markers. CONCLUSION: The data so far on miRNAs in esophageal carcinogenesis is promising but further work is required to determine whether miRNAs can be used as biomarkers, not only in the clinical setting or added to individualized treatment regimes but also in non-invasive test by making use of miRNAs identified in blood.
Asunto(s)
Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , MicroARNs/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/terapia , Animales , Esófago de Barrett/genética , Esófago de Barrett/metabolismo , Esófago de Barrett/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Neoplasias Esofágicas/terapia , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Pronóstico , Resultado del TratamientoRESUMEN
The apical membrane of intestinal epithelia expresses intermediate conductance K(+) channel (KCNN4), which provides the driving force for Cl(-) secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl(-) secretion via stimulation of Rap2-phospholipase Cε-[Ca(2+)]i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl(-) secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl(-) secretion and apical K(+) conductance (IK(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2'-O- methyladenosine 3',5'-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K(+) channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed â¼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate IK(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated IK(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.
Asunto(s)
Cloruros/metabolismo , Diarrea/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Toxina del Cólera , Colforsina/farmacología , AMP Cíclico/farmacología , Diarrea/inducido químicamente , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Microscopía Confocal , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Besides the glomerulus, the tubulointerstitium is often concomitantly affected in certain diseases, e.g., diabetic nephropathy, and activation of the renin-angiotensin system, to a certain extent, worsens its outcome because of perturbations in hemodynamics and possibly tubuloglomerular feedback. Certain studies suggest that pathobiology of the tubulointerstitium is influenced by small GTPases, e.g., Rap1. We investigated the effect of ANG II on inflammatory cytokines, while at the same time focusing on upstream effector of Rap1, i.e., Epac1, and some of the downstream tubular transport molecules, i.e., Na/H exchanger 3 (NHE3). ANG II treatment of LLC-PK1 cells decreased Rap1a GTPase activity in a time- and dose-dependent manner. ANG II treatment led to an increased membrane translocation of NHE3, which was reduced with Epac1 and PKA activators. ANG II-induced NHE3 translocation was notably reduced with the transfection of Rap1a dominant positive mutants, i.e., Rap1a-G12V or Rap1a-T35A. Transfection of cells with dominant negative Rap1a mutants, i.e., Rap1a-S17A, or Epac1 mutant, i.e., EPAC-ΔcAMP, normalized ANG II-induced translocation of NHE3. In addition, ANG II treatment led to an increased expression of inflammatory cytokines, i.e., IL-1ß, IL-6, IL-8, and TNF-α, which was reduced with Rap1a-G12V or Rap1a-T35A transfection, while it reverted to previous comparable levels following transfection of Rap1a-S17A or EPAC-ΔcAMP. ANG II-induced expression of cytokines was reduced with the treatment with NHE3 inhibitor S3226 or with Epac1 and PKA activators. These data suggest that this novel Epac1-Rap1a-NHE3 pathway conceivably modulates ANG II-induced expression of inflammatory cytokines, and this information may yield the impetus for developing strategies to reduce tubulointertstitial inflammation in various renal diseases.
Asunto(s)
Angiotensina II/farmacología , Citocinas/metabolismo , Inflamación/metabolismo , Túbulos Renales/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Factores de Intercambio de Guanina Nucleótido/metabolismo , Túbulos Renales/efectos de los fármacos , Masculino , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/fisiología , Transducción de Señal/efectos de los fármacos , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Porcinos , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor associated with Epstein-Barr virus (EBV) infection. Chemoradiotherapy is the mainstream treatment for locally advanced NPC, and chemotherapeutic drugs are an indispensable part of NPC treatment. However, the toxic side-effects of chemotherapy drugs limit their therapeutic value, and new chemotherapy drugs are urgently needed for NPC. Silvestrol, an emerging natural plant anticancer molecule, has shown promising antitumor activity in breast cancer, melanoma, liver cancer, and other tumor types by promoting apoptosis in cancer cells to a greater extent than in normal cells. However, the effects of silvestrol on NPC and its possible molecular mechanisms have yet to be fully explored. METHODS: Cell counting kit-8 (CCK-8), cell scratch, flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), and Western blot (WB) assays were used to evaluate the effects of silvestrol on the cell viability, cell cycle, apoptosis, and migration of NPC cells. RNA sequencing (RNA-Seq) was used to study the effect of extracellular signal-regulated kinase (ERK) inhibitors on the cell transcriptome, and immunohistochemistry (IHC) to assess protein expression levels in patient specimens. RESULTS: Silvestrol inhibited cell migration and DNA replication of NPC cells, while promoting the expression of cleaved caspase-3, apoptosis, and cell cycle arrest. Furthermore, silvestrol altered the level of ERK phosphorylation. The ERK-targeted inhibitor LY3214996 attenuated silvestrol-mediated inhibition of NPC cell proliferation but not migration. Analysis of RNA-Seq data and WB were used to identify and validate the downstream regulatory targets of silvestrol. Expression of GADD45A, RAP1A, and hexokinase-II (HK2) proteins was inhibited by silvestrol and LY3214996. Finally, IHC revealed that GADD45A, RAP1A, and HK2 protein expression was more abundant in cancer tissues than in non-tumor tissues. CONCLUSIONS: Silvestrol inhibits the proliferation of NPC cells by targeting ERK phosphorylation. However, the inhibition of NPC cell migration by silvestrol was independent of the Raf-MEK-ERK pathway. RAP1A, HK2, and GADD45A may be potential targets for the action of silvestrol.
Asunto(s)
Benzofuranos , Proteinas GADD45 , Hexoquinasa , Sistema de Señalización de MAP Quinasas , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Proteínas de Unión al GTP rap1 , Humanos , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Carcinoma Nasofaríngeo/tratamiento farmacológico , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patología , Hexoquinasa/genética , Hexoquinasa/metabolismo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Proteinas GADD45/genética , Proteinas GADD45/metabolismoRESUMEN
BACKGROUND: Optimized New Shengmai Powder (ONSMP) is a traditional Chinese medicine formula with significant anti-heart failure and myocardial fibrosis effects, but the specific molecular biological mechanisms are not fully understood. METHODS: In this study, we first used network pharmacology to analyze the ONSMP's active ingredients, core signaling pathways, and core targets. Second, calculate the affinity and binding modes of the ONSMP components to the core targets using molecular docking. Finally, the heart failure rat model was established by ligating the left anterior descending branch of the coronary artery and assessing the effect of ONSMP on myocardial fibrosis in heart failure using echocardiography, cardiac organ coefficients, heart failure markers, and pathological sections after 4 weeks of drug intervention. The cAMP level in rat myocardium was determined using Elisa, the α-SMA and FSP-1 positive expression determined by immunohistochemistry, and the protein and mRNA levels of the cAMP/Rap1A signaling pathway were detected by Western Blotting and quantitative real-time PCR, respectively. RESULTS: The result shows that the possible mechanism of ONSMP in reducing myocardial fibrosis also includes the use of 12 active ingredients such as baicalin, vitamin D, resveratrol, tanshinone IIA, emodin, 15,16-dihydrotanshinone-i to regulate ß1-AR, AC6, EPAC1, Rap1 A, STAT3, and CCND1 on the cAMP/Rap1A signaling pathway, thereby inhibiting the proliferation of cardiac fibroblasts and reduce the excessive secretion of collagen, effectively improve cardiac function and ventricular remodeling in heart failure rats. CONCLUSION: This research shows that ONSMP can inhibit myocardial fibrosis and delay heart failure through the cAMP/Rap1A signaling pathway.
RESUMEN
The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser(2113). Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.
Asunto(s)
Filaminas/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Línea Celular , AMP Cíclico/metabolismo , Filaminas/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , VasoconstricciónRESUMEN
Abnormal elevation of homocysteine (Hcy) level accelerates atherosclerosis through promote macrophage inflammation, while the precise mechanisms remain to be well elucidated. Previous study revealed that Rap1A is involved in the development of atherosclerosis, but little is known regarding the regulation of macrophage inflammation induced by Hcy and its potential mechanisms. In the present study, we demonstrated that Hcy upregulates Rap1A expression and knockdown of Rap1A inhibited pro-inflammatory cytokines IL-6 and TNF-α levels in ANA-1 cells. Mechanistically, DNMT3a-mediated DNA hypomethylation of Rap1A promoter accelerates Hcy-induced ANA-1 cells inflammation. Furthermore, FoxO1 transcriptionally activate Rap1A by direct binding to its promoter. More importantly, Hcy could enhance FoxO1 interaction with DNMT3a and synergistically promote the expression of Rap1A resulting in accelerate ANA-1 cells inflammation. These data indicate that Rap1A is a novel and important regulator in Hcy-induced ANA-1 cells inflammation.
Asunto(s)
Aterosclerosis , Homocisteína , Aterosclerosis/metabolismo , Células Cultivadas , Metilación de ADN , Proteína Forkhead Box O1/metabolismo , Homocisteína/farmacología , Inflamación/genética , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Animales , RatonesRESUMEN
BACKGROUND: Dedicator for cytokinesis 4 (DOCK4) is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. However, the functions of DOCK4 concerning the tumor microenvironment (TME) in colon adenocarcinoma (COAD) remain uncertain. METHODS: The TIMER and GEPIA databases were used to analyze the DOCK4 expression between COAD tissues and adjunct normal tissues. The PrognoScan database was used to assess the prognosis of DOCK4 expression in COAD. The co-expression networks of DOCK4 in COAD were constructed by the LinkedOmics website. Furthermore, the correlation between DOCK4 expression and TME of COAD was explored using TIMER and TISIDB databases. Finally, the clone formation assay was used to further verify the function of DOCK4 in COAD. The Western blotting assay was used to confirm the mechanism related to DOCK4 in COAD. RESULTS: The DOCK4 expression was different significantly in COAD tissues and paracancerous tissues. The DOCK4 was found to play a poor role in the prognosis of patients with COAD. The DOCK4 was found to participate in the TME by promoting immune evasion of COAD. The reduction of DOCK4 expression inhibited the clone formation and Ras-associated protein 1A (Rap1A) expression of HCT116 cells. CONCLUSIONS: DOCK4 potentially plays an important role in the regulation of TME in COAD. DOCK4 facilitates the development through the Rap1A pathway, thus becoming a novel prognostic biomarker in COAD.