ABSTRACT
AIMS: Exchange protein directly activated by cAMP 1 (EPAC1), a major isoform of guanine nucleotide exchange factors, is highly expressed in vascular endothelia cells and regulates angiogenesis in the retina. High intratumor microvascular densities (MVD) resulting from angiogenesis is responsible for breast cancer development. Downregulation of EPAC1 in tumor cell reduces triple-negative breast cancer (TNBC)-induced angiogenesis. However, whether Epac1 expressed in vascular endothelial cells contributes to angiogenesis and tumor development of TNBC remains elusive. MAIN METHODS: We employed NY0123, a previously identified potent EPAC inhibitor, to explore the anti-angiogenic biological role of EPAC1 in vitro and in vivo through vascular endothelial cells, rat aortic ring, Matrigel plug, and chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) assays, as well as the in vivo xenograft tumor models of TNBC in both chick embryo and mice. KEY FINDINGS: Inhibiting EPAC1 in vascular endothelial cells by NY0123 significantly suppresses angiogenesis and tumor growth of TNBC. In addition, NY0123 possesses a better inhibitory efficacy than ESI-09, a reported specific EPAC inhibitor tool compound. Importantly, inhibiting EPAC1 in vascular endothelia cells regulates the typical angiogenic signaling network, which is associated with not only vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR2) signaling, but also PI3K/AKT, MEK/ERK and Notch pathway. CONCLUSIONS: Our findings support that EPAC1 may serve as an effective anti-angiogenic therapeutic target of TNBC, and EPAC inhibitor NY0123 has the therapeutic potential to be developed for the treatment of TNBC.
Subject(s)
Guanine Nucleotide Exchange Factors , Neovascularization, Pathologic , Triple Negative Breast Neoplasms , Animals , Chick Embryo , Humans , Mice , Rats , Endothelial Cells/metabolism , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Phosphatidylinositol 3-Kinases , Triple Negative Breast Neoplasms/blood supply , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Vascular Endothelial Growth Factor A/metabolism , Neovascularization, Pathologic/drug therapyABSTRACT
P-Rex1 and P-Rex2 are guanine-nucleotide exchange factors (GEFs) that activate Rac small GTPases in response to the stimulation of G protein-coupled receptors and phosphoinositide 3-kinase. P-Rex Rac-GEFs regulate the morphology, adhesion and migration of various cell types, as well as reactive oxygen species production and cell cycle progression. P-Rex Rac-GEFs also have pathogenic roles in the initiation, progression or metastasis of several types of cancer. With one exception, all P-Rex functions are known or assumed to be mediated through their catalytic Rac-GEF activity. Thus, inhibitors of P-Rex Rac-GEF activity would be valuable research tools. We have generated a panel of small-molecule P-Rex inhibitors that target the interface between the catalytic DH domain of P-Rex Rac-GEFs and Rac. Our best-characterized compound, P-Rex inhibitor 1 (PREX-in1), blocks the Rac-GEF activity of full-length P-Rex1 and P-Rex2, and of their isolated catalytic domains, in vitro at low-micromolar concentration, without affecting the activities of several other Rho-GEFs. PREX-in1 blocks the P-Rex1 dependent spreading of PDGF-stimulated endothelial cells and the production of reactive oxygen species in fMLP-stimulated mouse neutrophils. Structure-function analysis revealed critical structural elements of PREX-in1, allowing us to develop derivatives with increased efficacy, the best with an IC50 of 2 µM. In summary, we have developed PREX-in1 and derivative small-molecule compounds that will be useful laboratory research tools for the study of P-Rex function. These compounds may also be a good starting point for the future development of more sophisticated drug-like inhibitors aimed at targeting P-Rex Rac-GEFs in cancer.
Subject(s)
Guanine Nucleotide Exchange Factors , Neoplasms , Animals , Mice , Endothelial Cells/metabolism , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Phosphatidylinositol 3-Kinases , Reactive Oxygen SpeciesABSTRACT
Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.
Subject(s)
COVID-19 , GTPase-Activating Proteins , Genome-Wide Association Study , Guanine Nucleotide Exchange Factors , Host Microbial Interactions , SARS-CoV-2 , Alleles , Animals , COVID-19/complications , COVID-19/genetics , COVID-19/immunology , COVID-19/physiopathology , Disease Models, Animal , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Genetic Predisposition to Disease , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Humans , Interferon Type I/genetics , Interferon Type I/immunology , Japan , Lung/pathology , Macrophages , Mesocricetus , Middle Aged , Pneumonia/complications , Pyrazoles/pharmacology , RNA-Seq , SARS-CoV-2/pathogenicity , Viral Load , Weight LossABSTRACT
Although PTX has been identified as an effective drug for nasopharyngeal carcinoma (NPC) therapy, it has serious side effects in the human body. Previous studies have shown that timosaponin AIII (TSAIII) can inhibit the malignant progression of NPC cells. This study investigated the active mechanism of the combination of TSAIII and paclitaxel (PTX) on NPC. Cellular viability, apoptosis, apoptotic factors, and RAP1 signaling regulators were detected in the PNC cells (CNE-1 and HNE-2) and the subcutaneous CNE-1 transplanted nude mice treated with PTX or/and TSAIII. The results showed that TSAIII notably strengthened the inhibitory effect of PTX on the proliferation of NPC cells CNE-1 and HNE-2; upregulated the expression of Bax B-cell lymphoma 2 (Bcl-2)/Bcl-xL-associated death promoter (Bad), and Ras-associated protein1 (RAP1) GTPase activating protein (Rap1GAP); inhibited the level of Bcl-2, RAP1, and Ras guanine nucleotide releasing protein (RasGRP2); and significantly enhanced the promoting effect of PTX on apoptosis in the CNE-1 and HNE-2 cells. Besides, TSAIII strengthened the inhibitory effect of PTX on xenograft tumor in nude mice without adverse reactions. In conclusion, the combination administration of TSAIII and PTX had a significantly therapeutic effect on NPC and avoided the PTX's side effects, which may have acted as a new direction for the study of therapeutic approaches for NPC clinically.
Subject(s)
Guanine Nucleotide Exchange Factors , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , Humans , Mice , Mice, Nude , Nasopharyngeal Carcinoma/drug therapy , Nasopharyngeal Carcinoma/metabolism , Nasopharyngeal Carcinoma/pathology , Nasopharyngeal Neoplasms/drug therapy , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/pathology , Paclitaxel/administration & dosage , Saponins/administration & dosage , Signal Transduction/drug effects , Steroids/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
Fibroblast growth factor 21 (FGF21) functions as a polypeptide hormone to regulate glucose and lipid metabolism, and its expression is regulated by cellular metabolic stress. Pyruvate is an important intermediate metabolite that acts as a key hub for cellular fuel metabolism. However, the effect of pyruvate on hepatic FGF21 expression and secretion remains unknown. Herein, we examined the gene expression and protein levels of FGF21 in human hepatoma HepG2 cells and mouse AML12 hepatocytes in vitro, as well as in mice in vivo. In HepG2 and AML12 cells, pyruvate at concentrations above 0.1 mM significantly increased FGF21 expression and secretion. The increase in cellular cAMP levels by adenylyl cyclase activation, phosphodiesterase (PDE) inhibition and 8-Bromo-cAMP administration significantly restrained pyruvate-stimulated FGF21 expression. Pyruvate significantly increased PDE activities, reduced cAMP levels and decreased CREB phosphorylation. The inhibition of exchange protein directed activated by cAMP (Epac) and cAMP response element binding protein (CREB) upregulated FGF21 expression, upon which pyruvate no longer increased FGF21 expression. The increase in plasma pyruvate levels in mice induced by the intraperitoneal injection of pyruvate significantly increased FGF21 gene expression and PDE activity with a reduction in cAMP levels and CREB phosphorylation in the mouse liver compared with the control. In conclusion, pyruvate activates PDEs to reduce cAMP and then inhibits the cAMP-Epac-CREB signaling pathway to upregulate FGF21 expression in hepatocytes.
Subject(s)
Cyclic AMP Response Element-Binding Protein , Fibroblast Growth Factors , Guanine Nucleotide Exchange Factors , Liver , Phosphoric Diester Hydrolases , Pyruvic Acid , Animals , Cyclic AMP Response Element-Binding Protein/antagonists & inhibitors , Cyclic AMP Response Element-Binding Protein/metabolism , Fibroblast Growth Factors/biosynthesis , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Gene Expression , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , Hep G2 Cells , Humans , Liver/enzymology , Liver/metabolism , Mice , Phosphoric Diester Hydrolases/metabolism , Pyruvic Acid/blood , Pyruvic Acid/metabolism , Pyruvic Acid/pharmacokinetics , Signal Transduction/physiologyABSTRACT
Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.
Subject(s)
ADP-Ribosylation Factor 1/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Infectious bursal disease virus/physiology , Secretory Pathway/physiology , Virus Replication/physiology , rab1 GTP-Binding Proteins/metabolism , ADP-Ribosylation Factor 1/genetics , Animals , Brefeldin A/pharmacology , Cell Line , Endosomes/metabolism , Golgi Apparatus/metabolism , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Host-Pathogen Interactions , Pyridines/pharmacology , Quinolines/pharmacology , Secretory Pathway/drug effects , Viral Replication Compartments/metabolism , Virus Replication/drug effects , rab1 GTP-Binding Proteins/geneticsABSTRACT
BACKGROUND: Propofol acts as an intravenous anesthetic cure which is widely used as a therapy for the craniocerebral injury that comprised surgical anesthesia as well as the sedation done in the intensive care units. Propofol is one of the most commonly used and efficient anesthetics where the painful effects are followed by an injection of propofol. In many cases, patients experience pain followed by anxiety, boredom, fear, and even myocardial ischemia. OBJECTIVE: This study was performed to investigate the underlying mechanism of propofol and its effect on regulating spinal glun2b-p38mapkepac1 pathways in chronic contractile injury. Material and Methods. Contractile injury was performed by ligation around the nerve of the thigh region postanesthesia. Rats were divided into three groups to analyze the changes like mechanical allodynia by the paw withdrawal threshold and histopathological analysis for assessing cellular degradation. L4-L6 from the spinal dorsal horns were isolated and harvested for studying protein expression, by the method of western blotting and immunofluorescence analysis. RESULTS: The pain caused due to mechanical allodynia in the paw region was highest at 1 hour postinduction and lasted for three days postinjury. Pain was significantly less in the group receiving propofol when compared with the isoflurane group for the first two hours of injury. In the propofol group, EPAC1, GluN2B, and p38 MAP K were significantly lower. CONCLUSION: In the rat model of induced chronic contractile injury, postsurgery there was a suppression of the GluN2B-p38MAPK/EPAC1 signaling pathway in the propofol group. As the p38MAPK/EPAC pathway has a significant role in the postoperative hyperalgesia, thus our experiment suggests that propofol has analgesic effects.
Subject(s)
Neuralgia/drug therapy , Propofol/pharmacology , Anesthetics, Intravenous/pharmacology , Animals , Computational Biology , Disease Models, Animal , Ganglia, Spinal/drug effects , Ganglia, Spinal/injuries , Ganglia, Spinal/metabolism , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Humans , Hyperalgesia/drug therapy , Hyperalgesia/etiology , Hyperalgesia/metabolism , Neuralgia/etiology , Neuralgia/metabolism , Rats , Rats, Wistar , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Signal Transduction/drug effects , Signal Transduction/physiology , Spinal Cord Dorsal Horn/drug effects , Spinal Cord Dorsal Horn/injuries , Spinal Cord Dorsal Horn/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in young children. It is also a significant contributor to upper respiratory tract infections, therefore, a major cause for visits to the pediatrician. High morbidity and mortality are associated with high-risk populations including premature infants, the elderly, and the immunocompromised. However, no effective and specific treatment is available. Recently, we discovered that an exchange protein directly activated by cyclic AMP 2 (EPAC2) can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, EPAC2 promotes RSV replication and pro-inflammatory cytokine/chemokine induction. However, the overall role of EPAC2 in the pulmonary responses to RSV has not been investigated. Herein, we found that EPAC2-deficient mice (KO) or mice treated with an EPAC2-specific inhibitor showed a significant decrease in body weight loss, airway hyperresponsiveness, and pulmonary inflammation, compared with wild-type (WT) or vehicle-treated mice. Overall, this study demonstrates the critical contribution of the EPAC2-mediated pathway to airway diseases in experimental RSV infection, suggesting the possibility to target EPAC2 as a promising treatment modality for RSV.
Subject(s)
Guanine Nucleotide Exchange Factors/physiology , Respiratory Syncytial Virus Infections/physiopathology , Airway Obstruction/etiology , Animals , Cyclic AMP/physiology , Cytokines/biosynthesis , Cytokines/genetics , Granulocyte Colony-Stimulating Factor/biosynthesis , Granulocyte Colony-Stimulating Factor/genetics , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/deficiency , Inflammation , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Respiratory Hypersensitivity/etiology , Respiratory Syncytial Virus Infections/complications , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Viruses/physiology , Specific Pathogen-Free Organisms , Virus Replication , Weight LossABSTRACT
P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons. Here, we used CRISPR/Cas9-mediated P-Rex1 deficiency in neuronal PC12 cells that stably overexpress the GPCR S1PR1, a receptor for sphingosine 1-phosphate (S1P), to investigate the role of P-Rex1 in neuronal GPCR signalling and cell responses. We show that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt, basal Rac3 activity, and constitutive cAMP production in PC12-S1PR1 cells. The constitutive cAMP production was not due to increased expression levels of major neuronal adenylyl cyclases, suggesting that P-Rex1 may regulate adenylyl cyclase activity. P-Rex1 was required for maintenance of neurite protrusions and spreading in S1P-stimulated PC12-S1PR1 cells, as well as for cell-cycle progression and proliferation. In summary, we identified novel functional roles of P-Rex1 in neuronal Rac, Akt and cAMP signalling, as well as in neuronal cell-cycle progression and proliferation.
Subject(s)
Cell Cycle , Guanine Nucleotide Exchange Factors/metabolism , Neurites/physiology , Neurons/physiology , Sphingosine-1-Phosphate Receptors/metabolism , Animals , Cell Movement , Cell Proliferation , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/genetics , Lysophospholipids/metabolism , Neurons/cytology , PC12 Cells , Rats , Signal Transduction , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Sphingosine-1-Phosphate Receptors/antagonists & inhibitors , Sphingosine-1-Phosphate Receptors/genetics , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
P2X1 receptors are adenosine triphosphate (ATP)-gated cation channels that are functionally important for male fertility, bladder contraction, and platelet aggregation. The activity of P2X1 receptors is modulated by lipids and intracellular messengers such as cAMP, which can stimulate protein kinase A (PKA). Exchange protein activated by cAMP (EPAC) is another cAMP effector; however, its effect on P2X1 receptors has not yet been determined. Here, we demonstrate that P2X1 currents, recorded from human embryonic kidney (HEK) cells transiently transfected with P2X1 cDNA, were inhibited by the highly selective EPAC activator 007-AM. In contrast, EPAC activation enhanced P2X2 current amplitude. The PKA activator 6-MB-cAMP did not affect P2X1 currents, but inhibited P2X2 currents. The inhibitory effects of EPAC on P2X1 were prevented by triple mutation of residues 21 to 23 on the amino terminus of P2X1 subunits to the equivalent amino acids on P2X2 receptors. Double mutation of residues 21 and 22 and single mutation of residue 23 also protected P2X1 receptors from inhibition by EPAC activation. Finally, the inhibitory effects of EPAC on P2X1 were also prevented by NSC23766, an inhibitor of Rac1, a member of the Rho family of small GTPases. These data suggest that EPAC is an important regulator of P2X1 and P2X2 receptors.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/pharmacology , Cyclic AMP/metabolism , Gene Expression Regulation , Guanine Nucleotide Exchange Factors/pharmacology , Kidney/metabolism , Receptors, Purinergic P2X1/metabolism , Receptors, Purinergic P2X2/metabolism , Adenosine Triphosphate , Aminoquinolines/pharmacology , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , HEK293 Cells , Humans , Kidney/drug effects , Pyrimidines/pharmacology , Receptors, Purinergic P2X1/genetics , Receptors, Purinergic P2X2/genetics , rac1 GTP-Binding Protein/antagonists & inhibitorsABSTRACT
The recently discovered exchange protein directly activated by cAMP (EPAC), compared with protein kinase A (PKA), is a fairly new family of cAMP effectors. Soon after the discovery, EPAC has shown its significance in many diseases including its emerging role in infectious diseases. In a recent study, we demonstrated that EPAC, but not PKA, is a promising therapeutic target to regulate respiratory syncytial virus (RSV) replication and its associated inflammation. In mammals, there are two isoforms of EPAC-EPAC1 and EPAC2. Unlike other viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) and Ebola virus, which use EPAC1 to regulate viral replication, RSV uses EPAC2 to control its replication and associated cytokine/chemokine responses. To determine whether EPAC2 protein has a broad impact on other respiratory viral infections, we used an EPAC2-specific inhibitor, MAY0132, to examine the functions of EPAC2 in human metapneumovirus (HMPV) and adenovirus (AdV) infections. HMPV is a negative-sense single-stranded RNA virus belonging to the family Pneumoviridae, which also includes RSV, while AdV is a double-stranded DNA virus. Treatment with an EPAC1-specific inhibitor was also included to investigate the impact of EPAC1 on these two viruses. We found that the replication of HMPV, AdV, and RSV and the viral-induced immune mediators are significantly impaired by MAY0132, while an EPAC1-specific inhibitor, CE3F4, does not impact or slightly impacts, demonstrating that EPAC2 could serve as a novel common therapeutic target to control these viruses, all of which do not have effective treatment and prevention strategies.
Subject(s)
Adenoviridae/physiology , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Metapneumovirus/physiology , Respiratory Syncytial Virus, Human/physiology , Virus Replication , A549 Cells , Cell Line , Chemokines/immunology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Epithelial Cells/drug effects , Epithelial Cells/virology , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , HEK293 Cells , Humans , Quinolines/pharmacologyABSTRACT
The present study investigated hyperalgesia during sickness syndrome in female rats. Hyperalgesia was induced by an intraperitoneal injection of lipopolysaccharide (LPS) or an intracerebroventricular injection of prostaglandin E2 (PGE2). No differences were found in basal mechanical and thermal thresholds or in LPS-induced hyperalgesia in sham-operated animals in the diestrus or proestrus phase or in ovariectomized (OVX) animals. However, higher levels of PGE2 where found in the cerebrospinal fluid of OVX animals compared to sham-operated females. Intracerebroventricular injection of PGE2 produced rapid mechanical hyperalgesia in sham-operated rats while these responses were observed at later times in OVX animals. The protein kinase A (PKA) inhibitor H-89 reduced mechanical PGE2-induced hyperalgesia in OVX female rats, whereas no effect was observed in sham-operated animals. In contrast, the exchange protein activated by cyclic adenosine monophosphate (cAMP; Epac) inhibitor ESI-09 reduced mechanical PGE2-induced hyperalgesia, whereas no effect was observed in OVX animals. PGE2 also induced thermal hyperalgesia in sham-operated and OVX female rats and a similar effect of ESI-09 was observed. These results suggest that PGE2-induced hyperalgesia that is observed during sickness syndrome has different signaling mechanisms in cycling and OVX female rats involving the activation of the cAMP-Epac or cAMP-PKA pathways, respectively.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Dinoprostone/pharmacology , Estrous Cycle/drug effects , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Illness Behavior/drug effects , Animals , Cyclic AMP/antagonists & inhibitors , Dinoprostone/administration & dosage , Disease Models, Animal , Female , Hydrazones/pharmacology , Isoquinolines/pharmacology , Isoxazoles/pharmacology , Lipopolysaccharides/pharmacology , Ovariectomy , Rats , Rats, Wistar , Signal Transduction/drug effects , Sulfonamides/pharmacologyABSTRACT
Despite the renal expression of P2Y12, the purinergic receptor for adenosine diphosphate, few data are available to discuss the renotherapeutic potential of ticagrelor, one of its reversible blockers. Indeed, the tonic inhibitory effect of this receptor has been linked to the activation of exchange protein activated by cyclic adenosine monophosphate-1 (Epac-1) protein through the cyclic adenosine monophosphate cascade. Epac-1 is considered a crossroad protein, where its activation has been documented to manage renal injury models. Hence, the current study aimed to investigate the possible therapeutic effectiveness of ticagrelor, against renal ischemia/reperfusion (I/R) model with emphasis on the involvement of Epac-1 signaling pathway using R-CE3F4, a selective Epac-1 blocker. Accordingly, rats were randomized into four groups; viz., sham-operated, renal I/R, I/R post-treated with ticagrelor for 3 days, and ticagrelor + R-CE3F4. Treatment with ticagrelor ameliorated the I/R-mediated structural alterations and improved renal function manifested by the reduction in serum BUN and creatinine. On the molecular level, ticagrelor enhanced renal Epac-1 mRNA expression, Rap-1 activation (Rap-1-GTP) and SOCS-3 level. On the contrary, it inhibited the protein expression of JAK-2/STAT-3 hub, TNF-α and MDA contents, as well as caspase-3 activity. Additionally, ticagrelor enhanced the protein expression/content of AKT/Nrf-2/HO-1 axis. All these beneficial effects were obviously antagonized upon using R-CE3F4. In conclusion, ticagrelor reno-therapeutic effect is partly mediated through modulating the Epac-1/Rap-1-GTP, AKT/Nrf-2/HO-1 and JAK-2/STAT-3/SOCS-3 trajectories, pathways that integrate to afford novel explanations to its anti-inflammatory, anti-oxidant, and anti-apoptotic potentials.
Subject(s)
Acute Kidney Injury/drug therapy , Guanine Nucleotide Exchange Factors/drug effects , Purinergic P2Y Receptor Antagonists/therapeutic use , Reperfusion Injury/drug therapy , Signal Transduction/drug effects , Ticagrelor/therapeutic use , rap1 GTP-Binding Proteins/drug effects , Animals , Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Ischemia , Kidney Function Tests , Male , Rats , Rats, Wistar , Renal Circulation/drug effects , rap1 GTP-Binding Proteins/antagonists & inhibitorsABSTRACT
Prolactin (PRL), mainly synthesized and secreted by the lactotrophs and somatolactotrophs of the anterior pituitary, is a pleiotropic hormone that regulates lactation. In the last decade, nesfatin-1 (NESF) and NESF-like peptide (NLP), encoded in nucleobindin 1 and 2 (NUCB1 and NUCB2), respectively, were characterized as metabolic factors with a potential role in the control of pituitary hormones. We hypothesized that NUCBs and their encoded peptides (NESF and NLP) suppress PRL transcription in the pituitary. The main objective of this research was to determine whether exogenous NESF and NLP, and/or endogenous NUCB1 and NUCB2 regulate the expression of prl and preb mRNAs. Using immortalized rat somatolactotrophs (GH3 cells), dose-response studies were performed to test whether NESF and NLP affect prl and preb. Moreover, the ability of these peptides to modulate the effects of the PRL stimulator thyrotropin releasing hormone (TRH) was studied. Besides, the effects of siRNA-mediated knockdown of endogenous NUCBs on prl and preb mRNAs were determined. NESF and NLP reduced the transcription of prl and preb in GH3 cells. Both NESF and NLP also prevented the stimulatory effects of TRH prl and preb expression. The knockdown of endogenous NUCB1 attenuates both basal prl and TRH-induced expression of prl and preb, while the silencing of NUCBs did not affect the actions of exogenous NESF or NLP. Overall, this work reveals that NUCBs and encoded-peptides are novel regulators of PRL. Future research should test whether the effects observed here in GH3 cells are preserved both in vivo and at the post-transcriptional level.
Subject(s)
DNA-Binding Proteins/genetics , Guanine Nucleotide Exchange Factors/genetics , Lactotrophs/drug effects , Nucleobindins/pharmacology , Peptides/pharmacology , Prolactin/genetics , Somatotrophs/drug effects , Transcription Factors/genetics , Animals , Cell Line, Transformed , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , Lactotrophs/cytology , Lactotrophs/metabolism , Nucleobindins/antagonists & inhibitors , Nucleobindins/genetics , Nucleobindins/metabolism , Prolactin/antagonists & inhibitors , Prolactin/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Signal Transduction , Somatotrophs/cytology , Somatotrophs/metabolism , Thyrotropin-Releasing Hormone/genetics , Thyrotropin-Releasing Hormone/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolismABSTRACT
RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer.
Subject(s)
Chromatin/metabolism , DNA Replication , GTPase-Activating Proteins/physiology , Guanine Nucleotide Exchange Factors/physiology , Replication Protein A/metabolism , Animals , Cell Line, Tumor , DNA Repair , Female , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/metabolism , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , HEK293 Cells , Humans , Mice , Mice, Nude , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Phosphorylation , Proteolysis , Signal Transduction , Stress, Physiological/genetics , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
The presence of adherens junctions and the associated protein ß-catenin are requirements for the development of glucose-stimulated insulin secretion (GSIS) in ß-cells. Evidence indicates that modulation of ß-catenin function in response to changes in glucose levels can modulate the levels of insulin secretion from ß-cells but the role of ß-catenin phosphorylation in this process has not been established. We find that a Ser552Ala version of ß-catenin attenuates glucose-stimulated insulin secretion indicating a functional role for Ser552 phosphorylation of ß-catenin in insulin secretion. This is associated with alterations F/G actin ratio but not the transcriptional activity of ß-catenin. Both glucose and GLP-1 stimulated phosphorylation of the serine 552 residue on ß-catenin. We investigated the possibility that an EPAC-PAK1 pathway might be involved in this phosphorylation event. We find that reduction in PAK1 levels using siRNA attenuates both glucose and GLP-1 stimulated phosphorylation of ß-catenin Ser552 and the effects of these on insulin secretion in ß-cell models. Furthermore, both the EPAC inhibitor ESI-09 and the PAK1 inhibitor IPA3 do the same in both ß-cell models and mouse islets. Together this identifies phosphorylation of ß-catenin at Ser552 as part of a cell signalling mechanism linking nutrient and hormonal regulation of ß-catenin to modulation of insulin secretory capacity of ß-cells and indicates this phosphorylation event is regulated downstream of EPAC and PAK1 in ß-cells.
Subject(s)
Guanine Nucleotide Exchange Factors/genetics , Insulin-Secreting Cells/metabolism , Insulin/genetics , Islets of Langerhans/metabolism , beta Catenin/genetics , p21-Activated Kinases/genetics , Actins/genetics , Actins/metabolism , Adherens Junctions/drug effects , Adherens Junctions/metabolism , Animals , Cell Line, Transformed , Disulfides/pharmacology , Gene Expression Regulation , Glucagon-Like Peptide 1/pharmacology , Glucose/metabolism , Glucose/pharmacology , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , Hydrazones/pharmacology , Insulin/metabolism , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Isoxazoles/pharmacology , Male , Mice , Naphthols/pharmacology , Phosphorylation , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Signal Transduction , Tissue Culture Techniques , beta Catenin/metabolism , p21-Activated Kinases/antagonists & inhibitors , p21-Activated Kinases/metabolismABSTRACT
In recent years, although Immune Checkpoint Inhibitors (ICIs) significantly improves survival both in local advanced stage and advanced stage of non-small cell lung cancer (NSCLC), the objective response rate of ICI monotherapy is still only about 20%. Thus, to identify the mechanisms of ICI resistance is critical to increase the efficacy of ICI treatments. By bioinformatics analysis, we found that the expression of regulator of chromosome condensation 1 (RCC1) in lung adenocarcinoma was significantly higher than that in normal lung tissue in TCGA and Oncomine databases. The survival analysis showed that high expression RCC1 was associated with the poor prognosis of NSCLC. And the expression of RCC1 was inversely related to the number of immune cell infiltration. In vitro, knockdown of RCC1 not only significantly inhibited the proliferation of lung adenocarcinoma cells but also increased the expression levels of p27kip1 and PD-L1, and decreased the expression level of CDK4 and p-Rb. In vivo, knockdown of RCC1 significantly slowed down the growth rate of tumour, and further reduced the volume and weight of tumour model after treated by PD-L1 monoclonal antibody. Therefore, RCC1 could up-regulate the expression level of PD-L1 by regulating p27kip1 /CDK4 pathway and decrease the resistance to ICIs. And this study might provide a new way to increase the efficacy of PD-L1 monoclonal antibody by inhibiting RCC1.
Subject(s)
B7-H1 Antigen/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Cycle Proteins/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Immunotherapy/methods , Nuclear Proteins/antagonists & inhibitors , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/immunology , Adenocarcinoma of Lung/metabolism , Adenocarcinoma of Lung/pathology , Adult , Aged , Animals , Apoptosis , B7-H1 Antigen/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Movement , Cell Proliferation , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase Inhibitor p27/genetics , Female , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Middle Aged , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Prognosis , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Young AdultABSTRACT
BACKGROUND: Targeted therapy has demonstrated high efficacy in the treatment of advanced cancer, and protein kinase inhibitors are a major focus of that therapy; therefore, our study aimed to identify a new protein kinase inhibitor that could be used in the treatment of advanced cancers. METHODS: We analyzed the expression profile of colorectal cancer (CRC), combined the driver gene and drug target databases, and identified protein kinase kalirin RhoGEF kinase (kalirin/KALRN) which is related to CRC metastasis. Based on the structure of kalirin, we screened for the small molecular compound ZINC65387069. We first compared the kinase inhibitory activities and molecular properties of ZINC65387069 and tyrosine kinase inhibitors (TKIs). We then determined the effects of ZINC65387069 on the phosphorylation of protein kinase B-Raf. Finally, we determined the effects of ZINC65387069 on migration and apoptosis of HCT116 cells as well as RKO cells. The cell cytoskeleton was also determined. RESULTS: Compared with traditional TKIs, ZINC65387069 had stronger kinase inhibitory activity, a simpler structure, higher water solubility, a smaller polar surface area, and lower molecular weight and volume. In CRC cells, ZINC65387069 could significantly inhibit the phosphorylation of B-Raf as well as inhibit cell migration, destroy the cell cytoskeleton, and promote cell apoptosis. CONCLUSION: ZINC65387069 is a newly identified protein kinase inhibitor that deserves additional research as a lead compound for drug development to help create targeted therapy against CRC.
Subject(s)
Protein Kinase Inhibitors/chemistry , Small Molecule Libraries/chemistry , Apoptosis/drug effects , Cell Movement/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cytoskeleton/drug effects , Databases, Chemical , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , HCT116 Cells , Humans , Phosphorylation/drug effects , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacologyABSTRACT
BACKGROUND: A keloid is a type of pathological scar often caused by abnormal tissue repair after a skin injury and is more common in genetically susceptible individuals. cAMP is a universal second messenger and regulates critical physiological processes, including calcium homeostasis, secretion, cell fate, and gene transcription, by affecting the expression of the exchange protein directly activated by cAMP (Epac). Epac has two isoforms, Epac1 (cAMP-GEF-1) and Epac2 (cAMP-GEF-II), which show varying expression levels depending on the tissue and cell type. The expression of Epac1 in keloids has not yet been investigated. MATERIALS AND METHODS: Keloid tissue and normal dermal skin tissue were analyzed by hematoxylin and eosin staining and immunofluorescence. Primary human keloid fibroblasts (HKFs) and human normal dermal fibroblasts were studied using immunofluorescence, wound healing tests, reverse transcription polymerase chain reaction, and western blot analysis with different concentrations of the Epac1 inhibitor ESI-09. RESULTS: Downregulation of Epac was performed using ESI-09, a specific Epac inhibitor. The proliferation and migration capacities of HKFs and human normal dermal fibroblasts showed an ESI-09 concentration-dependent decrease. Furthermore, the apoptosis rates were significantly different between fibroblasts treated with ESI-09 and control fibroblasts. In addition, the phosphorylation level of Akt was significantly decreased, indicating that ESI-09 reduces fibrosis and induces apoptosis through Akt signaling in HKFs. CONCLUSIONS: Our results illustrate the role of Epac1 in regulating fibroblast function during keloid pathogenesis and indicate that Epac1 may be a potential therapeutic target in keloid treatment.
Subject(s)
Fibroblasts/pathology , Guanine Nucleotide Exchange Factors/metabolism , Keloid/pathology , Proto-Oncogene Proteins c-akt/metabolism , Apoptosis/drug effects , Cells, Cultured , Dermis/cytology , Dermis/pathology , Down-Regulation , Fibrosis , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Humans , Hydrazones/pharmacology , Isoxazoles/pharmacology , Primary Cell Culture , Protein Isoforms/metabolism , Signal Transduction/drug effectsABSTRACT
Dedicator of cytokinesis 2 (DOCK2), an atypical Rac activator, has important anti-inflammatory properties in blepharitis, enteric bacterial infection and colitis. However, the roles of DOCK2 in macrophage activation and acute lung injury (ALI) are still poorly elucidated. In vitro studies demonstrated that DOCK2 was essential for the nucleotide-sensing Toll-like receptor (TLR) 4-mediated inflammatory response in macrophages. We also confirmed that exposure of macrophages to LPS induced Rac activation through a TLR4-independent, DOCK2-dependent mechanism. Phosphorylation of IκB kinase (IKK) ß and nuclear translocation of transcription factor nuclear factor kappa B (NF-κB) were impaired in Ad-shDOCK2-expressing macrophages, resulting in a decreased inflammatory response. Similar results were obtained when EHop-016 (a Rac inhibitor) was used to treat uninfected macrophages. In summary, these data indicate that the DOCK2-Rac signaling pathway acts in parallel with TLR4 engagement to control IKKß activation for inflammatory cytokine release. Next, we investigated whether pharmacological inhibition of DOCK2 protects against endotoxemia-induced lung injury in mice. Treatment with 4-[3'-(2â³-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP), a small-molecule inhibitor of DOCK2, reduced the severity of lung injury, as indicated by decreases in the lung injury score and myeloperoxidase (MPO) activity. Moreover, CPYPP attenuated LPS-induced proinflammatory cytokine release in mice. Our studies suggest that inhibition of DOCK2 may suppress LPS-induced macrophage activation and that DOCK2 may be a novel target for treating endotoxemia-related ALI.