scholarly journals Phosphorylation of GTP dissociation inhibitor by PKA negatively regulates RhoA

2008 ◽  
Vol 295 (5) ◽  
pp. C1161-C1168 ◽  
Author(s):  
Jing Qiao ◽  
Oksana Holian ◽  
Bao-Shiang Lee ◽  
Fei Huang ◽  
Jihang Zhang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Strong Support ◽  
Endothelial Permeability ◽  
Kinase Assay ◽  
Protective Mechanism ◽  
Rho Proteins ◽  
Inflammatory Injury ◽  
Rhoa Activity ◽  
Serum Response

The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDIα showed two potential protein kinase A (PKA) phosphorylation motifs, Ser174 and Thr182. Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDIα-GST fusion protein and PKA motif-containing GDIα peptide at Ser174, but not Thr182. Transfection of COS-7 cells with mutated full-length GDIα at Ser174 to Ala174 (GDIα-Ser174A) abrogated the ability of cAMP to phosphorylate GDIα. However, mutation of Thr182 to Ala182 (GDIα-Thr182A) did not abrogate, and cAMP increased phosphorylation of GDIα to a similar extent as wild-type GDIα transfectants. The mutant GDIα-Ser174A, but not GDIα-Thr182A, was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDIα-Ser174A was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIα binding with RhoA, suggesting that phosphorylation of Ser174 regulated preformed GDIα-RhoA complexes. The findings provide strong support that the selective phosphorylation at Ser174 by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury.

scholarly journals Lacidipine Ameliorates the Endothelial Senescence and Inflammatory Injury Through CXCR7/P38/C/EBP-β Signaling Pathway

2021 ◽  
Vol 8 ◽  
Author(s):  
Xing Liu ◽  
Zhuoshan Huang ◽  
Yuanyuan Zhang ◽  
Xing Shui ◽  
Fanmao Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Blood Pressure ◽  
Signaling Pathway ◽  
Cell Activity ◽  
Protective Role ◽  
Cell Counting ◽  
Control Group ◽  
Inflammatory Injury ◽  
Beneficial Effects

Background: Lacidipine, a third-generation calcium channel blocker, exerts beneficial effects on the endothelium of hypertensive patients in addition to blood pressure lowering. However, the detailed mechanism underlying Lacidipine-related endothelial protection is still elusive.Methods: Sixteen spontaneous hypertensive rats (SHRs) were randomly divided into two groups: Lacidipine-treated SHR group and saline-treated control group. Tail systolic blood pressure was monitored for four consecutive weeks. Endothelial cells (ECs) were pretreated with Lacidipine prior to being stimulated with H2O2, bleomycin, or Lipopolysaccharides (LPS) in vitro. Then, cell activity, migration, and senescence were measured by Cell Counting Kit-8 assay, transwell assay, and β-galactosidase staining, respectively. The fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was used to assess the intracellular reactive oxygen species (ROS). Related protein expression was detected by Western blotting and immunofluorescence.Results: Our data showed that Lacidipine treatment lowered the blood pressure of SHRs accompanied by the elevation of CXCR7 expression and suppression of P38 and CCAAT/enhancer-binding protein beta (C/EBP-β) compared with the control group. In vitro experiments further demonstrated that Lacidipine increased the cell viability and function of ECs under oxidative stress, cell senescence, and inflammatory activation via the CXCR7/P38/signaling pathway.Conclusions: Our results suggested that Lacidipine plays a protective role in EC senescence, oxidative stress, and inflammatory injury through the regulation of CXCR7/P38/C/EBP-β signaling pathway.

scholarly journals Activation of mTOR mediates hyperglycemia-induced renal glomerular endothelial hyperpermeability via the RhoA/ROCK/pMLC signaling pathway

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Xiaolin Chen ◽  
Jianhui Chen ◽  
Xianfan Li ◽  
Zengpu Yu
Keyword(s):  
Fluorescence Probe ◽  
Endothelial Permeability ◽  
Barrier Dysfunction ◽  
Actin Reorganization ◽  
Barrier Disruption ◽  
Rhoa Activity ◽  
Filtration Barrier ◽  
Activation Assay ◽  
Mlc Phosphorylation

Abstract Objective Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro. Materials and methods HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase–albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed. Results HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure. Conclusions Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

scholarly journals Berberine Exerts a Protective Effect on Gut-Vascular Barrier via the Modulation of the Wnt/Beta-Catenin Signaling Pathway During Sepsis

2018 ◽  
Vol 49 (4) ◽  
pp. 1342-1351 ◽  
Author(s):  
Yan He ◽  
Xiaoming Yuan ◽  
Hao Zuo ◽  
Ying Sun ◽  
Aiwen Feng
Keyword(s):  
Protective Effect ◽  
Vascular Permeability ◽  
Signaling Pathway ◽  
Cecal Ligation ◽  
Endothelial Permeability ◽  
Bacterial Invasion ◽  
Beta Catenin ◽  
Endotoxin Level

Background/Aims: The gut-vascular barrier (GVB) has recently been depicted to dampen the bacterial invasion of the bloodstream. The intestinal mucosa is a tissue rich in small vessels including capillaries. In this study, the protective effect of berberine on GVB in small bowel mucosa was investigated. Methods: The rat cecal ligation and puncture (CLP) sepsis model was employed to evaluate the effect of berberine on serum endotoxin level and intestinal vascular permeability to Evans blue in vivo. The rat intestinal microvascular endothelial cells (RIMECs) treated by lipopolysaccharide (LPS) were used to assess the effect of berberine on endothelial permeability to FITC-labeled dextran, transendothelial electrical resistance (TEER), and tight junction (TJ) and adherens junction (AJ) expression in vitro. Results: After 24-hr CLP operation the serum endotoxin concentration and gut vascular permeability were significantly increased, while berberine markedly reduced endotoxin level and vascular leakage. In vitro, LPS not only dramatically increased endothelial permeability of RIMECs to FITC-dextran, but also decreased TEER and inhibited claudin-12, beta-catenin and VE-cadherin expression. These effects of LPS were antagonized by berberine. In addition, our in vivo and vitro studies also confirmed that the effect of berberine on GVB could be partially abolished by ICG001. Conclusion: Berberine exerted a protective effect on GVB function in sepsis, which was strictly related to the modulation of the Wnt/beta-catenin signaling pathway.

scholarly journals Activation of mtor Mediates Hyperglycemia-Induced Renal Glomerular Endothelial Hyperpermeability via the RhoA/ROCK/pmlc Signaling Pathway

2021 ◽  
Author(s):  
Jianhui Chen ◽  
Xianfan Li ◽  
Xiaolin Chen ◽  
Zengpu Yu
Keyword(s):  
Fluorescence Probe ◽  
Endothelial Permeability ◽  
Barrier Dysfunction ◽  
Actin Reorganization ◽  
Barrier Disruption ◽  
Rhoa Activity ◽  
Filtration Barrier ◽  
Activation Assay ◽  
Mlc Phosphorylation

Abstract OBJECTIVE: Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro.MATERIALS AND METHODS: HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase–albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed.RESULTS: HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure.CONCLUSIONS: Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes

2002 ◽  
Vol 361 (2) ◽  
pp. 243-254 ◽  
Author(s):  
Marie-Annick FORGET ◽  
Richard R. DESROSIERS ◽  
Denis GINGRAS ◽  
Richard BÉLIVEAU
Keyword(s):  
Ionic Strength ◽  
Rat Brain ◽  
Small Gtpases ◽  
Rho Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rhoa Activity ◽  
Brain Membranes ◽  
Rat Brain Membranes ◽  
Gdp Dissociation Inhibitor

The Rho GDP dissociation inhibitor (RhoGDI) regulates the activation—inactivation cycle of Rho small GTPases, such as Cdc42 and RhoA, by extracting them from the membrane. To study the roles of Mg2+, phosphatidylinositol 4,5-bisphosphate (PIP2), ionic strength and phosphorylation on the interactions of RhoGDI with Cdc42 and RhoA, we developed a new, efficient and reliable method to produce prenylated Rho proteins using the yeast Saccharomyces cerevisiae. It has been previously reported that protein kinase A (PKA)-treatment of isolated membranes increased RhoA extraction from membranes by RhoGDI [Lang, Gesbert, Delespine-Carmagnat, Stancou, Pouchelet and Bertoglio (1996) EMBO J. 16, 510–519]. In the present study, we used an in vitro affinity chromatography system to show that phosphorylation of RhoA and Cdc42 significantly increased their interaction with RhoGDI under physiological conditions of ionic strength. This increase was independent of the nucleotide (GDP or guanosine 5′-[γ-thio]triphosphate) loaded on to the Rho proteins, as well as of Mg2+ and PIP2. Moreover, dephosphorylation of rat brain membranes by alkaline phosphatase significantly decreased the extraction of RhoA and Cdc42 by RhoGDI. Subsequent re-phosphorylation by PKA restored the extraction levels, indicating the reversibility of this process. These results clearly demonstrate that the phosphorylation states of Cdc42 and RhoA regulate their interactions with RhoGDI and, consequently, their extraction from rat brain membranes. We therefore suggest that phosphorylation is a mechanism of regulation of Cdc42 and RhoA activity that is independent of GDP—GTP cycling.

scholarly journals Comparison of the Effects of Essential Oil Obtained from the Crude and Bran-Processed Atractylodes lancea on Lipopolysaccharide-Induced Inflammatory Injury of Human Colonic Epithelial Cells by Downregulating the IKK/NF-κB Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Yan Yu ◽  
Zhenqi Wu ◽  
Yu Han ◽  
Yuan Yuan ◽  
Hui Fan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Essential Oil ◽  
Signaling Pathway ◽  
Underlying Mechanism ◽  
Colonic Epithelial Cells ◽  
Inflammatory Injury ◽  
Atractylodes Lancea ◽  
System Disease ◽  
Tnf Α

Background. Atractylodes lancea (AL) has been used in traditional Chinese medicine for the treatment of various diseases including digestive disorders. Ulcerative colitis (UC) is a common digestive system disease with a low cure rate and easy recurrence. However, it is still not clear whether AL is suitable for UC treatment. Currently, stir-baking with wheat bran is most commonly used to process AL. Here, we aimed to address the effects of the crude and bran-processed AL on UC in vitro and uncover the underlying mechanism based on regulating the IKK/NF-kappa B signaling pathway. Methods. Human colonic epithelial cells (HCoEpiC) were treated with lipopolysaccharide (LPS) to mimic the inflammatory injury of UC in vitro. The essential oil from crude and bran-processed AL was used to treat LPS-induced HcoEpiC cells. The cell viability was detected by an MTT assay. The levels of IL-4, IL-6, IL-8, IL-12, IL-1-β, TNF-α, and NO were determined by ELISA, and the mRNA expressions of IKK-α, NF-κB, IL-4, IL-6, IL-8, and TNF-α were determined by RT-PCR. Meanwhile, the expressions of IKK-α, p-IKK-α, p-IKK-β, NF-κB, IL-6, and IL-8 proteins were determined by Western blot. Results. The essential oil of AL, whether it was from crude or bran-processed AL, could significantly increase the viability of LPS-induced HCoEpiC cells. The treatment of AL essential oil also notably inhibited the productions of IL-6, IL-8, IL-12, IL-1-β, TNF-α, NO, p-IKK-α, p-IKK-β, and NF-κB and downregulated the mRNA expressions of NF-κB, IL-6, IL-8, and TNF-α. Meanwhile, IL-4 protein and mRNA expression were significantly stimulated by AL essential oil. Moreover, the essential oil from bran-processed AL was more effective than that from crude AL. Conclusion. Both kinds of AL essential oil had the anti-inflammatory effect on LPS-induced HCoEpiC, and the essential oil from bran-processed AL was more effective. The mechanism could be through the IKK/NF-κB signaling pathway in vitro.

scholarly journals Melatonin Protects Cardiomyocytes from Oxygen Glucose Deprivation And Reperfusion-Induced Injury by Inhibiting Rac1/JNK/Foxo3a/Bim Signaling Pathway

2021 ◽  
Author(s):  
Yulin Wang ◽  
Ying Jian ◽  
Xiaofu Zhang ◽  
Bin Ni ◽  
Mingwei Wang ◽  
...  
Keyword(s):  
Protective Effect ◽  
Signaling Pathway ◽  
Cell Injury ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Oxygen Glucose Deprivation ◽  
H9c2 Cell ◽  
Protective Mechanism ◽  
H9c2 Cells

Abstract Melatonin has been shown to exert protective effect during myocardial ischemia/reperfusion (I/R). However, the underlying mechanism is not completely understood. Using the oxygen-glucose deprivation and reperfusion (OGD/R) model of H9c2 cells in vitro, we found that melatonin alleviated OGD/R-induced H9c2 cell injury via inhibiting Foxo3a/Bim signaling pathway. Inhibition of Rac1 activation contributed to the protective effect of melatonin against OGD/R injury in H9c2 cells. Additionally, melatonin inhibited OGD/R-activated Foxo3a/Bim signaling pathway through inactivation of Rac1. Furthermore, JNK inactivation was responsible for Rac1 inhibition-mediated inactivation of Foxo3a/Bim signaling pathway and decreased cell injury in melatonin-treated H9c2 cells. Taken together, these findings identified a Rac1/JNK/Foxo3a/Bim signaling pathway in melatonin-induced protective effect against OGD/R injury in H9c2 cells. This study provided a novel insight into the protective mechanism of melatonin against myocardial I/R injury.

scholarly journals Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability

2003 ◽  
Vol 95 (5) ◽  
pp. 1889-1895 ◽  
Author(s):  
Fiemu E. Nwariaku ◽  
Patricia Rothenbach ◽  
Zijuan Liu ◽  
Xudong Zhu ◽  
Richard H. Turnage ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Endothelial Permeability ◽  
Kinase Assay ◽  
Western Blots ◽  
Mapk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Mapk Activity ◽  
Gtp Binding ◽  
Extracellular Signal

Our laboratory previously demonstrated that MAPK activation is an important signal during cytokine-induced endothelial permeability (Nwariaku FE, Liu Z, Terada L, Duffy S, Sarosi G, and Turnage R. Shock 18: 82-85, 2002). Because GTP-binding proteins have been implicated in MAPK activation, we now hypothesize that the GTP-binding protein Rho is a mediator of TNF-induced MAPK activation and increased endothelial permeability. Transmonolayer permeability was assessed in human lung microvascular cells by measuring transmonolayer electrical resistance. MAPK activity was assessed by using a phospho-specific immunoprecipitation kinase assay and by comparing Western blots for phospho-MAPK with total MAPK. MAPK inhibitors used were SB-202190 and PD-098059, whereas Clostridium botulinum C3 transferase was used as a Rho inactivator. Rho-associated coiled-coil kinase was inhibited with Y-27632. TNF increased pulmonary endothelial permeability in vitro and caused a rapid, sustained increase in endothelial p38 and extracellular signal-regulated kinase MAPK activity. Inhibition of p38 and extracellular signal-regulated kinase MAPK with SB-202190 and PD-098059, respectively, decreased TNF-induced endothelial permeability. C3 transferase attenuated TNF-induced MAPK activation and blocked TNF-induced endothelial permeability. Finally, inhibition of Rho-associated coiled-coil kinase with Y-27632 prevented both MAPK activation and TNF-induced decreases in transmonolayer resistance. Rho acts upstream of mitogen-activated protein kinases in mediating TNF-induced pulmonary endothelial leak.

Apigenin Nanoparticle Attenuates Renal Ischemia/Reperfusion Inflammatory Injury by Regulation of miR-140-5p/CXCL12/NF-κB Signaling Pathway

2021 ◽  
Vol 17 (1) ◽  
pp. 64-77
Author(s):  
Xiangfei He ◽  
Yibo Wen ◽  
Qingwei Wang ◽  
Yan Wang ◽  
Guoxian Zhang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Nuclear Translocation ◽  
Ischemia Reperfusion ◽  
Kidney Injury ◽  
Luciferase Reporter ◽  
Inflammatory Injury ◽  
Positive Effects ◽  
Hypoxia Reoxygenation

Apigenin as a natural flavonoid product has been proved previously to play a renoprotective effect during ischemia/reperfusion injury (IRI), but the particular mechanisms involving the positive effects of apigenin remain totally unclear. The study investigated apigenin's roles and underlying biological mechanisms in IR-induced acute kidney injury (AKI). Thirty-six mice received a right nephrectomy and clamping of the left renal artery for 30 minutes, and then perfusion for 24 h. Apigenin was loaded onto a biodegradable polymer carrier (nanoparticle) to enhance its bioavailability. Mice were subjected to intraperitoneally injection with apigenin (5, 10 or 20 mg/kg) for 24 h before surgery. For in vitro experiments, mouse renal tubular epithelial cells (mRTECs) and miR-140-5p mimic/inhibitor transfected mRTECs were subjected to hypoxia/reoxygenation in the presence or absence of apigenin. In vitro, we uncovered that hypoxia/reoxygenation stimulation caused inflammatory injury in mRTECs. Apigenin reduced the hypoxia/reoxygenation-induced cell inflammatory injury and NF- B p65 nuclear translocation from cytoplasm and activation. Moreover, apigenin reduced hypoxia/reoxygenationtriggered miR-140-5p down-regulation. What's more, the luciferase reporter system revealed that miR-140-5p negatively regulates CXCL12, which is its direct target of action. CXCL12 exhibited an inhibitory effect on the apigenin-induced inactivation of NF- B signaling pathway. Furthermore, we observed that apigenin pretreatment attenuated the IR-triggered up-regulation of serum creatinine and blood urea nitrogen, elevation of pro-inflammatory cytokines secretion and tubular cell apoptosis, enhancement of CXCL12 and decline of miR-140-5p in vivo. Our studies show that apigenin protects against IR-triggered renal cell inflammatory injury in vivo and in vitro by miR-140-5p up-regulation and CXCL12 downregulation via quenching the NF- B pathway activation. Apigenin may be an encouraging therapeutic agent for patients with IR-associated kidney injury.

scholarly journals Regulation of the cfos serum response element by C/EBPbeta.

1997 ◽  
Vol 17 (3) ◽  
pp. 1744-1755 ◽  
Author(s):  
L Sealy ◽  
D Malone ◽  
M Pawlak
Keyword(s):  
Signaling Pathway ◽  
Leucine Zipper ◽  
Site Directed Mutagenesis ◽  
3T3 Cells ◽  
Serum Response Element ◽  
Response Element ◽  
Nih 3T3 ◽  
Serum Response ◽  
Nih 3T3 Cells

Serum response element binding protein (SRE BP) is a novel binding factor present in nuclear extracts of avian and NIH 3T3 fibroblasts which specifically binds to the cfos SRE within a region overlapping and immediately 3' to the CArG box. Site-directed mutagenesis combined with transfection experiments in NIH 3T3 cells showed that binding of both serum response factor (SRF) and SRE BP is necessary for maximal serum induction of the SRE. In this study, we have combined size fractionation of the SRE BP DNA binding activity with C/EBPbeta antibodies to demonstrate that homodimers and heterodimers of p35C/EBPbeta (a transactivator) and p20C/EBPbeta (a repressor) contribute to the SRE BP complex in NIH 3T3 cells. Transactivation of the SRE by p35C/EBPbeta is dependent on SRF binding but not ternary complex factor (TCF) formation. Both p35C/EBPbeta and p20C/EBPbeta bind to SRF in vitro via a carboxy-terminal domain that probably does not include the leucine zipper. Moreover, SRE mutants which retain responsiveness to the TCF-independent signaling pathway bind SRE BP in vitro with affinities that are nearly identical to that of the wild-type SRE, whereas mutant SRE.M, which is not responsive to the TCF-independent pathway, has a nearly 10-fold lower affinity for SRE BP. We propose that C/EBPbeta may play a role in conjunction with SRF in the TCF-independent signaling pathway for SRE activation.

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