scholarly journals Disruption of the actin cytoskeleton regulates cytokine-induced iNOS expression

2001 ◽  
Vol 281 (3) ◽  
pp. C932-C940 ◽  
Author(s):  
Chenbo Zeng ◽  
Aubrey R. Morrison
Keyword(s):  
Nitric Oxide ◽  
Serum Response Factor ◽  
Mesangial Cells ◽  
Inos Expression ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells

Interleukin-1β (IL-1β) induces the inducible nitric oxide synthase (iNOS), resulting in the release of nitric oxide (NO) from glomerular mesangial cells. In this study, we demonstrated that disruption of F-actin formation by sequestration of G-actin with the toxin latrunculin B (LatB) dramatically potentiated IL-1β-induced iNOS protein expression in a dose-dependent manner. LatB by itself had little or no effect on iNOS expression. Staining of F-actin with nitrobenzoxadiazole (NBD)-phallacidin demonstrated that LatB significantly impaired F-actin stress fiber formation. Jasplakinolide (Jasp), which binds to and stabilizes F-actin, suppressed iNOS expression enhanced by LatB. These data strongly suggest that actin cytoskeletal dynamics regulates IL-1β-induced iNOS expression. We demonstrated that LatB decreases serum response factor (SRF) activity as determined by reporter gene assays, whereas Jasp increases SRF activity. The negative correlation between SRF activity and iNOS expression suggests a negative regulatory role for SRF in iNOS expression. Overexpression of a dominant negative mutant of SRF increases the IL-1β-induced iNOS expression, providing direct evidence that SRF inhibits iNOS expression.

Role of Rho and myosin phosphorylation in actin stress fiber assembly in mesangial cells

1997 ◽  
Vol 273 (2) ◽  
pp. F283-F288 ◽  
Author(s):  
J. I. Kreisberg ◽  
N. Ghosh-Choudhury ◽  
R. A. Radnik ◽  
M. A. Schwartz
Keyword(s):  
Cell Shape ◽  
Mesangial Cells ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Glomerular Mesangial Cells ◽  
Fiber Assembly ◽  
Camp Elevation ◽  
Actin Stress Fibers

Treatment of renal glomerular mesangial cells with adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents induces actin stress fiber disassembly, myosin light chain (MLC) dephosphorylation, loss of adhesion to the substratum and cell shape change [J. I. Kreisberg and M. A. Venkatachalam. Am. J. Physiol. 251 (Cell Physiol. 20): C505-C511, 1986]. Thrombin and vasopressin block the effects of cAMP. Because these agents are known to promote stress fiber formation via the small GTP-binding protein Rho, we investigated the effect of an activated variant of Rho on the response to cAMP elevation. Microinjecting V14-Rho completely blocked the effect of cAMP elevation on cell shape and the actin cytoskeleton, whereas inactivating Rho with botulinum C3 exoenzyme induced stress fiber disruption and cell retraction that was indistinguishable from that caused by elevations in intracellular levels of cAMP. Disruption of actin stress fibers by cAMP has previously been ascribed to MLC dephosphorylation; however, both C3 and cytochalasin D also caused dephosphorylation of MLC, whereas blocking MLC dephosphorylation failed to block the cAMP-induced loss of actin stress fibers. We conclude that Rho can modulate the effects of cAMP elevation and suggest that MLC dephosphorylation may be a consequence of actin stress fiber disassembly.

scholarly journals The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export

2002 ◽  
Vol 158 (2) ◽  
pp. 201-208 ◽  
Author(s):  
Igor Prudovsky ◽  
Cinzia Bagala ◽  
Francesca Tarantini ◽  
Anna Mandinova ◽  
Raffaella Soldi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Actin Stress Fiber ◽  
Fibroblast Growth ◽  
Real Time Analysis ◽  
Inner Surface

The release of signal peptideless proteins occurs through nonclassical export pathways and the release of fibroblast growth factor (FGF)1 in response to cellular stress is well documented. Although biochemical evidence suggests that the formation of a multiprotein complex containing S100A13 and Synaptotagmin (Syt)1 is important for the release of FGF1, it is unclear where this intracellular complex is assembled. As a result, we employed real-time analysis using confocal fluorescence microscopy to study the spatio-temporal aspects of this nonclassical export pathway and demonstrate that heat shock stimulates the redistribution of FGF1 from a diffuse cytosolic pattern to a locale near the inner surface of the plasma membrane where it colocalized with S100A13 and Syt1. In addition, coexpression of dominant-negative mutant forms of S100A13 and Syt1, which both repress the release of FGF1, failed to inhibit the stress-induced peripheral redistribution of intracellular FGF1. However, amlexanox, a compound that is known to attenuate actin stress fiber formation and FGF1 release, was able to repress this process. These data suggest that the assembly of the intracellular complex involved in the release of FGF1 occurs near the inner surface of the plasma membrane and is dependent on the F-actin cytoskeleton.

Nitric oxide synthesis in endothelial cells: evidence for a pathway inducible by TNF-alpha

1991 ◽  
Vol 261 (4) ◽  
pp. C634-C641 ◽  
Author(s):  
S. Lamas ◽  
T. Michel ◽  
B. M. Brenner ◽  
P. A. Marsden
Keyword(s):  
Nitric Oxide ◽  
Septic Shock ◽  
Endothelial Cells ◽  
Soluble Guanylate Cyclase ◽  
Mesangial Cells ◽  
Lung Fibroblasts ◽  
Tnf Alpha ◽  
Prolonged Release ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells

Nitric oxide (NO) release accounts for the biological activity of endothelium-derived relaxing factor. Given that tumor necrosis factor-alpha (TNF-alpha) has been implicated as an important mediator in septic shock, we explored whether TNF-alpha enhances L-arginine-dependent synthesis of NO and L-citrulline in endothelial cells. The release of NO was detected in a coincubation bioassay where measurement of guanosine 3',5'-cyclic monophosphate (cGMP) production in reporter monolayers, namely glomerular mesangial cells or fetal lung fibroblasts, reflected activation of soluble guanylate cyclase. Reporter monolayer cGMP content was greater in the presence of TNF-alpha-treated bovine aortic and renal artery endothelial cells than in the presence of vehicle-treated endothelial cells. TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were first evident at 8 h and maximal at 16-24 h. In addition, TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were abrogated by hemoglobin and methylene blue, blunted by N omega-nitro-L-arginine and augmented by superoxide dismutase and the calcium agonist bradykinin. These observations suggested that TNF-alpha enhanced release of NO. Furthermore, the formation of L-[14C]citrulline from L-[14C]arginine, as determined by quantitative cation-exchange chromatography and thin-layer chromatography, was enhanced by TNF-alpha in a time- and concentration-dependent manner. Thus it is evident that endothelial cells release NO for a prolonged period in response to TNF-alpha and transiently when stimulated with calcium agonists. The prolonged release of NO from TNF-alpha-stimulated endothelial cells may be implicated in the pathogenesis of septic shock.

AVP inhibits LPS- and IL-1β-stimulated NO and cGMP via V1 receptor in cultured rat mesangial cells

1999 ◽  
Vol 276 (3) ◽  
pp. F433-F441 ◽  
Author(s):  
Tetsuo Umino ◽  
Eiji Kusano ◽  
Shigeaki Muto ◽  
Tetsu Akimoto ◽  
Satoru Yanagiba ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mesangial Cells ◽  
Nuclear Factor Κb ◽  
Inos Mrna ◽  
No Production ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells ◽  
Inos Protein ◽  
Nitrite Production ◽  
Inhibitor Of Protein Synthesis

The present study examined how arginine vasopressin (AVP) affects nitric oxide (NO) metabolism in cultured rat glomerular mesangial cells (GMC). GMC were incubated with test agents and nitrite, and intracellular cGMP content, inducible nitric oxide synthase (iNOS) mRNA, and iNOS protein were analyzed by the Griess method, enzyme immunoassay, and Northern and Western blotting, respectively. AVP inhibited lipopolysaccharide (LPS)- and interleukin-1β (IL-1β)-induced nitrite production in a dose- and time-dependent manner, with concomitant changes in cGMP content, iNOS mRNA, and iNOS protein. This inhibition by AVP was reversed by V1- but not by oxytocin-receptor antagonist. Inhibition by AVP was also reproduced on LPS and interferon-γ (IFN-γ). Protein kinase C (PKC) inhibitors reversed AVP inhibition, whereas PKC activator inhibited nitrite production. Although dexamethasone and pyrrolidinedithiocarbamate (PDTC), inhibitors of nuclear factor-κB, inhibited nitrite production, further inhibition by AVP was not observed. AVP did not show further inhibition of nitrite production with actinomycin D, an inhibitor of transcription, or cycloheximide, an inhibitor of protein synthesis. In conclusion, AVP inhibits LPS- and IL-1β-induced NO production through a V1 receptor. The inhibitory action of AVP involves both the activation of PKC and the transcription of iNOS mRNA in cultured rat GMC.

Antioxidant PDTC induces stromelysin expression in mesangial cells via a tyrosine kinase-AP-1 pathway

1996 ◽  
Vol 270 (5) ◽  
pp. F806-F811 ◽  
Author(s):  
T. Yokoo ◽  
M. Kitamura
Keyword(s):  
Tyrosine Kinase ◽  
Blot Analysis ◽  
Tyrosine Kinases ◽  
Mesangial Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Pyrrolidine Dithiocarbamate ◽  
Glomerular Mesangial Cells ◽  
Nf Kappa B

We found that pyrrolidine dithiocarbamate (PDTC) induces the matrix metalloproteinase stromelysin in cultured glomerular mesangial cells. Although PDTC is a well-known inhibitor of nuclear factor-kappa B (NF-kappa B), this effect was independent of the NF-kappa B activity, since overexpression of a dominant negative mutant of p50 NF-kappa B subunit repressed activity of the kappa B site, whereas it failed to induce stromelysin. To elucidate the intracellular mechanisms involved, we focused on the role of activator protein 1 (AP-1), since its binding site, the 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TRE), is located in the 5'-flanking region of the stromelysin gene. Northern blot analysis revealed that PDTC upregulated expression of c-jun and c-fos before the expression of stromelysin. Transient transfection studies using a TRE-LacZ reporter plasmid elucidated that activity of AP-1 was significantly increased by PDTC. Stable transfection with a c-jun antisense cDNA or pretreatment with curcumin, a pharmacological inhibitor of c-Jun/AP-1, revealed that inactivation of AP-1 diminished the induction of stromelysin by PDTC. To identify the machinery involved upstream of AP-1 activation, the role of tyrosine kinases was investigated. Western blot analysis showed that PDTC induced phosphorylation of tyrosine kinases. Treatment of mesangial cells with tyrosine kinase inhibitors suppressed activation of AP-1 as well as induction of stromelysin by PDTC. These findings demonstrate that the antioxidant PDTC induces stromelysin expression via stimulation of the tyrosine kinase-AP-1 pathway independent of its suppressive action on NF-kappa B.

Platelet-activating factor and the kidney

1986 ◽  
Vol 251 (1) ◽  
pp. F1-F11 ◽  
Author(s):  
D. Schlondorff ◽  
R. Neuwirth
Keyword(s):  
De Novo ◽  
Mesangial Cells ◽  
Biological Activities ◽  
Platelet Activating Factor ◽  
Calcium Ionophore ◽  
Initial Step ◽  
Renal Physiology ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells ◽  
Isolated Kidney

Platelet-activating factor (PAF) represents a group of phospholipids with the basic structure of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine. A number of different cells are capable of producing PAF in response to various stimuli. The initial step of PAF formation is activation of phospholipase A2 in a calcium-dependent manner, yielding lyso-PAF. During this step arachidonic acid is also released and can be converted to its respective cyclooxygenase and lipoxygenase products. The lyso-PAF generated is then acetylated in position 2 of the glycerol backbone by a coenzyme A (CoA)-dependent acetyltransferase. An additional pathway may exist whereby PAF is generated de novo from 1-alkyl-2-acetyl-sn-glycerol by phosphocholine transferase. PAF inactivation in cells and blood is by specific acetylhydrolases. PAF exhibits a variety of biological activities including platelet and leukocyte aggregation and activation, increased vascular permeability, respiratory distress, decreased cardiac output, and hypotension. In the kidney PAF can produce decreases in blood flow, glomerular filtration, and fluid and electrolyte excretion. Intrarenal artery injection of PAF may also result in glomerular accumulation of platelets and leukocytes and mild proteinuria. PAF increases prostaglandin formation in the isolated kidney and in cultured glomerular mesangial cells. PAF also causes contraction of mesangial cells. Upon stimulation with calcium ionophore the isolated kidney, isolated glomeruli and medullary cells, and cultured mesangial cells are capable of producing PAF. The potential role for PAF in renal physiology and pathophysiology requires further investigation that may be complicated by 1) the multiple interactions of PAF, prostaglandins, and leukotrienes and 2) the autocoid nature of PAF, which may restrict its action to its site of generation.

scholarly journals Activation of Nuclear Factor κB and Induction of Inducible Nitric Oxide Synthase by Ureaplasma urealyticumin Macrophages

2000 ◽  
Vol 68 (12) ◽  
pp. 7087-7093 ◽  
Author(s):  
Y.-H. Li ◽  
Z.-Q. Yan ◽  
J. Skov Jensen ◽  
K. Tullus ◽  
A. Brauner
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Alveolar Macrophages ◽  
Nuclear Factor Κb ◽  
Inos Expression ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACT Chronic lung disease (CLD) of prematurity is an inflammatory disease with a multifactorial etiology. The importance ofUreaplasma urealyticum in the development of CLD is debated, and steroids produce some improvement in neonates with this disease. In the present study, the capability of U. urealyticum to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS), and activate nuclear factor κB (NF-κB) in vitro was characterized. The effect of NO on the growth of U. urealyticum was also investigated. In addition, the impact of dexamethasone and budesonide on these processes was examined. We found that U. urealyticum antigen (≥4 × 107 color-changing units/ml) stimulated alveolar macrophages to produce NO in a dose- and time-dependent manner (P < 0.05). This effect was further enhanced by gamma interferon (100 IU/ml; P < 0.05) but was attenuated by budesonide and dexamethasone (10−4 to 10−6 M) (P < 0.05). The mRNA and protein levels of iNOS were also induced in response to U. urealyticum and inhibited by steroids.U. urealyticum antigen triggered NF-κB activation, a possible mechanism for the induced iNOS expression, which also was inhibited by steroids. NO induced by U. urealyticum caused a sixfold reduction of its own growth after infection for 10 h. Our findings imply that U. urealyticum may be an important factor in the development of CLD. The host defense response againstU. urealyticum infection may also be influenced by NO. The down-regulatory effect of steroids on NF-κB activation, iNOS expression, and NO production might partly explain the beneficial effect of steroids in neonates with CLD.

scholarly journals Tumor necrosis factor alpha activates soluble guanylate cyclase in bovine glomerular mesangial cells via an L-arginine-dependent mechanism.

1990 ◽  
Vol 172 (6) ◽  
pp. 1843-1852 ◽  
Author(s):  
P A Marsden ◽  
B J Ballermann
Keyword(s):  
Guanylate Cyclase ◽  
Mesangial Cell ◽  
Soluble Guanylate Cyclase ◽  
Mesangial Cells ◽  
Tnf Alpha ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells ◽  
Concentration Dependent Manner ◽  
Necrosis Factor Alpha ◽  
Factor Alpha

Endothelium-derived nitric oxide (NO) causes vasodilatation by activating soluble guanylate cyclase, and glomerular mesangial cells respond to NO with elevations of intracellular guanosine 3',5'-cyclic monophosphate (cGMP). We explored whether mesangial cells can be stimulated to produce NO and whether NO modulates mesangial cell function in an autocrine or paracrine fashion. Tumor necrosis factor alpha (TNF-alpha) raised mesangial cell cGMP levels in a time- and concentration-dependent manner (threshold dose 1 ng/ml, IC50 13.8 ng/ml, maximal response 100 ng/ml). TNF-alpha-induced increases in mesangial cGMP content were evident at 8 h and maximal at 18-24 h. The TNF-alpha-induced stimulation of mesangial cell cGMP production was abrogated by actinomycin D or cycloheximide suggesting dependence on new RNA or protein synthesis. Hemoglobin and methylene blue, both known to inhibit NO action, dramatically reduced TNF-alpha-induced mesangial cell cGMP production. Superoxide dismutase, known to potentiate NO action, augmented the TNF-alpha-induced effect. Ng-monomethyl-L-arginine (L-NMMA) decreased cGMP levels in TNF-alpha-treated, but not vehicle-treated mesangial cells in a concentration-dependent manner (IC50 53 microM). L-arginine had no effect on cGMP levels in control or TNF-alpha-treated mesangial cells but reversed L-NMMA-induced inhibition. Interleukin 1 beta and lipopolysaccharide (LPS), but not interferon gamma, also increased mesangial cell cGMP content. Transforming growth factor beta 1 blunted the mesangial cell response to TNF-alpha. TNF-alpha-induced L-arginine-dependent increases in cGMP were also evident in bovine renal artery vascular smooth muscle cells, COS-1 cells, and 1502 human fibroblasts. These findings suggest that TNF-alpha induces expression in mesangial cell of an enzyme(s) involved in the formation of L-arginine-derived NO. Moreover, the data indicate that NO acts in an autocrine and paracrine fashion to activate mesangial cell soluble guanylate cyclase. Cytokine-induced formation of NO in mesangial and vascular smooth muscle cells may be implicated in the pathogenesis of septic shock.

scholarly journals High Glucose and Lipopolysaccharide Prime NLRP3 Inflammasome via ROS/TXNIP Pathway in Mesangial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Hong Feng ◽  
Junling Gu ◽  
Fang Gou ◽  
Wei Huang ◽  
Chenlin Gao ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Nlrp3 Inflammasome ◽  
High Glucose ◽  
Mesangial Cells ◽  
Central Component ◽  
Dependent Manner ◽  
Glomerular Mesangial Cells ◽  
Interacting Protein

While inflammation is considered a central component in the development in diabetic nephropathy, the mechanism remains unclear. The NLRP3 inflammasome acts as both a sensor and a regulator of the inflammatory response. The NLRP3 inflammasome responds to exogenous and endogenous danger signals, resulting in cleavage of procaspase-1 and activation of cytokines IL-1β, IL-18, and IL-33, ultimately triggering an inflammatory cascade reaction. This study observed the expression of NLRP3 inflammasome signaling stimulated by high glucose, lipopolysaccharide, and reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine in glomerular mesangial cells, aiming to elucidate the mechanism by which the NLRP3 inflammasome signaling pathway may contribute to diabetic nephropathy. We found that the expression of thioredoxin-interacting protein (TXNIP), NLRP3, and IL-1βwas observed by immunohistochemistry in vivo. Simultaneously, the mRNA and protein levels of TXNIP, NLRP3, procaspase-1, and IL-1βwere significantly induced by high glucose concentration and lipopolysaccharide in a dose-dependent and time-dependent manner in vitro. This induction by both high glucose and lipopolysaccharide was significantly inhibited by N-acetyl-L-cysteine. Our results firstly reveal that high glucose and lipopolysaccharide activate ROS/TXNIP/ NLRP3/IL-1βinflammasome signaling in glomerular mesangial cells, suggesting a mechanism by which inflammation may contribute to the development of diabetic nephropathy.

Phosphatidylinositol 3-kinase mediates integrin-dependent NF-(κ)B and MAPK activation through separate signaling pathways

2001 ◽  
Vol 114 (8) ◽  
pp. 1579-1589 ◽  
Author(s):  
M. Reyes-Reyes ◽  
N. Mora ◽  
A. Zentella ◽  
C. Rosales
Keyword(s):  
Signaling Pathways ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Monocytic Leukemia ◽  
Monocytic Cells ◽  
Mapk Activation

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, (β)1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via (β)1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor (κ)B (NF-(κ)B) activation was then analyzed. We found that integrins activated both NF-(κ)B and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-(κ)B activation. In contrast, a dominant negative mutant of Rac completely prevented NF-(κ)B activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-(κ)B is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Ρ augmented NF-(κ)B activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-(κ)B, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.

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