TNF‑α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR‑335‑5p

Allergic rhinitis (AR) is a nasal mucosal inflammatory disease mediated by environmental allergens. At present, the relationship between the IL-33/ST2 axis, ERK1/2 pathway and AR progression needs further exploration. In our study, an AR model was constructed in vitro by treating HNEpC cells with Der p1. qRT-PCR was applied to assess the mRNA levels of IL-33, ST2, TNF-α, IL-6, and IL-8. Western blotting was used to measure the protein levels of IL-33, ST2, and the downstream proteins p-ERK1/2, ERK1/2, p-RSK, and RSK. IL-6, IL-8, IL-33, and TNF-α protein levels in cell supernatants were evaluated by ELISA. Flow cytometry was performed to check cell apoptosis of HNEpC in the presence or absence of Der p1. Our results indicate that the relative levels of IL-33, ST2, TNF-α, IL-6, and IL-8 were increased significantly in the AR model group. The above effects were notably reversed after transfection with shIL-33 or shST2. IL-33 stimulation further resulted in the increase in both ST2 and inflammation-associated cytokines, and these effects were restored after shST2 treatment. Also, the levels of inflammatory factors induced by IL-33 stimulation or ST2 overexpression were reversed after applying an ERK1/2 pathway blocker. In conclusion, IL-33/ST2 mediated inflammation of nasal mucosal epithelial cells by inducing the ERK1/2 pathway.

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“Subclinical” gentamicin nephrotoxicity: a potential risk factor for exaggerated endotoxin-driven TNF-α production

AJP Renal Physiology ◽

10.1152/ajprenal.00144.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. F43-F49 ◽

Cited By ~ 6

Author(s):

Richard A. Zager

Keyword(s):

Antimicrobial Effect ◽

Specific Response ◽

Mrna Levels ◽

Gram Negative ◽

Protein Levels ◽

Target Organs ◽

Gentamicin Treatment ◽

Tnf Α ◽

Oxide Synthase ◽

And Control

This study sought to determine whether gentamicin, a mainstay in treating Gram-negative sepsis, alters endotoxin (lipopolysaccharide; LPS)-driven TNF-α increases. CD-1 mice received 1 day of gentamicin treatment. Either 0, 24, or 72 h later, gentamicin-treated and control mice were injected with LPS. Renal cortical and plasma TNF-α, as well as MCP-1, protein levels were measured 2 or 24 h post-LPS injection. Renal cortical mRNAs for TNF-α, MCP-1, IL-10, and inducible nitric oxide synthase (iNOS) were also determined. Finally, gentamicin's potential impact(s) on TNF-α/MCP-1 mRNA levels in nontraditional “target” organs (liver, spleen) was assessed. Gentamicin, when administered alone, slightly increased renal cortical TNF-α and MCP-1 mRNAs, but without changing plasma or renal TNF-α/MCP-1 protein levels. The gentamicin protocol induced no overt renal damage (assessed by blood urea nitrogen, creatinine, and histology). Nevertheless, gentamicin augmented LPS responsiveness, as manifested, in part, by a doubling of LPS-induced plasma TNF-α increases (vs. LPS injection alone). Plasma and renal cortical MCP-1 protein levels were also selectively enhanced. Gentamicin augmented LPS-driven renal mRNA increases (TNF-α, MCP-1, IL-10, iNOS). However, this was not an entirely renal-specific response, since gentamicin also enhanced basal and LPS-stimulated hepatic TNF-α mRNA levels. Subclinical gentamicin toxicity can potentiate LPS-driven TNF-α increases. Alterations in multiple proinflammatory (TNF-α; MCP-1; iNOS) and anti-inflammatory (IL-10) genes in the kidney, and possibly in extrarenal organs, may be involved. Thus gentamicin's activity in Gram-negative sepsis may extend beyond its traditional antimicrobial effect.

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Significance of changes in TNF-α and IL-10 levels in the progression of heart failure subsequent to myocardial infarction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01327.2005 ◽

2006 ◽

Vol 291 (1) ◽

pp. H106-H113 ◽

Cited By ~ 74

Author(s):

K. Kaur ◽

A. K. Sharma ◽

P. K. Singal

Keyword(s):

Myocardial Infarction ◽

Heart Failure ◽

Coronary Artery ◽

Cardiac Function ◽

Interleukin 10 ◽

Mrna Levels ◽

Protein Ratio ◽

Protein Levels ◽

Tnf Α ◽

Membrane Bound

We tested whether a decrease in the ratio of interleukin-10 (IL-10) to tumor necrosis factor-α (TNF-α) correlates with the decrease in cardiac function in heart failure. It has been suggested that TNF-α plays a role in the progression of heart failure, and the effect of TNF-α in many tissues is modulated by IL-10. Any relation of these two cytokines to heart failure has never been examined. Cardiac function was assessed by echocardiographic and hemodynamic techniques in coronary artery-ligated rats at 1, 4, 8, and 16 wk after myocardial infarction (MI). Membrane-bound and soluble fractions of TNF-α and IL-10 proteins, the ratio of TNF-α to IL-10, and TNF-α and IL-10 mRNA levels were analyzed. Losartan was used to modify cardiac function in rats 4 wk after MI to further validate the relation between the IL-10-to-TNF-α ratio and cardiac function. Cardiac function deteriorated with time in all coronary artery-ligated groups, with severe failure at 16 wk after MI. Membrane-bound and soluble TNF-α protein fractions were increased 1 and 4 wk after MI, whereas TNF -α mRNA was increased 4 and 8 wk after MI. Membrane-bound IL-10 protein and mRNA levels were decreased 4, 8, and 16 wk after MI. The decrease in the IL-10-to-TNF-α protein ratio in all coronary artery-ligated groups correlated with the depressed cardiac function. Losartan improved cardiac function, membrane-bound and soluble TNF-α and IL-10 protein levels, the ratio of IL-10 to TNF-α, and IL-10 mRNA. This study suggests that a decrease in IL-10 and IL-10-to-TNF-α ratio correlates with depressed cardiac function.

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GRIM-19, a gene associated with retinoid-interferon-induced mortality, affects endometrial receptivity and embryo implantation

Reproduction Fertility and Development ◽

10.1071/rd16104 ◽

2017 ◽

Vol 29 (7) ◽

pp. 1447 ◽

Cited By ~ 2

Author(s):

Yang Yang ◽

Yanyan Sun ◽

Laiyang Cheng ◽

Anna Li ◽

Yanjun Shen ◽

...

Keyword(s):

Immune Tolerance ◽

Embryo Implantation ◽

Pregnant Mouse ◽

Endometrial Receptivity ◽

Mrna Levels ◽

Protein Levels ◽

Tnf Α ◽

Pregnant Mice ◽

Adhesion Rate

GRIM-19 is associated with apoptosis, abnormal proliferation, immune tolerance and malignant transformation, and it also plays an important role in early embryonic development. Although the homologous deletion of GRIM-19 causes embryonic lethality in mice, the precise role of GRIM-19 in embryo implantation has not been elucidated. Here we show that GRIM-19 plays an important role in endometrial receptivity and embryo implantation. Day 1 to Day 6 pregnant mouse uteri were collected. Immunohistochemistry studies revealed the presence of GRIM-19 on the luminal epithelium and glandular epithelium throughout the implantation period in pregnant mice. The protein and mRNA levels of GRIM-19 were markedly decreased on Day 4 of pregnancy in pregnant mice, but there was no change in GRIM-19 levels in a group of pseudopregnant mice. Overexpression of GRIM-19 decreased the adhesion rate of RL95–2–BeWo co-cultured spheroids and increased apoptosis. Furthermore, STAT3 and IL-11 mRNA and protein levels were reduced by overexpressing GRIM-19, but protein and mRNA levels of TNF-α were increased. These findings indicate the involvement of GRIM-19 in the embryo implantation process by regulating adhesion, apoptosis and immune tolerance.

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Role of the TNF pathway in the progression of diabetic nephropathy in KK-Ay mice

AJP Renal Physiology ◽

10.1152/ajprenal.00509.2013 ◽

2014 ◽

Vol 306 (11) ◽

pp. F1335-F1347 ◽

Cited By ~ 32

Author(s):

Keisuke Omote ◽

Tomohito Gohda ◽

Maki Murakoshi ◽

Yu Sasaki ◽

Saiko Kazuno ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Early Stage ◽

Mrna Levels ◽

Monocyte Chemoattractant Protein 1 ◽

Protein Levels ◽

Tnf Α ◽

Soluble Tnf Receptor

Chronic inflammation promotes the progression of diabetic nephropathy (DN). However, the role of TNF-α remains unclear. The objectives of the present study were to examine whether TNF-α inhibition with a soluble TNF receptor (TNFR)2 fusion protein, i.e., etanercept (ETN), improves the early stage of DN in the type 2 diabetic model of the KK-Ay mouse and to also investigate which TNF pathway, TNFR1 or TNFR2, is predominantly involved in the progression of this disease. ETN was injected intraperitoneally into mice for 8 wk. Renal damage was evaluated by immunohistochemistry, Western blot analysis, and/or real-time PCR. In vitro, mouse tubular proximal cells were stimulated by TNF-α and/or high glucose (HG) and treated with ETN. ETN dramatically improved not only albuminuria but also glycemic control. Renal mRNA and/or protein levels of TNFR2, but not TNF-α and TNFR1, in ETN-treated KK-Ay mice were significantly decreased compared with untreated KK-Ay mice. mRNA levels of ICAM-1, VCAM-1, and monocyte chemoattractant protein-1 and the number of F4/80-positive cells were all decreased after treatment. Numbers of cleaved caspase-3- and TUNEL-positive cells in untreated mice were very few and were not different from ETN-treated mice. In vitro, stimulation with TNF-α or HG markedly increased both mRNA levels of TNFRs, unlike in the in vivo case. Furthermore, ETN partly recovered TNF-α-induced but not HG-induced TNFR mRNA levels. In conclusion, it appears that ETN may improve the progression of the early stage of DN predominantly through inhibition of the anti-inflammatory action of the TNF-α-TNFR2 pathway.

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Pitfalls in AR42J-model of cerulein-induced acute pancreatitis

PLoS ONE ◽

10.1371/journal.pone.0242706 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0242706

Author(s):

Marcus Hollenbach ◽

Sebastian Sonnenberg ◽

Ines Sommerer ◽

Jana Lorenz ◽

Albrecht Hoffmeister

Keyword(s):

Acute Pancreatitis ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Mrna Levels ◽

Amylase Release ◽

Protein Levels ◽

Qrt Pcr ◽

Transfection Efficacy ◽

Tnf Α ◽

Amylase Production

Background AR42J are immortalized pancreatic adenocarcinoma cells that share similarities with pancreatic acinar cells. AR42J are often used as a cell-culture model of cerulein (CN)-induced acute pancreatitis (AP). Nevertheless, it is controversial how to treat AR42J for reliable induction of AP-like processes. Gene knockout and/or overexpression often remain challenging, as well. In this study, we demonstrate conditions for a reliable induction of proinflammatory markers upon CN treatment in AR42J and high transfection efficacy using Glyoxalase-I (Glo-I) as a target of interest. Methods Effects of dexamethasone (dexa) and CN on cell morphology and amylase secretion were analyzed via ELISA of supernatant. IL-6, TNF-α and NF-κB-p65 were measured via qRT-PCR, ELISA and Western Blot (WB). Transfection efficacy was determined by WB, qRT-PCR and immune fluorescence of pEGFP-N1-Glo-I-Vector and Glo-I-siRNA. Results Treatment of AR42J with 100 nm dexa is mandatory for differentiation to an acinar-cell-like phenotype and amylase production. CN resulted in secretion of amylase but did not influence amylase production. High levels of CN-induced amylase secretion were detected between 3 and 24 hours of incubation. Treatment with LPS alone or in combination with CN did not influence amylase release compared to control or CN. CN treatment resulted in increased TNF-α production but not secretion and did not influence IL-6 mRNA. CN-induced stimulation of NF-κB was found to be highest on protein levels after 6h of incubation. Transient transfection was able to induce overexpression on protein and mRNA levels, with highest effect after 12 to 24 hours. Gene-knockdown was achieved by using 30 pmol of siRNA leading to effective reduction of protein levels after 72 hours. CN did not induce amylase secretion in AR42J cell passages beyond 35. Conclusion AR42J cells demonstrate a reliable in-vitro model of CN-induced AP but specific conditions are mandatory to obtain reproducible data.

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Soluble guanylyl cyclase expression is reduced in LPS-induced lung injury

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00341.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. R1448-R1455 ◽

Cited By ~ 11

Author(s):

Constantinos Glynos ◽

Anastasia Kotanidou ◽

Stylianos E. Orfanos ◽

Zongmin Zhou ◽

Davina C. M. Simoes ◽

...

Keyword(s):

Smooth Muscle ◽

Lung Injury ◽

Guanylyl Cyclase ◽

Muscle Tone ◽

Soluble Guanylyl Cyclase ◽

Mrna Levels ◽

Subunit Protein ◽

Protein Levels ◽

Lps Challenge ◽

Tnf Α

Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme implicated in the control of smooth muscle tone that also regulates platelet aggregation. Moreover, sGC activation has been shown to reduce leukocyte adherence to the endothelium. Herein, we investigated the expression of sGC in a murine model of LPS-induced lung injury and evaluated the effects of sGC inhibition in the context of acute lung injury (ALI). Lung tissue sGC α1 and β1 subunit protein levels were determined by Western blot and immunohistochemistry, and steady-state mRNA levels for the β1 subunit were assessed by real-time PCR. LPS inhalation resulted in a decrease in β1 mRNA levels, as well as a reduction in both sGC subunit protein levels. Decreased α1 and β1 expression was observed in bronchial smooth muscle and epithelial cells. TNF-α was required for the LPS-triggered reduction in sGC protein levels, as no change in α1 and β1 levels was observed in TNF-α knockout mice. To determine the effects of sGC blockade in LPS-induced lung injury, mice were exposed to 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-l-one (ODQ) prior to the LPS challenge. Such pretreatment led to a further increase in total cell number (mainly due to an increase in neutrophils) and protein concentration in the bronchoalveoalar lavage fluid; the effects of ODQ were reversed by a cell-permeable cGMP analog. We conclude that sGC expression is reduced in LPS-induced lung injury, while inhibition of the enzyme with ODQ worsens lung inflammation, suggesting that sGC exerts a protective role in ALI.

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Activation of ubiquitin-proteasome pathway is involved in skeletal muscle wasting in a rat model with biliary cirrhosis: potential role of TNF-α

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00186.2004 ◽

2005 ◽

Vol 288 (3) ◽

pp. E493-E501 ◽

Cited By ~ 35

Author(s):

Shih-Yi Lin ◽

Wen-Yin Chen ◽

Fa-Yauh Lee ◽

Ching-Jang Huang ◽

Wayne Huey-Herng Sheu

Keyword(s):

Muscle Wasting ◽

Ex Vivo ◽

Muscle Protein ◽

Biliary Cirrhosis ◽

Mrna Levels ◽

Male Adult ◽

Protein Levels ◽

Duct Ligation ◽

Tnf Α ◽

Ubiquitin Proteasome

Hepatic cirrhosis is associated with negative nitrogen balance and loss of lean body mass. This study aimed to identify the specific proteolytic pathways activated in skeletal muscles of cirrhotic rats. TNF-α can stimulate muscle proteolysis; therefore, a potential relationship between TNF-α and muscle wasting in liver cirrhosis was also evaluated. Cirrhosis was induced by bile duct ligation (BDL) in male adult Sprague-Dawley rats. mRNA and protein levels of various targets were determined by RT-PCR and Western blotting, respectively. The proteolytic rate was measured ex vivo using isolated muscles. Compared with sham-operated controls, BDL rats had an increased degradation rate of muscle proteins and enhanced gene expression of ubiquitin, 14-kDa ubiquitin carrier protein E2, and the proteasome subunits C2 and C8 ( P < 0.01). The muscle protein levels of free ubiquitin and conjugated ubiquitin levels were also elevated ( P < 0.01). However, there was no difference between the two groups with regard to cathepsin and calpain mRNA levels. Cirrhotic muscle TNF-α levels were increased and correlated positively with free and conjugated ubiquitin ( P < 0.01). We conclude that the ubiquitin-proteasome system is involved in muscle wasting of rats with BDL-induced cirrhosis. TNF-α might play a role in mediating activation of this proteolytic pathway, probably through a local mechanism.

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Carbon Monoxide-Releasing Molecule-Derived CO Regulates Tissue Factor, Plasminogen Activator Inhibitor Type 1, and Thrombomodulin Production by Human Endothelial Cells.

Blood ◽

10.1182/blood.v116.21.1146.1146 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1146-1146

Author(s):

Keiko Maruyama ◽

Eriko Morishita ◽

Shigeki Ohtake ◽

Akihiro Yachie ◽

Shinji Nakao

Keyword(s):

Plasminogen Activator Inhibitor ◽

Mapk Signaling ◽

Mrna Levels ◽

Protein Levels ◽

Tnf Α ◽

Inhibitor Type ◽

Activator Inhibitor Type ◽

Activator Inhibitor ◽

Pai 1

Abstract Abstract 1146 Background and purpose: Heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). HO-1 is rapidly induced by various oxidative stresses and inflammation, thereby playing an important role in the self defence system. Recently, Yachie, et al. reported the first human case of HO-1 deficiency. This patient showed prominent signs of intravascular hemolysis, endothelial cell injury, and abnormalities in the coagulation / fibrinolysis system, suggesting the involvement of HO-1 or HO-1 products, such as CO, regulation of coagulation / fibrinolysis system. The current study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberated CO in the presence of dimethyl sulfoxide (DMSO), modulates the expression of tissue factor (TF), plasminogen activator inhibitor type 1 (PAI-1) and thrombomodulin (TM) in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were pretreated with CORM-2 at the concentration of 50 μM for 3h, washed and stimulated with tumor necrosis factor-α(TNF-α, 10 ng/ml) for additional 2 or 5h. The mRNA and protein levels of TF, PAI-1 and TM in the cultured HUVECs were determined by real-time reverse transcriptase polymerase chain reaction and Western blotting. To determine whether CORM-2 affects the MAPK signaling pathways, the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) in the HUVECs were analyzed with Western blotting. Results: After TNF-α stimulation, TF mRNA levels were approximately 6-fold and PAI-1 mRNA levels were approximately 8-fold increased, and TM mRNA levels were decreased by 20% compared to the control. Similarly to the mRNA expression, TF and PAI-1 protein levels were increased while the TM protein level was decreased. On the other hand, pretreatment of HUVECs with CORM-2 significantly decreased TF mRNA levels (approximately 80% suppression), and PAI-1 mRNA levels (approximately 90% suppression) while increased TM mRNA levels by 3-fold as compared to the TNF-α-stimulated group (p<0.05; Figure 1). Similarly, the pretreatment with CORM-2 inhibited TNF-α-induced TF and PAI-1 protein up-regulation and TM protein down-regulation. CORM-2 inhibited TNF-α-induced activation of p38MAPK and ERK1/2 by 50% compared to the control. Conclusions: These results indicate that CO liberated by CORM-2 suppresses the TNF-α-induced TF and PAI-1 up-regulation and prevents the TNF-α-induced TM down-regulation in HUVECs via MAPK signaling pathways. Thus, the modulation of endothelial function by CO may offer a novel antithrombotic option for treatment of the hypercoagulable state associated with inflammation. Disclosures: No relevant conflicts of interest to declare.

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Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.280.2.l272 ◽

2001 ◽

Vol 280 (2) ◽

pp. L272-L278 ◽

Cited By ~ 44

Author(s):

Masao Takata ◽

Galina Filippov ◽

Heling Liu ◽

Fumito Ichinose ◽

Stefan Janssens ◽

...

Keyword(s):

Smooth Muscle ◽

Pulmonary Artery ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

No Production ◽

Mrna Levels ◽

Protein Levels ◽

Subunit Mrna ◽

Tnf Α ◽

Pulmonary Artery Smooth Muscle

Exposure of rat pulmonary artery smooth muscle cells (rPASMC) to cytokines leads to nitric oxide (NO) production by NO synthase 2 (NOS2). NO stimulates cGMP synthesis by soluble guanylate cyclase (sGC), a heterodimer composed of α1- and β1-subunits. Prolonged exposure of rPASMC to NO decreases sGC subunit mRNA and protein levels. The objective of this study was to determine whether levels of NO produced endogenously by NOS2 are sufficient to decrease sGC expression in rPASMC. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) increased NOS2 mRNA levels and decreased sGC subunit mRNA levels. Exposure of rPASMC to IL-1β and TNF-α for 24 h decreased sGC subunit protein levels and NO-stimulated sGC enzyme activity.l- N 6-(1-iminoethyl)lysine (NOS2 inhibitor) or 1 H-[1,2,4]oxadiazolo-[4,3- a]quinoxalin-1-one (sGC inhibitor) partially prevented the cytokine-mediated decrease in sGC subunit mRNA levels. However, cytokines also decreased sGC subunit mRNA levels in PASMC derived from NOS2-deficient mice. These results demonstrate that levels of NO and cGMP produced in cytokine-exposed PASMC are sufficient to decrease sGC subunit mRNA levels. In addition, cytokines can decrease sGC subunit mRNA levels via NO-independent mechanisms.

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