scholarly journals Ubiquitin-Specific Protease 2 Modulates the Lipopolysaccharide-Elicited Expression of Proinflammatory Cytokines in Macrophage-like HL-60 Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-15 ◽  
Author(s):  
Hiroshi Kitamura ◽  
Takeshi Ishino ◽  
Yoshinori Shimamoto ◽  
Jun Okabe ◽  
Tomomi Miyamoto ◽  
...  
Keyword(s):  
Proinflammatory Cytokines ◽  
Peritoneal Macrophages ◽  
Human Macrophage ◽  
Mrna Levels ◽  
Mrna Accumulation ◽  
Protein Levels ◽  
Protein Ubiquitination ◽  
Specific Protease ◽  
Tlr4 Activation ◽  
Mouse Peritoneal Macrophages

We investigated the regulatory roles of USP2 in mRNA accumulation of proinflammatory cytokines in macrophage-like cells after stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Human macrophage-like HL-60 cells, mouse macrophage-like J774.1 cells, and mouse peritoneal macrophages demonstrated negative feedback to USP2 mRNA levels after LPS stimulation, suggesting that USP2 plays a significant role in LPS-stimulated macrophages.USP2knockdown (KD) by short hairpin RNA in HL-60 cells promoted the accumulation of transcripts for 25 of 104 cytokines after LPS stimulation. In contrast, limited induction of cytokines was observed in cells forcibly expressing the longer splice variant of USP2 (USP2A), or in peritoneal macrophages isolated fromUsp2atransgenic mice. An ubiquitin isopeptidase-deficient USP2A mutant failed to suppress LPS-induced cytokine expression, suggesting that protein ubiquitination contributes to USP2-mediated cytokine repression. Although USP2 deficiency did not accelerate TNF receptor-associated factor (TRAF) 6-nuclear factor-κB (NF-κB) signaling, it increased the DNA binding ratio of the octamer binding transcription factor (Oct)-1 to Oct-2 inTNF,CXCL8,CCL4, andIL6promoters. USP2 decreased nuclear Oct-2 protein levels in addition to decreasing the polyubiquitination of Oct-1. In summary, USP2 modulates proinflammatory cytokine induction, possibly through modification of Oct proteins, in macrophages following TLR4 activation.

Hypoxia enhances lysosomal TNF-α degradation in mouse peritoneal macrophages

2008 ◽  
Vol 295 (1) ◽  
pp. C2-C12 ◽  
Author(s):  
Nitza Lahat ◽  
Michal A. Rahat ◽  
Amalia Kinarty ◽  
Lea Weiss-Cerem ◽  
Sigalit Pinchevski ◽  
...  
Keyword(s):  
Peritoneal Macrophages ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Mrna Levels ◽  
Tnf Α ◽  
Primary Mouse ◽  
Factor Α ◽  
Secretory Lysosomes ◽  
Mouse Peritoneal Macrophages ◽  
Necrosis Factor

Infection, simulated by lipopolysaccharide (LPS), is a potent stimulator of tumor necrosis factor-α (TNF-α) production, and hypoxia often synergizes with LPS to induce higher levels of the secreted cytokine. However, we show that in primary mouse peritoneal macrophages and in three mouse peritoneal macrophage cell lines (RAW 264.7, J774A.1, and PMJ-2R), hypoxia (O2 < 0.3%) reduces the secretion of LPS-induced TNF-α ( P < 0.01). In RAW 264.7 cells this reduction was not regulated transcriptionally as TNF-α mRNA levels remained unchanged. Rather, hypoxia and LPS reduced the intracellular levels of TNF-α by twofold ( P < 0.01) by enhancing its degradation in the lysosomes and inhibiting its secretion via secretory lysosomes, as shown by confocal microscopy and verified by the use of the lysosome inhibitor Bafilomycin A1. In addition, although hypoxia did not change the accumulation of the soluble receptor TNF-RII, it increased its binding to the secreted TNF-α by twofold ( P < 0.05). We suggest that these two posttranslational regulatory checkpoints coexist in hypoxia and may partially explain the reduced secretion and diminished biological activity of TNF-α in hypoxic peritoneal macrophages.

scholarly journals The P-body component USP52/PAN2 is a novel regulator of HIF1A mRNA stability

2013 ◽  
Vol 451 (2) ◽  
pp. 185-194 ◽  
Author(s):  
John S. Bett ◽  
Adel F. M. Ibrahim ◽  
Amit K. Garg ◽  
Van Kelly ◽  
Patrick Pedrioli ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Mrna Stability ◽  
Cellular Response ◽  
Hypoxia Inducible Factor ◽  
Tail Length ◽  
Mrna Levels ◽  
P Bodies ◽  
Protein Levels ◽  
Specific Protease ◽  
Processing Body

HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52 (ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3′-UTR (untranslated region)-dependent poly(A)-tail-length-independent destabilization in HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body (processing body) components and we confirmed further that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.

scholarly journals Analysis of Chemokines and Receptors Expression Profile in the Myelin MutantTaiepRat

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Guadalupe Soto-Rodriguez ◽  
Juan-Antonio Gonzalez-Barrios ◽  
Daniel Martinez-Fong ◽  
Victor-Manuel Blanco-Alvarez ◽  
Jose R. Eguibar ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Expression Profile ◽  
Proinflammatory Cytokines ◽  
Mrna Levels ◽  
Pcr Array ◽  
Sprague Dawley ◽  
Protein Levels ◽  
Chronic Neuroinflammation ◽  
Sprague Dawley Rats ◽  
Old Rats

Taieprat has a failure in myelination and remyelination processes leading to a state of hypomyelination throughout its life. Chemokines, which are known to play a role in inflammation, are also involved in the remyelination process. We aimed to demonstrate that remyelination-stimulating factors are altered in the brainstem of 1- and 6-month-oldtaieprats. We used a Rat RT2Profiler PCR Array to assess mRNA expression of 84 genes coding for cytokines, chemokines, and their receptors. We also evaluated protein levels of CCL2, CCR1, CCR2, CCL5, CCR5, CCR8, CXCL1, CXCR2, CXCR4, FGF2, and VEGFA by ELISA. Sprague-Dawley rats were used as a control. PCR Array procedure showed that proinflammatory cytokines were not upregulated in thetaieprat. In contrast, some mRNA levels of beta and alpha chemokines were upregulated in 1-month-old rats, but CXCR4 was downregulated at their 6 months of age. ELISA results showed that CXCL1, CCL2, CCR2, CCR5, CCR8, and CXCR4 protein levels were decreased in brainstem at the age of 6 months. These results suggest the presence of a chronic neuroinflammation process with deficiency of remyelination-stimulating factors (CXCL1, CXCR2, and CXCR4), which might account for the demyelination in thetaieprat.

Interleukin-10 Suppresses IP-10 Gene Transcription by Inhibiting the Production of Class I Interferon

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4742-4749 ◽  
Author(s):  
Julie M. Tebo ◽  
Hee Sun Kim ◽  
Jing Gao ◽  
David A. Armstrong ◽  
Thomas A. Hamilton
Keyword(s):  
Gene Transcription ◽  
Interleukin 10 ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
Class I ◽  
Cytokine Gene Expression ◽  
Cytokine Gene ◽  
Mrna Accumulation ◽  
Mouse Peritoneal Macrophages ◽  
Gene Expression Levels

Interleukin-10 (IL-10) selectively inhibited lipopolysaccharide (LPS)-induced chemoattractant cytokine gene expression: levels of IP-10 mRNA were markedly suppressed in IL-10–treated mouse peritoneal macrophages, whereas the expression of the RANTES mRNA was only modestly reduced. IL-10 inhibited IP-10 mRNA accumulation by reducing IP-10 gene transcription as demonstrated by nuclear run-on analysis. Interestingly, the ability of IL-10 to inhibit expression of IP-10 was dependent on the inducing stimulus; IL-10 did not suppress interferon γ (IFNγ)- or IFNβ-stimulated IP-10 transcription or mRNA accumulation. These results suggested that IL-10 might act indirectly to suppress IP-10 expression by inhibiting LPS-induced class I IFN production. This hypothesis was supported by the following observations. First, LPS-induced IP-10 mRNA expression was blocked in cells cotreated with cycloheximide. Second, IL-10 inhibited the production of IFN/β-mediated antiviral activity. Finally, the IL-10–mediated suppression of LPS-stimulated IP-10 production could be rescued by cotreatment with IFNβ.

Interleukin-10 Suppresses IP-10 Gene Transcription by Inhibiting the Production of Class I Interferon

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4742-4749 ◽  
Author(s):  
Julie M. Tebo ◽  
Hee Sun Kim ◽  
Jing Gao ◽  
David A. Armstrong ◽  
Thomas A. Hamilton
Keyword(s):  
Gene Transcription ◽  
Interleukin 10 ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
Class I ◽  
Cytokine Gene Expression ◽  
Cytokine Gene ◽  
Mrna Accumulation ◽  
Mouse Peritoneal Macrophages ◽  
Gene Expression Levels

Abstract Interleukin-10 (IL-10) selectively inhibited lipopolysaccharide (LPS)-induced chemoattractant cytokine gene expression: levels of IP-10 mRNA were markedly suppressed in IL-10–treated mouse peritoneal macrophages, whereas the expression of the RANTES mRNA was only modestly reduced. IL-10 inhibited IP-10 mRNA accumulation by reducing IP-10 gene transcription as demonstrated by nuclear run-on analysis. Interestingly, the ability of IL-10 to inhibit expression of IP-10 was dependent on the inducing stimulus; IL-10 did not suppress interferon γ (IFNγ)- or IFNβ-stimulated IP-10 transcription or mRNA accumulation. These results suggested that IL-10 might act indirectly to suppress IP-10 expression by inhibiting LPS-induced class I IFN production. This hypothesis was supported by the following observations. First, LPS-induced IP-10 mRNA expression was blocked in cells cotreated with cycloheximide. Second, IL-10 inhibited the production of IFN/β-mediated antiviral activity. Finally, the IL-10–mediated suppression of LPS-stimulated IP-10 production could be rescued by cotreatment with IFNβ.

scholarly journals Expression and regulation of sterol 27-hydroxylase (CYP27A1) in human macrophages: a role for RXR and PPARγ ligands

2005 ◽  
Vol 385 (3) ◽  
pp. 823-830 ◽  
Author(s):  
Carmel M. QUINN ◽  
Wendy JESSUP ◽  
Jenny WONG ◽  
Leonard KRITHARIDES ◽  
Andrew J. BROWN
Keyword(s):  
Human Monocyte ◽  
Cell Types ◽  
Human Macrophage ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Macrophage Cell ◽  
Peroxisome Proliferator Activated Receptor ◽  
Protein Levels ◽  
Cholesterol Levels ◽  
Elimination Pathway

CYP27A1 (sterol 27-hydroxylase) catalyses an important sterol elimination pathway in the human macrophage, and consequently may protect against atherosclerosis. We studied the expression and regulation of CYP27A1 in a human macrophage-like cell-line, THP-1, and primary HMDMs (human monocyte-derived macrophages). In both macrophage cell types, we found that CYP27A1 expression is independent of cellular cholesterol levels and of LXR (liver X receptor)-dependent control of transcription. However, the RXR (retinoid X receptor) ligand, 9-cis-retinoic acid, upregulates CYP27A1 expression. Of the RXR heterodimeric partners tested, PPAR (peroxisome-proliferator-activated receptor) γ ligands significantly increased CYP27A1 mRNA levels. Its reversal by a PPARγ antagonist demonstrated the specificity of this effect. Interestingly, HMDMs express markedly higher levels of CYP27A1 than THP-1 macrophages, and this difference was reflected in both protein levels and enzyme activities between the two cell types. In conclusion, stimulation of CYP27A1 by PPARγ may represent a key previously unrecognized mechanism by which PPARγ protects against atherosclerosis.

Liver X receptor β increases aquaporin 2 protein level via a posttranscriptional mechanism in renal collecting ducts

2017 ◽  
Vol 312 (4) ◽  
pp. F619-F628 ◽  
Author(s):  
Wen Su ◽  
Shi-Zheng Huang ◽  
Min Gao ◽  
Xiao-Mu Kong ◽  
Jan-Åke Gustafsson ◽  
...  
Keyword(s):  
Mrna Levels ◽  
Water Homeostasis ◽  
Protein Levels ◽  
Aquaporin 2 ◽  
Protein Ubiquitination ◽  
Collecting Ducts ◽  
X Receptors ◽  
Aqp2 Gene ◽  
Lxr Agonists

Liver X receptors (LXRs) including LXRα and LXRβ are nuclear receptor transcription factors and play an important role in lipid and glucose metabolism. It has been previously reported that mice lacking LXRβ but not LXRα develop a severe urine concentrating defect, likely via a central mechanism. Here we provide evidence that LXRβ regulates water homeostasis through increasing aquaporin 2 (AQP2) protein levels in renal collecting ducts. LXRβ−/− mice exhibited a reduced response to desmopressin (dDAVP) stimulation, suggesting that the diabetes insipidus phenotype is of both central and nephrogenic origin. AQP2 protein abundance in the renal inner medulla was significantly reduced in LXRβ−/− mice but with little change in AQP2 mRNA levels. In vitro studies showed that AQP2 protein levels were elevated upon LXR agonist treatment in both primary cultured mouse inner medullary duct cells (mIMCD) and the mIMCD3 cell line with stably expressed AQP2. In addition, LXR agonists including TO901317 and GW3965 failed to induce AQP2 gene transcription but diminished its protein ubiquitination in primary cultured mIMCD cells, thereby inhibiting its degradation. Moreover, LXR activation-induced AQP2 protein expression was abolished by the protease inhibitor MG132 and the ubiquitination-deficient AQP2 (K270R). Taken together, the present study demonstrates that activation of LXRβ increases AQP2 protein levels in the renal collecting ducts via a posttranscriptional mechanism. As such, LXRβ represents a key regulator of body water homeostasis.

scholarly journals Hoveniae Semen Seu Fructus Ethanol Extract Exhibits Anti-Inflammatory Activity via MAPK, AP-1, and STAT Signaling Pathways in LPS-Stimulated RAW 264.7 and Mouse Peritoneal Macrophages

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Yun Hee Jeong ◽  
You-Chang Oh ◽  
Won-Kyung Cho ◽  
Nam-Hui Yim ◽  
Jin Yeul Ma
Keyword(s):  
Signaling Pathways ◽  
Proinflammatory Cytokines ◽  
Inflammatory Responses ◽  
Peritoneal Macrophages ◽  
Inflammatory Activity ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Anti Inflammatory ◽  
Main Components ◽  
Mouse Peritoneal Macrophages

Hoveniae semen seu fructus (HSF, fruit and seed of Hovenia dulcis Thunb) is an important traditional herbal medicine and food supplement in East Asia for the treatment of liver diseases, alcohol poisoning, obesity, allergy, and cancer. HSF has also been reported to have anti-inflammatory activity, but the cellular mechanism of action is not fully understood. We assessed the anti-inflammatory properties of an HSF ethanol (HSFE) extract and explored its precise mechanism. The ability of HSFE to suppress inflammatory responses was investigated in a murine macrophage cell line, RAW 264.7, and mouse primary macrophages. Secretions of NO, proinflammatory cytokines, inflammatory factors, and related proteins were measured using the Griess assay, ELISA, Western blot analysis, and real-time PCR, respectively. In addition, the main components of HSFE were analyzed by HPLC, and their anti-inflammatory activity was confirmed. Our results showed that pretreatment of HSFE markedly reduced the expression of NO and iNOS without causing cytotoxicity and significantly attenuated secretion of proinflammatory cytokines, including TNF-α, IL-6, and IL-1β. In addition, HSFE strongly suppressed phosphorylation of MAPK and decreased the activation of AP-1, JAK2/STAT, and NF-κB in LPS-stimulated RAW 264.7 cells in a concentration-dependent manner. Furthermore, HSFE strongly suppressed the inflammatory cytokine levels in mouse peritoneal macrophages. Also, as a result of HPLC analysis, three main components, ampelopsin, taxifolin, and myricetin, were identified in the HSFE extract, and each compound effectively inhibited the secretion of inflammatory mediators induced by LPS. These findings show that HSFE exerts anti-inflammatory effects by suppressing the activation of MAPK, AP-1, JAK2/STAT, and NF-κB signaling pathways in LPS-stimulated macrophages. In addition, the anti-inflammatory efficacy of HSFE appears to be closely related to the action of the three main components. Therefore, HSFE appears to be a promising candidate for the treatment of inflammatory diseases.

Intermedin1-53 Ameliorates Homocysteine-Promoted Atherosclerotic Calcification by Inhibiting Endoplasmic Reticulum Stress

2019 ◽  
Vol 25 (3) ◽  
pp. 251-264 ◽  
Author(s):  
Jin-Ling Ren ◽  
Yue-Long Hou ◽  
Xian-Qiang Ni ◽  
Qing Zhu ◽  
Yao Chen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
High Fat Diet ◽  
Peritoneal Macrophages ◽  
Human Aorta ◽  
High Fat ◽  
Protein Levels ◽  
Cardiovascular Morbidity And Mortality ◽  
Mouse Peritoneal Macrophages

Aim: Vascular calcification (VC) is thought to be an independent predictor of cardiovascular morbidity and mortality. Intermedin1-53 (IMD) is a cardiovascular protective peptide and can inhibit vascular medial calcification in rats. In this study, we investigated the effect of IMD on atherosclerotic calcification induced by a high-fat diet plus homocysteine (Hcy) and the potential mechanisms. Methods: ApoE−/− mice were fed a high-fat diet with Hcy in drinking water to induce atherosclerotic calcification. Results: As compared to the high-fat diet alone, Hcy treatment significantly increased atherosclerotic lesion areas and the number of calcified nodules in aortic roots and was reduced by IMD infusion or 4-phenylbutyric acid (PBA) treatment. In vitro, as compared to calcifying medium alone, Hcy treatment further increased alkaline phosphatase activity, calcium content, and calcium nodule number in human aorta vascular smooth muscle cells (HA-VSMCs), all blocked by IMD or PBA pretreatment. Mechanistically, IMD or PBA significantly alleviated endoplasmic reticulum stress (ERS) activation compared with Hcy treatment. In parallel, IMD or PBA attenuated the messenger RNA levels of osteogenic markers and inflammatory cytokines in aortas and their protein levels in lesions of aortic roots. In vitro, Hcy treatment significantly increased the protein levels of osteoblast-like cell markers in primary rat VSMCs and inflammation markers in mouse peritoneal macrophages, all decreased with IMD or PBA pretreatment. Intermedin1-53 pretreatment also markedly reduced the protein levels of ERS markers in rat VSMCs and mouse peritoneal macrophages. Conclusions: Intermedin1-53 protects against Hcy-promoted atherosclerotic calcification in ApoE−/− mice by inhibiting ERS.

scholarly journals Differential Expression of Caveolin-1 in Lipopolysaccharide-Activated Murine Macrophages

2000 ◽  
Vol 68 (9) ◽  
pp. 5084-5089 ◽  
Author(s):  
Mei G. Lei ◽  
David C. Morrison
Keyword(s):  
Principal Component ◽  
Subtractive Hybridization ◽  
Peritoneal Macrophages ◽  
Caveolin 1 ◽  
Protein Levels ◽  
Reverse Transcription Pcr ◽  
Important Regulator ◽  
Lps Signaling ◽  
Mouse Peritoneal Macrophages

ABSTRACT Five reciprocal cycles of subtractive hybridization using cDNA generated from fibroblasts with normal lipopolysaccharide (LPS) responsiveness (lps n) and from hyporesponsive (lps d) fibroblasts have led to the finding that caveolin-1 is expressed at markedly higher levels of mRNA inlps d than in lps nfibroblasts. Caveolin-1 message can also be readily detected via reverse transcription-PCR in the RAW264.7 and J774.1 macrophage-like cell lines as well as in primary thioglycolate (TG)-elicited mouse peritoneal macrophages. In RAW264.7 cells, both caveolin-1 mRNA and protein levels are down-regulated by LPS. In TG-elicited C3HeB/FeJ peritoneal macrophages, in contrast, expression of both caveolin-1 protein and mRNA is up-regulated in vitro in response to LPS stimulation. The up-regulation of caveolin-1 protein expression in C3HeB/FeJ peritoneal macrophages can be demonstrated at concentrations as low as 1.0 pg of LPS/ml. However, LPS concentrations approximately 4 orders of magnitude higher (104 pg/ml) were required to stimulate the LPS-hyporesponsive C3H/HeJ mice peritoneal macrophages such that significant caveolin-1 protein up-regulation was detected. Caveolin-1, a principal component of plasmalemmal caveolae, has been reported as a potentially important regulator for signal transduction during cellular stimulation. The results described in this report suggest that caveolin-1 expression may be associated with LPS signaling/internalization.

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