Down-regulation of transcobalamin receptor TCblR/CD320 by siRNA inhibits cobalamin uptake and proliferation of cells in culture

The clinical phenotype of cobalamin (Cbl) deficiency is dictated by the essential role of this vitamin in two key enzymatic reactions. Multiple proteins and receptors participate in the absorption, transport and delivery of this vitamin to tissue cells. Cellular uptake of Cbl is mediated by transcobalamin (TC), a plasma protein and a transmembrane receptor (TCblR) with high affinity for TC saturated with Cbl. Knockdown of TCblR with siRNA results in decreased TC–Cbl uptake. The ensuing Cbl deficiency leads to an increase in doubling time and decreased proliferation of these cells. The study confirms the seminal role of this receptor in the cellular uptake of Cbl and its down-regulation as a potential strategy to inhibit proliferation of cancer cells.     

Tumor necrosis factor receptor superfamily members 1a and 1b contribute to exacerbation of atherosclerosis by Chlamydia pneumoniae in mice

The host immune responses that mediate Chlamydia-induced chronic disease sequelae are incompletely understood. The role of TNF-α, TNF receptor 1 (TNFR1), and TNF receptor 2 (TNFR2), in Chlamydia pneumoniae (CPN)-induced atherosclerosis was studied using the high-fat diet-fed male C57BL/6J mouse model. Following intranasal CPN infection, TNF-α knockout (KO), TNFR1 KO, TNFR2 KO, and TNFR 1/2 double-knockout, displayed comparable serum anti-chlamydial antibody response, splenic antigen-specific cytokine response, and serum cholesterol profiles compared to wild type (WT) animals. However, atherosclerotic pathology in each CPN-infected KO mouse group was reduced significantly compared to WT mice, suggesting that both TNFR1 and TNFR2 promote CPN-induced atherosclerosis.     

Effects of protease activated receptor (PAR)2 blocking peptide on endothelin-1 levels in kidney tissues in endotoxemic rat mode

Aims

Septic shock, the severe form of sepsis, is associated with development of progressive damage in multiple organs. Kidney can be injured and its functions altered by activation of coagulation, vasoactive-peptide and inflammatory processes in sepsis. Endothelin (ET)-1, a potent vasoconstrictor, is implicated in the pathogenesis of sepsis and its complications. Protease-activated receptors (PARs) are shown to play an important role in the interplay between inflammation and coagulation. We examined the time-dependent alterations of ET-1 and inflammatory cytokine, such as tumor necrosis factor (TNF)-α in kidney tissue in lipopolysaccharide (LPS)-induced septic rat model and the effects of PAR2 blocking peptide on the LPS-induced elevations of renal ET-1 and TNF-α levels.

Main methods

Male Wistar rats at 8 weeks of age were administered with either saline solution or LPS at different time points (1, 3, 6 and 10 h). Additionally, we treated LPS-administered rats with PAR2 blocking peptide for 3 h to assess whether blockade of PAR2 has a regulatory role on the ET-1 level in septic kidney.

Key findings

An increase in ET-1 peptide level was observed in kidney tissue after LPS administration time-dependently. Levels of renal TNF-α peaked (around 12-fold) at 1 h of sepsis. Interestingly, PAR2 blocking peptide normalized the LPS-induced elevations of renal ET-1 and TNF-α levels.

Significance

The present study reveals a distinct chronological expression of ET-1 and TNF-α in LPS-administered renal tissues and that blockade of PAR2 may play a crucial role in treating renal injury, via normalization of inflammation, coagulation and vaso-active peptide.     

Exercise training increases membrane bound form of tumor necrosis factor-alpha receptors with decreases in the secretion of soluble forms of receptors in rat adipocytes

We examined the effects of exercise training (treadmill running over 9 weeks) on the ability of isolated adipocytes to secrete tumor necrosis factor-alpha (TNF-alpha) and type 1 soluble TNF receptor (sTNFR1) in vitro in Wistar rats. We also examined the effects of exercise training on the expression of membrane bound forms of type 1 TNF receptor (mTNFR1) in adipocyte crude membranes of the same rat subjects. Exercise training significantly increased the secretions of TNF-alpha from isolated adipocytes. Treatment with a cyclooxygenase inhibitor, either indomethacin (100 microM) or eicosatetraynoic acid (100 microM), significantly blocked the release of TNF-alpha from adipocytes in both exercise-trained rat group and sedentary control rat group, suggesting that some cyclooxygenase metabolite(s) acts as a ligand in TNF-alpha synthesis. Decreased amounts of TNF-alpha were found to be significantly greater in both exercise-trained rat group than in sedentary control rat group after incubation with inhibitors. Thus, the inhibitory effect of both indomethacin and eicosatetraynoic acid was significantly greater in adipocytes from exercise-trained rats. Both plasma sTNFR1 levels and adipocytes-derived sTNFR1 were found to be significantly less in the exercise-trained rat group. Western blot analysis revealed that exercise training remarkably increased the expressions of mTNFR1 in adipocyte crude membrane. Thus, exercise training enhanced the ability of isolated adipocytes to secrete TNF-alpha with reduced secretion of sTNFR1, and provoked the greater expressions of mTNFR1 in adipocyte crude membrane. These alterations may induce enhanced the autocrine effects of TNF-alpha within adipocytes in exercise-trained rats.     

The prostaglandin EP4 receptor is a master regulator of the expression of PGE2 receptors following inflammatory activation in human monocytic cells

Prostaglandin E₂ (PGE₂) is responsible for inflammatory symptoms. However, PGE₂ also suppresses pro-inflammatory cytokine production. There are at least 4 subtypes of PGE₂ receptors, EP1–EP4, but it is unclear which of these specifically control cytokine production. The aim of this study was to determine which of the different receptors, EP1R–EP4R modulate production of tumor necrosis factor-α (TNF-α) in human monocytic cells.Human blood, or the human monocytic cell line THP-1 were stimulated with LPS. The actions of PGE₂, alongside selective agonists of EP1–EP4 receptors, were assessed on LPS-induced TNF-α, IL-1β and IL-10 release. The expression profiles of EP2R and EP4R in monocytes and THP-1 cells were characterised by RT-qPCR. In addition, the production of cytokines was evaluated following knockdown of the receptors using siRNA and over-expression of the receptors by transfection with constructs.PGE₂ and also EP2 and EP4 agonists (but not EP1 or EP3 agonists) suppressed TNF-α production in blood and THP-1 cells. LPS also up regulated expression of EP2R and EP4R but not EP1 or EP3. siRNA for either EP2R or EP4R reversed the suppressive actions of PGE₂ on cytokine production and overexpression of EP2R and EP4R enhanced the suppressive actions of PGE₂.This indicates that PGE₂ suppression of TNF-α by human monocytic cells occurs via EP2R and EP4R expression. However EP4Rs also control their own expression and that of EP2 whereas the EP2R does not affect EP4R expression. This implies that EP4 receptors have an important master role in controlling inflammatory responses.     

Relationships between cobalamin,epidermal growth factor,and normal prions in the myelin maintenance of central nervous system

Cobalamin (Cbl), epidermal growth factor (EGF), and prions (PrPs) are key molecules for myelin maintenance in the central and peripheral nervous systems. Cbl and EGF increase normal prion (PrP^C) synthesis and PrP^C levels in rat spinal cord (SC) and elsewhere. Cbl deficiency increases PrP^C levels in rat SC and cerebrospinal fluid (CSF), and decreases PrP^C-mRNA levels in rat SC. The administration of anti-octapeptide repeat PrP^C region antibodies (Abs) to Cbl-deficient (Cbl-D) rats prevents SC myelin lesions and a local increase in tumor necrosis factor (TNF)-α levels, whereas anti-TNF-α Abs prevent SC myelin lesions and the increase in SC and CSF PrP^C levels. As it is known that both Cbl and EGF regulate SC PrP^C synthesis independently, and that Cbl regulates SC EGF synthesis, EGF may play both Cbl-independent and Cbl-dependent roles. When Cbl-D rats undergo Cbl replacement therapy, SC PrP^C levels are similar to those observed in Cbl-D rats. In rat frontal cortex (which is marginally affected by Cbl deficiency in histological terms), Cbl deficiency decreases PrP^C levels and the increase induced by Cbl replacement leads to their normalization. Increased nerve PrP^C levels are detected in the myelin lesions of the peripheral neuropathy of Cbl-D rats, and CSF PrP^C levels are also increased in Cbl-D patients (but not in patients with Cbl-unrelated neurological diseases). Various common steps in the downstream signaling pathway of Cbl, EGF, and PrP^C underlines the close relationship between the three molecules in keeping myelin normal.     

NT FRZ 基因多态性与SEL 的相关性研究

本研究通过调查中国南方SLE人群和健康对照人群中TNFR2基因两个位点（nt587,nt694）的多态性频率,探讨TNFR2基因多态性是否与中国汉族SLE人群的易感性相关。结果发现128例SLE中,nt587G的等位基因频率为54个（21．1％）;而135例健康人群中nt587G的频率为35个（13．0％）;SLE组明显高于健康对照人群（P〈0．05）,携带nt587G的个体SLE发病危险性大。同时128例SLE中,舶94A的等位基因频率为41个（16．0％）;而健康人群中舶94A的频率为32个（11．9％）;两组比较无显著差异（P=0．149）。提示TNFR2基因nt587的多态性与中国南方SLE人群相关,可能通过影响TNFR2的表达而参与SLE的发病,而nt694（G—A）的多态性与中国南方SLE人群不相关。     

Parathyroid hormone and the regulation of cell cycle in colon adenocarcinoma cells

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In this study, we investigated the role of PTH in the regulation of the cell cycle in human colon adenocarcinoma Caco-2 cells. Flow cytometry analysis revealed that PTH (10^− 8 M, 12-24 h) treatment increases the number of cells in the G0/G1 phase and diminishes the number in both phases S and G2/M. In addition, analysis by Western blot showed that the hormone increases the expression of the inhibitory protein p27Kip1 and diminishes the expression of cyclin D1, cyclin D3 and CDK6. However, the amounts of CDK4, p21Cip1, p15INK4B and p16INK4A were not different in the absence or presence of PTH. Inhibitors of PKC (Ro-318220, bisindolylmaleimide and chelerythine), but not JNK (SP600125) and PP2A (okadaic acid and calyculin A), reversed PTH response in Caco-2 cells. Taken together, our results suggest that PTH induces G0/G1 phase arrest of Caco-2 intestinal cells and changes the expression of proteins involved in cell cycle regulation via the PKC signaling pathway.     

Effects of nitrobenzylthioinosine on neuronal injury, adenosine levels, and adenosine receptor activity in rat forebrain ischemia

Adenosine levels increase in brain during cerebral ischemia, and adenosine has receptor-mediated neuroprotective effects. This study was performed to test the hypothesis that nitrobenzylthioinosine (NBMPR), a selective and potent inhibitor of one adenosine transporter subtype termed ENT1, or es, can protect against ischemic neuronal injury by enhancing adenosine levels and potentiating adenosine receptor-mediated effects, including attenuation of the cellular production and release of tumor necrosis factor-alpha (TNF-alpha). In rats, the phosphorylated prodrug form of NBMPR, NBMPR-phosphate, or saline was administered by intracerebroventricular injection 30 min before forebrain ischemia. Seven days following the ischemic episode, rats were killed, and neuronal damage in the CA1 region of the hippocampus was assessed. The number of pyramidal neurons was significantly (p < 0.001) greater in the NBMPR-P treatment group. A trend toward protection was still evident at 28 days postreperfusion. Adenosine increased significantly during ischemia to levels eight- to 85-fold above basal. NBMPR-P treatment did not cause statistically significant increases in ischemic adenosine levels; however, this treatment tended to increase adenosine levels in all brain regions at 7 min postreperfusion. Ischemia-induced expression of TNF-alpha was not altered by NBMPR-P treatment, and the nonselective adenosine receptor antagonist 8-(p-sulfophenyl) theophylline did not abolish the neuroprotective effects of NBMPR-P treatment. These data indicate that NBMPR can protect CA1 pyramidal neurons from ischemic death without statistically significant effects on adenosine levels or adenosine receptor-mediated inhibition of the proinflammatory cytokine TNF-alpha.     

Matrix metalloproteinase-2 contributes to tumor necrosis factor alpha induced apoptosis in cultured rat cardiac myocytes

Tumor necrosis factor alpha (TNFalpha) is associated with a higher risk of cardiovascular disease. Matrix metalloproteinase-2 (MMP-2) has been implicated in the pathophysiology of ischemic heart disease. However, the role of interactions between MMP-2 and TNFalpha, associated with cardiac apoptosis, is unknown. We hypothesized that MMP-2 will contribute to TNFalpha-induced myocardial apoptosis. After treatment with TNFalpha (1-20 ng/ml) for 24 h, or with TNFalpha (10 ng/ml) for 0, 6, 12, 24, or 48 h, MMP-2 activity, percent of TUNEL-positive myocytes, and DNA fragmentation dose, and time-dependently increased compared to control. However, TNFalpha blockade (neutralizing antibodies against human TNFalpha, 25 microg/ml) significantly reduced the activity of MMP-2 and markers of apoptosis induced by TNFalpha. Interestingly, MMP-2 antibody (30 microg/ml), or the MMP-2 inhibitors Doxycycline (Dox, 1-50 micromol/l) or GM6001 (GM, 10 micromol/l), prior to TNFalpha insult, decreased myocardial MMP-2 activity and reduced the percent of TUNEL-positive myocytes and DNA fragmentation. Moreover, MMP-2 inhibition reduced Bax expression and caspase3 activity, as well as increasing Bcl2 expression. MMP-2 inhibition was associated with decreased cardiac MMP-2 activity and decreased myocardial apoptosis induced by TNFalpha. These results suggest that MMP-2 contributes to TNFalpha-induced apoptosis in cultured rat cardiac myocytes.     

Role of caspases in TNF-mediated regulation of cPLA(2)

A major part of the proinflammatory activity of tumor necrosis factor (TNF) is brought about by cytosolic phospholipase A(2) (cPLA(2)) that generates arachidonic acid, the precursor for the production of leukotrienes and prostaglandins. The activation of cPLA(2) and induction of proinflammatory lipid mediators is in striking contrast to the teleologic meaning of apoptosis, which is to avoid an inflammatory reaction. In this review we highlight the evidence for a caspase-mediated cleavage and inactivation of cPLA(2), which seems to be an important mechanism by which TNF downregulates cPLA(2) activity in cells undergoing apoptosis.     

The new MATH: homology suggests shared binding surfaces in meprin tetramers and TRAF trimers

Although apparently functionally unrelated, intracellular TRAFs and extracellular meprins share a region with conserved meprin and traf homology, MATH¹. Both TRAFs and meprins require subunit assembly for function. By structural analysis of the sequences, we provide an explanation of how meprins, which form tetramers, and TRAF molecules, which form trimers, can share homology. Our analysis suggests it is highly likely that the same oligomerization surface is used. The analysis has implications for the widely distributed group of proteins containing MATH domains.     

妊娠高血压综合征患者血清瘦素、TNF-α水平测定及其临床意义

目的检测妊娠高血压综合征患者血清中瘦素、TNF-α的水平,并探讨其与妊高征发病的关系。方法用放射免疫法测定35例妊高征患者外周血中瘦素及TNF-α的水平,并以21例正常晚期妊娠妇女(正常妊娠组)作比较。结果(1)妊高征组血清瘦素水平为(32.9±11.5)ng/ml,明显高于正常妊娠组的(18.9±3.0)ng/ml,P<0.05;轻度妊高征组血清瘦素水平为(22.0±4.8)ng/ml,与正常妊娠组比较,差异无显著性,P>0.05;中、重度妊高征组血清瘦素水平分别为(31.1±5.2)、(41.8±10.9)ng/ml,与轻度妊高征组及正常妊娠组比较,差异有显著性,P均<0.05;轻、中、重妊高征组之间血清瘦素水平差异有显著性(P均<0.05)。(2)妊高征组血清TNF-α水平为(22.7±11.5)fmol/ml,明显高于正常妊娠组的(9.2±2.1)fmol/ml,P<0.05,轻度妊高征组血清TNF-α水平为(11.5±4.1)fmol/ml,与正常妊娠组比较,差异无显著性,P>0.05;中、重度妊高征组血清TNF-α水平分别为(19.5±7.4)、(32.9±8.3)fmol/ml,与轻度妊高征组及正常妊娠组比较,差异有显著性(P均<0.05);轻、中、重妊高征组之间血清TNF-α水平差异有显著性(P均<0.05)。(3)妊高征组血清瘦素水平与TNF-α水平呈正相关(分别为r=0.56,P<0.01)。结论妊高征患者血清TNF-α水平增高可能是导致血清瘦素水平升高的原因之一。     

T3JAM, a novel protein that specifically interacts with TRAF3 and promotes the activation of JNK(1)

Previous studies suggest that localization of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family members is important for regulating their signal transduction. During a screen for TRAF3-associated proteins that potentially alter TRAF3 subcellular localization and enable signal transduction, we identified a novel protein, T3JAM (TRAF3-interacting Jun N-terminal kinase (JNK)-activating modulator). This protein associates specifically with TRAF3 but not other TRAF family members. Coexpression of T3JAM with TRAF3 recruits TRAF3 to the detergent-insoluble fraction. More importantly, T3JAM and TRAF3 synergistically activate JNK but not nuclear factor (NF)-kappaB. Our studies indicate that T3JAM may function as an adapter molecule that specifically regulates TRAF3-mediated JNK activation.     

Stimulation of endothelin B receptor modulates the inflammatory activation of rat astrocytes

Inside the brain tissue, endothelins play numerous important biological roles. One of the targets, astrocytes, predominantly display endothelin receptor subtype B (ET(B)). On cultured primary rat astroglial cells, we analyzed the effect of IRL1620, a selective ET(B) receptor agonist, on the production of nitric oxide (NO) and the synthesis of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. We performed these experiments in the presence or absence of interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS). IRL1620 decreases NO production under basal conditions and after IFN-gamma stimulation. However, during LPS-induced NO production, IRL1620 enhances this release. The basal IL-6 secretion and especially the LPS-induced synthesis are enhanced by the IRL1620 stimulation. The LPS-dependent TNF-alpha production is increased by the ET(B) stimulation. The IRL1620-induced decrease of basal NO production is not dependent on Ca2+ entry or on phospholipase C (PLC) activation, as shown by the use of LaCl3 and U73122, respectively. In the presence of LPS, the IRL1620 potentiation of NO production is inhibited by LaCl3 and U73122. The IRL1620-induced increase of IL-6 is dependent on PLC activation. These results suggest that endothelins can have dual effects depending on the costimulatory factors present. Endothelins thus have important immunomodulatory functions in the brain.