Tumour necrosis factor-alpha receptor 1 polymorphisms and serum soluble TNFR1 in early spontaneous miscarriage

OBJECTIVES: The study investigated the association of TNFR1 gene polymorphism with early recurrent spontaneous miscarriage (ERSM) in Chinese women, and soluble TNFR1 (sTNFR1) expression in ERSM women. STUDY DESIGN: Two single nucleotide polymorphisms (SNPs) located at -383 (AGA to AGC) in the promoter region and +36 (CCA to CCG) in exon 1 of TNFR1 were investigated in 188 non-pregnant ERSM Chinese women. The serum sTNFR1 was measured by the ELISA method. RESULTS: Both SNPs were not associated with ERSM. The non-pregnant ERSM women had significantly higher levels of serum sTNFR1, compared with the non-pregnant, normal women (1.84+/-0.54 ng/ml versus 1.62+/-0.38 ng/ml; t=-2.053; p<0.05). CONCLUSIONS: The data do not provide evidence that TNFR1 gene polymorphism is etiologically important for ERSM in Chinese women. But, a significantly raised sTNFR1 level in non-pregnant ERSM women was recorded compared to women with normal pregnancies. The result suggests that pregnancy failure is associated with an increase of sTNFR1.     

Biomechanical strain regulates TNFR2 but not TNFR1 in TMJ cells

We sought to examine whether cyclic tensile strain (CTS) regulates the gene expression of tumor necrosis factor (TNF)-alpha, its receptors TNFR1 and TNFR2, and inducible nitric oxide synthase (iNOS) under inflammatory conditions, and whether these effects of CTS are sustained. Rat temporomandibular joint disc cells (TDC) were exposed to CTS in the presence or absence of interleukin (IL)-1beta for 4 and 24h. Cells were also stimulated with IL-1beta for 24h while being subjected to CTS only for the initial 1, 2, 4, 8, and 12h or the entire 24h incubation time. Furthermore, cells were incubated with IL-1beta for 24, 36, or 48 h while being exposed to CTS only for the initial 8h. Gene expression of TNF-alpha, its receptors, and iNOS was analyzed by RT-PCR, whereas protein synthesis was determined by ELISA for TNF-alpha, immunofluorescence for TNFRs, and Griess reaction for nitric oxide. CTS inhibited the IL-1beta-stimulated synthesis of TNF-alpha, TNFR2, and iNOS. TNFR1 was constitutively expressed but not regulated by IL-1beta or CTS. Application of CTS for only 1 or 2h during a 24h incubation with IL-1beta was sufficient to inhibit IL-1beta-induced upregulation of TNF-alpha, TNFR2, and iNOS. However, for maximal inhibition of these genes a longer exposure of CTS was required. These findings are the first to show that biomechanical signals regulate the expression of TNFR2 but not TNFR1 under inflammatory conditions. Furthermore, the antiinflammatory effects of biomechanical signals on TDC are maintained for prolonged periods of time but are transient.     

Retracted: TNF-α–TNFR signal pathway inhibits autophagy and promotes apoptosis of alveolar macrophages in coal worker's pneumoconiosis

Objective : Exposure to coal dust causes the development of coal worker's pneumoconiosis (CWP), which is associated with accumulating macrophages in the lower respiratory tract. This study was performed to investigate the effect of tumor necrosis factor-α (TNF-α)–tumor necrosis factor receptor (TNFR) signal pathway on autophagy and apoptosis of alveolar macrophages (AMs) in CWP. Methods: AMs from controls exposed to coal dust and CWP patients were collected, in which expressions of TNF-α and TNFR1 were determined. Autophagy was observed by transmission electron microscopy, and apoptosis by light microscope and using terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. AMs in CWP patients were treated with TNF-α or anti-TNF-α antibody. Besides, expressions of autophagy marker proteins, apoptosis-related factors, FAS, caspase-8, and receptor-interacting serine–threonine-protein kinase 3 (RIPK3) were determined by western Blot. Activities of caspase-3 and caspase-8 were determined by a fluorescence kit. Flow cytometry was applied to measure the expression of TNFR1 on the surface of the AM. Results: TNF-α expression and TNFR1 expression on the surface of AM, as well as autophagy and apoptotic index were significantly increased in AMs of CWP patients. In response to the treatment of TNF-α, TNF-α expression and TNFR1 expression on the surface of AM as well as LC3I expression were increased, autophagy was decreased, and LC3, LC3II, Beclin1 and B-cell lymphoma 2 expressions decreased, whereas FAS expression and activity and expression of caspase-3 and caspase-8 increased, and apoptotic index increased. Moreover, the situations were reversed with the treatment of anti-TNF-α antibody. Conclusion: TNF-α–TNFR signal pathway was involved in the occurrence and development of CWP by activating FAS–caspase-8 and thus inhibiting autophagy while promoting apoptosis of AM.     

Tumor necrosis factor-alpha inhibits aquaporin 5 expression in mouse lung epithelial cells

Aquaporin 5 (AQP5), the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down-regulated during pulmonary inflammation and edema. The mechanisms that underlie this decrease in AQP5 levels are therefore of considerable interest. Here we show that AQP5 expression in cultured lung epithelial cells is decreased 2-fold at the mRNA level and 10-fold at the protein level by the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Treatment of murine lung epithelial cells (MLE-12) with TNF-alpha results in a concentration- and time-dependent decrease in AQP5 mRNA and protein expression. Activation of the p55 TNF-alpha receptor (TNFR1) with an agonist antibody is sufficient to cause decreased AQP5 expression, demonstrating that the TNF-alpha effect is mediated through TNFR1. Inhibition of nuclear factor kappaB (NF-kappaB) translocation to the nucleus blocks the effect of TNF-alpha on AQP5 expression, indicating that activation of NF-kappaB is required, whereas inhibition of extracellular signal-regulated or p38 mitogen-activated protein kinases showed no effect. These data show that TNF-alpha decreases AQP5 mRNA and protein expression and that the molecular pathway for this effect involves TNFR1 and activated NF-kappaB. The ability of inflammatory cytokines to decrease aquaporin expression may help explain the connection between inflammation and edema.     

EphrinA5-EphA7 complex induces apoptotic cell death via TNFR1

A previous study showed that the EphA7 receptor regulates apoptotic cell death during early brain development. In this study, we provide evidence that the EphA7 receptor interacts with death receptors such as tumor necrosis factor receptor 1 (TNFR1) to decrease cell viability. We showed that ephrinA5 stimulates EphA7 to activate the TNFR1-mediated apoptotic signaling pathway. In addition, a pull-down assay using biotinylated ephrinA5-Fc revealed that ephrinA5-EphA7 complexes recruit TNFR1 to form a multi-protein complex. Immunocytochemical staining analysis showed that EphA7 was co-localized with TNFR1 on the cell surface when cells were incubated with ephrinA5 at low temperatures. Finally, both the internalization motif and death domain of TNFR1 was important for interacting with an intracytoplasmic region of EphA7; this interaction was essential for inducing the apoptotic signaling cascade. This result suggests that a distinct multi-protein complex comprising ephrinA5, EphA7, and TNFR1 may constitute a platform for inducing caspase-dependent apoptotic cell death.     

Phosphatidic acid as a regulator of matrix metalloproteinase-9 expression via the TNF-alpha signaling pathway

Phosphatidic acid (PA) is implicated in pathophysiological processes associated with cellular signaling events and inflammation, which include the expressional regulation of numerous genes. Here, we show that PA stimulation increases matrix metalloproteinase-9 (MMP-9) expression in macrophages through tumor necrosis factor (TNF)-alpha signaling. We performed antibody array analysis on proteins from macrophages stimulated with PA. PA was found to induce the production of TNF-alpha, but not of TNF receptor (TNFR)1 and TNFR2 in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. PA induced the phosphorylations of both ERK1/2 and p38, but not of c-jun amino-terminal kinase. Moreover, only ERK1/2 inhibition by U0126 suppressed PA-induced TNF-alpha production and MMP-9 expression. Neutralizing TNF-alpha, TNFR1 or TNFR2 antibodies significantly suppressed PA-induced MMP-9 expression, suggesting that the production of TNF-alpha in response to PA preceded the expression of MMP-9. Moreover, lipopolysaccharide-induced PA also led to TNF-alpha release and resulted in MMP-9 expression. Taken together, these observations suggest that PA may play a role in MMP-9 regulation through ERKs/TNF-alpha/TNFRs-dependent signaling pathway.     

Regulation and dysregulation of tumor necrosis factor receptor-1

TNF is an essential regulator of the immune system. Dysregulation of TNF plays a role in the pathology of many auto-immune diseases. TNF-blocking agents have proven successful in the treatment of such diseases. Development of novel, safer or more effective drugs requires a deeper understanding of the regulation of the pro-inflammatory activities of TNF and its receptors. The ubiquitously expressed TNFR1 is responsible for most TNF effects, while TNFR2 has a limited expression pattern and performs immune-regulatory functions. Despite extensive knowledge of TNFR1 signaling, the regulation of TNFR1 expression, its modifications, localization and processing are less clear and the data are scattered. Here we review the current knowledge of TNFR1 regulation and discuss the impact this has on the host.     

Cell death in the gastrulating chick embryo: potential roles for tumor necrosis factor-alpha (TNF-alpha)

We have examined the expression of TNF-alpha and its receptors, TNFR1 and TNFR2, during gastrulation in the chick embryo, and have investigated the possible role of this factor in the control of cell death at this early stage of development. TNF-alpha, immunoreactive at approximately 17 kD, was found both in vivo and in vitro, most intensely associated with the cell surface and cytoskeleton of endoderm cells. TNFR2 was especially immunoreactive in endoderm cells of the marginal zone. TNFR1 was found in nuclei throughout the embryo. Embryos also showed widespread expression of both the bcl-2 and Bax gene products, which are both associated with cell death pathways. Intact embryos in culture were sensitive to the addition of TNF-alpha (approx. 110 ng/ml), responding by significantly increasing the incidence of DNA fragmentation in cells from all tissues of the embryo. This effect was abrogated by immunological pre-absorption of the cytokine. Cultured cells from these embryos also responded to the addition of agonistic antibodies to TNF-alpha receptors by increasing DNA fragmentation. A similar response to TNF-alpha antiserum by cultured cells appeared to be related to a concomitant decrease in cell-substratum adhesion caused by the antibody. Decreased cell adhesion, induced non-specifically with anti-integrin antiserum, also resulted in increased DNA fragmentation. TNF-alpha, synthesized and secreted by the embryo itself, may be able to exert a paracrine effect on the incidence of cell death in tissues of the embryo, and the cell death process may be related to the expression of bcl-2 and Bax gene products. The influence of TNF-alpha may be exerted by the activation of cell death signalling pathways directly, or indirectly through perturbation of the cytoskeleton or of integrin-mediated cell adhesion.     

Critical role of Lin28‐TNFR2 signalling in cardiac stem cell activation and differentiation

Tumour necrotic factor receptor‐2 (TNFR2) has been to be cardiac‐protective and is expressed in cardiac progenitor cells. Our goal is to define the mechanism for TNFR2‐mediated cardiac stem cell activation and differentiation. By employing a protocol of in vitro cardiac stem cell (CSC) differentiation from human inducible pluripotent stem cell (hiPSC), we show that expression of TNFR2 precedes expression of CSC markers followed by expression of mature cardiomyocyte proteins. Activation of TNFR2 by a specific agonist promotes whereas inhibition of TNFR2 by neutralizing antibody diminishes hiPSC‐based CSC differentiation. Interestingly, pluripotent cell factor RNA‐binding protein Lin28 enhances TNFR2 protein expression in early CSC activation by directly binding to a conserved Lin28‐motif within the 3'UTR of Tnfr2 mRNA. Furthermore, inhibition of Lin28 blunts TNFR2 expression and TNFR2‐dependent CSC activation and differentiation. Our study demonstrates a critical role of Lin28‐TNFR2 axis in CSC activation and survival, providing a novel strategy to enhance stem cell‐based therapy for the ischaemic heart diseases.     

Activation of toll-like receptor-9 induces matrix metalloproteinase-9 expression through Akt and tumor necrosis factor-alpha signaling

CpG oligodeoxunucleotide (ODN) plays an important role in immune cell function. The present study examined whether temporal control of toll-like receptor (TLR)-9 by CpG ODN can regulate the expression of matrix metalloproteinase-9 (MMP-9). CpG ODN induced the release of tumor necrosis factor (TNF)-alpha and the expression of TNF receptor (TNFR)-II, but not of TNFR-I, in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. The endosomal acidification inhibitors, chloroquine or bafilomycin A, inhibited CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expression. CpG ODN induced the phosphorylation of Akt, and the inhibition of Akt by LY294002 suppressed CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expressions. Moreover, neutralizing TNF-alpha antibody significantly suppressed CpG ODN-induced MMP-9 expression, suggesting the involvement of TNF-alpha. These observations suggest that CpG ODN may play important roles in macrophage activation by regulating the expression of MMP-9 via a TLR-9/Akt/TNF-alpha-dependent signaling pathway.     

Construction of mTNFR1shRNA plasmid and its biological effects on MHV-3 induced fulminant hepatitis in BALB/cJ mice

Previous study on TNFR1-mediated hepatocyte apoptosis has been implicated in the development of fulminant viral hepatitis. To interfere with the potentially effective target, plasmid named p-mTNFR1shRNA complimentary to the sequence responsible for mTNFR1 was also constructed and further confirmed by sequence analysis. To investigate the effect of mTNFR1shRNA plasmid on mTNFR1 expression in vivo and the disease progress in MHV-3 induced fulminant hepatitis mice model. By hydrodynamic injection of mTNFR1shRNA plasmid, the survival rate of mice, hepatic pathological change were examined and compared between mice with/without mTNFR1shRNA plasmid intervention. The expression of mTNFR1 was detected by Real-time PCR, immunohistochemistry staining. The mTNFR1shRNA plasmid significantly reduced mTNFR1 expression in vivo, markedly ameliorates inflammatory infiltration, prolonged the survival time period and elevated the survival rate from 0 up to 13.3% in Balb/cJ mice with MHV-3 induced fulminant hepatitis. This study was designed to explore the opportunity of RNA interference technique in inhibiting TNFR1 expression, which has been reported to be involved in the development of a variety of diseases including fulminant viral hepatitis and severe chronic hepatitis B.     

Activation and distribution of inflammatory cells in the mouse uterus during the preimplantation period.

Northern blot analysis of mouse uterine RNA showed that IL-1 (alpha and beta), and TNF-alpha mRNA were abundant on day (D) 1 of pregnancy, reduced on D2, and remained basal throughout the remainder of the preimplantation period (D3 and D4). Elevated IL-1 beta and TNF-alpha mRNA levels on D1 were accompanied by increased levels of immunoreactive protein in uterine cytosol preparations as determined by ELISA. In situ hybridization detected IL-1 beta mRNA in cells located in the endometrial stroma and concentrated in subepithelial regions on D1. Immunocytochemical localization of IL-1 beta and TNF-alpha identified cells scattered throughout the endometrial stroma, but more concentrated in the subepithelial region on D1. On D3 and D4, cytokine-immunopositive cells decreased in number and became located predominantly at the endometrial-myometrial junction. Histochemical localization of peroxidase as a marker predominantly for eosinophils showed an abundance of these cells in the D1 uterus. The distribution of peroxidase-positive cells in the uterus followed the same temporal and spatial changes as cytokine-immunopositive cells during the preimplantation period. These data document the occurrence of an inflammatory response in the uterus on D1 of pregnancy, and demonstrate that as the preimplantation period progresses the distribution of inflammatory cells changes from the subepithelial region of the endometrial stroma to the periphery of the uterus at the endometrial-myometrial junction. Mechanisms regulating the uterine inflammatory response on D1 were investigated. Cytokine mRNA levels were not significantly elevated during the estrous cycle or after treatment of adult ovariectomized mice with estradiol-17 beta. In contrast, mating with vasectomized males resulted in an inflammatory response on D1 of pseudopregnancy similar to that on D1 of normal pregnancy, whereas mechanical stimulation of the uterine cervix failed to elicit such a response. These results strongly suggest a role for some factor(s) in the ejaculate, other than spermatozoa, in the initiation of a uterine inflammatory response after mating, but an effect of the act of mating cannot be excluded.     

A chemotactic peptide from laminin alpha 5 functions as a regulator of inflammatory immune responses via TNF alpha-mediated signaling

Tissue injury triggers inflammatory responses that may result in release of degradation products or exposure of cryptic domains of extracellular matrix components. Previously, we have shown that a cryptic peptide (AQARSAASKVKVSMKF) in the alpha-chain of laminin-10 (alpha5beta1gamma1), a prominent basement membrane component, is chemotactic for both neutrophils (PMNs) and macrophages (Mphis) and induces matrix metalloproteinase-9 (MMP-9) production. To determine whether AQARSAASKVKVSMKF has additional effects on inflammatory cells, we performed microarray analysis of RNA from RAW264.7 Mphis stimulated with AQARSAASKVKVSMKF. Several cytokines and cytokine receptors were increased >3-fold in response to the laminin alpha5 peptide. Among these were TNF-alpha and one of its receptors, the p75 TNFR (TNFR-II), increasing 3.5- and 5.7-fold, respectively. However, the peptide had no effect on p55 TNFR (TNFR-I) expression. Corroborating the microarray data, the protein levels of TNF-alpha and TNFR-II were increased following stimulation of RAW264.7 cells with AQARSAASKVKVSMKF. In addition, we determined that the production of TNF-alpha and TNFR-II in response to AQARSAASKVKVSMKF preceded the production of MMP-9. Furthermore, using primary Mphis from mice deficient in TNFR-I, TNFR-II, or both TNF-alpha receptors (TNFRs), we determined that AQARSAASKVKVSMKF induces MMP-9 expression by Mphis through a pathway triggered by TNFR-II. However, TNF-alpha signaling is not required for AQARSAASKVKVSMKF-induced PMN release of MMP-9 or PMN emigration. These data suggest that interactions of inflammatory cells with basement membrane components may orchestrate immune responses by inducing expression of cytokines, recruitment of inflammatory cells, and release of proteinases.     

An association between RBMX, a heterogeneous nuclear ribonucleoprotein, and ARTS-1 regulates extracellular TNFR1 release

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released to the extracellular space by two mechanisms, the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains. Both pathways appear to be regulated by an interaction between TNFR1 and ARTS-1 (aminopeptidase regulator of TNFR1 shedding). Here, we sought to identify ARTS-1-interacting proteins that modulate TNFR1 release. Co-immunoprecipitation identified an association between ARTS-1 and RBMX (RNA-binding motif gene, X chromosome), a 43-kDa heterogeneous nuclear ribonucleoprotein. RNA interference attenuated RBMX expression, which reduced both the constitutive release of TNFR1 exosome-like vesicles and the IL-1β-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1β-mediated inducible shedding of TNFR1 ectodomains. This identifies RBMX as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains.     

TNFR1 promoter -329G/T polymorphism results in allele-specific repression of TNFR1 expression

Tumor necrosis factor (TNF) and the TNF receptor (TNFR) superfamily play very important roles for cell death as well as normal immune regulation. Dysregulation of TNF-TNFR superfamily gene expression will influence many biological processes, and contributes to human diseases, including cancer. We investigated the genetic alterations of the TNF-TNFR superfamily genes in hepatocellular carcinoma (HCC). Several genetic alterations were detected in the 44 TNF-TNFR superfamily genes by sequencing hepatocellular carcinoma DNA samples. In particular, we found that the TNFR1 promoter −329G/T polymorphism was strongly associated with primary HCC (odds ratio [OR] = 5.22, p = 0.0007). We also observed frequent loss of heterozygosity at the polymorphic TNFR1 −329G/T site in the primary tumor tissues, indicating that the polymorphic TNFR1 −329G/T site is very susceptible to genetic alterations in HCC. Furthermore, in the polymorphic TNFR1 −329G/T site, the T allele resulted in the repression of TNFR1 expression. Therefore, our results suggest that TNFR1 −329G/T polymorphism may play an important role in the development of HCC.     

Expression of TWEAK and its receptor Fn14 in human subcutaneous adipose tissue. Relationship with other inflammatory cytokines in obesity

TWEAK, a cytokine of the TNF family, has been found to be expressed under different inflammatory conditions but no data is available concerning the expression of this cytokine and its receptor (Fn14) in human obesity. In the present work we have evaluated the expression of many pro-inflammatory TNF system cytokines (TNF-alpha, TWEAK and their respective receptors, TNFR1, TNFR2 and Fn14) in human adipose tissue of 84 subjects some with different degree of obesity and type 2 diabetes, and its relation with inflammation by also measuring the expression of macrophage marker CD68. We detected expression of TWEAK and Fn14 in isolated mature adipocytes and in the stromovascular fraction. Additionally, we found that LPS upregulates the expression of both genes on THP-1 human monocytic cell line. TWEAK was expressed in adipose tissue of all studied subjects with no differences between obesity group, and was associated with Fn14 expression in morbid obese, mainly in women with type 2 diabetes. The data obtained here also showed that TNF-alpha and TNFR2 mRNAs were significantly more expressed in subcutaneous adipose tissue of subjects with morbid obesity compared to obese and non-obese subjects. In contrast, TNFR1 gene expression was negatively associated with BMI. Our results suggest that the expression of TNF-derived pro-inflammatory cytokines are increased in severe obesity, where macrophage infiltrate could modulate the inflammatory environment through activation of its receptors.