LIND/ABIN-3 is a novel lipopolysaccharide-inducible inhibitor of NF-kappaB activation

Recognition of lipopolysaccharide (LPS) by Toll-like receptor (TLR)4 initiates an intracellular signaling pathway leading to the activation of nuclear factor-kappaB (NF-kappaB). Although LPS-induced activation of NF-kappaB is critical to the induction of an efficient immune response, excessive or prolonged signaling from TLR4 can be harmful to the host. Therefore, the NF-kappaB signal transduction pathway demands tight regulation. In the present study, we describe the human protein Listeria INDuced (LIND) as a novel A20-binding inhibitor of NF-kappaB activation (ABIN) that is related to ABIN-1 and -2 and, therefore, is further referred to as ABIN-3. Similar to the other ABINs, ABIN-3 binds to A20 and inhibits NF-kappaB activation induced by tumor necrosis factor, interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate. However, unlike the other ABINs, constitutive expression of ABIN-3 could not be detected in different human cells. Treatment of human monocytic cells with LPS strongly induced ABIN-3 mRNA and protein expression, suggesting a role for ABIN-3 in the LPS/TLR4 pathway. Indeed, ABIN-3 overexpression was found to inhibit NF-kappaB-dependent gene expression in response to LPS/TLR4 at a level downstream of TRAF6 and upstream of IKKbeta. NF-kappaB inhibition was mediated by the ABIN-homology domain 2 and was independent of A20 binding. Moreover, in vivo adenoviral gene transfer of ABIN-3 in mice reduced LPS-induced NF-kappaB activity in the liver, thereby partially protecting mice against LPS/D-(+)-galactosamine-induced mortality. Taken together, these results implicate ABIN-3 as a novel negative feedback regulator of LPS-induced NF-kappaB activation.     

Expression of APOBEC2 is transcriptionally regulated by NF-kappaB in human hepatocytes

Apolipoprotein B mRNA-editing enzyme catalytic subunit 2 (APOBEC2) is a member of the nucleic-acid-editing enzymes. However, the physiological function of APOBEC2 remains unclear. We demonstrate that APOBEC2 expression is strongly enhanced in response to both tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta. Inhibition of NF-kappaB activation invariably blocks TNF-alpha-induced APOBEC2 expression. The promoter region of APOBEC2 contains functional NF-kappaB response elements in the 5' untranslated region of the gene at -625/-616. These results show that APOBEC2 expression is regulated by pro-inflammatory cytokines via NF-kappaB activation and suggest a possible role of APOBEC2 in the pathophysiology of hepatic inflammation.     

Expression of toll-like receptor 4 is down-regulated during progression of cervical neoplasia

Chronic infection and inflammation are among the most important factors contributing to cancer development and growth. Toll-like receptors (TLRs) are important families of pattern recognition receptors, which recognize conserved components of microbes and trigger the immune response against invading microorganisms. TLR4 is the signaling receptor for lipopolysaccharide (LPS), the endotoxic component of Gram-negative bacteria. Recent studies demonstrate that TLRs are expressed in some tumor cells, and that the expression of TLRs in these cells is associated with tumorigenesis. Cervical intraepithelial neoplasia (CIN) is a key stage in the development of cervical cancer and human papillomavirus (HPV) infection is an essential factor in cervical carcinogenesis. As the cervix is in constant contact with bacteria, especially Gram-negative bacteria, we hypothesize that TLR4-mediated bacterial stimulation may be involved in the tumorigenesis of cervical cancer. In the present study, the expression and distribution of TLR4 in CIN and cervical squamous carcinoma were investigated by immunohistochemistry. To our surprise, we observed a decrease in the expression of TLR4 during the progression of cervical neoplasia and this down-regulation of TLR4 appeared to be associated with the expression of \textP ^{1 6\textINK4A} , {\text{P}}^{{ 1 6^{\text{INK4A}} }} , which is a crucial marker of HPV integration into host cells. These data offer further insight regarding the association of HPV infection and TLR signaling during the carcinogenesis of cervical cancer.     

Expression of the cyclin-dependent kinase inhibitor Dacapo is regulated by cyclin E

The Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) has been implicated in mediating cell cycle arrest prior to terminal differentiation. In many instances, increased expression of CKIs immediately precedes mitotic arrest. However, the mechanism that activates CKI expression in cells that are about to stop dividing has remained elusive. Here we have addressed this issue by investigating the expression pattern of dacapo, a Cip/Kip CKI in Drosophila. We show that the accumulation of dacapo RNA and protein requires Cyclin E and that increased expression of Cyclin E can induce dacapo expression. We also show that the oscillation of the Cyclin E and Dacapo proteins are tightly coupled during ovarian endocycles. Our results argue for a mechanism where Cyclin E/Cdk activity induces Dacapo expression but only within certain windows that are permissive for dacapo expression.     

MEKK4 stimulation of p38 and JNK activity is negatively regulated by GSK3beta

The MAPK kinase kinase MEKK4 is required for neurulation and skeletal patterning during mouse development. MEKK4 phosphorylates and activates MKK4/MKK7 and MKK3/MKK6 leading to the activation of JNK and p38, respectively. MEKK4 is believed to be auto-inhibited, and its interaction with other proteins controls its dimerization and activation. TRAF4, GADD45, and Axin each bind and activate MEKK4, with TRAF4 and Axin binding to the kinase domain and GADD45 binding within the N-terminal regulatory domain. Here we show that similar to the interaction with TRAF4 and Axin, the kinase domain of MEKK4 interacts with the multifunctional serine/threonine kinase GSK3beta. GSK3beta binding to MEKK4 blocks MEKK4 dimerization that is required for MEKK4 activation, effectively inhibiting MEKK4 stimulation of the JNK and p38 MAPK pathways. Inhibition of GSK3beta kinase activity with SB216763 results in enhanced MEKK4 kinase activity and increased JNK and p38 activation, indicating that an active state of GSK3beta is required for binding and inhibition of MEKK4 dimerization. Furthermore, GSK3beta phosphorylates specific serines and threonines in the N terminus of MEKK4. Together, these findings demonstrate that GSK3beta binds to the kinase domain of MEKK4 and regulates MEKK4 dimerization. However, unlike TRAF4, Axin, and GADD45, GSK3beta inhibits MEKK4 activity and prevents its activation of JNK and p38. Thus, control of MEKK4 dimerization is regulated both positively and negatively by its interaction with specific proteins.     

Cutting edge: Immune stimulation by neisserial porins is toll-like receptor 2 and MyD88 dependent

The immunopotentiating activity of neisserial porins, the major outer membrane protein of the pathogenic Neisseria, is mediated by its ability to stimulate B cells and up-regulate the surface expression of B7-2. This ability is dependent on MyD88 and Toll-like receptor (TLR)2 expression, as demonstrated by a lack of a response by B cells from MyD88 or TLR2 knockout mice to the porins. Using previously described TLR2-dependent reporter constructs, these results were confirmed and were shown to be due to induction of NF-kappaB nuclear translocation. This is the first demonstration of known vaccine adjuvant to stimulate immune cells via TLR2.     

Expression of c-erbB-4/HER4 is regulated in T47D breast carcinoma cells by retinoids and vitamin D3.

Nuclear steroid/retinoid and memgbrane c-erbB receptor tyrosine kinase signaling control proliferation and differentiation of mammary epithelial cells. Recently, we reported that retinoic acids are efficient repressors of c-erbB-2 and -3, but not of c-erbB-1 gene expresson. Here we demonstrate that retinoid acid- mediated growth inhibition is accompanied with reduced expression of c-erbB-4/HER4 in T47D breast cancer cells as determined by FACS, Western, and RT-PCR. All-trans and 9-cis retinoic acid reduce c-erbB-4 expression to 30%-60% of control, depending on the concentration. Dexamethasone (Dex) is inactive on D3 (D3), in contrast, acts as a strong inducer, elevation more that twofold at the mRNA, but does not significantly affect cell growth. We concolude that retinoic acids are efficient growth inhibitors and repressors of cell growth and c-erbB-4, whereas D3 represents a highly efficient inducer of c-erbB-4 expression with affecting cell proliferation.     

The toll-like receptor 3-mediated antiviral response is important for protection against poliovirus infection in poliovirus receptor transgenic mice

RIG-I-like receptors and Toll-like receptors (TLRs) play important roles in the recognition of viral infections. However, how these molecules contribute to the defense against poliovirus (PV) infection remains unclear. We characterized the roles of these sensors in PV infection in transgenic mice expressing the PV receptor. We observed that alpha/beta interferon (IFN-α/β) production in response to PV infection occurred in an MDA5-dependent but RIG-I-independent manner in primary cultured kidney cells in vitro. These results suggest that, similar to the RNA of other picornaviruses, PV RNA is recognized by MDA5. However, serum IFN-α levels, the viral load in nonneural tissues, and mortality rates did not differ significantly between MDA5-deficient mice and wild-type mice. In contrast, we observed that serum IFN production was abrogated and that the viral load in nonneural tissues and mortality rates were both markedly higher in TIR domain-containing adaptor-inducing IFN-β (TRIF)-deficient and TLR3-deficient mice than in wild-type mice. The mortality rate of MyD88-deficient mice was slightly higher than that of wild-type mice. These results suggest that multiple pathways are involved in the antiviral response in mice and that the TLR3-TRIF-mediated signaling pathway plays an essential role in the antiviral response against PV infection.     

Platelet-derived toll-like receptor 4 (tlr-4) is sufficient to promote microvascular thrombosis in endotoxemia

Endotoxin (lipopolysaccharide, LPS) produced by gram-negative bacteria initiates a host of pro-inflammatory effects through Toll-like receptor 4 (TLR-4). We reported previously that LPS enhances microvascular thrombosis in cremaster venules of wild-type mice, but had no effect in mice deficient in TLR-4. Since TLR-4 is expressed on various cell types, the cellular origin of TLR-4 responsible for the LPS-enhanced thrombosis remains undetermined. Platelets are known to express functional TLR-4. Platelet-derived TLR-4 has been suggested to mediate various inflammatory responses in endotoxemia, including production of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β), two cytokines reported to enhance microvascular thrombosis. We determined whether platelet-derived TLR-4 was sufficient to mediate the enhanced thrombosis induced by endotoxin and whether these responses were accompanied by systemic increases in TNF-α and IL-1β. We isolated platelets from wild-type mice and transfused them into either of two strains of TLR-4-deficient mice (C57BL/10ScN and B6.B10ScN-TLR-4(lps-del)/Jth). The mice were then injected with LPS or saline, and the kinetics of thrombosis were studied 4 hours later. Transfusion of wild-type platelets restored responsiveness to LPS in TLR-4-deficient mice with regards to microvascular thrombosis but not to plasma levels of TNF-α or IL-1β. The accelerated rates of microvascular thrombosis induced by platelet transfusions were specific to TLR-4, since isolation and transfusion of platelets derived from TLR-4-deficient donors did not restore responsiveness to LPS. These studies demonstrate that platelet-derived TLR-4 is sufficient to promote microvascular thrombosis in endotoxemia, independent of systemic increases in TNF-α or IL-1β.     

外周血Toll样受体2与早产的关系

目的:探讨早产产妇外周血Toll样受体2(toll-like receptor2,TLR2)mRNA的表达水平及与亚临床绒毛膜羊膜炎的关系.方法:选择早产自然分娩组15例,足月妊娠自然分娩组20例,足月要求剖宫产10例.RT.PCR检测母血中TLR2 mRNA的表达及与胎盘亚临床绒毛膜羊膜炎的关系.结果:①早产自然分娩组产妇TLR2高表达,明显高于足月自然分娩组,差异有统计学意义(P<0.05).②妊娠合并亚临床绒毛膜羊膜炎组TLR2较无亚临床绒毛膜羊膜炎组高,有统计学意义(P<0.05).结论:孕妇外周血表达TLR2,与分娩方式及感染情况有关;可能参与了分娩发动,尤其是早产分娩发动.     

Placental toll-like receptor 3 and toll-like receptor 7/8 activation contributes to preeclampsia in humans and mice

Preeclampsia (PE) is a pregnancy-specific hypertensive syndrome characterized by excessive maternal immune system activation, inflammation, and endothelial dysfunction. Toll-like receptor (TLR) 3 activation by double-stranded RNA (dsRNA) and TLR7/8 activation by single-stranded RNA (ssRNA) expressed by viruses and/or released from necrotic cells initiates a pro-inflammatory immune response; however it is unknown whether viral/endogenous RNA is a key initiating signal that contributes to the development of PE. We hypothesized that TLR3/7/8 activation will be evident in placentas of women with PE, and sufficient to induce PE-like symptoms in mice. Placental immunoreactivity and mRNA levels of TLR3, TLR7, and TLR8 were increased significantly in women with PE compared to normotensive women. Treatment of human trophoblasts with the TLR3 agonist polyinosine-polycytidylic acid (poly I:C), the TLR7-specific agonist imiquimod (R-837), or the TLR7/8 agonist CLO97 significantly increased TLR3/7/8 levels. Treatment of mice with poly I:C, R-837, or CLO97 caused pregnancy-dependent hypertension, endothelial dysfunction, splenomegaly, and placental inflammation. These data demonstrate that RNA-mediated activation of TLR3 and TLR7/8 plays a key role in the development of PE.     

The amino-terminal region of toll-like receptor 4 is essential for binding to MD-2 and receptor translocation to the cell surface

Toll-like receptor 4 (TLR4) and MD-2 are pivotal components that elicit inflammatory responses to lipopolysaccharide (LPS). They have been shown to form a physical complex on the cell surface that responds directly to LPS. However, the functional region of TLR4 required for association with MD-2 and LPS responsiveness is poorly understood. To identify the region of TLR4, we created a series of mutants with deletions in the extracellular domain and examined their activities in human embryonic kidney 293 cells. A mutant with a 317-amino acid deletion from the membrane proximal region of TLR4 was capable of associating with MD-2, while only a 9-amino acid truncation of the N terminus severely impaired the interaction. The association between the two molecules was well correlated with TLR4 maturation into an endoglycosidase H-resistant form and the cell surface expression. Mouse MD-2 bound to human TLR4, but its activity to facilitate the cell surface expression of TLR4 and confer LPS responsiveness was much weaker than that of human MD-2, indicating species specificity. A chimeric receptor composed of the N-terminal region of human TLR4 and the adjacent region of mouse TLR4 showed preference for human MD-2 in its transport to the cell surface and responsiveness to LPS. Taken together, the N-terminal region of TLR4 is essential for association with MD-2, which is required for the cell surface expression and hence the responsiveness to LPS.     

Role of the toll-like receptor 4 in neuroinflammation in Alzheimer's disease.

Microglial activation is a key feature in Alzheimer's disease and is considered to contribute to progressive neuronal injury by release of neurotoxic products. The innate immune receptor Toll-like-receptor 4 (TLR4), localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms. Here, we show that a spontaneous loss-of-function mutation in the Tlr4 gene strongly inhibits microglial and monocytic activation by aggregated Alzheimer amyloid peptide resulting in a significantly lower release of the inflammatory products IL-6, TNFalpha and nitric oxide. Treatment of primary murine neuronal cells with supernatant of amyloid peptide-stimulated microglia demonstrates that Tlr4 contributes to amyloid peptide-induced microglial neurotoxicity. In addition, stimulation experiments in transfected HEK293 cells allowed to define a tri-molecular receptor complex consisting of TLR4, MD-2 and CD14 necessary for full cellular activation by aggregated amyloid peptide. A clinical relevance of these findings is supported by a marked upregulation of Tlr4 mRNA in APP transgenic mice and by an increased expression of TLR4 in Alzheimer's disease brain tissue associated with amyloid plaque deposition. Together, these observations provide the first evidence for a role of the key innate immune receptor, TLR4, in neuroinflammation in Alzheimer's disease.     

Role of nod2 in the response of macrophages to toll-like receptor agonists

Nod2 (CARD15) is a macrophage-specific protein containing two CARD domains, a large nucleotide binding domain and leucine-rich repeats. Human genetic studies have linked mutations in NOD2/CARD15 with Crohn's disease, although the mechanisms involved are unknown. However, Nod2 has been proposed to directly bind bacterial lipopolysaccharide (LPS) and subsequently act as an activator of NF-kappaB via the association of the CARD domains with Rip2/RICK/CARDIAK. This is hypothesized to constitute a pathogen recognition pathway distinct from Toll-like receptor 4-mediated recognition of LPS. Using targeted mutagenesis, we introduced a mutation to delete the CARD domains of mouse Nod2. Mice lacking Nod2 were indistinguishable from controls and showed no signs of intestinal pathology. Macrophages responded normally to multiple Toll-like receptor agonists in terms of NF-kappaB target activation, mitogen-activated protein kinase activation, and cytokine secretion. However, Nod2(-/-) mice were significantly protected in endotoxin challenge experiments, and Nod2(-/-) macrophages were refractory to muramyl dipeptide stimulation. These results argue that Nod2 does not play an essential, nonredundant role in the response of macrophages to bacterial products but rather plays unexpected roles in regulating systemic responses to pathogens.