Chronic inflammation and protection from acute hepatitis in transgenic mice expressing TNF in endothelial cells

Endothelial activation is an important feature of many inflammatory diseases and has been implicated as the cause of vascular complications in disorders such as diabetes, atherosclerosis, and transplant rejection. One of the most potent activators of the endothelium is TNF, which can also be expressed by endothelial cells, causing a permanent, autocrine stimulatory signal. To establish a model of continuous endothelial activation and to elucidate the role of endothelial derived TNF in vivo, we generated transgenic mice expressing a noncleavable transmembrane form of TNF under the control of the endothelial-specific tie2 promoter. Adult tie2-transmembrane TNF-transgenic mice developed chronic inflammatory pathology in kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. Along with the infiltrate, an up-regulation of the adhesion molecules ICAM-1 and VCAM-1, but not E-selectin, in the endothelium was observed. Despite predisposition to chronic inflammation these mice were protected from immune-mediated liver injury in a model of Con A-induced acute hepatitis. Although the blood levels of soluble TNF and IFN-gamma were increased in transgenic animals after challenge with Con A, no damage of hepatocytes could be detected, as assessed by the lack of increase in plasma transaminase activities and the absence of TUNEL staining in the liver. We conclude that expression of transmembrane TNF in the endothelium causes continuous endothelial activation, leading to both proinflammatory and protective events.     

Tumor necrosis factor receptor cross-talk

Extensive research has been performed to unravel the mechanistic signaling pathways mediated by tumor necrosis factor receptor 1 (TNFR1), by contrast there is limited knowledge on cellular signaling upon activation of TNFR2. Recently published data have revealed that these two receptors not only function independently, but also can influence each other via cross-talk between the different signaling pathways initiated by TNFR1 and TNFR2 stimulation. Furthermore, the complexity of this cross-talk is also dependent on the different signaling kinetics between TNFR1 and TNFR2, by which a delicate balance between cell survival and apoptosis can be maintained. Some known signaling factors and the kinetics that are involved in the receptor cross-talk between TNFR1 and TNFR2 are the topic of this review.     

Neuronal AKAP150 coordinates PKA and Epac-mediated PKB/Akt phosphorylation

In diverse neuronal processes ranging from neuronal survival to synaptic plasticity cyclic adenosine monophosphate (cAMP)-dependent signaling is tightly connected with the protein kinase B (PKB)/Akt pathway but the precise nature of this connection remains unknown. In the current study we investigated the effect of two mainstream pathways initiated by cAMP, cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) on PKB/Akt phosphorylation in primary cortical neurons and HT-4 cells. We demonstrate that PKA activation leads to a reduction of PKB/Akt phosphorylation, whereas activation of Epac has the opposite effect. This effect of Epac on PKB/Akt phosphorylation was mediated by Rap activation. The increase in PKB/Akt phosphorylation after Epac activation could be blocked by pretreatment with Epac2 siRNA and to a somewhat smaller extent by Epac1 siRNA. PKA, PKB/Akt and Epac were all shown to establish complexes with neuronal A-kinase anchoring protein150 (AKAP150). Interestingly, activation of Epac increased phosphorylation of PKB/Akt complexed to AKAP150. From experiments using PKA-binding deficient AKAP150 and peptides disrupting PKA anchoring to AKAPs, we conclude that AKAP150 acts as a key regulator in the two cAMP pathways to control PKB/Akt phosphorylation.     

Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels

It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-d-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the nucleus basalis has not yet been investigated. Here, by means of choline acetyl transferase and NR2B or NR2C double staining, we demonstrate that mice express both the NR2C and NR2B subunits in nucleus basalis cholinergic cells. We generated NR2C-2B mutant mice in which an insertion of NR2B cDNA into the gene locus of the NR2C gene replaced NR2C by NR2B expression throughout the brain. This NR2C-2B mutant was used to examine whether a subunit exchange in cholinergic neurons would affect acetylcholine (ACh) content in several brain structures. We found increased ACh levels in the frontal cortex and amygdala in the brains of NR2C-2B mutant mice. Brain ACh has been implicated in neuroplasticity, novelty-induced arousal and encoding of novel stimuli. We therefore assessed behavioral habituation to novel environments and objects as well as object recognition in NR2C-2B subunit exchange mice. The behavioral analysis did not indicate any gross behavioral alteration in the mutant mice compared with the wildtype mice. Our results show that the NR2C by NR2B subunit exchange in mice affects ACh content in two target areas of the nucleus basalis.     

Tumor necrosis factor (TNF)-mediated neuroprotection against glutamate-induced excitotoxicity is enhanced by N-methyl-D-aspartate receptor activation. Essential role of a TNF receptor 2-mediated phosphatidylinositol 3-kinase-dependent NF-kappa B pathway

We have previously shown that two tumor necrosis factor (TNF) receptors (TNFR) exhibit antagonistic functions during neurodegenerative processes in vivo with TNFR1 aggravating and TNFR2 reducing neuronal cell loss, respectively. To elucidate the neuroprotective signaling pathways of TNFR2, we investigated glutamate-induced excitotoxicity in primary cortical neurons. TNF-expressing neurons from TNF-transgenic mice were found to be strongly protected from glutamate-induced apoptosis. Neurons from wild type and TNFR1(-/-) mice prestimulated with TNF or agonistic TNFR2-specific antibodies were also resistant to excitotoxicity, whereas TNFR2(-/-) neurons died upon glutamate and/or TNF exposures. Both protein kinase B/Akt and nuclear factor-kappa B (NF-kappa B) activation were apparent upon TNF treatment. Both TNFR1 and TNFR2 induced the NF-kappa B pathway, yet with distinguishable kinetics and upstream activating components, TNFR1 only induced transient NF-kappa B activation, whereas TNFR2 facilitated long term phosphatidylinositol 3-kinase-dependent NF-kappa B activation strictly. Glutamate-induced triggering of the ionotropic N-methyl-D-aspartate receptor was required for the enhanced and persistent phosphatidylinositol 3-kinase-dependent NF-kappa B activation by TNFR2, indicating a positive cooperation of TNF and neurotransmitter-induced signal pathways. TNFR2-induced persistent NF-kappa B activity was essential for neuronal survival. Thus, the duration of NF-kappa B activation is a critical determinant for sensitivity toward excitotoxic stress and is dependent on a differential upstream signal pathway usage of the two TNFRs.