Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.

Continuous activation of gp130, a signal-transducing receptor component for interleukin 6-related cytokines, causes myocardial hypertrophy in mice.

To investigate the physiological roles of gp130 in detail and to determine the pathological consequence of abnormal activation of gp130, transgenic mice having continuously activated gp130 were created. This was carried out by mating mice from interleukin 6 (IL-6) and IL-6 receptor (IL-6R) transgenic lines. Offspring overexpressing both IL-6 and IL-6R showed constitutive tyrosine phosphorylation of gp130 and a downstream signaling molecule, acute phase response factor/signal transducer and activator of transcription 3. Surprisingly, the distinguishing feature of such offspring was hypertrophy of ventricular myocardium and consequent thickened ventricular walls of the heart, where gp130 is also expressed, in adulthood. Transgenic mice overexpressing either IL-6 or IL-6R alone did not show detectable myocardial abnormalities. Neonatal heart muscle cells from normal mice, when cultured in vitro, enlarged in response to a combination of IL-6 and a soluble form of IL-6R. The results suggest that activation of the gp130 signaling pathways leads to cardiac hypertrophy and that these signals might be involved in physiological regulation of myocardium.

A single phosphotyrosine residue of the prolactin receptor is responsible for activation of gene transcription.

Members of the cytokine/growth hormone/prolactin (PRL) receptor superfamily are associated with cytoplasmic tyrosine kinases of the Jak family. For the PRL receptor (PRLR), after PRL stimulation, both the kinase Jak2 and the receptor undergo tyrosine phosphorylation. To assess the role of tyrosine phosphorylation of the PRLR in signal transduction, several mutant forms of the PRLR in which various tyrosine residues were changed to phenylalanine were constructed and their functional properties were investigated. We identified a single tyrosine residue located at the C terminus of the PRLR to be necessary for in vivo activation of PRL-responsive gene transcription. This clearly indicates that a phosphotyrosine residue in the cytoplasmic domain of a member of the cytokine/growth hormone/PRL receptor superfamily is directly involved in signal transduction.

Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and anti-viral immunity

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group l etabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

The activation of the CGRP receptor

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. CLR is an example of a family B GPCR. Unlike family A GPCRs, little is known about how these receptors are activated by their endogenous ligands. This review considers what is known about the activation of family B GPCRs and then considers how this might be applied to CLR, particularly in light of new knowledge of the crystal structures of family A GPCRs.

Pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC1) gene is suppressed by transglutaminase 2 activation

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via toll-like receptor 2

To determine whether non-enterobacterial endotoxins, which are likely to constitute the majority of the circulating endotoxin pool, may stimulate coronary artery endothelial cell activation. Interleukin-8 secretion, monocyte adhesion, and E-selectin expression were measured in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) challenged in vitro with highly purified endotoxins of common host colonisers Escherichia coli, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Bacteroides fragilis. HCAECs but not HUVECs expressed Toll-like receptor (TLR)-2 and were responsive to non-enterobacterial endotoxins. Transfection of TLR-deficient HEK-293 cells with TLR2 or TLR4/MD2 revealed that while E. coli endotoxin utilised solely TLR4 to signal, the endotoxins, deglycosylated endotoxins (lipid-A), and whole heat-killed bacteria of the other species stimulated TLR2-but not TLR4-dependent cell-signalling. Blockade of TLR2 with neutralizing antibody prevented HCAEC activation by non-enterobacterial endotoxins. Comparison of each endotoxin with E. coli endotoxin in limulus amoebocyte lysate assay revealed that the non-enterobacterial endotoxins are greatly underestimated by this assay, which has been used in all previous studies to estimate plasma endotoxin concentrations. Circulating non-enterobacterial endotoxins may be an underestimated contributor to endothelial activation and atherosclerosis in individuals at risk of increased plasma endotoxin burden.

GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells

The proteinase-activated receptor 2 (PAR-2) expression is increased in endothelial cells derived from women with preeclampsia, characterized by widespread maternal endothelial damage, which occurs as a consequence of elevated soluble vascular endothelial growth factor receptor-1 (sVEGFR-1; commonly known as sFlt-1) in the maternal circulation. Because PAR-2 is upregulated by proinflammatory cytokines and activated by blood coagulation serine proteinases, we investigated whether activation of PAR-2 contributed to sVEGFR-1 release. PAR-2–activating peptides (SLIGRL-NH2 and 2-furoyl-LIGRLO-NH2) and factor Xa increased the expression and release of sVEGFR-1 from human umbilical vein endothelial cells. Enzyme-specific, dominant-negative mutants and small interfering RNA were used to demonstrate that PAR-2–mediated sVEGFR-1 release depended on protein kinase C-ß1 and protein kinase C-e, which required intracellular transactivation of epidermal growth factor receptor 1, leading to mitogen-activated protein kinase activation. Overexpression of heme oxygenase 1 and its gaseous product, carbon monoxide, decreased PAR-2–stimulated sVEGFR-1 release from human umbilical vein endothelial cells. Simvastatin, which upregulates heme oxygenase 1, also suppressed PAR-2–mediated sVEGFR-1 release. These results show that endothelial PAR-2 activation leading to increased sVEGFR-1 release may contribute to the maternal vascular dysfunction observed in preeclampsia and highlights the PAR-2 pathway as a potential therapeutic target for the treatment of preeclampsia.