Hypoxia Accelerate β-Actin Expression through Transcriptional Activation of ACTB by Nuclear Respiratory Factor-1

Molecular Biology - Cytoskeletal protein β-actin is abundant both in the cytoplasm and the nucleus, its mRNA is commonly utilized an internal control for gene expression analysis. Recent...     

RAPGEF5 Regulates Nuclear Translocation of β-Catenin

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Endogenous and Exogenous Regulation of Human α- and β-Adrenergic Receptors

Abstract

Regulation of human β2-adrenergic receptors in lymphocytes (determined by (±)-125 iodocyanopindolol (ICYP) binding) and α₂-adrenergic receptors in platelets (determined by ³H-yohimbine binding) was studied. While α₂-adrenergic receptor number did not change with age, a significant negative correlation between the number of α₂-adrenergic receptors and age was found; plasma catecholamines, on the contrary, were elevated in the elderly.In healthy women during normal menstrual cycle the number of α₂-adrenergic receptors decreased with increasing plasma estradiol levels.Incubation of lymphocyte membranes with isoprenaline (100 μM) and of platelet membranes with clonidine (1-100 μM) led to a reduction of the number of β₂- and α₂-receptors, respectively, without changes in the K_D-values. Treatment of hypertensive patients with clonidine (3x150 μg/die) for 7 days reduced the number of α₂-adrenergic receptors in platelets. In platelet membranes from such treated patients inhibition of ³H-yohimbine binding by clonidine and adrenaline was not affected by 10-⁴MGTP. It is concluded, that human α- and β-adrenergic receptors undergo regulatory mechanisms similar to those recently described for adrenergic receptors in a variety of animal models.     

Exercise Protects against Chronic β-Adrenergic Remodeling of the Heart by Activation of Endothelial Nitric Oxide Synthase

Extensive data have shown that exercise training can provide cardio-protection against pathological cardiac hypertrophy. However, how long the heart can retain cardio-protective phenotype after the cessation of exercise is currently unknown. In this study, we investigated the time course of the loss of cardio-protection after cessation of exercise and the signaling molecules that are responsible for the possible sustained protection. Mice were made to run on a treadmill six times a week for 4 weeks and then rested for a period of 0, 1, 2 and 4 weeks followed by isoproterenol injection for 8 days. Morphological, echocardiographic and hemodynamic changes were measured, gene reactivation was determined by real-time PCR, and the expression and phosphorylation status of several cardio-protective signaling molecules were analyzed by Western-blot. HW/BW, HW/TL and LW/BW decreased significantly in exercise training (ER) mice. The less necrosis and lower fetal gene reactivation induced by isoproterenol injection were also found in ER mice. The echocardiographic and hemodynamic changes induced by β-adrenergic overload were also attenuated in ER mice. The protective effects can be sustained for at least 2 weeks after the cessation of the training. Western-blot analysis showed that the alterations in the phosphorylation status of endothelial nitric oxide synthase (eNOS) (increase in serine 1177 and decrease in threonine 495) continued for 2 weeks after the cessation of the training whereas increases of the phosphorylation of Akt and mTOR disappeared. Further study showed that L-NG-Nitroarginine methyl ester (L-NAME) treatment abolished the cardio-protective effects of ER. Our findings demonstrate that stimulation of eNOS in mice through exercise training provides acute and sustained cardioprotection against cardiac hypertrophy.     

Regulation of β-Adrenergic Receptor Trafficking and Lung Microvascular Endothelial Cell Permeability by Rab5 GTPase

Rab5 GTPase modulates the trafficking of the cell surface receptors, including G protein-coupled β-adrenergic receptors (β-ARs). Here, we have determined the role of Rab5 in regulating the internalization of β-ARs in lung microvascular endothelial cells (LMECs) and in maintaining the integrity and permeability of endothelial cell barrier. Our data demonstrate that lipopolysaccharide (LPS) treatment disrupts LMEC barrier function and reduces the cell surface expression of β-ARs. Furthermore, the activation of β-ARs, particularly β2-AR, is able to protect the LMEC permeability from LPS injury. Moreover, siRNA-mediated knockdown of Rab5 inhibits both the basal and agonist-provoked internalization of β-ARs, therefore, enhancing the cell surface expression of the receptors and receptor-mediated ERK1/2 activation. Importantly, knockdown of Rab5 not only inhibits the LPS-induced effects on β-ARs but also protects the LMEC monolayer permeability. All together, these data provide strong evidence indicating a crucial role of Rab5-mediated internalization of β-ARs in functional regulation of LMECs.     

Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFNγ-Induced STAT1 Transcriptional Activity

The protozoan Toxoplasma gondii actively modulates cytokine-induced JAK/STAT signaling pathways to facilitate survival within the host, including blocking IFNγ-mediated STAT1-dependent proinflammatory gene expression. We sought to further characterize inhibition of STAT1 signaling in infected murine dendritic cells (DC) because this cell type has not previously been examined, yet is known to serve as an early target of in vivo infection. Unexpectedly, we discovered that T. gondii infection alone induced sustained STAT1 phosphorylation and nuclear translocation in DC in a parasite strain-independent manner. Maintenance of STAT1 phosphorylation required active invasion but intracellular parasite replication was dispensable. The parasite rhoptry protein ROP16, recently shown to mediate STAT3 and STAT6 phosphorylation, was not required for STAT1 phosphorylation. In combination with IFNγ, T. gondii induced synergistic STAT1 phosphorylation and binding of aberrant STAT1-containing complexes to IFNγ consensus sequence oligonucleotides. Despite these findings, parasite infection blocked STAT1 binding to the native promoters of the IFNγ-inducible genes Irf-1 and Lrg47, along with subsequent gene expression. These results reinforce the importance of parasite-mediated blockade of IFNγ responses in dendritic cells, while simultaneously showing that T. gondii alone induces STAT1 phosphorylation.     

The Inhibitory Role of Lactacystin and β-Lactacystinon T-cell Activation and Proliferation

To evaluate the effects of proteasome inhibitors lactacystin (LAC) and beta-lactacystin (beta-LAC) on the proliferation and activation of T lymphocytes, flow cytometry was used to analyze the proliferation and the expression of CD69, CD25 and CD3 of T lymphocytes activated by PHA. Furthermore, the expressions of PA28 and IL-2 mRNA were assayed by competitive RT-PCR. The results indicated that: (1) LAC and beta-LAC significantly decreased the incorporation of BrdU and inhibited T lymphocytes proliferation in T lymphocytes activated by PHA; (2) although LAC and beta-LAC did not affect the expression of CD69 at any time, they significantly inhibited the expression of CD25 (48 h, 72 h, P<0.05); (3) in comparison with control, LAC and beta--LAC significantly down-regulated the expression of PA28 and IL-2 mRNA (48 h, 72 h, P<0.05). LAC and beta-LAC significantly inhibited the proliferation and activation of T cells. Mechanisms involved are inhibition of CD25 and down-regulation of PA28 and IL-2 mRNA expressions.     

ERK5 Activation by Gq-Coupled Muscarinic Receptors Is Independent of Receptor Internalization and β-Arrestin Recruitment

G-protein-coupled receptors (GPCRs) are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK) pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII) failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.     

Localization of β1-Adrenergic Receptors in the Cochlea and the Vestibular Labyrinth

Sympathetic activation in a “fight or flight reaction” may put the sensory systems for hearing and balance into a state of heightened alert via β₁-adrenergic receptors (β₁-AR). The aim of the present study was to localize β₁-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize β₁-AR by Western immunoblots, and to identify β₁-AR pharmacologically by measurements of cAMP production. Staining for β₁-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated β₁-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, β₁-AR are present and functional in inner ear epithelial cells that are involved in K⁺ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.