phosphothreonine and Inflammation

Hyperphosphorylated tau is the main component of neurofibrillary tangles and involved in the pathogenesis of Alzheimer's disease (AD). Increasing evidences suggest close associations between Porphyromonas gingivalis (P. gingivalis) and AD, but the relationship between P. gingivalis and tau hyperphosphorylation is still unclear. In this study, we investigated whether peripheral infection with P. gingivalis caused tau hyperphosphorylation by using wild Sprague-Dawley (SD) rats and HT-22 cells. The rats were injected with P. gingivalis suspension or phosphate-buffered saline 3 times per week. After 4 weeks or 12 weeks, the rats were sacrificed for analyzing systemic inflammation, neuroinflammation, and tau hyperphosphorylation. The results showed that the severity of phosphorylated tau at the AD-related sites Thr181 and Thr231 and the number of activated astrocytes were notably greater in the hippocampus of rats with P. gingivalis injection. And the levels of the inflammatory cytokines interleukin (IL)-1β and IL-6 and tumor necrosis factor-α in serum and hippocampus were also increased in the rats with P. gingivalis injection. In addition, the activity of protein phosphatase 2A (PP2A) was significantly inhibited in the hippocampus of rats with P. gingivalis injection. In vitro, IL-1β induced tau hyperphosphorylation by inhibiting the activity of PP2A in HT-22 cells and application of the PP2A promoter efficiently attenuated IL-1β-induced tau hyperphosphorylation in HT-22 cells. These results indicated that P. gingivalis could induce tau hyperphosphorylation via, in part, attenuating the activity of PP2A through triggering systemic inflammation and neuroinflammation in wild-type SD rats.

Topics: Alzheimer Disease; Animals; Astrocytes; Bacteremia; Bacteroidaceae Infections; Cell Line; Cytokines; Disease Models, Animal; Enzyme Activation; Hippocampus; Inflammation; Male; Nerve Tissue Proteins; Neurons; Phosphorylation; Phosphothreonine; Porphyromonas gingivalis; Protein Phosphatase 2; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Specific Pathogen-Free Organisms; tau Proteins; Tumor Necrosis Factor-alpha

The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKβ, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied.. Molecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry.. We show that IKKα and IKKβ bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3β, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers.. Our data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; HEK293 Cells; Humans; I-kappa B Kinase; Inflammation; Male; Mice; Models, Biological; Mutation; Phosphorylation; Phosphoserine; Phosphothreonine; Prostate; Protein Binding; Protein Stability; Proto-Oncogene Proteins c-myc; Transcription, Genetic

Smooth muscle (SM) 22α, an actin-binding protein, is down-regulated in atherosclerotic arteries. Disruption of SM22α promotes arterial inflammation through activation of reactive oxygen species (ROS)-mediated nuclear factor (NF)-κB pathways. This study aimed to investigate the mechanisms by which SM22α regulates vascular inflammatory response. The ligation injury model of SM22α(-/-) mice displayed up-regulation of inflammatory molecules MCP-1, VCAM-1, and ICAM-1 in the carotid arteries. Similar results were discovered in human atherosclerotic samples. In vitro studies, overexpression of SM22α attenuated TNF-α-induced IκBα phosphorylation and degradation, accompanied by decreased NF-κB activity and reduced inflammatory molecule expression. Using coimmunoprecipitation, we found that SM22α interacted with and stabilized IκBα in quiescent VSMCs. Upon TNF-α stimulation, SM22α was phosphorylated by casein kinase (CK) II at Thr139, leading to dissociation of SM22α from IκBα, followed by IκBα degradation and NF-κB activation. Our findings demonstrate that SM22α is a phosphorylation-regulated suppressor of IKK-IκBα-NF-κB signaling cascades. SM22α may be a novel therapeutic target for human vascular diseases and other inflammatory conditions.

Topics: Aged; Animals; Casein Kinase II; Cell Nucleus; DNA; HEK293 Cells; Humans; I-kappa B Proteins; Inflammation; Male; Mice, Knockout; Microfilament Proteins; Muscle Proteins; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Phosphothreonine; Protein Binding; Protein Stability; Protein Transport; Proteolysis; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

The aim of this study was to determine the role of AMP-activated protein kinase (AMPK) in lipopolysaccharide (LPS)-induced lung endothelial barrier dysfunction and lung injury in vivo. Both cultured human pulmonary artery endothelial cells (HPAECs) and experimental animals [AMPK subunit α-deficient mice and wild-type (WT) control mice (C57BL/6J)] were used. In cultured HPAECs, LPS increased endothelial permeability in parallel with a decrease in AMPK activity. Consistent with this observation, AMPK activation with the potent AMPK activator 5-aminoimidazole-4-carboxamide-1-d-ribofuranoside (AICAR) attenuated LPS-induced endothelial hyperpermeability in vitro. Intratracheal administration of LPS (1 mg/kg) in WT mice reduced AMPK phosphorylation at Thr172 in lung tissue extracts, increased protein content and cell count in bronchial alveolar lavage fluid, and increased Evans Blue dye infiltration into the lung. These same attributes were similarly enhanced in AMPKα-knockout mice, compared with WT mice. Pretreatment with AICAR reduced these lung injury indicators in LPS-treated WT mice. AMPK activation with AICAR attenuated LPS-induced endothelial hyperpermeability by activating the Rac/Cdc42/PAK pathway, with concomitant inhibition of the Rho pathway, and decreased VE-cadherin phosphorylation at Tyr658. We conclude that AMPK activity supports normal endothelial barrier function and that LPS exposure inhibits AMPK, thereby contributing to endothelial barrier dysfunction and lung injury.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Antigens, CD; Blood Vessels; Cadherins; Cattle; cdc42 GTP-Binding Protein; Cell Membrane Permeability; Cells, Cultured; Endothelial Cells; Enzyme Activation; Humans; Inflammation; Lipopolysaccharides; Lung; Lung Injury; Mice; Mice, Inbred C57BL; Myosin Light Chains; p21-Activated Kinases; Phosphoprotein Phosphatases; Phosphorylation; Phosphothreonine; Protein Phosphatase 2C; Protein Serine-Threonine Kinases; rac1 GTP-Binding Protein; Ribonucleotides; Signal Transduction

The pulmonary collectin surfactant protein (SP)-A has a pivotal role in anti-inflammatory modulation of lung immunity. The mechanisms underlying SP-A-mediated inhibition of LPS-induced NF-kappaB activation in vivo and in vitro are only partially understood. We previously demonstrated that SP-A stabilizes IkappaB-alpha, the primary regulator of NF-kappaB, in alveolar macrophages (AM) both constitutively and in the presence of LPS. In this study, we show that in AM and PBMC from IkappaB-alpha knockout/IkappaB-beta knockin mice, SP-A fails to inhibit LPS-induced TNF-alpha production and p65 nuclear translocation, confirming a critical role for IkappaB-alpha in SP-A-mediated LPS inhibition. We identify atypical (a) protein kinase C (PKC) zeta as a pivotal upstream regulator of SP-A-mediated IkappaB-alpha/NF-kappaB pathway modulation deduced from blocking experiments and confirmed by using AM from PKCzeta-/- mice. SP-A transiently triggers aPKCThr(410/403) phosphorylation, aPKC kinase activity, and translocation in primary rat AM. Coimmunoprecipitation experiments reveal that SP-A induces aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory macrophage activation via IkappaB-alpha by SP-A critically depends on PKCzeta activity, and thus attribute a novel, stimulus-specific signaling function to PKCzeta in SP-A-modulated pulmonary immune response.

Topics: Active Transport, Cell Nucleus; Animals; Cell Membrane; Cells, Cultured; Enzyme Activation; I-kappa B Kinase; Inflammation; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Transgenic; Mutation; NF-kappa B; Phosphothreonine; Protein Binding; Protein Kinase C; Pulmonary Surfactant-Associated Protein A; Rats; Tumor Necrosis Factor-alpha

Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Chlorocebus aethiops; Enzyme Activation; HeLa Cells; Humans; Inflammation; Interleukin-1; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Weight; Osmolar Concentration; Phosphorylation; Phosphothreonine; Phosphotyrosine; Recombinant Proteins; Sequence Deletion; Stress, Physiological; Subcellular Fractions; Substrate Specificity; Threonine; Transfection; Tumor Necrosis Factor-alpha; Tyrosine