Redox regulation of protein kinases

Abstract

Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous hydrogen peroxide (H₂O₂) by membrane-bound NADPH oxidases. In turn, H₂O₂ can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H₂O₂ regarding kinase activity, as well as the components involved in H₂O₂ production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H₂O₂ through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiological and pathological H₂O₂ responses.     

Role of receptor and nonreceptor protein tyrosine kinases in H2O2-induced PKB and ERK1/2 signaling

Excessive generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of many diseases, including atherosclerosis, hypertension, and vascular complications of diabetes. However, the precise mechanisms by which ROS contribute to the development of these diseases are not fully characterized. Hydrogen peroxide (H₂O₂), a ROS, has been shown to activate several signaling protein kinases, such as extracellular signal-regulated kinase (ERK)1/2 and protein kinase B (PKB) in different cell types, notably in vascular smooth muscle cells. Because these pathways regulate cellular mitogenesis, migration, proliferation, survival, and death responses, their aberrant activtion has been suggested to be a potential mechanism of ROS-induced pathologies. The upstream elements responsible for H₂O₂-induced ERK1/2 and PKB activation remain poorly characterized, but a potential role of receptor and nonreceptor protein tyrosine kinases (PTKs) as triggers that initiate such events has been postulated. Therefore, the aim of this review is to highlight the involvement of receptor and nonreceptor PTKs in modulating H₂O₂-induced ERK1/2 and PKB signaling.     

NEK2 overexpression aggravates IL-22-induced keratinocyte proliferation and cytokine level increases and IMQ-induced psoriasis-like dermatitis

BackgroundPsoriasis is a common inflammatory skin disease characterized by the excessive proliferation and abnormal differentiation of keratinocytes. Protein kinases could act on intracellular signaling pathways associated with cell proliferation.ObjectiveIdentifying more hub protein kinases affecting cellular and molecular processes in psoriasis, and exploring the dynamic effects of baicalin and NEK2 on the IL-22-induced cellular inflammation and IMQ-induced psoriasis-like mice.Methods and resultsIn this study, differentially expressed protein kinases playing a hub role in psoriasis initiation and development were identified using integrative bioinformatics analyses, and NEK2 has been chosen. NEK2 was significantly up-regulated in psoriatic samples according to online datasets and experimental analyses. In IL-22-induced cellular inflammation model in HaCaT cells, NEK2 overexpression promoted, whereas NEK2 knockdown partially abolished IL-22-induced alterations in cell viability, DNA synthesis, cytokine levels, as well as STAT3 phosphorylation and p-RB, cyclin D1, CDK4, and CDK6 protein contents. Baicalin treatment partially suppressed IL-22-induced HaCaT cell viability, DNA synthesis, and increases in cytokine levels, whereas NEK2 overexpression significantly abolished Baicalin-induced protection against cellular inflammation. In IMQ-induced psoriasis-like skin inflammation model in mice, baicalin markedly ameliorated IMQ-induced psoriasis-like symptoms and local skin inflammation, whereas NEK2 overexpression partially eliminated the therapeutic effects of baicalin.ConclusionNEK2, up-regulated in psoriatic lesion skin, could aggravate IMQ-induced psoriasis-like dermatitis and attenuate the therapeutic efficiency of baicalin through promoting keratinocyte proliferation and cytokine levels. The STAT3 signaling might be involved.     

Asthmatics exhibit altered oxylipin profiles compared to healthy individuals after subway air exposure

Background

Asthma is a chronic inflammatory lung disease that causes significant morbidity and mortality worldwide. Air pollutants such as particulate matter (PM) and oxidants are important factors in causing exacerbations in asthmatics, and the source and composition of pollutants greatly affects pathological implications.

Objectives

This randomized crossover study investigated responses of the respiratory system to Stockholm subway air in asthmatics and healthy individuals. Eicosanoids and other oxylipins were quantified in the distal lung to provide a measure of shifts in lipid mediators in association with exposure to subway air relative to ambient air.

Methods

Sixty-four oxylipins representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of bronchoalveolar lavage (BAL)-fluid. Validations through immunocytochemistry staining of BAL-cells were performed for 15-LOX-1, COX-1, COX-2 and peroxisome proliferator-activated receptor gamma (PPARγ). Multivariate statistics were employed to interrogate acquired oxylipin and immunocytochemistry data in combination with patient clinical information.

Results

Asthmatics and healthy individuals exhibited divergent oxylipin profiles following exposure to ambient and subway air. Significant changes were observed in 8 metabolites of linoleic- and α-linolenic acid synthesized via the 15-LOX pathway, and of the COX product prostaglandin E₂ (PGE₂). Oxylipin levels were increased in healthy individuals following exposure to subway air, whereas asthmatics evidenced decreases or no change.

Conclusions

Several of the altered oxylipins have known or suspected bronchoprotective or anti-inflammatory effects, suggesting a possible reduced anti-inflammatory response in asthmatics following exposure to subway air. These observations may have ramifications for sensitive subpopulations in urban areas.     

Mitogen-activated protein kinases as key players in osmotic stress signaling

BackgroundOsmotic stress arises from the difference between intracellular and extracellular osmolality. It induces cell swelling or shrinkage as a consequence of water influx or efflux, which threatens cellular activities. Mitogen-activated protein kinases (MAPKs) play central roles in signaling pathways in osmotic stress responses, including the regulation of intracellular levels of inorganic ions and organic osmolytes.Scope of reviewThe present review summarizes the cellular osmotic stress response and the function and regulation of the vertebrate MAPK signaling pathways involved. We also describe recent findings regarding apoptosis signal-regulating kinase 3 (ASK3), a MAP3K member, to demonstrate its regulatory effects on signaling molecules beyond MAPKs.Major conclusionsMAPKs are rapidly activated by osmotic stress and have diverse roles, such as cell volume regulation, gene expression, and cell survival/death. There is significant cell type specificity in the function and regulation of MAPKs. Based on its activity change during osmotic stress and its regulation of the WNK1-SPAK/OSR1 pathway, ASK3 is expected to play important roles in osmosensing mechanisms and cellular functions related to osmoregulation.General significanceMAPKs are essential for various cellular responses to osmotic stress; thus, the identification of the upstream regulators of MAPK pathways will provide valuable clues regarding the cellular osmosensing mechanism, which remains elusive in mammals. The elucidation of in vivo MAPK functions is also important because osmotic stress in physiological and pathophysiological conditions often results from changes in the intracellular osmolality. These studies potentially contribute to the establishment of therapeutic strategies against diseases that accompany osmotic perturbation.     

In vitro effect of air pollutants on human bronchi

A useful approach for constructing dose–response relationships and for studying the underlying mechanisms by which a xenobiotic agent enhances airway reactivity is to measure the response of an isolated airway following ex vivo exposure to a pollutant. We have in this way determined the dose–response relationship between ex vivo exposure to pollutants such as nitrogen dioxide (NO₂), the aldehyde acrolein, and ozone (O₃) and the reactivity to agonists in human isolated bronchial smooth muscle. We have also investigated the underlying alteration in the cellular mechanisms of airway smooth-muscle contraction induced by such exposure and found that it is related to alteration in calcium signaling at the site of the airway smooth-muscle cell. Finally, although there is epidemiological evidence that an increase in allergic diseases such as asthma may be linked to air pollution, there are few experimental data to address this issue. The final aim of this study was therefore to investigate the interaction between passive sensitization and exposure to pollutants in human isolated airways. We have examined (i) the effect of a pre-exposure to pollutants on the contraction of sensitized bronchi in response to a specific antigen and (ii) the effect of passive sensitization on the contraction in response to nonspecific agonists in bronchi pre-exposed to pollutants. The results indicate a combined effect of immunological sensitization and exposure to pollutants; that is, passive sensitization and exposure to pollutants act in a synergistic manner on human bronchial smooth-muscle reactivity in response to both specific antigens and nonspecific agonists. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of SO(2) and O(3) on Production of Antioxidants in Conifers

Production of antioxidants was investigated in needles of fir (Abies alba Mill.) and spruce (Picea abies (L.) Karst) after exposure to low concentrations of SO₂, O₃, and a combination of both pollutants. Glutathione reacted most sensitively to pollutants followed by vitamin E and vitamin C. In spruce needles, the overall increase of antioxidants after exposure to air pollutants was lower than in needles of fir. SO₂ was more potent than O₃. Maximum increase of antioxidants was found in needles after exposure of trees to SO₂ + O₃.     

Production of reactive oxygen species and induction of signaling pathways for the ACO gene expressions in tomato plants triggered by the volatile organic compound ether

Diethyl ether (ether), a volatile organic compound, is widely used as an industrial solvent and easily released to the environment. Acute exposure of tomato plants to high concentrations of ether caused young leaves to curl. Histochemical analyses revealed that superoxide anion (^•O₂ ⁻) and hydrogen peroxide were formed sequentially by ether, and that ^•O₂ ⁻ was the major ROS produced in response to ether exposure. We observed cell death by microscopic inspection of Evans blue-stained samples, following fumigation with ether for 6 h. The ethylene biosynthetic gene, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), was induced as early as 15–30 min after ether fumigation and could be activated at ether concentration as low as 1 μL/L. Induction of ACO gene expression occurred simultaneously with ROS accumulation and coincided with the occurrence of cell death. Simultaneous treatment of tomato plants with mechanical wounding and ether induced differential expression of the ACO gene family. Ether strongly induced ACO4 and moderately induced ACO1, whereas mechanical wounding strongly induced ACO1 and slightly induced ACO4. Induction of the ACO gene family by ether occurred via different signaling pathways. While the ACO1 gene was induced via protein phosphorylation, the ACO4 gene was induced through protein dephosphorylation. Induction of ACO1 and ACO4 might be through MPK1, MPK2, MPK3, and PP2Ac1. These results suggest that the cellular responses of tomato plants to ether are different from the plant responses to ozone, and that tomato plants respond to different air pollutants through different perceptions and downstream signaling pathways.     

Transcriptomic Analyses of the Biological Effects of Airborne PM2.5 Exposure on Human Bronchial Epithelial Cells

Epidemiological studies have associated high levels of airborne particulate matter (PM) with increased respiratory diseases. In order to investigate the mechanisms of air pollution-induced lung toxicity in humans, human bronchial epithelial cells (16HBE) were exposed to various concentrations of particles smaller than 2.5 μm (PM2.5) collected from Beijing, China. After observing that PM2.5 decreased cell viability in a dose-dependent manner, we first used Illumina RNA-seq to identify genes and pathways that may contribute to PM2.5-induced toxicity to 16HBE cells. A total of 539 genes, 283 up-regulated and 256 down-regulated, were identified to be significantly differentially expressed after exposure to 25 μg/cm² PM2.5. PM2.5 induced a large number of genes involved in responses to xenobtiotic stimuli, metabolic response, and inflammatory and immune response pathways such as MAPK signaling and cytokine-cytokine receptor interaction, which might contribute to PM2.5-related pulmonary diseases. We then confirmed our RNA-seq results by qPCR and by analysis of IL-6, CYP1A1, and IL-8 protein expression. Finally, ELISA assay demonstrated a significant association between exposure to PM2.5 and secretion of IL-6. This research provides a new insight into the mechanisms underlying PM2.5-induced respiratory diseases in Beijing.     

The P2Y6 receptor signals through Gαq/Ca2+/PKCα and Gα13/ROCK pathways to drive the formation of membrane protrusions and dictate cell migration

Cell migration is a ubiquitous process necessary to maintain and restore tissue functions. However, in cancer, cell migration leads to metastasis development and thus worsens the prognosis. Although the mechanism of cell migration is well understood, the identification of new targets modulating cell migration and deciphering their signaling events could lead to new therapies to restore tissue functions in diseases, such as inflammatory bowel disease, or to block metastatic development in different forms of cancer. Previous research has identified the G-protein-coupled P2Y₆ receptor as an innovative target that could dictate cell migration under normal and pathological conditions. Surprisingly, there is little information on the cellular events triggered by activated P2Y₆ during cell migration. Here, we demonstrated that P2Y₆ activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y₆ increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gα_q/calcium/protein kinases C (PKC) and Gα₁₃/RHO-associated protein kinase-dependent pathways that dictate the formation of the migratory structures. These pathways led to the stabilization of the actin cytoskeleton through a PKC-dependent phosphorylation of cofilin. These results support the idea that the P2Y₆ receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways.     

Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D₁-like family of dopamine receptors and inputs of aversion coming from neurons containing the D₂-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D₁ reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D₂ receptors function, we present evidence of σ₁ receptor molecular and functional interaction with dopamine D₂ receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D₂ receptors (the long isoform of the D₂ receptor) can complex with σ₁ receptors, a result that is specific to D₂ receptors, as D₃ and D₄ receptors did not form heteromers. We demonstrate that the σ₁-D₂ receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ₁ -D₂ receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ₁ knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D₂ receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D₁ and D₂ receptor containing neurons in the brain.     

Peroxyacetyl nitrate-induced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line

It has long been concerned that some secondary air pollutants such as smog components, ozone (O₃) and peroxyacetyl nitrate (PAN), are highly phytotoxic even at low concentrations. Compared with the biology of O₃, we largely lack the information on the toxicity model for PAN at the cellular signaling levels. Here, we studied the cell-damaging impact of PAN using suspension culture of smog-sensitive tobacco variety (Bel-W3). The cells were exposed to freshly synthesized PAN and the induced cell death was assessed under microscope after staining with Evans blue. Involvement of reactive oxygen species (ROS) in PAN toxicity was suggested by PAN-dependently increased intracellular H₂O₂ and also by the cell-protective effects of ROS scavengers and related inhibitors. Calcium chelator also lowered the level of PAN-induced cell death, indicating that Ca²⁺ is also involved. Using a transgenic cell line expressing aequorin, an increase in cytosolic Ca²⁺ concentration responsive to the pulse of PAN, but sensitive to Ca²⁺ channel blockers, was recorded, indicating that Ca²⁺ channels are activated by PAN or PAN-derived signals. Above data show some similarity between the signaling mechanisms responsive to O₃ and PAN.     

A traditional herbal formula,Deng-Shi-Qing-Mai-Tang,regulates TLR4/NF-κB signaling pathway to reduce inflammatory response in PM2.5-induced lung injury

BackgroundStudies revealed that particulate matter 2.5 (PM2.5) enters the lung through the respiratory tract and can damage lung tissue resulting in lung injury primarily via imbalance between pro-inflammatory and anti-inflammatory responses. Moreover, TLR4/NF-κB signaling pathways are reported to play a role in PM2.5-induced inflammation and lung injury, which is closely related to the inflammatory responses. Therefore, the traditional herbal formula, Deng-Shi-Qing-Mai-Tang (DSQMT), has been applied to improve patients' clinical symptoms with lung injury induced by PM2.5. It can reduce inflammatory reactions in lung injury and relieve cough and phlegm. However, the underlying mechanism of DSQMT treatment is still exclusive.PurposeTo clarify the preventive and therapeutic effects of DSQMT on PM2.5-induced lung injury and explore its underlying mechanism.Study designPM2.5-induced lung injury rat model was established, and DSQMT was administered.MethodsFirst, PM2.5 was collected, and PM2.5 suspension was prepared. Then, a rat model with PM2.5-induced lung injury was established, and the effects of DSQMT were evaluated in vivo. Finally, the roles of DSQMT in inhibiting the TLR4/NF-κB signaling pathway were investigated in vitro using the NR8383 cell line via Western blot analysis, real-time PCR, electrophoretic mobility shift assay (EMSA), and immunofluorescence staining, respectively and analyzed.ResultsWe found that DSQMT significantly attenuated pathological lung tissue damage and inflammatory responses in PM2.5-induced lung injury. We also found that after PM2.5 stimulation in vitro, DSQMT regulates the expression of TLR4, MyD88, IKK, IκB-α, NF-κB p65 in the TLR4/NF-κB signaling pathway. It also constrains activated NF-κB entry into the nucleus and further limits its binding to target DNA. In addition, we revealed that DSQMT down-regulated interleukin (IL)-1β, IL-6, IL-10, THF-α, NO, PGE₂ to reduce the inflammatory response.ConclusionWe demonstrated that DSQMT has preventive and therapeutic effects on PM2.5-induced lung injury by down-regulation of the TLR4/NF-κB signaling pathway. Therefore, the efficacy of traditional Chinese medicine (TCM) in PM2.5 lung injury can be taken into consideration and may be improved in the future through further researches.     

Specificity of Signaling by Hematopoietic Cytokine Receptors: Instructive Versus Permissive Effects

Abstract

The helical cytokines constitute a family of proteins with a common three-dimensional structure. They exert a wide variety of biological effects with a preference for the hematopoietic system. The effects of helical cytokines are mediated by cell surface receptors, which belong to the cytokine receptor superfamily and signal by activating cytoplasmic tyrosine kinases of the Janus kinase (Jak) family and other downstream signaling pathways. The relevance of each of these pathways for eliciting a specific cellular response remains to be determined. This review will focus on cytokine receptors which play a role in the regulation of hematopoiesis and summarize data that address the question how specificity of signaling is achieved.