Usamah S. Kayyali

Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia

The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre- and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Posttranslational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased (∼50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an ∼4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.

Cytoskeletal Changes in the Brains of Mice Lacking Calcineurin Aα

Abstract: Hyperphosphorylated τ, the major component of the paired helical filaments of Alzheimers disease, was found to accumulate in the brains of mice in which the calcineurin Aα gene was disrupted [calcineurin Aα knockout (CNAα−/−)]. The hyperphosphorylation involved several sites on τ, especially the Ser396 and/or Ser404 recognized by the PHF‐1 monoclonal antibody. The increase in phosphorylated τ content occurred primarily in the mossy fibers of the CNAα−/− hippocampus, which contained the highest level of calcineurin in brains of wild‐type mice. The CNAα−/− mossy fibers also contained less neurofilament protein than normal, although the overall level of neurofilament phosphorylation was unchanged. In the electron microscope, the mossy fibers of CNAα−/− mice exhibited abnormalities in their cytoskeleton and a lower neurofilament/microtubule ratio than those of wild‐type animals. These findings indicate that hyperphosphorylated τ can accumulate in vivo as a result of reduced calcineurin activity and is accompanied by cytoskeletal changes that are likely to have functional consequences on the affected neurons. The CNAα−/− mice were found in a separate study to have deficits in learning and memory that may result in part from the cytoskeletal changes in the hippocampus.

Smooth Muscle α-Actin Expression and Myofibroblast Differentiation by TGFβ are Dependent Upon MK2

Fibroblasts play a major role in processes such as wound repair, scarring, and fibrosis. Differentiation into myofibroblasts, characterized by upregulation of smooth muscle α‐actin (smα) in response to profibrotic agents such as TGFβ is believed to be an important step in fibrosis. Therefore, elucidating mechanisms of myofibroblast differentiation might reveal novel targets in treating diseases such as idiopathic pulmonary fibrosis (IPF). MK2 is a kinase substrate of p38 MAP kinase that mediates some effects of p38 activation on the actin cytoskeleton. Using mouse embryonic fibroblasts (MEF) from MK2 knockout (MK2−/−) mice, we demonstrate that disrupting expression of MK2 expression reduces filamentous actin and stress fibers. It also causes MK2−/− MEF to express less smα than their corresponding wild‐type (WT) MEF at baseline and in response to TGFβ. Furthermore, TGFβ causes downregulation of smα in MK2−/− MEF, instead of upregulation observed in WT MEF. Expression of other fibroblast markers, such as collagen, is not altered in MK2−/− MEF. Our results further suggest that downregulation of smα in MK2−/− MEF is not due to lack of activation of serum responsive promoter elements, but probably due to reduced smα message stability in these cells. These results indicate that MK2 plays a key role in regulation of smα expression, and that targeting MK2 might present a therapeutic approach in managing conditions such as pulmonary fibrosis. J. Cell. Biochem. 100: 1581–1592, 2007.

Regulation of vimentin intermediate filaments in endothelial cells by hypoxia

Hypoxia triggers responses in endothelial cells that play roles in many conditions including high-altitude pulmonary edema and tumor angiogenesis. Signaling pathways activated by hypoxia modify cytoskeletal and contractile proteins and alter the biomechanical properties of endothelial cells. Intermediate filaments are major components of the cytoskeleton whose contribution to endothelial physiology is not well understood. We have previously shown that hypoxia-activated signaling in endothelial cells alters their contractility and adhesiveness. We have also linked p38-MAP kinase signaling pathway leading to HSP27 phosphorylation and increased actin stress fiber formation to endothelial barrier augmentation. We now show that vimentin, a major intermediate filament protein in endothelial cells, is regulated by hypoxia. Our results indicate that exposure of endothelial cells to hypoxia causes vimentin filament networks to initially redistribute perinuclearly. However, by 1 hour hypoxia these networks reform and appear more continuous across cells than under normoxia. Hypoxia also causes transient changes in vimentin phosphorylation, and activation of PAK1, a kinase that regulates vimentin filament assembly. In addition, exposure to 1 hour hypoxia increases the ratio of insoluble/soluble vimentin. Overexpression of phosphomimicking mutant HSP27 (pmHSP27) causes changes in vimentin distribution that are similar to those observed in hypoxic cells. Knocking-down HSP27 destroys the vimentin filamentous network, and disrupting vimentin filaments with acrylamide increases endothelial permeability. Both hypoxia- and pmHSP27 overexpression-induced changes are reversed by inhibition of phosphatase activity. In conclusion hypoxia causes redistribution of vimentin to a more insoluble and extensive filamentous network that could play a role in endothelial barrier stabilization. Vimentin redistribution appears to be mediated through altering the phosphorylation of the protein and its interaction with HSP27.

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HVT MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

Stress induced gene expression: a direct role for MAPKAP kinases in transcriptional activation of immediate early genes

Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in a signal specific manner. Stress-activated p38α MAP kinase is implicated in transcriptional regulation of IEGs via MSK-mediated CREB phosphorylation. The protein kinases downstream to p38, MAPKAP kinase (MK) 2 and MK3 have been identified to regulate gene expression at the posttranscriptional levels of mRNA stability and translation. Here, we analyzed stress-induced IEG expression in MK2/3-deficient cells. Ablation of MKs causes a decrease of p38α level and p38-dependent IEG expression. Unexpectedly, restoration of p38α does not rescue the full-range IEG response. Instead, the catalytic activity of MKs is necessary for the major transcriptional activation of IEGs. By transcriptomics, we identified MK2-regulated genes and recognized the serum response element (SRE) as a common promoter element. We show that stress-induced phosphorylation of serum response factor (SRF) at serine residue 103 is significantly reduced and that induction of SRE-dependent reporter activity is impaired and can only be rescued by catalytically active MK2 in MK2/3-deficient cells. Hence, a new function of MKs in transcriptional activation of IEGs via the p38α-MK2/3-SRF-axis is proposed which probably cooperates with MKs’ role in posttranscriptional gene expression in inflammation and stress response.

Upregulation of xanthine oxidase by tobacco smoke condensate in pulmonary endothelial cells

Tobacco smoking has been causally linked to the development of chronic obstructive pulmonary disease. It has been reported that the reactive oxygen species (ROS)- generating enzyme xanthine dehydrogenase/oxidase (XO) is increased in smoking-related stomach ulcers and that gastric mucosal damage caused by tobacco smoke can be blocked by the XO inhibitor allopurinol. In order to test the hypothesis that tobacco may cause the upregulation of XO in the lung, cultured rat pulmonary microvascular endothelial cells were exposed to tobacco smoke condensate (TSC). TSC at a concentration of 20 microg/mL significantly upregulated XO activity after 24 h of exposure. Longer exposure (1 week) to a lower concentration of TSC (2 microg/mL) also caused an increase in XO activity. Unlike hypoxia, TSC treatment did not alter the phosphorylation of XO. However, TSC treatment increased XO mRNA expression and the XO gene promoter activity. Furthermore, actinomycin D blocked the activation of XO by TSC. In conclusion, our results indicate that tobacco smoke condensate causes upregulation of XO transcription and activity.

The involvement of proteases, protease inhibitors, and an acute phase response in Alzheimer's disease.

Proteases participate in many key processes in cellular and organismic physiology. They are an extremely versatile, heterogeneous collection of proteins directed against a large number of targets, and controlled by a whole family of protease inhibitors. I t is also becoming increasingly clear that proteases and their inhibitors are involved in many aspects of the formation of the amyloid deposits of Alzheimer’s disease. Indeed, experiments in our and other laboratories suggest that the pathology in Alzheimer’s disease may result from an imbalance in the normal neuronal-glial and protease-antiprotease interactions in the brain, leading to amyloid formation and neuronal cell death. In this paper we shall review the data supporting this conclusion, and present new results suggesting that these imbalances reflect an “acute phase resp0nse”that is elicited in certain areas of the brain by the accumulation of p/A4 amyloid deposits and involve both proteases and protease inhibitors produced by nonneuronal cells. The importance of proteases and protease inhibitors in Alzheimer’s disease first became apparent when it was found that the &’A4 protein, the major component of the Alzheimer amyloid deposits, is proteolytically derived from a precursor protein, /3-APP.’-5 At the same time, we found that the protease inhibitor a,-antichymotrypsin (ACT) was an integral component of the amyloid deposits.6 It was then discovered that, through alternative splicing, some forms of fl-APP contained an extra domain with Kunitz-type protease inhibitory activity.’-’ Clearly, the study of Alzheimer amyloid formation would require a basic understanding of proteases and their inhibitors. There are a number ofways that proteases and protease inhibitors might be involved in the formation of the amyloid deposits. First, at least one protease, and probably two, were involved in the cleavage of &’A4 from its precursor. Significantly, the cleavage at the N-terminus ofB/A4 occurs following a methionine-a target of chymotrypsin-like proteases. The presence of ACT, a protease inhibitor directed against

Modulation of HSP27 alters hypoxia‐induced endothelial permeability and related signaling pathways

This manuscript describes how the permeability of pulmonary artery microvascular endothelial cell (RPMEC) monolayer is elevated by hypoxia and the role played by HSP27 phosphorylation. p38 MAP kinase activation leading to HSP27 phosphorylation was previously shown by our laboratory to alter the actin cytoskeleton and tethering properties of RPMEC. This effect was independent of hypoxia‐induced contractility which was ROCK‐dependent rather than HSP27‐dependent. Results described here show that increased HSP27 phosphorylation not only does not underlie hypoxia‐induced permeability, but may actually augment the endothelial barrier. Hypoxia causes gap formation between RPMEC and increases MLC2 phosphorylation. The phosphorylation of MYPT1, which inhibits MLC2 phosphatase, is also increased in hypoxia. In addition, FAK phosphorylation, which alters focal adhesion signaling, is increased in hypoxia. Overexpressing phosphomimicking HSP27 (pmHSP27), which induces significant actin stress fiber formation, surprisingly renders RPMEC resistant to hypoxia‐ or TGFβ‐induced permeability. siRNA against pmHSP27 reverses the increased actin stress fiber formation in pmHSP27‐overexpressing cells, and disrupting actin stress fibers in pmHSP27‐overexpressing RPMEC renders them more susceptible to hypoxia. Finally, hypoxia‐induced gap formation, as well as phosphorylation of MLC2, MYPT1 and FAK are almost abolished by overexpressing pmHSP27 in RPMEC. These effects of pmHSP27 overexpression might represent decreased cytoskeletal plasticity and increased tethering which counteracts permeability‐inducing contractility. Thus hypoxia activates two pathways one leading to contractility and increased permeability, the other leading to actin stress fibers, stronger adhesion, and reduced permeability. Altering HSP27 phosphorylation, which tips the balance towards decreased permeability, might be targeted in managing endothelial barrier dysfunction. J. Cell. Physiol. 220: 600–610, 2009.

Anthrax lethal toxin disrupts the endothelial permeability barrier through blocking p38 signaling.

Exposure to anthrax causes life‐threatening disease through the action of the toxin produced by the Bacillus anthracis bacteria. Lethal factor (LF), an anthrax toxin component which causes severe vascular leak and edema, is a protease which specifically degrades MAP kinase kinases (MKK). We have recently shown that p38 MAP kinase activation leading to HSP27 phosphorylation augments the endothelial permeability barrier. We now show that treatment of rat pulmonary microvascular endothelial cells with anthrax lethal toxin (LeTx), which is composed of LF and the protective antigen, increases endothelial barrier permeability and gap formation between endothelial cells through disrupting p38 signaling. LeTx treatment increases MKK3b degradation and in turn decreases p38 activity at baseline as well as after activation of p38 signaling. Consequently, LeTx treatment decreases activation of the p38 substrate kinase, MK2, and the phosphorylation of the latters substrate, HSP27. LeTx treatment disrupts other signaling pathways leading to suppression of Erk‐mediated signaling, but these effects do not correlate with LeTx‐induced barrier compromise. Overexpressing phosphomimicking (pm)HSP27, which protects the endothelial permeability barrier against LeTx, blocks LeTx inactivation of p38 and MK2, but it does not block MKK3b degradation or Erk inactivation. Our results suggest that LeTx might cause vascular leak through inactivating p38‐MK2‐HSP27 signaling and that activating HSP27 phosphorylation specifically restores p38 signaling and blocks anthrax LeTx toxicity. The fact that barrier integrity could be restored by pmHSP27 overexpression without affecting degradation of MKK3b, or inactivation of Erk, suggests a specific and central role for p38‐MK2‐HSP27 in endothelial barrier permeability regulation. J. Cell. Physiol. 227: 1438–1445, 2012.