Ischemia-activated microglia induces neuronal injury via activation of gp91phox NADPH oxidase

Although glial cells play a major role in the pathogenesis of many neurological diseases by exacerbating neuronal and non-neuronal cell death, the mechanisms involved are unclear. We examined the effects of microglia-(MCM) or astrocyte-(ACM) conditioned media obtained by chemical ischemia on the neuronal injury in SH-SY5Y cells. Chemical ischemia was induced by the treatment with NaN₃ and 2-deoxy-d-glucose for 2 h. MCM-treated SH-SY5Y cells showed reduced the viability, increased caspase-3 activity, decreased Bcl-2/Bax ratio, and increased cytochrome c release, increased inflammatory cytokines, and increased reactive oxygen species (ROS) generation. MCM also increased gp91phox nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which was inhibited by NADPH oxidase inhibitor, apocynin, and gp91phox siRNA. However, ACM did not show any significant changes. The results suggest that microglia activated by ischemic insult may increase reactive oxygen species generation via activation of gp91phox NADPH oxidase, resulting in neuronal injury.     

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.     

Macrophage colony-stimulating factor induces the expression of mitogen-activated protein kinase phosphatase-1 through a protein kinase C-dependent pathway.

M-CSF triggers the activation of extracellular signal-regulated protein kinases (ERK)-1/2. We show that inhibition of this pathway leads to the arrest of bone marrow macrophages at the G0/G1 phase of the cell cycle without inducing apoptosis. M-CSF induces the transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), which correlates with the inactivation of ERK-1/2. Because the time course of ERK activation must be finely controlled to induce cell proliferation, we studied the mechanisms involved in the induction of MKP-1 by M-CSF. Activation of ERK-1/2 is not required for this event. Therefore, M-CSF activates ERK-1/2 and induces MKP-1 expression through different pathways. The use of two protein kinase C (PKC) inhibitors (GF109203X and calphostin C) revealed that M-CSF induces MKP-1 expression through a PKC-dependent pathway. We analyzed the expression of different PKC isoforms in bone marrow macrophages, and we only detected PKCbetaI, PKCepsilon, and PKCzeta. PKCzeta is not inhibited by GF109203X/calphostin C. Of the other two isoforms, PKCepsilon is the best candidate to mediate MKP-1 induction. Prolonged exposure to PMA slightly inhibits MKP-1 expression in response to M-CSF. In bone marrow macrophages, this treatment leads to a complete depletion of PKCbetaI, but only a partial down-regulation of PKCepsilon. Moreover, no translocation of PKCbetaI or PKCzeta from the cytosol to particulate fractions was detected in response to M-CSF, whereas PKCepsilon was constitutively present at the membrane and underwent significant activation in M-CSF-stimulated macrophages. In conclusion, we remark the role of PKC, probably isoform epsilon, in the negative control of ERK-1/2 through the induction of their specific phosphatase.     

T cell antigen receptor engagement stimulates c-raf phosphorylation and induces c-raf-associated kinase activity via a protein kinase C-dependent pathway

The c-raf kinase has been shown to be activated following stimulation of several tyrosine kinase growth factor receptors. We examined changes in c-raf following engagement of the T cell receptor for antigen (TCR), a stimulus which activates both a non-receptor tyrosine kinase and protein kinase C (PKC). We found that activation of the T-cell receptor on the T cell hybridoma 2B4 causes a rapid and stoichiometric hyperphosphorylation of c-raf and an increase in c-raf-associated kinase activity. Phosphoamino acid analysis showed that the phosphorylation was entirely on serine residues. High-resolution phosphopeptide mapping showed the appearance of a single major new phosphopeptide with TCR stimulation. That phosphopeptide was shown to comigrate with the major new phosphopeptide induced in response to phorbol ester. When cells were depleted of PKC by pretreatment with high concentrations of phorbol ester, TCR stimulation was no longer capable of inducing c-raf-associated kinase activity. To determine whether activation of the tyrosine kinase alone would activate c-raf, we examined the 2B4 variant cell line FL.8. In response to Thy-1 stimulation, these cells activate the tyrosine kinase but not protein kinase C due to a deficiency in TCR eta chain expression. We found that in contrast to Thy-1 stimulation of 2B4 cells, stimulation of FL.8 cells does not lead to the induction of c-raf-associated kinase activity, although phorbol ester activates the kinase to an equivalent degree in both cells. We conclude that T cell receptor activation of c-raf occurs via phosphorylation by the serine/threonine kinase PKC. Activation of c-raf through PKC represents a mechanism distinct from that reported for tyrosine kinase growth factor receptors.     

PU.1 is dominant and HAF-1 supplementary for activation of the gp91(phox) promoter in human monocytic PLB-985 cells

Gp91(phox) is a key component of the phagocyte NADPH oxidase. Mutations of its promoter found in patients with chronic granulomatous disease cause deficient binding of PU.1 and HAF-1. Because the two factors bind to the same site (Pu box) of the promoter, we attempted to clarify their relative in vivo contributions to activation of the gp91(phox) promoter in monocytically differentiated PLB-985 cells using a dual luciferase reporter assay and a gel shift competition assay. We found that the activity of a series of single-point-mutated promoters increases or decreases according to an increase or decrease, respectively, in the affinity of the promoters to PU.1 but not to HAF-1. Two of 7 mutants showing weak binding affinity to PU.1 exhibited moderate promoter activity and normal binding affinity for HAF-1. These results suggest PU.1 is the dominant activator and HAF-1 is supplementary. The increased promoter activity of single-, double-, and triple-point-mutated constructs with sequences closer to that of the Ets-binding element correlates with their binding affinity to PU.1 but not to HAF-1, supporting that PU.1 is a more efficient activator than HAF-1. In contrast to co-expressed wild-type PU.1, dominant-negative PU.1 significantly inhibited the activity of a PU.1-optimised gp91(phox) promoter construct. Therefore, we conclude that PU.1 and HAF-1 binding to the Pu box is dominant and supplementary, respectively, for activation of the gp91(phox) promoter in human monocytic cells.     

Endothelin-1 induces tyrosine phosphorylation in human blood monocytes

Mediators including the neuropeptide endothelin-1 (ET-1), which are released in response to injury, modulate the expression of cell adhesion molecules on leukocytes and endothelial cells. The mechanisms underlying this process are not clear. In this study we investigated the effect of endothelin-1 on the expression of tyrosine phosphorylated proteins in human blood monocytes. Endothelin-1 caused an increase in tyrosine phosphorylated proteins in monocytes in a time-dependent and dose-dependent manner, the Mr 60, 80 and 110 kDa proteins being the most prominent. This effect was blocked by pre-incubating the monocytes with the selective tyrosine kinase inhibitors genistein or herbimycin A. Endothelin-1-induced upregulation of tyrosine phosphorylated proteins appears to be mediated by the ET_Areceptor. Unlike our previously reported studies in endothelial cells, immunoprecipitation with anti-src or anti-JAK antibodies followed by immunoblotting with PY20 in human blood monocytes revealed that these proteins of Mr 60, 80 and 110 kDa were not related to src or JAK kinases. These findings suggest that ET-1 exerts its effect on monocytes by a pathway involving tyrosine kinases other than src or JAK kinases.     

Thrombin induces nitric-oxide synthase via Galpha12/13-coupled protein kinase C-dependent I-kappaBalpha phosphorylation and JNK-mediated I-kappaBalpha degradation

An imbalance between thrombin and antithrombin III contributed to vascular hyporeactivity in sepsis, which can be attributed to excess NO production by inducible nitric-oxide synthase (iNOS). In view of the importance of the thrombin-activated coagulation pathway and excess NO as the culminating factors in vascular hyporeactivity, this study investigated the effects of thrombin on the induction of iNOS and NO production in macrophages. Thrombin induced iNOS protein in the Raw264.7 cells, which was inhibited by a thrombin inhibitor, LB30057. Thrombin increased NF-kappaB DNA binding, whose band was supershifted with anti-p65 and anti-p50 antibodies. Thrombin elicited the phosphorylation and degradation of I-kappaBalpha prior to the nuclear translocation of p65. The NF-kappaB-mediated iNOS induction was stimulated by the overexpression of activated mutants of Galpha(12/13) (Galpha(12/13)QL). Protein kinase C depletion inhibited I-kappaBalpha degradation, NF-kappaB activation, and iNOS induction by thrombin or the iNOS induction by Galpha(12/13)QL. JNK, p38 kinase, and ERK were all activated by thrombin. JNK inhibition by the stable transfection with a dominant negative mutant of JNK1 (JNK1(-)) completely suppressed the NF-kappaB-mediated iNOS induction by thrombin. Conversely, the inhibition of p38 kinase enhanced the expression of iNOS. In addition, JNK and p38 kinase oppositely controlled the NF-kappaB-mediated iNOS induction by Galpha(12/13)QL. Hence, iNOS induction by thrombin was regulated by the opposed functions of JNK and p38 kinase downstream of Galpha(12/13). In the JNK1(-) cells, thrombin did not increase either the NF-kappaB binding activity or I-kappaBalpha degradation despite I-kappaBalpha phosphorylation. These results demonstrated that thrombin induces iNOS in macrophages via Galpha(12) and Galpha(13), which leads to NF-kappaB activation involving the protein kinase C-dependent phosphorylation of I-kappaBalpha and the JNK-dependent degradation of phosphorylated I-kappaBalpha.     

Protein kinase C delta is required for p47phox phosphorylation and translocation in activated human monocytes

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCdelta, -epsilon, -theta;, and -eta) and conventional PKC (including PKCalpha and -beta), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCepsilon, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCdelta, -theta;, or -eta is required. Furthermore, rottlerin (at doses that inhibit PKCdelta activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O2 production at similar doses. In addition to pharmacological inhibitors, PKCdelta-specific antisense oligodeoxyribonucleotides were used. PKCdelta antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCdelta in vitro. Furthermore, lysate-derived PKCdelta from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCdelta plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.     

IL-12 induces monocyte IL-18 binding protein expression via IFN-gamma

IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-gamma production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-gamma, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-gamma derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-gamma production. The IFN-gamma/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-gamma and probably plays a key role in the modulation of both innate and adaptive immunity.     

Adhesion of epithelial cells to fibronectin or collagen I induces alterations in gene expression via a protein kinase C-dependent mechanism

Adhesion of human salivary gland (HSG) epithelial cells to fibronectin- or collagen I gel-coated substrates, mediated by beta1 integrins, has been shown to upregulate the expression of more than 30 genes within 3-6 h. Adhesion of HSG cells to fibronectin or collagen I for 6 h also enhanced total protein kinase C (PKC) activity by 1.8-2.3-fold. HSG cells expressed PKC-alpha, gamma, delta, epsilon, mu, and zeta. Adhesion of HSG cells to fibronectin or collagen I specifically activated PKC-gamma and PKC-delta. Cytoplasmic PKC-gamma and PKC-delta became membrane-associated, and immunoprecipitated PKC-gamma and PKC-delta kinase activities were enhanced 2.5-4.0-fold in HSG cells adherent to fibronectin or collagen I. In addition, adhesion of fibronectin-coated beads to HSG monolayers co-aggregated beta1 integrin and PKC-gamma and PKC-delta but not other PKC isoforms. Thus, integrin-dependent adhesion of HSG cells to fibronectin or collagen I activated PKC-gamma and PKC-delta. The role of this PKC upregulation on adhesion-responsive gene expression was then tested. HSG cells were treated with the specific PKC inhibitor bisindolylmaleimide I, cultured on non-precoated, fibronectin- or collagen I-coated substrates, and analyzed for changes in adhesion-responsive gene expression. Bisindolylmaleimide I strongly inhibited the expression of seven adhesion-responsive genes including calnexin, decorin, S-adenosylmethionine decarboxylase, steroid sulfatase, and 3 mitochondrial genes. However, the expression of two adhesion-responsive genes was not affected by bisindolylmaleimide I. Treatment with bisindolylmaleimide I did not affect cell spreading and did not significantly affect the actin cytoskeleton. These data suggest that adhesion of HSG cells to fibronectin or collagen I induces PKC activity and that this induction contributes to the upregulation of a variety of adhesion-responsive genes.     

Colony-stimulating factor 1 activates protein kinase C in human monocytes.

Colony-stimulating factor 1 (CSF-1) is required for the survival, proliferation and differentiation of monocytes. We previously demonstrated that the CSF-1 receptor is linked to a pertussis toxin-sensitive G protein and that the induction of Na+ influx by CSF-1 is a pertussis toxin-sensitive event. The present studies have examined activation of protein kinase C as a potential intracellular signaling event induced by the activated CSF-1 receptor. The results demonstrate that CSF-1 stimulates translocation of protein kinase C activity from the cytosol to membrane fractions. This activation of protein kinase C was sensitive to pretreatment of the monocytes with pertussis toxin. Lipid distribution studies demonstrated that phosphatidylcholine (PC) is the major phospholipid in human monocytes. Moreover, the results indicate that CSF-1 stimulation is associated with decreases in PC, but not in phosphatidylinositol (PI), levels. The absence of an effect of CSF-1 on PI turnover was confirmed by the lack of changes in inositol phosphate production. In contrast, CSF-1 stimulation was associated with increased hydrolysis of PC to phosphorylcholine and diacylglycerol (DAG) in both intact monocytes and cell-free assays. Furthermore, the increase in PC turnover induced by CSF-1 was sensitive to pertussis toxin. The results also demonstrate that the induction of Na+ influx by CSF-1 is inhibited by the protein kinase C inhibitors staurosporine and the isoquinoline derivative H7, but not by HA1004.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4

The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expression of nox4 remained unchanged. This was associated with a significant increase in the NADPH oxidase activity in membrane fractions of diabetic heart and aortic tissue. Furthermore, we observed a decreased sensitivity of diabetic vessels to acetylcholine and nitroglycerin and a decrease in both acetylcholine-stimulated NO production and phosphorylation of VASP, despite an increase in endothelial NO synthase (NOSIII) expression. In addition, xanthine oxidase activity was markedly increased in plasma and 100,000 g supernatant of cardiac tissue of diabetic rats, while myocardial mitochondrial superoxide formation was only weakly enhanced. We conclude that in addition to phagocytic NADPH oxidase, also nonphagocytic, vascular NADPH oxidase subunit nox1, uncoupled NOSIII, and plasma xanthine oxidase contribute to endothelial dysfunction in the setting of diabetes mellitus.     

The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils

Phosphorylation of p47 phagocyte oxidase, (p47(phox)), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47(phox) is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47(phox) phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47(phox). However, PD98059 weakly affected PMA-induced p47(phox) phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47(phox) induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47(phox) phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47(phox) phosphorylation either directly or indirectly via other kinases. In PMA-induced p47(phox) phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47(phox) phosphorylation. However, in fMLP-induced p47(phox) phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47(phox) when tested alone, and exerted additive inhibitory effects on p47(phox) phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47(phox). Furthermore, they strongly suggest that p47(phox) is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.