Switch activation of PI-PLC downstream signals in activated macrophages with wortmannin

Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) has been known to serve as a substrate for phosphatidylinositol 3-kinase (PI(3)K) and phosphoinositide-specific phospholipase C (PI-PLC), which can produce PtdIns(3,4,5)P(3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG), respectively. In this study, we elucidated the role of PI-PLC during the LPS-activated mouse macrophages RAW264.7 treated with PI(3)K inhibitor wortmannin. First, wortmannin treatment enhanced Ins(1,4,5)P(3) production and iNOS expression in LPS-activated macrophages. Inhibition of PI(3)K by p85 siRNA also showed an enhancement of iNOS expression. On the other hand, overexpression of PI(3)K by ras-p110 expression plasmid significantly decreased iNOS expression in LPS-activated macrophages. In addition, overexpression of wild-type or dominant-negative Akt expression plasmid did not affect the iNOS expression in LPS-activated macrophages. Second, treatment of PI-PLC inhibitor U73122 reversed the enhancement of iNOS expression, the increase of phosphorylation level of ERK, JNK and p38, and the increase of AP-1-dependent gene expression in wortmannin-treated and LPS-activated macrophages. However, NF-kappaB activity determined by EMSA assay and reporter plasmid assay did not change during LPS-activated macrophages with or without wortmannin. We propose that the inhibition of PI(3)K by wortmannin in mouse macrophages enhances the PI-PLC downstream signals, and subsequently increases the LPS induction of iNOS expression independently of Akt pathway.     

Phosphatidylinositol 3-kinase inhibitor suppresses inducible nitric oxide synthase expression in bronchiole epithelial cells in asthmatic rats

Inducible nitric oxide synthase (iNOS) is known to produce nitric oxide (NO), which is a main contributor to asthmatic airway inflammation. Recent studies have shown that phosphatidylinositol 3-kinase (PI3K) is ubiquitously expressed in airway epithelial cells and its inhibition could relieve airway inflammation and hyperresponsiveness. This study aimed to explore the interaction of PI3K and NO signaling in allergic asthma. We investigated the effects of PI3K inhibitor wortmannin on iNOS expression in bronchiole epithelial cells and NO, IL-4 and IFN-γ levels in lung tissues of asthmatic rat model, which was prepared by 10% OVA solution sensitization and 1% OVA aerosol challenge. Our results showed that the ratio of eosinophils to total cells in BALF, PI3K activity, NO and IL-4 levels in lung tissues was increased after OVA sensitization and challenge, but then was attenuated by the administration of wortmannin. In contrast, IFN-γ level in lung tissues was decreased after OVA sensitization and challenge and increased after the administration of wortmannin. The expression of iNOS protein in bronchiole epithelial cells, iNOS mRNA level and iNOS activity in lung tissues was markedly upregulated after OVA sensitization and challenge, but the upregulation was significantly antagonized by wortmannin. Taken together, these data provide evidence that PI3K functions upstream to modulate iNOS/NO signaling, which then promotes the development of airway inflammation in asthmatic animal model. PI3K inhibitor wortmannin could lead to reduced iNOS expression and NO production, therefore inhibiting airway inflammatory responses.     

A p85 subunit-independent p110alpha PI 3-kinase colocalizes with p70 S6 kinase on actin stress fibers and regulates thrombin-stimulated stress fiber formation in swiss 3T3 cells

The signaling pathways linking receptor activation to actin stress fiber rearrangements during growth factor-induced cell shape change are still to be determined. Recently our laboratory demonstrated the involvement of p70 S6 kinase (p70(s6k)) activation in thrombin-induced stress fiber formation in Swiss 3T3 cells. The present work shows that thrombin-induced p70(s6k) activation is inhibited by the PI 3-kinase inhibitors wortmannin and LY-294002. These inhibitors also significantly reduced thrombin-induced stress fiber formation, demonstrating a role for PI 3-kinase activity in this process, most likely upstream of p70(s6k). Furthermore, the p110alpha form of PI 3-kinase was localized to actin stress fibers, as was previously shown for p70(s6k), as well as to a golgi-like distribution. In contrast, PI 3-kinase p110gamma colocalized with microtubules. The PI 3-kinase p85 subunit, known to be capable of association with p110alpha, was present in a predominantly golgi-like distribution with no presence on actin filaments, suggesting the existence of distinctly localized PI 3-kinase pools. Immunodepletion of p85 from cell lysates resulted in only partial depletion of p110alpha and p110alpha-associated PI 3-kinase activity, confirming the presence of a p85-free p110alpha pool located on the actin stress fibers. Our data, therefore, point to the importance of subcellular localization of PI 3-kinase in signal transduction and to a novel action of p85 subunit-independent PI 3-kinase p110alpha in the stimulation by thrombin of p70(s6k) activation and actin stress fiber formation.     

Activation of phosphoinositide 3-kinase in response to inflammation and nitric oxide leads to the up-regulation of cyclooxygenase-2 expression and subsequent cell proliferation in mesangial cells

In this study, we showed that nitric oxide (NO) donors induced the mesangial cell proliferation and cyclooxygenase-2 (COX-2) protein expression in murine mesangial cells. An inflammatory condition [lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)] could also induce cell proliferation and significantly enhance inducible nitric oxide synthase (iNOS) and COX-2 expression. Phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, inhibited these responses. LPS/IFN-gamma-induced COX-2 expression in mesangial cells could be inhibited by iNOS inhibitor, aminoguanidine. Selective COX-2 inhibitor, NS398, was capable of inhibiting NO donor- or LPS/IFN-gamma-induced mesangial cell proliferation. Both NO donor and LPS/IFN-gamma markedly activated the PI3K activity and the phosphorylation of Akt and nuclear factor (NF)-kappaB DNA binding activity in mesangial cells, which could be inhibited by LY294002 and transfection of dominant-negative vectors of PI3K/p85 and Akt. These results indicate that a PI3K/Akt-dependent pathway involved in the NO-regulated COX-2 expression and cell proliferation in mesangial cells under inflammatory condition.     

PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy

Hepatic resection is associated with rapid proliferation and regeneration of the remnant liver. Phosphatidylinositol 3-kinase (PI3K), composed of a p85alpha regulatory and a p110alpha catalytic subunit, participates in multiple cellular processes, including cell growth and survival; however, the role of PI3K in liver regeneration has not been clearly delineated. In this study, we used the potent PI3K inhibitor wortmannin and small interfering RNA (siRNA) targeting the p85alpha and p110alpha subunits to determine whether total or selective PI3K inhibition would abrogate the proliferative response of the liver after partial hepatectomy in mice. Hepatic resection is associated with an induction in PI3K activity; total PI3K blockade with wortmannin and selective inhibition of p85alpha or p110alpha with siRNA resulted in a significant decrease in hepatocyte proliferation, especially at the earliest time points. Fewer macrophages and Kupffer cells were present in the regenerating liver of mice treated with wortmannin or siRNA to p85alpha or p110alpha, as reflected by a paucity of F4/80-positive cells. Additionally, PI3K inhibition led to an aberrant architecture in the regenerating hepatocytes characterized by vacuolization, lipid deposition, and glycogen accumulation; these changes were not noted in the sham livers. Our data demonstrate that PI3K/Akt pathway activation plays a critical role in the early regenerative response of the liver after resection; inhibition of this pathway markedly abrogates the normal hepatic regenerative response, most likely by inhibiting macrophage infiltration and cytokine elaboration and thus hepatocyte priming for replication.     

Hepatocyte growth factor induced human trophoblast motility involves phosphatidylinositol-3-kinase,mitogen-activated protein kinase,and inducible nitric oxide synthase

Hepatocyte growth factor (HGF) increases human trophoblast motility and invasion, an effect which is abrogated when inducible nitric oxide synthase (iNOS) is inhibited. In this study we have investigated the pathways involved in the regulation of trophoblast motility. Both basal and HGF-stimulated motility of the extravillous trophoblast cell line, SGHPL-4, were inhibited in a dose-dependent manner by the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, LY294002. HGF-stimulated iNOS expression was also inhibited by LY294002 and direct activation of PI3-kinase, using the peptide 740Y-P, led to an increase in iNOS expression and cell motility. Pretreatment with rapamycin, which acts at a point distal to PI3-kinase activation, also inhibited basal and HGF-stimulated motility. Inhibition of the p42/p44 mitogen activated protein kinase (MAPK) pathway but not the p38 MAPK pathway had significant inhibitory effects on HGF-stimulated but not basal trophoblast motility. Inhibition of p42/p44 MAPK also inhibited HGF-induced iNOS expression. This data demonstrate that the PI3-kinase signaling pathway is involved in basal trophoblast motility and that both MAPK and PI3-kinase signaling pathways are important in HGF-stimulated motility and iNOS expression.     

Identification of wortmannin-sensitive targets in 3T3-L1 adipocytes. DissociationoOf insulin-stimulated glucose uptake and glut4 translocation.

The current studies investigated the contribution of phosphatidylinositol 3-kinase (PI3-kinase) isoforms to insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation. Experiments involving the microinjection of antibodies specific for the p110 catalytic subunit of class I PI3-kinases demonstrated an absolute requirement for this form of the enzyme in GLUT4 translocation. This finding was confirmed by the demonstration that the PI3-kinase antagonist wortmannin inhibits GLUT4 and insulin-responsive aminopeptidase translocation with a dose response identical to that required to inhibit another class I PI3-kinase-dependent event, activation of pp70 S6-kinase. Interestingly, wortmannin inhibited insulin-stimulated glucose uptake at much lower doses, suggesting the existence of a second, higher affinity target of the drug. Subsequent removal of wortmannin from the media shifted this dose-response curve to one resembling that for GLUT4 translocation and pp70 S6-kinase. This is consistent with the lower affinity target being p110, which is irreversibly inhibited by wortmannin. Wortmannin did not reduce glucose uptake in cells stably expressing Myr-Akt, which constitutively induced GLUT4 translocation to the plasma membrane; this demonstrates that wortmannin does not inhibit the transporters directly. In addition to elucidating a second wortmannin-sensitive pathway in 3T3-L1 adipocytes, these studies suggest that the presence of GLUT4 on the plasma membrane is not sufficient for activation of glucose uptake.     

Extracellular Signal-regulated Kinase Mediates Expression of Arginase II but Not Inducible Nitric-oxide Synthase in Lipopolysaccharide-stimulated Macrophages

The mitogen-activated protein kinases (MAPK) have been shown to participate in iNOS induction following lipopolysaccharide (LPS) stimulation, while the role of MAPKs in the regulation of arginase remains unclear. We hypothesized that different MAPK family members are involved in iNOS and arginase expression following LPS stimulation. LPS-stimulated RAW 264.7 cells exhibited increased protein and mRNA levels for iNOS, arginase I, and arginase II; although the induction of arginase II was more robust than that for arginase I. A p38 inhibitor completely prevented iNOS expression while it only attenuated arginase II induction. In contrast, a MEK1/2 inhibitor (ERK pathway) completely abolished arginase II expression while actually enhancing iNOS induction in LPS-stimulated cells. Arginase II promoter activity was increased by ∼4-fold following LPS-stimulation, which was prevented by the ERK pathway inhibitor. Arginase II promoter activity was unaffected by a p38 inhibitor or JNK pathway interference. Transfection with a construct expressing a constitutively active RAS mutant increased LPS-induced arginase II promoter activity, while transfection with a vector expressing a dominant negative ERK2 mutant or a vector expressing MKP-3 inhibited the arginase II promoter activity. LPS-stimulated nitric oxide (NO) production was increased following siRNA-mediated knockdown of arginase II and decreased when arginase II was overexpressed. Our results demonstrate that while both the ERK and p38 pathways regulate arginase II induction in LPS-stimulated macrophages, iNOS induction by LPS is dependent on p38 activation. These results suggest that differential inhibition of the MAPK pathway may be a potential therapeutic strategy to regulate macrophage phenotype.     

Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway.

The signaling events which mediate activation of c-Jun N-terminal kinase (JNK) are not yet well characterized. To broaden our understanding of upstream mediators which link extracellular signals to the JNK pathway, we investigated the role of phosphatidylinositol (PI) 3-kinase in epidermal growth factor (EGF)-mediated JNK activation. In this report we demonstrate that a dominant negative form of PI 3-kinase as well as the inhibitor wortmannin blocks EGF-induced JNK activation dramatically. However, wortmannin does not have an effect on JNK activation induced by UV irradiation or osmotic shock. In addition, a membrane-targeted, constitutively active PI 3-kinase (p110beta) was shown to produce in vivo products and to activate JNK, while a kinase-mutated form of this protein showed no activation. On the basis of these experiments, we propose that PI 3-kinase activity plays a role in EGF-induced JNK activation in these cells.