Wortmannin Research Articles

Regulation of D6 chemokine scavenging activity by ligand- and Rab11-dependent surface up-regulation

AbstractThe decoy receptor D6 plays a nonredundant role in the control of inflammatory processes through scavenging of inflammatory chemokines. However it remains unclear how it is regulated. Here we show that D6 scavenging activity relies on unique trafficking properties. Under resting conditions, D6 constitutively recycled through both a rapid wortmannin (WM)–sensitive and a slower brefeldin A (BFA)–sensitive pathway, maintaining low levels of surface expression that required both Rab4 and Rab11 activities. In contrast to “conventional” chemokine receptors that are down-regulated by cognate ligands, chemokine engagement induced a dose-dependent BFA-sensitive Rab11-dependent D6 re-distribution to the cell membrane and a corresponding increase in chemokine degradation rate. Thus, the energy-expensive constitutive D6 cycling through Rab11 vesicles allows a rapid, ligand concentration–dependent increase of chemokine scavenging activity by receptor redistribution to the plasma membrane. D6 is not regulated at a transcriptional level in a variety of cellular contexts, thus ligand-dependent optimization of its scavenger performance represents a rapid and unique mechanism allowing D6 to control inflammation.

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca 2+ concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.

High LET heavy ion radiation induces lower numbers of initial chromosome breaks with minimal repair than low LET radiation in normal human cells

We investigated the earliest possible chromosome break and repair process in normal human fibroblasts irradiated with low and high LET (linear energy transfer) heavy ion radiation using the modified premature chromosome condensation (PCC) technique utilizing wortmannin (WM) during the fusion incubation period [M. Okada, S. Saito, R. Okayasu, Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortannin to G1-type PCC fusion incubation, Mutat. Res., 562 (2004) 11–17]. The initial numbers of breaks were approximately 10/cell/Gy in X-irradiated samples, followed by carbon (LET: 70 keV/μm), neon, and the number was around 5/cell/Gy in silicon (LET: 70 and 200 keV/μm) and iron (LET: 200 keV/μm) samples. If WM was not used, the initial numbers of breaks with silicon and iron were higher than those of X-rays. To quantify these data, we used initial repair ratio (IRR) defined as the number of G1 PCC breaks with WM divided by the number of breaks without WM. X-irradiation gave the maximum IRR (∼2.0), while iron as well as silicon irradiation showed the minimum IRR (∼1.0), suggesting almost no rejoining at the initial stage. Although there is a comparatively good correlation between the IRR value and the cell survival, the survival fraction with the repair data at 2 or 6 h correlates better statistically. Our data indicate that high LET heavy ion irradiation induces a lower number of initial chromosome breaks with minimal repair when compared with low LET irradiation. These results at the chromosome level substantiate and extend the notion that high LET radiation produces complex-type DNA double strand breaks (DSBs).

Activation of the Small GTPase Rac2 via the B Cell Receptor Regulates B Cell Adhesion and Immunological-Synapse Formation

The integrin leukocyte function-associated antigen-1 (LFA-1) is important in the promotion of B cell adhesion, thereby facilitating immunological synapse (IS) formation and B cell activation. Despite this significance, the associated signaling mechanisms regulating LFA-1 activation remain elusive. Here, we show that both isoforms of the small GTPase Rac expressed by primary B cells, Rac1 and Rac2, were activated rapidly downstream of Src-family kinases, guanine-nucleotide exchange factors Vav1 and Vav2, and phosphoinositide-3 kinase (PI3K) after BCR engagement. We identify Rac2, but not Rac1, as critical for B cell adhesion to intercellular adhesion molecule-1 (ICAM-1) and IS formation. Furthermore, B cells expressing constitutively active Rac2 are highly adhesive. We observe that Rac2-deficient B cells exhibit lower amounts of Rap1-GTP and severe actin polymerization defects, identifying a potential mechanism underlying their behavior. We postulate that this critical role for Rac2 in mediating B cell adhesion and IS formation might apply in all lymphocytes.

Phosphatidylinositol 3‐ and 4‐phosphate modulate actin filament reorganization in guard cells of day flower

Phosphatidylinositol 3-kinases (PtdIns 3-kinases) that produce phosphatidylinositol (3,4,5) triphosphate (PtdIns(3,4,5)P(3)) are considered to be important regulators of actin dynamics in animal cells. In plants, neither PtdIns(3,4,5)P(3) nor the enzyme that produces this lipid has been reported. However, a PtdIns 3-kinase that produces phosphatidylinositol 3-phosphate (PtdIns3P) has been identified, suggesting that PtdIns3P, instead of PtdIns(3,4,5)P(3), regulates actin dynamics in plant cells. Phosphatidylinositol 4-kinase (PtdIns 4-kinase) is closely associated with the actin cytoskeleton in plant cells, suggesting a role for this lipid kinase and its product phosphatidylinositol 4-phosphate (PtdIns4P) in actin-related processes. Here, we investigated whether or not PtdIns3P or PtdIns4P plays a role in actin reorganization induced by a plant hormone abscisic acid (ABA) in guard cells of day flower (Commelina communis). ABA-induced changes in actin filaments were inhibited by LY294002 (LY) and wortmannin (WM), inhibitors of PtdIns3P and PtdIns4P synthesis. Expression of PtdIns3P- and PtdIns4P-binding domains also inhibited ABA-induced actin reorganization in a manner similar to LY and WM. These results suggest that PtdIns3P and PtdIns4P regulate actin dynamics in guard cells. Furthermore, we demonstrate that PtdIns3P exerts its effect on actin dynamics, at least in part, via generation of reactive oxygen species (ROS) in response to ABA.

IN THIS ISSUE

![Figure][1]</img> There is growing evidence that PI3Ks are involved in chemotaxis and motility of B and T cells. On p. [2261][2] , Matheu et al. saw that fluorescent-tagged B and T cells treated with wortmannin (WMN), the covalent PI3K catalytic domain inhibitor, had less migration from peripheral

Mixed polymeric micelles for combination cancer chemotherapy through the concurrent delivery of multiple chemotherapeutic agents

As a novel drug nanocarrier for combination delivery of multiple anticancer agents, mixed polymeric micelles were developed and characterized in this study. A DNA-damaging anthracycline agent doxorubicin (DOX) and a phosphatidylinositol-3 kinase inhibitor wortmannin (WOR) were conjugated alone or combination onto poly(ethylene glycol)-poly(aspartate hydrazide) block copolymers through a hydrazone bond. Polymer–drug conjugates assembled into a unimodal micelle structure with a < 100 nm particle size, in which the drug mixing ratios between DOX/WOR were precisely controlled. Cytotoxicity assay against a human breast cancer MCF-7 cell line showed that drug nanocarrier based on mixed polymeric micelles can reduce DOX required for cytotoxicity while maintaining the biological activity of the independent polymeric micelles. It is postulated that WOR enhances the efficacy of DOX through an efficient combination delivery accompanied by synergistic drug action. These findings, therefore, bring an effective drug delivery methodology that might reduce the effective dose as well as toxicity in vivo compared to the conventional drug formulations.

Role of PI3-Kinase and PI4-Kinase in Actin Polymerization During Bovine Sperm Capacitation1

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP(2(4,5)), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 microM, respectively. PI4K activity and PIP(2(4,5)) production were blocked by 10 microM WT but not by 10 nM WT, whereas PI3K activity and PIP(3(3,4,5)) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP(2(4,5)) and F-actin formation. The increases in PIP(2(4,5)) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP(2(4,5)), PIP(3(3,4,5)), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP(2(4,5)) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP(3(3,4,5)) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.

Inhibition of protein kinase B by palmitate in the insulin signaling of HepG2 cells and the preventive effect of arachidonic acid on insulin resistance

Elevated plasma levels of free fatty acids (FFAs) may contribute to insulin resistance (IR) that is characteristic of type 2 diabetes mellitus. In this study, we investigated the effects of two fatty acids, palmitate (PA) and arachidonic acid (AA) on glycogenesis under insulin signaling in HepG2cells, a transformed hepatic carcinoma cell line. In the presence of 200 μmol of palmitate, insulin (10(-7) mol/L) stimulation of glycogenesis was inhibited, as evidenced by increased glucose in the medium and decreased intracellular glycogen. Wortmannin (WM), a specific inhibitor of PI3K, dramatically decreased the amount of intracellular glycogen in cells without PA incubation. However, glycogen in PA treated cells was not significantly changed by WM, indicating that PA may also act on PI3K. Interestingly, AA restored the effects of WM inhibition on glycogenesis in PA cells. Western blot analysis demonstrated that PA in the absence of WM increased phosphorylated glycogen synthase (inactive form of GS) and decreased phosphorylated protein kinase B (active form of PKB), causing a reduction of intracellular glycogen. AA, however, reversed the effects of PA on GS and PKB. Furthermore, inhibition of protein kinase C (PKC) by a specific inhibitor chelerythrine chloride (CC) abolished the inhibitory effect of PA on glycogen synthesis by decreasing phosphorylated GS and increasing phosphorylated PKB. However, the effect of CC in the presence of PA disappeared when AA was also present.Our results suggest that there is a disruption of the insulin signaling pathway between PKB and GS when the cells were exposed to PA, contributing to IR. PA may also interrupt the PKC signaling pathway. In contrast, AA could rescue glycogenesis impaired by PA.

Protein kinase B inhibitor enhance sensitivity of gastric cancer cell to etoposide

To observe the effects of etoposide on protein kinase B (PKB) activity in distinct differentiated gastric cancer cell lines and the change of sensitivity to etoposide after pretreatment by wortmannin, a PKB inhibitor. To explore the relationship between PKB activity in gastric cancer cells and their sensitivity to etoposide chemotherapy. Four distinct differentiated gastric cancer cell lines, including MKN-28 (well differentiated), SGC-7901 (moderate differentiated), BGC-823 (poorly differentiated) and HGC-27 (undifferentiated), were studied. The PKB activities of these cell lines were detected by nonradioactive protein-kinase assay at different time points after etoposide treatment for 0,3,6,12,24 h with or without wortmannin pretreatment. Cell viabilities were assayed by MTT and cell apoptosis was analyzed by flow cytometry. Poorer differentiated gastric cancer cell lines had higher PKB activities. Etoposide treatment resulted in increase in PKB activity and apoptosis rate,and decrease in cell survival rate in a time-dependent manner in gastric cancer cell lines. Wortmannin pretreatment abolished PKB activity completely in gastric cancer cells,and decreased survival rate and increased apoptosis rate in SGC-7901, BGC-823, and HGC-27 cell lines. Etoposide can induce the PKB activity in gastric cancer cell lines. Wortmannin pretreatment enhances sensitivity of median and low differentiated gastric cancer cells to etoposide chemotherapy.

Expression of phosphatidylinositol 3-kinase and effects of wortmannin on the expression of tumor necrosis factor-alpha in ulcerative colitis

To elucidate the role of phosphatidylinositol 3-kinase (PI3K)/Akt in the pathogenesis of ulcerative colitis (UC) and provide experimental evidence that PI3K inhibitor wortmannin can be used as a possible novel approach for treatment of UC. Samples of intestinal mucosa were collected from 30 UC patients, 22 males and 8 females, aged 35 +/- 11, during enteroscopy. Samples of normal intestinal mucosa 10 cm beyond the cancerous tissues were collected from 15 patients with intestinal cancer, 9 males and 6 females, aged 40 +/- 9, as normal controls. The samples underwent pathological examination and immunohistochemistry. Another tissues of intestinal mucosa were cultured and divided into 3 groups: UC + wortmannin group, (n = 10, wortmannin, an inhibitor of PI3K/Akt pathway, of the concentration of 0.002 nmol/microl was added), UC control group (n = 10, without addition of wortmannin), and peri-cancer normal intestinal tissue group (n = 10). 4.5 hours after the culture, immunohistochemistry was used to detect the expression of phosphorylated Akt (p-Akt) in the intestinal mucosa and ELISA was used to detect the content of tumor necrosis factor (TNF-alpha) in intestinal mucosa. (1) The A value of p-Akt in the intestinal mucosa of the UC control group was 73.6 +/- 5.2, significantly higher than that of the normal control group (18.0 +/- 2.6, P < 0.05), the positive area of the UC control group was 720 +/- 58, significantly larger than that of the normal control group (133 +/- 29, P < 0.05). (2) The level of TNF-alpha in intestinal mucosa of the UC + wortmannin group was 135 +/- 11, significantly lower than that of the UC control group (296 +/- 39, P < 0.05), however, still significantly higher than that of the normal control group (26 +/- 5, P < 0.05). (3) The A value of p-Akt in the intestinal mucosa biopsy specimens of the UC + wortmannin group was 35.3 +/- 5.6, significantly lower than that of the UC control group (72.3 +/- 6.2, P < 0.05), however, still significantly higher than that of the normal control group (18.0 +/- 2.2, P < 0.05); and the positive area of the UC + wortmannin group was 351 +/- 50, significantly lower than that of the UC control group (716 +/- 94, P < 0.05), however, still significantly higher than that of the normal control group (129 +/- 30, P < 0.05). (1) PI3K/Akt signal transduction pathway is a critical factor in regulating the expression of pro-inflammatory cytokine, and plays a role in the pathogenesis of UC. (2) Decreasing the levels of relevant cytokines in UC by inhibiting PI3K/Akt signal transduction pathway, wortmannin may be a novel approach for the treatment of UC.

Ca2+‐independent thr18 regulatory myosin light chain phosphorylation and contraction in vascular smooth muscle

Evidence for a Ca2+-independent contraction in vascular smooth muscle (VSM) involving phosphorylation of regulatory myosin light chain (rMLC) by kinases other than myosin light chain kinase (MLCK) has been suggested. In this study we used both immunoblot and tandem mass spectrometry (FT-MS) techniques to investigate non-MLCK-dependent contraction in rat mesenteric VSM. Comparison of signals obtained using non-specific rMLC antibody, and specific phos-ser19 and phos-thr18 rMLC antibodies, indicated that Ca2+-dependent VSM contraction was accompanied primarily by increases in ser19 rMLC phosphorylation, which was sensitive to the MLCK inhibitor wortmannin (WOR). In contrast, Ca2+-independent contraction in the presence of the MLC phosphatase inhibitor calyculin A was accompanied primarily by increases in thr18 rMLC phosphorylation which was insensitive to WOR, but sensitive to the non-specific kinase inhibitor staurosporine (STR). Calyculin A, in either Ca2+or Ca2+ free conditions, also resulted in prominent diphos-thr18/ser19 rMLC bands, which were sensitive only to STR. Analysis of the FT-MS data obtained from in situ trypsin digested rMLC antibody-identified bands confirmed the identity of the rMLC proteins, as well as phos-ser19 in the presence of Ca2+ and phos-thr18 in the absence of Ca2+. These data suggest that a Ca2+- and MLCK-independent contraction pathway involving thr18 phosphorylation of rMLC is unmasked in the presence of calyculin. This work was supported by NIH R01GM055849 (to WAB).

SPHINGOSINE‐1‐PHOSPHATE INDUCED SMOOTH MUSCLE CELL MIGRATION REQUIRES RHO DEPENDENT AKT ACTIVATION

Background: S-1-P stimulates migration of smooth muscle cells in vitro through Gαi G-proteins and MAPK activation and has been shown to utilize the small GTPase, rho. S-1-P will activate akt, which can regulates multiple cellular functions including migration. akt activation is downstream of phosphatidyl - inositol 3 kinase (PI3 - K). The control of akt signaling during migration in response to S-1-P is poorly understood. Methods: Murine arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P with and without an akt inhibitor (aktI). Western blotting was performed for PI3-K, akt and GSK3β activation in the presence of the PI3-K inhibitors, Wortmannin (WN) and LY294002 (LY), aktI, the rho inhibitor, C3 and the rho kinase inhibitor, Y27632. Additional experiments were performed with transfections of siRNA to rho to decrease rho expression in SMC. Results: S-1-P produced a dose-dependent cell migration in both migration assays and induced PI3-K and akt activation. The migratory responses in both assays to S-1-P were blocked by aktI. Inhibition of PI3-K significantly reduced akt activation. Activation of akt and dephosphorylation of its downstream kinase, GSK3β, were inhibited by aktI. S-1-P activated rho and this activation was inhibited by C3, but not by Y27632. In the presence of C3, the rho inhibitor, PI3-K activation and akt phosphorylation were significantly reduced in response to S-1-P. Inhibition of rho kinase had no effect. siRNA to rho decreased rho expression by >80% without affecting cell viability and resulted in significantly decreased PI3-K activation and akt phosphorylation in response to S-1-P. Conclusion: S-1-P mediated migration is akt dependent and is is controlled by rho dependent PI3-K activation.

G-CSF and Cardioprotection—Deeper Experimental Insights

Myocardial infarction is the most common cause of cardiac morbidity and mortality in many countries. Several cytokines including G-CSF and erythropoietin have already demonstrated beneficial effects on cardiac remodeling after myocardial infarction [1–4]. In an experimental setting G-CSF improved cardiac function and mortality after myocardial infarction in mice, possibly by unknown regenerative process including the induction of proliferation, survival and differentiation of hematopoietic cells, as well as mobilization of bone marrow cells. Although bone marrow cells were claimed to differentiate into cardiomyocytes and vascular cells, thereby contributing to regeneration of myocardium and angiogenesis in ischemic hearts, accumulating evidence has questioned this concept [5]. Conversely, there is emerging evidence for other molecular mechanisms of G-CSF such as enhanced activation of Stat3 in the infarcted heart [6]. The role of G-CSF-induced Stat3 activation was investigated in transgenic mice with expression of dominant-negative Stat3 in cardiomyocytes under the control of the !-myosin heavy chain promoter (dnStat3-Tg). Administration of G-CSF was started at the time of coronary artery ligation until day 4 (Tg-G mice), while a control group of dnStat3-Tg mice given myocardial infarction received saline (Tg-cont). In this setting G-CSF resulted in smaller infarcts and less left ventricular remodeling at two weeks. The beneficial effects of GCSF on cardiac function were dose dependent and most pronounced with early undelayed treatments [6]. Takahama et al. investigated the acute effects of a clinical relevant dose of G-CSF on ischemia/reperfusion injury including both lethal arrhythmia and infarct size in canine hearts in a setting likely to simulate the human infarction reperfusion setting. For proof of concept intracoronary administration of wortmannin (WTMN), a PI3K inhibitor, was selectively administered into the infarct artery (1.5 2g/kg/min) for 60 min after the onset of reperfusion (GCSF + WTMN group, n = 7) with the effect of counteracting the antiapoptotic effects of phosphorylation and enhanced expression of Akt proteins [7]. Moreover, suppression of ventricular tachycardias by G-CSF is a novel aspect of this interesting study [7] and has been recently demonstrated for the combination of G-SCF/SCF; inducibility of ventricular tachycardias during programmed stimulation was reduced 5 weeks after G-CSF/SCF treatment. G-CSF/SCF increased cardiomyocyte diameter, arteriogenesis, and expression of connexin43 in the border zone of the infarction [8]. In summary the group of Takahama could elegantly demonstrate in a vivo model that acute cardioprotective effects of G-CSF applicated in a clinical dose at the onset of reperfusion relies on the PI3K/Aktpathway. Unfortunately, the potential risk of aggravated instent restenosis rate [9] by G-CSF was not investigated in this study. But there is mounting evidence that GCSF accelerates reendothelialization and inhibits neointimal thickening after vascular injury [10, 11]. In parallel, clinical studies on safety and feasibility of G-CSF in acute myocardial infarction have surfaced [12–16]. Considering those encouraging findings, the application of G-CSF in a reperfusion setting could be a non-invasive option to ameliorate post-infarction remodelling. Although this approach is very promising it is still a far way. We need further investigation to clarify the molecular targets of signallings activated by G-CSF and in parallel the scrutiny of double-blinded Cardiovasc Drugs Ther (2006) 20: 155–156 DOI 10.1007/s10557-006-8799-0

Hydrogen peroxide is involved in methyl jasmonate‐induced senescence of rice leaves

The role of H2O2 in the senescence of detached rice leaves induced by methyl jasmonate (MJ) was investigated. MJ treatment resulted in H2O2 production in detached rice leaves, which was prior to the occurrence of leaf senescence. Dimethylthiourea, a chemical trap of H2O2, was observed to be effective in inhibiting MJ‐induced senescence and MJ‐increased malondialdehyde (MDA) content in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented MJ‐induced H2O2 production, suggesting that NADPH oxidase is a H2O2‐generating enzyme in MJ‐treated detached rice leaves. DPI and IMD also inhibited MJ‐promoted senescence and MJ‐increased MDA content in detached rice leaves. Phosphatidylinositol 3‐kinase inhibitors wortmannin (WM) or LY 294002 (LY) inhibited MJ‐induced H2O2 production and senescence of detached rice leaves. Exogenous H2O2 reversed the inhibitory effect of WM or LY. In terms of leaf senescence, it was observed that rice seedlings of cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)‐sensitive and those of cultivar Tainung 67 (TNG67) are JA‐insensitive. On treatment with JA, H2O2 accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Evidence was also provided to show that MJ‐induced H2O2 production in detached rice leaves is abscisic acid (ABA)‐independent. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ‐ and ABA‐induced H2O2 production and senescence of detached rice leaves, suggesting that the action of MJ and ABA is ethylene‐dependent.

Enhancement of cytokine-mediated NF-κB activation by phosphatidylinositol 3-kinase inhibitors in monocytic cells

Nuclear factor-κB (NF-κB) is a transcription factor that plays crucial roles in inflammation and immunity. Understanding the positive and negative regulation of NF-κB activity is therefore of fundamental importance. A few previous studies reported that inhibition of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway enhances lipopolysaccharide (LPS)-induced activation of NF-κB. However, many aspects of the PI3K negative regulation of NF-κB activation remain to be clarified. The present study was conducted to shed light on cell-type specificity, stimulus specificity, and upstream mechanisms of the enhanced NF-κB activation by PI3K inhibitors. Gel shift assays showed that LY294002 (LY29) potently increased interleukin (IL)-1-induced NF-κB DNA binding in human monocytic THP-1 cells. Moreover, another PI3K inhibitor 3-methyladenine also strongly enhanced IL-1-induced NF-κB DNA binding, while LY303511, an inactive analogue of LY29, did not increase the NF-κB DNA binding. Compared with LY29, wortmannin (WM) effected only a marginal enhancement of NF-κB DNA binding. LY29 treatment also augmented tumor necrosis factor (TNF)-mediated NF-κB DNA binding. Furthermore, LY29, but not WM, increased cyclooxygenase (COX)-2 mRNA expression by IL-1 or TNF in THP-1 cells. Likewise, prostaglandin E 2 production by IL-1 was increased by LY29, but not by WM. Western blot analysis demonstrated that IκB kinase (IKK) activation as well as IκB-α degradation and NF-κB nuclear translocation was elevated by LY29 and WM. Among the tested cell lines (HL-60, ECV304, Hep-2, and Molt-4), only HL-60, a promyelocytic cell line, showed enhanced NF-κB DNA binding by LY29. These results suggest that pharmacological inhibition of PI3K enhances the NF-κB-activating pathways by IL-1 through augmentation of IKK activation in myeloid/monocytic cells and the NF-κB enhancement is more robustly achieved by LY29 than by WM.

Inhibition of the phosphatidylinositol 3′-kinase signaling pathway leads to decreased insulin-stimulated adiponectin secretion from 3T3-L1 adipocytes

Adiponectin is a protein secreted by adipocytes, which modulates insulin resistance and is thought to confer protection from atherosclerosis. Decreased circulating adiponectin is seen in states of insulin resistance, yet the cause of this decrease remains unclear. We investigated the role of insulin in adiponectin secretion and the effect of selective insulin resistance on insulin-stimulated adiponectin secretion by 3T3-L1 adipocytes. Inhibition of the phosphatidylinositol 3′-kinase (PI3K) insulin-signaling pathway was induced with wortmannin (WT) or with a kinase-inactive Akt adenoviral construct (Akt-KD), and inhibition of the mitogen-activated protein kinase pathway was induced with PD98059 or with a dominant-negative ras adenoviral construct (DNras). The PI3K pathway was activated with a constitutively active Akt adenoviral construct (Akt-myr). Adiponectin was measured by Western blot, and adiponectin messenger RNA (mRNA) levels were determined by real-time reverse transcription–polymerase chain reaction. Insulin treatment increased adiponectin secretion and decreased intracellular adiponectin. Treatment with 100 nmol/L insulin for 24 hours resulted in a 78% increase in secreted adiponectin ( P < .05). Insulin had no effect on adiponectin mRNA. WT or Akt-KD, but not PD98059 or DNras, inhibited insulin-stimulated adiponectin secretion ( P < .05). Activation of the PI3K pathway resulted in increased insulin-independent adiponectin secretion. Inhibition of the PI3K- or mitogen-activated protein kinase–dependent pathway decreased adiponectin mRNA by 50% ( P < .01). We demonstrate a decrease in insulin-stimulated adiponectin secretion with selective inhibition of the PI3K pathway. These results suggest a mechanism for the observed decreased adiponectin levels associated with insulin resistance, when defects in the PI3K-dependent insulin-signaling pathway lead to decreased adiponectin production, inadequate adiponectin secretion, and therefore low circulating adiponectin levels.

Overexpression of regucalcin suppresses cell proliferation of cloned normal rat kidney proximal tubular epithelial NRK52E cells.

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell proliferation was investigated by using the cloned normal rat kidney proximal tubular epithelial NRK52E cells over-expressing regucalcin. NRK52E cells were transfected with regucalcin (RC) pCXN2 vector and the multiple neomycin-resistant clones that stably overexpress regucalcin were selected. The regucalcin content of RC/pCXN2-transfected cells used in this study was about 21-fold as compared with that of the parental wild-type NRK52E cells. Wild-type NRK52E cells, pCXN2 vector-transfected cells (mock-type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of bovine serum (5%). The cell numbers of wild- and mock-type were significantly increased with the time course of the culture. Cell numbers of transfectants were significantly suppressed as compared with that of wild- and mock-type. The decrease in cell number of wild-type cultured for 72 h in the presence of butyrate (8.3 x 10(-6) or 8.3 x 10(-5) M), rescovitine (10(-8) or 10(-7) M), or sulforaphane (10(-9) M), which is an inhibitor of the cell cycle, was not observed in transfectants. The effect of PD98059 (10(-8) M), staurosporine (10(-10) M) or dibucaine (10(-8)-10(-6) M), which is an inhibitor of protein kinases, in decreasing cell number of wild-type was not seen in transfectants. Moreover, the culture with wortmannin (10(-8) or 10(-7) M), an inhibitor of phosphatidylinositol 3 (PI3)-kinase, or Bay K 8644 (10(-8) or 10(-7) M), an agonist of calcium entry in cells, caused a significant decrease in cell number of the wild-type. This decrease was not observed in transfectants. The result of reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers showed that c-jun and chk2 mRNA levels were significantly decreased in transfectant. p53 mRNA level was significantly increased in transfectants. The expression of c-myc, c-fos, cdc2, p21, and G3PDH mRNAs in transfectants was not significantly changed. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell proliferation, which is mediated through various signaling pathways, in the cloned normal rat kidney proximal tubular epithelial NRK52E cells.

Activation of glycogen synthase kinase-3 induces Alzheimer-like tau hyperphosphorylation in rat hippocampus slices in culture

Formation of neurofibrillary tangle from hyperphosphorylated tau is one of the hallmark lesions seen in Alzheimer's disease (AD) brain, and neuronal deregulation of glycogen synthase kinase-3 (GSK-3) activity plays key role in tau hyperphosphorylation. In the present study, the role of GSK-3 on tau phosphorylation in hippocampus slice culture was examined by incubating the slice with wortmannin (WT), an inhibitor of phosphatidylinositol 3-kinase (PI3K) and GF-109203X (GFX), an inhibitor of protein kinase C (PKC). It was found that treatment of the slices with GFX or WT separately induced tau hyperphosphorylation both at Ser396/Ser404 (PHF-1) and Ser199/Ser202 (Tau-1) sites. The phosphorylation rate of tau at PHF-1 and Tau-1 epitopes was further increased when GFX and WT were used in combination, and at this condition, AD-like tau accumulation was observed. GSK-3 activity was significantly increased with a concurrently decreased level of inactivated form of GSK-3. Lithium chloride (LiCl), a GSK-3 inhibitor, prevented tau from WT- and GFX-induced hyperphosphorylation. It suggests that GSK-3 is regulated through PI3K and PKC pathway, and activation of GSK-3 not only induces hyperphosphorylation of tau but also leads to accumulation of tau in cultured rat brain slice.

514. Effect of DNA-PKcs on AAV Replication

Top of pageAbstract DNA dependent protein kinase (DNA-PK) is a DNA repair enzyme with multiple functions. It has been shown that it plays an important role in determining the molecular fate of the rAAV genome in mouse muscle and liver. DNA-PKcs inhibits AAV integration in vitro and in vivo. In the present study, we sought to determine the effect of DNA-PKcs on recombinant adeno-associated virus (rAAV) replication. We co-infected 293 cells with rAAV vector (UF5) and recombinant herpes simplex virus (rHSV) helper virus. The replicated forms were detected by southern blot analysis. When cells were treated with a DNA-PKcs inhibitor, wortmannin (WM), rAAV replication is significantly reduced in a dose-dependent manner. Similar results were obtained from MO59K (DNA-PKcs positive) cells. In order to confirm this observation, we have employed small interference RNA (siRNA) to target DNA-PKcs. Transfection of this synthetic siRNA resulted in 70% to 90% reduction of the mRNA and the protein levels of DNA-PKcs. Remarkably, this treatment significantly decreased rAAV replication. In order to avoid the possible side effects by viral infection on DNA-PK activity, we co-transfected the siRNA treated 293 cells with vector (pUF5) and helper (pDG) plasmids. Results from these experiments again showed that targeting of DNA-PKcs decreased rAAV replication by at least two folds compared to the controls. Our results demonstrated that inhibition of DNA-PKcs by a DNA-PK inhibitor or siRNA decreased rAAV replication suggesting the important role of this cellular enzyme in AAV replication.