The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-speci?c inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.     

Insulin-like growth factor-1 induces the phosphorylation of PRAS40 via the PI3K/Akt signaling pathway in PC12 cells

Insulin-like growth factor-1 (IGF-1) is a polypeptide tropic factor that plays an important role in the survival and differentiation of both neuronal and non-neuronal cells. Numerous studies have demonstrated that IGF-1 promotes neuronal cell survival via the PI3K/Akt signaling pathway. Proline-rich Akt substrate of 40kDa (PRAS40) is a recently discovered downstream target of Akt. However, the relationship between IGF-1 and PRAS40 is not known. In this study, we characterized the phosphorylation of PRAS40 induced by IGF-1 in PC12 cells and explored the signaling pathway responsible for the effect of IGF-1. IGF-1 induced the phosphorylation of Akt at Thr473 and PRAS40 at Thr246 in PC12 cells. The phosphorylation of Akt and PRAS40 induced by IGF-1 (100ng/ml) was inhibited by the phosphatidylinositide 3-kinase (PI3K) specific inhibitor LY294002 (50μM), while no inhibitory effect was observed for a MAPK kinase pathway specific inhibitor PD98059 nor a p38 MAPK inhibitor PD169316, suggesting that the phosphorylation of PRAS40 induced by IGF-1 is mediated by the PI3K pathway in PC12 cells and primary cultured neurons. In further support this hypothesis, an Akt kinase specific inhibitor, Akt inhibitor VIII, attenuated IGF-1-induced phosphorylation of PRAS40 at the concentration that blocked the phosphorylation of Akt induced by IGF-1. Taken together, these data demonstrate that IGF-1 stimulates the phosphorylation of PRAS40 at Thr246 in neuronal cells and the effect of IGF-1 is mediated, at least in part, by the PI3K/Akt signaling pathway.     

A dual effect of porcine reproductive and respiratory syndrome virus replication on the phosphatidylinositol-3-kinase-dependent Akt pathway

We have recently shown that porcine reproductive and respiratory syndrome virus (PRRSV) can undergo a productive replication in porcine monocyte-derived dendritic cells (Mo-DCs). Here, we further demonstrate that PRRSV activates the host’s phosphatidylinositol-3-kinase (PI3K)-dependent Akt pathway (PI3K/Akt) to facilitate its replication in Mo-DCs at 90 min and 4 h after infection. Inhibition of PI3K/Akt by treatment with a PI3K-specific inhibitor (LY294002) prior to PRRSV infection reduced virus replication. Furthermore, inhibition of PI3K/Akt by LY294002 at 90 min and 8 h after virus infection still significantly reduced virus production, suggesting that virus replication may be dependent on the activation of PI3K/Akt. Interestingly, PRRSV inhibited PI3K/Akt at 12 h after infection. Heat-inactivated virus failed to inhibit PI3K/Akt, indicating that virus replication is essential for this inhibition. Overall, PRRSV replication exhibits a dual effect on the PI3K/Akt pathway in which both time-dependent activation and inhibition of PI3K/Akt are observed.     

ET-1介导损伤气道上皮诱导成纤维细胞活化的信号机制

目的： 探讨p38丝裂原活化蛋白激酶(MAPK)、磷脂酰肌醇3-激酶(PI3K/Akt)在内皮素(ET)-1介导损伤气道上皮诱导上皮下成纤维细胞活化过程中的作用及对白细胞介素(IL)-6的影响。方法：将正常或经多聚左旋精氨酸(PLA)刺激的人气道上皮细胞与原代气道成纤维细胞共培养并分别加入p38 MAPK、PI3K特异性抑制剂SB203580、LY294002或ET受体A阻断剂BQ123,应用免疫组化、免疫印迹技术或ELISA检测成纤维细胞α-平滑肌肌动蛋白（α-SMA）表达、p38 MAPK、Akt的活化及成纤维细胞上清IL-6水平;制备成纤维细胞胶原凝胶并与不同方法处理的上皮细胞共培养,测量各组凝胶面积变化以了解上皮细胞对成纤维细胞收缩反应的诱导及其上述处理因素的影响。结果：与损伤上皮细胞共培养的成纤维细胞上清中ET-1、IL-6水平［（13.69±1.36） ng/L、(56.7±10.7) ng/L］明显高于与正常上皮细胞共培养的成纤维细胞上清［（3.79±0.64） ng/L、（15.5±3.2）ng/L］,BQ123、SB203580或LY294002皆不同程度减弱损伤上皮细胞诱导的IL-6释放［分别为(27.2±3.1) ng/L、(31.5±3.6) ng/L、(41.3±3.2) ng/L］;成纤维细胞与损伤气道上皮细胞共培养后p38 MAPK、Akt先后激活,BQ123减弱磷酸化p38 MAPK、Akt水平,SB203580浓度依赖性减弱Akt磷酸化水平,而LY294002对磷酸化p38 MAPK水平影响很小。与损伤气道上皮细胞共培养后成纤维细胞α-SMA表达增加,并且胶原收缩百分比明显大于与正常气道上皮共培养的成纤维细胞［(61.2±2.7)% vs (15.4±7.3)%］;BQ123、SB203580及LY294002皆不同程度减弱成纤维细胞α-SMA表达与凝胶收缩且BQ123、SB203580抑制凝胶收缩作用较LY294002更明显。结论：ET-1通过激活p38 MAPK、PI3K/Akt信号通路并促进IL-6分泌在损伤气道上皮诱导成纤维细胞活化过程中发挥关键作用。     

Testosterone up-regulates seladin-1 expression by iAR and PI3-K/Akt signaling pathway in C6 cells

The previous study indicated that DHCR24/seladin-1 was an important neuroprotective effector. However, the molecular mechanisms that androgen modulates the expression of seladin-1 remain incompletely defined. In this paper, we showed that the expression of seladin-1 was significantly increased by testosterone at all concentrations tested at the protein and mRNA levels in C6 cells, the selective AR antagonist flutamide obviously inhibited the effect in a concentration-dependent manner. Furthermore, we found that testosterone significantly increased the phosphorylation level of V-akt murine thymoma viral oncogene (Akt), a key effector of the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway, while a specific PI3-K inhibitor LY294002 obviously prevented the activation of Akt phosphorylation. In addition, the PI3-K inhibitor LY294002 also markedly blocked the up-regulation expression of seladin-1 gene induced by testosterone at the protein and mRNA levels. Collectively, the above results suggested that testosterone regulated the expression of seladin-1 by the intracellular androgen receptor (iAR)-mediated genomic signaling pathway and the non-genomic PI3-K/Akt signaling pathway in C6 glial cells.     

Phosphatidylinositol-3-kinase and Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS-1

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pattern-recognition receptor that recognizes viruses and triggers anti-viral immune responses. Activation of intracellular RIG-I signalling is mediated through interferon-β (IFN-β) promoter stimulator-1 (IPS-1), an adaptor of RIG-I, which induces IFN regulatory factor (IRF) 3 activation and type I IFN expression. The phosphatidylinositol-3-kinase (PI3K) and Akt pathway is activated in host immune cells upon viral infection. However, the mechanism as to how they work in RIG-I signalling has not been fully elucidated. Therefore, we investigated the role of PI3K and Akt in the regulation of RIG-I-mediated IRF3 activation and type I IFN expression in macrophages. Our results show that Sendai virus infection, which is recognized by RIG-I, led to IRF3 activation and IFN-β expression and these responses were attenuated by the PI3K inhibitor (LY294002) and an Akt dominant-negative mutant in the macrophage cell line(RAW264.7). IRF3 phosphorylation and dimerization as well as IFN-β expression induced by a synthetic RIG-I agonist, short poly(I:C), were suppressed by LY294002 or siRNA-Akt in bone marrow-derived macrophages. Suppression of PI3K and Akt using a dominant-negative mutant and siRNA knockdown resulted in attenuation of IRF3 activation and IFN-β expression induced by RIG-I itself or its adaptor, IPS-1. Association of Akt with IPS-1 increased with short poly(I:C) stimulation and required the pleckstrin homology domain of Akt and caspase-recruitment domain in IPS-1. Collectively, our results show that PI3K and Akt are required downstream of IPS-1 for RIG-I-mediated anti-viral immune responses. The results describe a novel, interactive relationship between RIG-I downstream signalling molecules resulting in efficient anti-viral immunity.     

S100A6通过PI3K/Akt信号通路促进人骨肉瘤细胞143B增殖和迁移

 目的：研究PI3K/Akt信号通路在S100A6介导的人骨肉瘤细胞143B的增殖和迁移中的作用。方法：首先制备重组的人S100A6蛋白（recombinant human S100A6, rhS100A6）;rhS100A6与PI3K抑制剂（LY294002和wortmannin）单独或同时处理143B细胞,其中rhS100A6的终浓度为30 mg/L,LY294002和wortmannin的终浓度分别为10 μmol/L和05 μmol/L;采用Western blotting分析143B细胞中PI3K/Akt信号通路相关分子总Akt（total Akt, t-Akt）及磷酸化Akt（phosphorylation of Akt, p-Akt）蛋白的表达变化,MTT检测细胞增殖,Transwell检测细胞迁移。 结果：（1）成功制备rhS100A6蛋白,rhS100A6显著增强143B细胞的增殖和迁移能力（P<005）;（2）rhS100A6上调143B细胞中Akt的磷酸化;（3）与rhS100A6组相比,rhS100A6与LY294002或wortmannin联合处理组143B细胞的p-Akt减少（P<005）,细胞的增殖和迁移能力降低,在不同时点细胞的增殖率下降103%～697%,细胞迁移率下降379%～416%,差异均有统计学意义（P<005）。 结论：S100A6促进人骨肉瘤细胞143B增殖和迁移的作用至少部分是通过激活PI3K/Akt信号通路实现的。     

Regulation of CD38 expression in human airway smooth muscle cells: role of class I phosphatidylinositol 3 kinases

The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose, a Ca(2+)-mobilizing agent. In human airway smooth muscle (HASM) cells, TNF-α mediates CD38 expression through mitogen-activated protein kinases and NF-κB and AP-1. The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in TNF-α signaling and contributes to airway hyperresponsiveness and airway remodeling. We hypothesized that PI3Ks mediate CD38 expression and are involved in the differential induction of CD38 by TNF-α in asthmatic HASM cells. HASM cells were treated with pan-PI3K inhibitors (LY294002 or wortmannin) or class I-selective (GDC0941) or isoform-selective PI3K inhibitors (p110α-PIK-75 and p110β-TGX-221) with or without TNF-α. HASM cells were transfected with a catalytically active form of PI3K or phosphatase and tensin homolog (PTEN) or nontargeting or p110 isoform-targeting siRNAs before TNF-α exposure. CD38 expression and activation of Akt, NF-κB, and AP-1 were determined. LY294002 and wortmannin inhibited TNF-α-induced Akt activation, whereas only LY294002 inhibited CD38 expression. P110 expression caused Akt activation and basal and TNF-α-induced CD38 expression, whereas PTEN expression attenuated Akt activation and CD38 expression. Expression levels of p110 isoforms α, β, and δ were comparable in nonasthmatic and asthmatic HASM cells. Silencing of p110α or -δ, but not p110β, resulted in comparable attenuation of TNF-α-induced CD38 expression in asthmatic and nonasthmatic cells. NF-κB and AP-1 activation were unaltered by the PI3K inhibitors. In HASM cells, regulation of CD38 expression occurs by specific class I PI3K isoforms, independent of NF-κB or AP-1 activation, and PI3K signaling may not be involved in the differential elevation of CD38 in asthmatic HASM cells.     

Neuregulin-1β attenuates sepsis-induced diaphragm atrophy by activating the PI3K/Akt signaling pathway

The aim of this study was to investigate the protective effects of neuregulin-1β (NRG-1β) on sepsis-induced diaphragm atrophy and the possible underlying mechanisms. Sprague–Dawley rats were randomly divided into sham, sepsis and NRG groups. Sepsis was induced by cecal ligation and puncture (CLP). In the NRG group, rats received tail vein injections of NRG-1β (10 μg/kg) every 12 h for 72 h after CLP. At 3 days after surgery, diaphragm contractile forces were measured by determining the force-frequency curve and muscle fiber areas by hematoxylin–eosin staining. Moreover, the NRG-1 expression level in the diaphragm was detected by Western blotting. Furthermore, the proteins in the PI3K/Akt signaling pathway and its downstream Akt-mTOR and Akt-FOXO axes were detected by Western blotting analysis. In L6 myotubes treated with lipopolysaccharide (LPS) and NRG-1β, PI3K/Akt signaling pathway-related protein expression was further determined using the PI3K inhibitor LY294002. Exogenous NRG-1β could compensate for sepsis-induced diminished NRG-1 in the diaphragm and attenuate the reduction in diaphragm contractile forces and muscle fiber areas during sepsis. Moreover, NRG-1β treatment could activate the PI3K/Akt signaling pathway in the diaphragm during sepsis. The inhibition of p70S6K and 4E-BP1 on the Akt-mTOR axis and the increased expression of Murf1 on the Akt-FOXO axis were reversed after NRG-1 treatment. In addition, NRG-1β could activate the PI3K/Akt signaling pathway in L6 myotubes treated with LPS, while the PI3K inhibitor LY294002 blocked the effects of NRG-1β. NRG-1 expression in the diaphragm was reduced during sepsis, and exogenously administered recombinant human NRG-1β could attenuate sepsis-induced diaphragm atrophy by activating the PI3K/Akt signaling pathway.

     

Serotonin-induced growth of pulmonary artery smooth muscle requires activation of phosphatidylinositol 3-kinase/serine-threonine protein kinase B/mammalian target of rapamycin/p70 ribosomal S6 kinase 1

We have previously found that both mitogen-activated protein kinase (MAPK)- and Rho kinase (ROCK)-related signaling pathways are necessary for the induction of pulmonary artery smooth muscle cell (SMC) proliferation by serotonin (5-hydroxytryptamine [5-HT]). In the present study, we investigated the possible additional participation of a phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K1) pathway in this growth response. We found transient activation of Akt (Ser473) and more prolonged activation of S6K1 by 5-HT. Inhibition of PI3K with Wortmannin and LY294002 completely blocked these activations, but not that of MAPK or the ROCK substrate myosin phosphatase targeting subunit. Similarly, inhibition of MAPK and ROCK failed to block the Akt activation. Inhibition of Akt with NL-71-101 and downregulation of Akt expression with Akt small interfering RNA blocked 5-HT-induced S6K1 phosphorylation. Wortmannin, LY294002, and NL-71-101 dose-dependently inhibited 5-HT-induced SMC proliferation. 5-HT stimulated mTOR phosphorylation and the mTOR inhibitor, rapamycin, blocked activations of S6K1 and S6 ribosomal protein, and inhibited 5-HT-induced SMC proliferation. Akt phosphorylation and cell proliferation were also blocked by the antioxidants, N-acetyl-l-cysteine, Ginko biloba 501, and tiron, the reduced nicotinamide adenine dinucleotide phosphate oxidase inhibitor, diphenyleneiodonium, and the 5-HT2 receptor antagonists ketanserin and mianserin, but not by the 5-HT serotonin transporter or 5-HT 1B/1D receptor antagonists. We conclude from these studies that a parallel PI3K- and reactive oxygen species-dependent Akt/mTOR/S6K1 pathway participates independently from MAPK and Rho/ROCK in the mitogenic effect of 5-HT on pulmonary artery SMCs. From these and other studies, we postulate that independent signaling pathways leading to 5-HT-induced SMC proliferation are initiated through multiple 5-HT receptors and serotonin transporter at the cell surface.     

Resveratrol inhibits the development of obesity-related osteoarthritis via the TLR4 and PI3K/Akt signaling pathways

ABSTRACT

Aim of the study: Obesity leads to mild, chronic inflammation which is a primary risk factor for osteoarthritis (OA). Resveratrol exerts a protective effect on OA through its anti-inflammatory properties, but the precise mechanism remains unknown. The present study aimed to investigate the mechanism by which resveratrol alleviates obesity-related OA, and whether it is linked to the TLR4 and PI3K/Akt signaling pathways.

Materials and methods: C57BL/6J male mice were fed a high-fat diet (HFD) with or without resveratrol treatment and knee joints were collected for analysis. In addition, IL-1β-induced SW1353 cells were used to study in vitro the reciprocal effects of TLR4 and PI3K/Akt pathways.

Results: Resveratrol inhibited the development of OA in mice fed a HFD. TLR4 and PI3K/Akt signaling pathways were both activated in the articular cartilage; resveratrol treatment down-regulated TLR4 but up-regulated PI3K/Akt signaling. Further in vitro results showed that the effect of resveratrol alone on activation of PI3K/Akt was attenuated but not abolished by the TLR4 inhibitor CLI-095, and resveratrol failed to reduce TLR4 protein expression in IL-1β stimulated cells pretreated with the PI3K inhibitor LY294002.

Conclusions: Resveratrol may exert an anti-osteoarthritic effect by inhibiting TLR4 via the activation of PI3K/Akt signaling pathways. Resveratrol has potential as a drug for OA prevention.     

Lipoteichoic acid induces nuclear factor-kappaB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages

We previously demonstrated that lipoteichoic acid (LTA) might activate phosphatidylcholine-phospholipase C (PC-PLC) and phosphatidylinositol-phospholipase C (PI-PLC) to induce protein kinase C activation, which in turn initiates nuclear factor-kappaB (NF-kappaB) activation and finally induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release in RAW 264.7 macrophages. In this study, we further investigated the roles of tyrosine kinase, phosphatidylinositiol 3-kinase (PI3K)/Akt, and p38 mitogen-activated protein kinase (MAPK) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Tyrosine kinase inhibitors (genistein and tyrphostin AG126), PI3K inhibitors (wortmannin and LY 294002), and a p38 MAPK inhibitor (SB 203580) attenuated LTA-induced iNOS expression and NO release in concentration-dependent manners. Treatment of RAW 264.7 macrophages with LTA caused time-dependent activations of Akt and p38 MAPK. The LTA-induced Akt activation was inhibited by wortmannin, LY 294002, genistein, and tyrphostin AG126. The LTA-induced p38 MAPK activation was inhibited by genistein, tyrphostin AG126, wortmannin, LY 294002, and SB 203580. The LTA-induced formation of an NF-kappaB-specific DNA-protein complex in the nucleus was inhibited by wortmannin, LY 294002, genistein, tyrphostin AG126, and SB 203580. Treatment of macrophages with LTA caused an increase in kappaB-luciferase activity, and this effect was inhibited by tyrphostin AG126, wortmannin, LY 294002, the Akt dominant negative mutant (AktDN), and SB 203580. Based on those findings, we suggest that LTA might activate the PI3K/Akt pathway through tyrosine kinase to induce p38 MAPK activation, which in turn initiates NF-kappaB activation, and ultimately induces iNOS expression and NO release in RAW 264.7 macrophages.     

Activation of the PI3K/Akt/mTOR/p70S6K Pathway is Involved in S100A4-induced Viability and Migration in Colorectal Cancer Cells

The S100 protein family member S100A4 regulates various cellular functions. Previous studies have shown that elevated expression of S100A4 is associated with progression and metastasis of colorectal cancer (CRC). However, little is known about whether and how S100A4 contributes to CRC development. In our present study, the elevated expression of S100A4 in CRC tissues compared to matched adjacent normal tissues was confirmed by immunohistochemistry, semi-quantitative RT-PCR and Western blot. Adenovirus-mediated S100A4 overexpression obviously enhanced viability and migration of CRC cells, which was detected by MTT assay and transwell assay, respectively. Additionally, S100A4 overexpression increased the phosphorylation levels of Akt, mTOR and p70S6K. These effects of S100A4 were abolished by treatment with either the specific PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, or the specific mTOR/p70S6K inhibitor rapamycin. Furthermore, overexpression of S100A4 resulted in upregulation of VEGF and downregulation of E-cadherin, which were strongly reversed by either {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or rapamycin. Altogether, our results demonstrate that activation of the PI3K/Akt/mTOR/p70S6K signaling pathway is involved in S100A4-induced viability, migration, upregulation of VEGF and downregulation of E-cadherin in CRC cells.     

Epithelial phosphatidylinositol-3-kinase signaling is required for β-catenin activation and host defense against Citrobacter rodentium infection

Citrobacter rodentium infection of mice induces cell-mediated immune responses associated with crypt hyperplasia and epithelial β-catenin signaling. Recent data suggest that phosphatidylinositol-3-kinase (PI3K)/Akt signaling cooperates with Wnt to activate β-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-β-catenin(552)). Our aim was to determine whether epithelial PI3K/Akt activation is required for β-catenin signaling and host defense against C. rodentium. C57BL/6 mice were infected with C. rodentium and treated with dimethyl sulfoxide (DMSO) (vehicle control) or with the PI3K inhibitor LY294002 or wortmannin. The effects of infection on PI3K activation and β-catenin signaling were analyzed by immunohistochemistry. The effects of PI3K inhibition on host defense were analyzed by the quantification of splenic and colon bacterial clearance, and adaptive immune responses were measured by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Increased numbers of P-β-catenin(552)-stained epithelial cells were found throughout expanded crypts in C. rodentium colitis. We show that the inhibition of PI3K signaling attenuates epithelial Akt activation, the Ser552 phosphorylation and activation of β-catenin, and epithelial cell proliferative responses during C. rodentium infection. PI3K inhibition impairs bacterial clearance despite having no impact on mucosal cytokine (gamma interferon [IFN-γ], tumor necrosis factor [TNF], interleukin-17 [IL-17], and IL-1β) or chemokine (CXCL1, CXCL5, CXCL9, and CXCL10) induction. The results suggest that the host defense against C. rodentium requires epithelial PI3K activation to induce Akt-mediated β-catenin signaling and the clearance of C. rodentium independent of adaptive immune responses.     

The phosphoinositide-3-kinase-Akt signaling pathway is important for Staphylococcus aureus internalization by endothelial cells

Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)-Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3β on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3β, and NF-κB.     

Interleukin-32α production is regulated by MyD88-dependent and independent pathways in IL-1β-stimulated human alveolar epithelial cells

Interleukin (IL)-32 is a recently described cytokine that appears to play a critical role in a variety of inflammatory diseases including chronic obstructive pulmonary disease (COPD). However, thus far, the regulation of IL-32 production has not been fully established. Here, we report on signaling pathways that regulate the production of IL-32α, the most abundant isoform, in the human alveolar epithelial cell line, A549. IL-32α was expressed and secreted by IL-1β. The IL-32 expression was attenuated by PP2 (a Src-family kinase [SFK] inhibitor), rottlerin (a protein kinase [PK] Cδ inhibitor), and LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor). Furthermore, the overexpression of Fgr rather than other SFKs upregulated IL-32α expression, while Fgr small interfering RNA (siRNA) transfection downregulated it. The suppression of Fgr with PP2 and Fgr siRNA inhibited activating phosphorylation of PKCδ and PI3K/Akt, but not IL-1 receptor-associated kinase (IRAK)1, a well-known MyD88-dependent signaling molecule, and Erk1/2, p38, and JNK. Rottlerin and PKCδ siRNA also inhibited expression of IL-32α and activation of PI3K/Akt, but not of IRAK1 and mitogen activation protein (MAP) kinases. MyD88 siRNA suppressed the expression of IL-32α and the phosphorylation of IRAK1, PI3K, and MAP kinases, but not of PKCδ. Of interest, both Fgr/PKCδ and MyD88-dependent signals regulated PI3K/Akt, suggesting that it is a crosstalk molecule. Among MyD88-dependent MAP kinases, only p38 regulated IL-32α expression and PI3K/Akt activation. With these results, we demonstrated that the expression and secretion of IL-32α are regulated by MyD88-dependent IRAK1/p38/PI3K and independent Fgr/PKCδ/PI3K pathways, and that Fgr and PKCδ are critical for the MyD88-independent IL-32α production.     

Staurosporine-induced apoptosis in astrocytes is prevented by A1 adenosine receptor activation

Astrocyte apoptosis occurs in acute and chronic pathological processes at the central nervous system and the prevention of astrocyte death may represent an efficacious intervention in protecting neurons against degeneration. Our research shows that rat astrocyte exposure to 100 nM staurosporine for 3 h caused apoptotic death accompanied by caspase-3, p38 mitogen-ed protein kinase (MAPK) and glycogen synthase kinase-3β (GSK3β) activation. N⁶-chlorocyclopentyladenosine (CCPA, 2.5–75 nM), a selective agonist of A₁ adenosine receptors, added to the cultures 1 h prior to staurosporine, induced a dose-dependent anti-apoptotic effect, which was inhibited by the A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. CCPA also caused a dose- and time-dependent phosphorylation/activation of Akt, a downstream effector of cell survival promoting phosphatidylinositol 3-kinase (PI3K) pathway, which in turn led to inhibition of staurosporine-induced GSK3β and p38 MAPK activity. Accordingly, the anti-apoptotic effect of CCPA was abolished by culture pre-treatment with LY294002, a selective PI3K inhibitor, pointing out the prevailing role played by PI3K pathway in the protective effect exerted by A₁ receptor activation. Since an abnormal p38 and GSK3β activity is implicated in acute (stroke) and chronic (Alzheimer's disease) neurodegenerative diseases, the results of the present study provide a hint to better understand adenosine relevance in these disorders.     

LY294002 inhibits LPS-induced NO production through a inhibition of NF-kappaB activation: independent mechanism of phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinase (PI3K) is critical player in cell proliferation and survival. The effects of LY294002 and wortmannin, inhibitors of PI3K, on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipoploysaccharide (LPS)-induced Raw 264.7 cells were investigated. Significant inhibition of LPS-induced protein kinase B (PKB, Akt) phosphorylation occurred at 25 microM LY294002 or 0.5 microM wortmannin. At the same concentrations, LY294002, but not wortmannin, significantly inhibited NO production and iNOS expression. LY303511, an inactive analogue of LY294002, also inhibited NO production and iNOS expression. In addition, LY294002 and LY303511 significantly inhibited the DNA binding activity of NF-kappaB and NF-kappaB dependent reporter gene expression. These results suggest that LY294002 inhibits iNOS expression at least in part via inhibition of NF-kappaB activation, independent of PI3K.     

Activation of phosphatidylinositol 3-kinase/Akt-mammalian target of Rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine-induced place preference in rats

Hippocampus is a critical structure for the acquisition of morphine-induced conditioned place preference (CPP), which is a usual learning paradigm for assessing drug reward. However, the precise mechanisms remain largely unknown. Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt, mammalian target of Rapamycin (mTOR) and 70-kDa ribosomal S6 kinase (p70S6K), are critical molecules implicated in learning and memory. Here, we tested the role of PI3K/Akt-mTOR-p70S6K signaling pathway in morphine-induced CPP in the hippocampus. Our results showed that the acquisition of morphine CPP increased phosphorylation of Akt in the hippocampal CA3, but not in the nucleus accumbens (NAc), the ventral tegmental area (VTA) or the CA1. Moreover, the phosphorylated Akt exclusively expressed in the CA3 neurons. Likewise, levels of phosphorylated mTOR and p70S6K were significantly enhanced in the CA3 following morphine CPP. The alterations of these phosphorylated proteins are positively correlated with the acquisition of morphine CPP. More importantly, microinjection of PI3K inhibitor (LY294002) or mTOR inhibitor (Rapamycin) into the CA3 prevented the acquisition of CPP and inhibited the activation of PI3K-Akt signaling pathway. In addition, pre-infusion of β-FNA (β-funaltrexamine hydrochloride), a selective irreversible μ opioid receptor antagonist, into CA3 significantly prevented the acquisition of CPP and impaired Akt phosphorylation. All these results strongly implied that the PI3K-Akt signaling pathway activated by μ opioid receptor in hippocampal CA3 plays an important role in acquisition of morphine-induced CPP.     

Akt/GSK-3β介导高糖上调肾小管上皮细胞Snail1的表达

 目的：观察高糖对原代肾小管上皮细胞Snail1和蛋白激酶B/糖原合成酶激酶3β（Akt/GSK-3β）信号通路的影响,探讨糖尿病肾病时Snail1表达的调节机制。方法：原代培养大鼠肾小管上皮细胞（RTECs）,随机分为正常糖对照组、高渗组和高糖组。Western blotting检测不同处理组 RTECs不同培养时点（30 min、2 h、12 h、24 h、48 h和72 h）Snail1、Akt1、GSK-3β、磷酸化Akt（p-Akt,Ser473）和磷酸化GSK-3β（p-GSK-3β,Ser9）蛋白的水平。RT-PCR检测Snail1、Akt1和GSK3β mRNA的表达。RTECs以磷脂酰肌醇3-激酶（PI3K）抑制剂LY294002（25 μmol/L）预处理50 min,再与高糖共同培养24 h,Western blotting检测上述指标蛋白的表达。结果：与正常糖对照组比较,高糖组RTECs Snail1和Akt1蛋白和mRNA的表达上调,p-Akt及p-GSK-3β蛋白表达增加,但总GSK-3β蛋白和mRNA表达无变化。以LY294002处理后,高糖组RTECs Snail1、p-Akt及p-GSK-3β蛋白表达水平较未处理高糖组明显下降,但LY294002不影响总Akt1和GSK-3β蛋白表达。结论：Akt/GSK-3β可能介导了高糖诱导的RTECs锌指转录因子Snail1的表达上调。