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PubMed2005

Astrocyte-derived transforming growth factor-{beta} mediates the neuroprotective effects of 17{beta}-estradiol: involvement of nonclassical genomic signaling pathways.

17beta-Estradiol (E2) and selective estrogen receptor modulators (SERMs), such as tamoxifen, mediate numerous effects in the brain, including neurosecretion, neuroprotection, and the induction of synaptic plasticity. Astrocytes, the most abundant cell type in the brain, influence many of these same functions and thus may represent a mediator of estrogen action. The present study examined the regulatory effect and underlying cell signaling mechanisms of E2-induced release of neurotropic growth factors from primary rat cortical astrocyte cultures. The results revealed that E2 (0.5, 1, and 10 nm) and tamoxifen (1 mum) increased both the expression and release of the neuroprotective cytokines, TGF-beta1 and TGF-beta2 (TGF-beta), from cortical astrocytes. The stimulatory effect of E2 was attenuated by the estrogen receptor (ER) antagonist, ICI182,780, suggesting ER dependency. The effect of E2 also appeared to involve mediation by the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway, because E2 rapidly induced Akt phosphorylation, and pharmacological or molecular inhibition of the PI3K/Akt pathway prevented E2-induced release of TGF-beta. Additionally, the membrane-impermeant conjugate, E2-BSA, stimulated the release of TGF-beta, suggesting the potential involvement of a membrane-bound ER. Finally, E2, tamoxifen, and E2-BSA were shown to protect neuronal-astrocyte cocultures from camptothecin-induced neuronal cell death, effects that were attenuated by ICI182,780, Akt inhibition, or TGF-beta immunoneutralization. As a whole, these studies suggest that E2 induction of TGF-beta release from cortical astrocytes could provide a mechanism of neuroprotection, and that E2 stimulation of TGF-beta expression and release from astrocytes occurs via an ER-dependent mechanism involving mediation by the PI3K/Akt signaling pathway.

Animals↗
PubMed2006

Insulin resistance accelerates muscle protein degradation: Activation of the ubiquitin-proteasome pathway by defects in muscle cell signaling.

Conditions such as acidosis, uremia, and sepsis are characterized by insulin resistance and muscle wasting, but whether the insulin resistance associated with these disorders contributes to muscle atrophy is unclear. We examined this question in db/db mice with increased blood glucose despite high levels of plasma insulin. Compared with control littermate mice, the weights of different muscles in db/db mice and the cross-sectional areas of muscles were smaller. In muscle of db/db mice, protein degradation and activities of the major proteolytic systems, caspase-3 and the proteasome, were increased. We examined signals that could activate muscle proteolysis and found low values of both phosphatidylinositol 3 kinase (PI3K) activity and phosphorylated Akt that were related to phosphorylation of serine 307 of insulin receptor substrate-1. To assess how changes in circulating insulin and glucose affect muscle protein, we treated db/db mice with rosiglitazone. Rosiglitazone improved indices of insulin resistance and abnormalities in PI3K/Akt signaling and decreased activities of caspase-3 and the proteasome in muscle leading to suppression of proteolysis. Underlying mechanisms of proteolysis include increased glucocorticoid production, decreased circulating adiponectin, and phosphorylation of the forkhead transcription factor associated with increased expression of the E3 ubiquitin-conjugating enzymes atrogin-1/MAFbx and MuRF1. These abnormalities were also corrected by rosiglitazone. Thus, insulin resistance causes muscle wasting by mechanisms that involve suppression of PI3K/Akt signaling leading to activation of caspase-3 and the ubiquitin-proteasome proteolytic pathway causing muscle protein degradation.

3T3-L1 Cells↗
PubMed2002

Follicle-stimulating hormone amplifies insulin-like growth factor I-mediated activation of AKT/protein kinase B signaling in immature rat Sertoli cells.

FSH and IGF-I are both important determinants of testicular development and Sertoli cell function. The present studies were performed to determine the actions of FSH and IGF-I on PI3K/AKT protein kinase signaling in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were prepared from 10-d-old rats. After 7 d in culture, Sertoli cells were treated with IGF-I, FSH, or IGF-I plus FSH. In some experiments cultures were treated with 8-bromo-cAMP (40 microM), (Bu)(2)cAMP (40 microM), or forskolin (10 microM). After treatments, cell lysates were prepared, and the activation state of AKT and cAMP response element-binding protein (CREB) was determined by Western blot analysis using phosphorylation site-specific antibodies. IGF-I had little effect on CREB phosphorylation, but rapidly increased the phosphorylation of AKT in a concentration-dependent manner. Maximal stimulatory effects of IGF-I were observed at 10-20 ng/ml. Treatment with FSH (0.9 IU/ml) or forskolin for 20 min increased CREB phosphorylation, but had little effect on AKT phosphorylation. However, FSH caused a concentration-dependent increase in IGF-I-induced AKT phosphorylation. Longer incubations (1-4 h) with FSH alone resulted in the elevation of AKT phosphorylation concomitant with an increased secretion of IGF-I and decreased production of IGF-binding protein-3, implicating endogenous IGF-I in the action of FSH on AKT phosphorylation. IGF-I- and FSH-dependent AKT phosphorylation was inhibited by LY29400 (10 microM), a PI3K inhibitor, and by IGF-binding protein 3, but not by a PKA inhibitor (H89). The present study demonstrates that immature rat Sertoli cells possess multiple protein kinase signaling cascades that are regulated by FSH. Furthermore, FSH amplifies IGF-I-mediated PI3K/AKT signaling in Sertoli cells. The results provide evidence for intracellular signaling mechanisms that may be required for the proliferation and differentiation of Sertoli cells.

Animals↗
PubMed2003

Cell cycle progression and activation of Akt kinase are required for insulin-like growth factor I-mediated suppression of apoptosis in granulosa cells.

Ovarian follicle development is dependent on growth factors that stimulate cell proliferation and act as survival factors to prevent apoptosis of follicle cells. We examined the mechanism of the protective effect of IGF-I against Fas ligand-induced apoptosis of granulosa cells and its relationship to cell proliferation. IGF-I activated both the phosphoinositide 3'-OH kinase (PI3K) and the MAPK pathways. Experiments using specific inhibitors of these pathways showed that protection by IGF-I was mediated by the PI3K pathway and not the MAPK pathway. Recombinant adenoviruses were used to test whether the downstream target of PI3K activation, Akt kinase, was required for protection against apoptosis. Expression of dominant negative Akt prevented protection by IGF-I whereas expression of constitutively active Akt (myrAkt) mimicked the effect of IGF-I. Treatment with IGF-I, or expression of myrAkt, increased progression from G(0)/G(1) to S phase of the cell cycle whereas expression of dominant negative Akt inhibited G(0)/G(1) to S phase progression and prevented the stimulatory effect of IGF-I. We tested whether cell cycle progression was required for protection from apoptosis using the cyclin-dependent kinase-2 inhibitor roscovitine, which blocks cells at the G(1)/S transition. Roscovitine prevented the protective effect of IGF-I and myrAkt expression against apoptosis. Therefore, activation of Akt is not sufficient to protect granulosa cells from apoptosis in the absence of cell cycle progression. In summary, IGF-I protects granulosa cells from apoptosis by activation of the PI3K/Akt pathway. This protective effect can occur only when progression from G(1) to S phase of the cell cycle regulated by the PI3K/Akt pathway is unperturbed.

Animals↗
PubMed2004

Hepatocyte growth factor stimulates nitric oxide production through endothelial nitric oxide synthase activation by the phosphoinositide 3-kinase/Akt pathway and possibly by mitogen-activated protein kinase kinase in vascular endothelial cells.

Hepatocyte growth factor (HGF) has recently been the focus of attention due to its angiogenic effects, which are similar to those of vascular endothelial growth factor (VEGF); because of these effects, HGF is considered to be a novel therapeutic agent against vascular disorders, including atherosclerotic angiopathies. Although nitric oxide (NO), which is derived from vascular endothelial cells (ECs), is also involved in angiogenesis, little is known regarding the interactions between HGF and NO. We therefore examined the effects of HGF on NO production as well as endothelial NO synthase (eNOS) phosphorylation, and investigated their mechanisms. In bovine aortic ECs, HGF induced a rapid (5 min) increase of NO production measured by diaminofluorescein-2 diacetate. Moreover, HGF rapidly (2.5 min) stimulated eNOS phosphorylation (Ser-1179) as determined by Western immunoblot analyses. Both of these effects were almost completely suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and were partially suppressed by the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126. HGF also stimulated Akt phosphorylation (Ser-473), which was completely suppressed by LY294002 and was partially suppressed by U0126. Moreover, HGF stimulated extracellular signal-regulated kinase 1/2 phosphorylation (Thr-202/Tyr-204), which was completely suppressed by U0126 and was partially suppressed by LY294002. Taken together, these results indicate that HGF not only phosphorylates eNOS through the PI3K/Akt pathway, but also partially through the MAPK pathway, and that these two pathways may interact. Compared with VEGF, HGF was more potent in both NO production and eNOS phosphorylation. Our study thus demonstrates a novel activity of HGF-the stimulation of NO production-which occurs via eNOS phosphorylation that may in turn be mediated by cross-talk between the PI3K/Akt and MAPK pathways.

Animals↗
PubMed2005

Regulation of osteoclast apoptosis and motility by small GTPase binding protein Rac1.

UNLABELLED: The role of Rac1 in osteoclast survival and bone-resorbing activity was examined using adenovirus vector expression systems. Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling primarily through PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity, probably by regulating the motility of osteoclasts. INTRODUCTION: Rac1 is a member of Rho family small G-proteins, and recent studies have revealed that it mediates anti-apoptotic signals in some types of cells. Rac1 is reported to be required for the cytoskeletal organization and bone-resorbing activity of osteoclasts, but their roles in osteoclast survival and function are not fully elucidated. MATERIALS AND METHODS: We constructed the adenovirus vector carrying cDNA of either the dominant negative Rac1 (Rac1(DN)) or constitutively active Rac1 (Rac1(CA)) gene, and osteoclast-like cells (OCLs) generated in mouse co-culture system were infected with these viruses. To examine the role of Rac1 in osteoclast survival and function, we performed pit formation assays, survival assays, and Western blotting, including an activated-Rac1 pull-down assay using adenovirus-infected OCLs. To further clarify the mechanism of Rac1 regulation in osteoclast survival, some specific inhibitors and adenovirus vectors of signal transduction molecules were used. To quantify membrane movement before and after macrophage colony-stimulating factor (M-CSF) treatment, OCLs expressing either enhanced green fluorescent protein (EGFP) or Rac1(DN) were recorded with a time-lapse video microscope. RESULTS: Adenovirus vector-mediated dominant negative Rac1 (Rac1(DN)) expression significantly reduced pit formation, and promoted their apoptosis. M-CSF rapidly activated Rac1, and the prosurvival effect of M-CSF for OCLs was abrogated by Rac1(DN) overexpression. Constitutively active Rac1 enhanced OCL survival, which was completely suppressed by phosphatidylinositol 3'-kinase (PI3K) inhibitors, whereas a Mek inhibitor had only partial effect. Rac1(DN) also partially blocked the activation of Akt induced by the overexpressing catalytic subunit of PI3K. Using time-lapse video microscopy, we found that Rac1(DN) expression reduced membrane ruffling and the spreading of OCLs in response to M-CSF. CONCLUSIONS: Small guanosine triphosphatase (GTPase) Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling of osteoclasts primarily by modulating PI3K/Akt pathways. Rac1 also plays a significant role in the bone resorptive activity of cells, probably by regulating the motility of osteoclasts.

Actins↗
PubMed2006

Macrophage pro-inflammatory response to Francisella novicida infection is regulated by SHIP.

Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL)-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP) is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida-induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida-induced cytokine production through the inhibition of NFkappaB. Consistently, macrophages lacking SHIP displayed enhanced NFkappaB-driven gene transcription, whereas overexpression of SHIP led to decreased NFkappaB activation. Thus, we propose that SHIP negatively regulates F. novicida-induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFkappaB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia.

Animals↗
PubMed2004

Regulation of Cu/Zn-superoxide dismutase expression via the phosphatidylinositol 3 kinase/Akt pathway and nuclear factor-kappaB.

Aerobic cells adjust the expression of antioxidant enzymes to maintain reactive oxygen species within tolerable levels. In addition, phosphatidylinositol 3 kinase (PI3K) and its downstream protein kinase effector Akt adapt cells to survive in the presence of oxidative stress. Here we provide evidence for an association between these two defense systems via transcriptional regulation of Cu/Zn-superoxide dismutase (Cu/Zn-SOD). PC12 pheochromocytoma cells expressing active Akt1 exhibit lower ROS levels in response to hydrogen peroxide, as determined with the superoxide-sensitive probe hydroethidine. Transfection of constitutive or 4-hydroxytamoxifen-inducible versions of Akt1 results in higher messenger RNA and protein levels of Cu/Zn-SOD. Luciferase reporter constructs, carrying different length fragments of the human sod1 gene promoter, have identified a region between -552 and -355 that is targeted by PI3K and Akt and that contains a putative site of regulation by nuclear factor-kappaB (NF-kappaB). Nerve growth factor (NGF) and Akt augment the transactivating activity and produce higher nuclear levels of p65-NF-kappaB. Electrophoretic mobility shift assays indicate that the putative NF-kappaB regulatory sequence binds p65-NF-kappaB more efficiently in nuclear extracts from these cells. A dominant-negative mutant of IkappaBalpha further demonstrates that the PI3K/Akt axis targets the sod1 promoter at the level of the newly characterized NF-kappaB site. These results illustrate a new mechanism by which the PI3K/Akt pathway protects cells against oxidative stress, involving the upregulation of Cu/Zn-SOD gene expression, and the results identify NF-kappaB as a key mediator in the regulation of this gene.

Animals↗
PubMed2005

Molecular aspects of gefitinib antiproliferative and pro-apoptotic effects in PTEN-positive and PTEN-negative prostate cancer cell lines.

To date, no effective therapeutic treatment allows abrogation of the progression of prostate cancer (PCa) to more invasive forms. One of the major targets for the therapy in PCa can be epidermal growth factor receptor (EGFR), which signals via the phosphoinositide 3'-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways, among others. Despite multiple reports of overexpression in PCa, the reliance on activated EGFR and its downstream signalling to the PI3K and/or MAPK/extracellular signal-regulated kinase (ERK) pathways has not been fully elucidated. We reported that the EGFR-selective tyrosine kinase inhibitor gefitinib (ZD1839; Iressa) is able to induce growth inhibition, G(1) arrest and apoptosis in PCa cells and that its effectiveness is associated primarily with phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression (and thus Akt activity). In fact PTEN-negative PCa cells are slowly sensitive to gefitinib treatment, because this molecule is unable to downregulate PI3K/Akt activity. PI3K inhibition, by LY294002 or after PTEN transfection, restores EGFR-stimulated Akt signalling and sensitizes the cells to pro-apoptotic action of gefitinib. The MAPK pathway seems to be involved primarily on cell-growth modulation because dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition (both not cell apoptosis) in PTEN-positive PCa cells and reduced EGF-mediated growth in PTEN-negative cells. Thus the effectiveness of gefitinib requires growth factor receptor-stimulated PI3K/Akt and MAPK signalling to be intact and functional. The loss of the PTEN activity leads to uncoupling of this signalling pathway, determining a partial gefitinib resistance. Moreover, gefitinib sensitivity may be maintained in these cells through its inhibitory potential in MAPK/ERK pathway activity, modulating proliferative EGFR-triggered events. Therefore, our data suggest that the inhibition of EGFR signalling can result in a significant growth reduction and in increased apoptosis in EGFR-overexpressing PCa cells with different modalities, which are regulated by PTEN status, and this may have relevance in the clinical setting of PCa.

Antineoplastic Agents↗
PubMed2004

c-Myc protein is stabilized by fibroblast growth factor 2 and destabilized by ACTH to control cell cycle in mouse Y1 adrenocortical cells.

ACTH is the hormone known to control adrenal cortex function and maintenance in the intact animal but, in culture, it inhibits proliferation of adrenocortical cells from different mammalian species, a puzzle that has remained unsolved for nearly 30 years. In this paper we compare ACTH and fibroblast growth factor 2 (FGF2) antagonistic effects on the cell cycle in the Y1 cell line, a functional lineage of mouse adreno-cortical tumor cells. This cell line displays chronic high levels of c-Ki-Ras-GTP, high active constitutive levels of phosphatidylinositol 3-OH kinase/Protein Kinase B (PI3K/AKT) and low constitutive basal expression of c-Myc, which accounts for a minor deregulation of the cell cycle. In G0/G1-arrested Y1 cells, over-expression of the dominant negative mutant HaRasN17 drastically reduces c-Ki-Ras-GTP levels, eliminating basal c-Myc expression and basal S phase entry. PI3K/Akt seems to be the downstream pathway from c-Ki-ras for deregulation of c-Myc basal expression, since wortmannin abolishes c-Myc expression in serum-starved, G0/G1-arrested Y1 cells. FGF2 is a strong mitogen for Y1 cells, promoting -- in a manner dependent on the MEK/ERK pathway -- c-myc transcription induction, c-Myc protein stabilization and S phase entry in G0/G1-arrested Y1 cells. On the other hand, ACTH causes c-Myc protein destabilization, partially blocking S phase entry induced by FGF2, by a process dependent on the cAMP/protein kinase A (PKA) pathway. The whole pathway activated by ACTH to destabilize c-Myc protein in Y1 cells might comprise the following steps: ACTH receptor -->cAMP/PKA --> Akt deactivation -->GSK3 activity liberation --> c-Myc Thr58 phosphorylation. We demonstrate that c-Myc regulation is a central key in the cell cycle control by these factors, since enforced expression of c-Myc through the MycER chimera abrogates the ACTH inhibitory effect over FGF2-induced S phase entry.

Adrenal Cortex↗
PubMed2006

Role of the Akt/FoxO3a pathway in TGF-beta1-mediated mesangial cell dysfunction: a novel mechanism related to diabetic kidney disease.

Diabetic nephropathy (DN) is characterized by mesangial cell (MC) expansion and accumulation of extracellular matrix proteins. TGF-beta is increased in MC under diabetic conditions and in DN and activates key signaling pathways, including the phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway. FoxO transcription factors play roles in cell survival and oxidative stress and are negatively regulated by Akt-mediated phosphorylation. We tested whether phosphorylation-mediated inactivation of FoxO3a by TGF-beta can mediate MC survival and oxidative stress. TGF-beta treatment significantly increased levels of p-Akt (activation) and p-FoxO3a (inactivation) in cultured MC. This FoxO3a inactivation was accompanied by significant decreases in the expression of two key FoxO3a target genes, the proapoptotic Bim and antioxidant manganese superoxide dismutase in MC. TGF-beta treatment triggered the nuclear exclusion of FoxO3a, significantly inhibited FoxO3a transcriptional activity, and markedly protected MC from apoptosis. A PI3K inhibitor blocked these TGF-beta effects. It is interesting that p-Akt and p-FoxO3A levels also were increased in renal cortical tissues from rats and mice at 2 wk after the induction of diabetes by streptozotocin, thus demonstrating in vivo significance. In summary, TGF-beta and diabetes can increase FoxO3a phosphorylation and transcriptional inactivation via PI3K/Akt. These new results suggest that Akt/FoxO pathway regulation may be a novel mechanism by which TGF-beta can induce unopposed MC survival and oxidant stress in early DN, thereby accelerating renal disease.

Animals↗
PubMed2026

PCSK9 as a Key Gene of Metastasis in Lung Adenocarcinoma: A Multi-omics and Experimental Validation Study.

BACKGROUND: Lung adenocarcinoma (LUAD) is the most common form of lung cancer. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is abnormally expressed in various tumor tissues and is associated with malignant phenotypes. However, the clinical significance, function, and mechanism of LUAD invasion and metastasis remain unclear. METHODS: We retrospectively enrolled 100 patients with LUAD in this study. Initially, qRT-PCR was performed to detect PCSK9 levels in clinical tissues. Subsequently, bioinformatics analysis of scRNA-seq and The Cancer Genome Atlas Program (TCGA) datasets was performed to predict the role of PCSK9 in tumor cell malignancy and its potential downstream pathways. These predictions were validated experimentally using the CCK-8 assay, TUNEL staining, wound healing, transwell invasion assay, and an in vivo lung metastasis model. Finally, Western blotting and an AKT inhibitor (MK2206) were used to verify the underlying mechanism. RESULTS: PCSK9 was significantly upregulated in LUAD tissues compared to paracancerous tissues and was associated with poorer OS and DFS. Bioinformatics analysis of scRNA-seq data and TCGA analysis predicted that PCSK9 is highly enriched in tumor cells and is involved in EMT, and that the PI3K/AKT pathway plays a significant role in LUAD development. Experiments confirmed that PCSK9 markedly promoted LUAD cell proliferation, migration, and invasion in vitro and lung metastasis in vivo. PCSK9 overexpression significantly upregulated p-AKT, p-PI3K, and p-mTOR levels. Furthermore, the AKT inhibitor, MK2206, reversed the promoting effects of PCSK9. CONCLUSIONS: PCSK9 expression is associated with the prognosis and diagnosis of LUAD. This molecule activates the PI3K/AKT signaling pathway, thereby driving invasion, metastasis, and proliferation in LUAD.

Humans↗
PubMed2002

IL-12 provides proliferation and survival signals to murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway.

IL-12 is a pleiotropic cytokine that plays an important role in innate and adaptive immunity. IL-12 induces T cell proliferation and IFN-gamma secretion from activated T cells. It was also reported that IL-12 prevents apoptosis of CD4(+) T cells. However, the signaling mechanism that regulates these IL-12-induced responses is poorly understood yet. In this study, we demonstrated that IL-12 activates phosphatidylinositol 3-kinase (PI3K)/Akt pathway in murine CD4(+) T cells, and that this signaling pathway is required for IL-12-induced T cell proliferation and antiapoptotic function, but not for IFN-gamma induction. Through PI3K/Akt pathway, IL-12 up-regulates the expression of cell cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular inhibitors of apoptosis proteins-2, followed by down-regulation of active caspase-3. These results suggest that PI3K/Akt pathway is critical for mediating IL-12-induced CD4(+) T cell responses such as T cell proliferation and survival.

Animals↗
PubMed2003

Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression.

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.

3T3 Cells↗
PubMed2005

IL-18 induces monocyte chemotactic protein-1 production in macrophages through the phosphatidylinositol 3-kinase/Akt and MEK/ERK1/2 pathways.

Macrophages are activated during an inflammatory response and produce multiple inflammatory cytokines. IL-18 is one of the most important innate cytokines produced from macrophages in the early stages of the inflammatory immune response. Monocyte chemoattractant protein (MCP-1) is expressed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, and its expression is correlated with the severity of the disease. Both IL-18 and MCP-1 have been shown to be involved in inflammatory immune responses. However, it has been unclear whether IL-18 is involved in the induction of MCP-1. This investigation was initiated to determine whether IL-18 can induce MCP-1 production, and if so, by which signal transduction pathways. We found that IL-18 induced the production of MCP-1 in macrophages, which was IL-12-independent and was not mediated by autocrine cytokines such as IFN-gamma or TNF-alpha. We then examined signal transduction pathways involved in IL-18-induced MCP-1 production. We found that IL-18 did not activate the IkappaB kinase/NF-kappaB pathway, evidenced by no degradation of IkappaBalpha and no translocation of NF-kappaB p65 to the nucleus in IL-18-stimulated macrophages. Instead, IL-18 activated the PI3K/Akt and MEK/ERK1/2 pathways. Inhibition of either of these pathways attenuated MCP-1 production in macrophages, and inhibition of both signaling pathways resulted in the complete inhibition of MCP-1 production. On the basis of these observations, we conclude that IL-18 induces MCP-1 production through the PI3K/Akt and MEK/ERK1/2 pathways in macrophages.

Animals↗
PubMed2005

G(i)-protein-dependent inhibition of IL-12 production is mediated by activation of the phosphatidylinositol 3-kinase-protein 3 kinase B/Akt pathway and JNK.

Ligands for certain G(i)-protein-coupled receptors (GiPCRs) potently inhibit the production of IL-12 by human monocytes. We addressed the intracellular signaling mechanisms by which this occurs using primary human cells. Stimulation with the GiPCR ligands C5a and 1-deoxy-1-[6-[(3-iodophenyl)methyl]amino]-9H-purine-9-y1]-N-methyl-beta-D-ribofuranuronamide (IB-MECA) blocked the production of IL-12 p70 by human monocytes stimulated with LPS and IFN-gamma. In addition, C5a reduced the expression of mRNA for IL-12 p35, p40, IL-23 p19, and IL-27 p28. This effect was due neither to a down-regulation of TLR4 or IFN-gamma receptor on the cell surface nor to interference with IFN-gamma signaling, because IFN-gamma-induced up-regulation of HLA-DR and CD40 were unaffected. C5a or IB-MECA activated the PI3K/Akt signaling pathway and induced the phosphorylation of the MAPK p38, ERK, and JNK. Inhibition of the PI3K/Akt signaling pathway with wortmannin or an inhibitor of Akt activity, and inhibition of JNK but not ERK prevented IL-12 and IL-23 suppression by C5a. These data extend observations on IL-12 suppression by C5a to IL-23 and IL-27, and are the first to demonstrate the intracellular signaling events leading to IL-12 and IL-23 inhibition after GiPCR activation.

Adenosine↗
PubMed2006

A novel role of hypoxia-inducible factor in cobalt chloride- and hypoxia-mediated expression of IL-8 chemokine in human endothelial cells.

Tissue hypoxemia is common in several pathological diseases, including vaso-occlusion in sickle cell disease and myocardial infarction. One finds increased presence of leukocytes during lung injury and at sites of inflammation in vascular endothelium. In this study, we used human pulmonary microvascular endothelial cells and human dermal microvascular endothelial immortalized cell line to delineate the cellular signaling mechanism of hypoxia- and CoCl2 (a mimetic of hypoxia)-induced IL-8 expression, and the latter's role in chemotaxis of polmorphonuclear neutrophils. We show that hypoxia- and CoCl2-induced IL-8 mRNA and protein expression involved activation of PI3K/Akt and p38 MAPK, but not MEK kinase. Analysis of some transcription factors associated with IL-8 promoter revealed that hypoxia and CoCl2 increased DNA-binding activity of hypoxia-inducible factor-1alpha (HIF-1alpha), NF-kappaB, and AP-1. In addition, we show that hypoxia- and CoCl2-induced IL-8 expression requires activation of HIF as demonstrated by the following: 1) EMSA; 2) transfection studies with IL-8 promoter reporter constructs with mutation in HIF-1alpha binding site; 3) attenuation of IL-8 expression by both HIF-1alpha small interfering RNA and R59949; 4) augmentation of IL-8 expression by either transfection with HIF-prolyl hydroxylase-2 small interfering RNA or overexpression of HIF-1alpha; and 5) chromatin immunoprecipitation analysis. Moreover, conditioned medium from hypoxia-treated endothelial cells augmented chemotaxis of neutrophils, due to release of IL-8. These data indicate that hypoxia-induced signaling in vascular endothelium for transcriptional activation of IL-8 involves PI3K/Akt, p38 MAPK, and HIF-1alpha. Pharmacological agents, which inhibit HIF-1alpha, may possibly ameliorate inflammation associated with hypoxia in pathological diseases.

Base Sequence↗
PubMed2004

Activating FOXO3a, NF-kappaB and p53 by targeting IKKs: an effective multi-faceted targeting of the tumor-cell phenotype?

Tumor cells frequently recruit the PI3K/Akt pathway in order to evade cell death, terminal differentiation and replicative inhibition. A wealth of targets for the PI3K/Akt pathway involved in these processes has been described. Among others, targets for the Akt-kinase include certain members of the Forkhead Box Class O (FOXO) transcription factors, involved in DNA damage repair, apoptosis, cell cycle progression and arrest. Akt regulates the sub-cellular localization of FOXO3a by phosphorylation thereby preventing the protein to translocate to the nucleus and regulate transcription. Constitutive Akt-activation is frequently correlated with cytoplasmatic FOXO3a in breast tumors and this is associated with decreased patient survival. In a recent paper (Hu MC, et al. Cell 2004; 117:225-237) FOXO3a was found in the cytoplasm in the absence of activated Akt. Instead, IKKbeta was shown to interact with and phosphorylate FOXO3a. The recent findings suggest that the IKKs might serve as a potential drug target in anti-cancer therapy since multiple signal transduction pathways inhibiting proliferation and facilitating cell death could be activated.

DNA-Binding Proteins↗