Involvement of phosphatidylinositol 3-kinase-mediated up-regulation of I kappa B alpha in anti-inflammatory effect of gemfibrozil in microglia

The present study underlines the importance of PI3K in mediating the anti-inflammatory effect of gemfibrozil, a prescribed lipid-lowering drug for humans, in mouse microglia. Gemfibrozil inhibited LPS-induced expression of inducible NO synthase (iNOS) and proinflammatory cytokines in mouse BV-2 microglial cells and primary microglia. By overexpressing wild-type and dominant-negative constructs of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in microglial cells and isolating primary microglia from PPAR-alpha-/- mice, we have demonstrated that gemfibrozil inhibits the activation of microglia independent of PPAR-alpha. Interestingly, gemfibrozil induced the activation of p85alpha-associated PI3K (p110beta but not p110alpha) and inhibition of that PI3K by either chemical inhibitors or dominant-negative mutants abrogated the inhibitory effect of gemfibrozil. Conversely, overexpression of the constitutively active mutant of p110 enhanced the inhibitory effect of gemfibrozil on LPS-induced expression of proinflammatory molecules. Similarly, gemfibrozil also inhibited fibrillar amyloid beta (Abeta)-, prion peptide (PrP)-, dsRNA (poly IC)-, HIV-1 Tat-, and 1-methyl-4-phenylpyridinium (MPP+)-, but not IFN-gamma-, induced microglial expression of iNOS. Inhibition of PI3K also abolished the inhibitory effect of gemfibrozil on Abeta-, PrP-, poly IC-, Tat-, and MPP+-induced microglial expression of iNOS. Involvement of NF-kappaB activation in LPS-, Abeta-, PrP-, poly IC-, Tat-, and MPP+-, but not IFN-gamma-, induced microglial expression of iNOS and stimulation of IkappaBalpha expression and inhibition of NF-kappaB activation by gemfibrozil via the PI3K pathway suggests that gemfibrozil inhibits the activation of NF-kappaB and the expression of proinflammatory molecules in microglia via PI3K-mediated up-regulation of IkappaBalpha.     

Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance

Estrogen receptors (ERs) mediate most of the biological effects of estrogen in mammary and uterine epithelial cells by binding to estrogen response elements in the promoter region of target genes or through protein-protein interactions. Anti-estrogens such as tamoxifen inhibit the growth of ER-positive breast cancers by reducing the expression of estrogen-regulated genes. However, anti-estrogen-resistant growth of ER-positive tumors remains a significant clinical problem. Here we show that phosphatidylinositol (PI) 3-kinase and AKT activate ERalpha in the absence of estrogen. Although PI 3-kinase increased the activity of both estrogen-independent activation function 1 (AF-1) and estrogen-dependent activation function 2 (AF-2) of ERalpha, AKT increased the activity of only AF-1. PTEN and a catalytically inactive AKT decreased PI 3-kinase-induced AF-1 activity, suggesting that PI 3-kinase utilizes AKT-dependent and AKT-independent pathways in activating ERalpha. The consensus AKT phosphorylation site Ser-167 of ERalpha is required for phosphorylation and activation by AKT. In addition, LY294002, a specific inhibitor of the PI 3-kinase/AKT pathway, reduced phosphorylation of ERalpha in vivo. Moreover, AKT overexpression led to up-regulation of estrogen-regulated pS2 gene, Bcl-2, and macrophage inhibitory cytokine 1. We demonstrate that AKT protects breast cancer cells from tamoxifen-induced apoptosis. Taken together, these results define a molecular link between activation of the PI 3-kinase/AKT survival pathways, hormone-independent activation of ERalpha, and inhibition of tamoxifen-induced apoptotic regression.     

Simvastatin treatment promotes proliferation of human dental pulp stem cells via modulating PI3K/AKT/miR-9/KLF5 signalling pathway

Simvastatin serves as an effective therapeutic potential in the treatment of dental disease via alternating proliferation of dental pulp stem cells. First, western-blot and real-time quantitative PCR were used to detect the effect of simvastatin or LY294002 on the expression levels of AKT, miR-9 and KLF5, or determine the effect of miR-9. Simvastatin, KLF5 and AKT significantly enhanced the proliferation of pulp stem cells, whilst this effect induced by simvastatin was suppressed by LY294002, AKT siRNA, KLF5 siRNA and miR-9, and simvastatin dose-dependently upregulated the expression of PI3K. Furthermore, simvastatin upregulated PI3K and p-AKT expression in a concentration-dependent manner. LY294002 abrogated the upregulation of p-AKT expression levels induced by simvastatin, and LY294002 induced the miR-9 expression and simvastatin dose-dependently inhibited the expression of miR-9, by contrast, LY294002 reduced the KLF5 expression and simvastatin dose-dependently promoted the expression of KLF5. And using computational analysis, KLF5 was found to be a candidate target gene of miR-9, and which was further verified using luciferase assay. Finally, the level of KLF5 in cells was much lower following the transfection with miR-9 and KLF5 siRNA, and the level of AKT mRNA in cells was significantly inhibited after transfection with AKT siRNA than control. These findings suggested simvastatin could promote the proliferation of pulp stem cells, possibly by suppressing the expression of miR-9 via activating the PI3K/AKT signalling pathway, and the downregulation of miR-9 upregulated the expression of its target gene, KLF5, which is directly responsible for the enhanced proliferation of pulp stem cells.     

CTLA-4 Activation of Phosphatidylinositol 3-Kinase (PI 3-K) and Protein Kinase B (PKB/AKT) Sustains T-Cell Anergy without Cell Death

The balance of T-cell proliferation, anergy and apoptosis is central to immune function. In this regard, co-receptor CTLA-4 is needed for the induction of anergy and tolerance. One central question concerns the mechanism by which CTLA-4 can induce T-cell non-responsiveness without a concurrent induction of antigen induced cell death (AICD). In this study, we show that CTLA-4 activation of the phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death. CTLA-4 ligation induced PI 3K activation as evidenced by the phosphorylation of PKB/AKT that in turn inactivated GSK-3. The level of activation was similar to that observed with CD28. CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL. In keeping with this, CD3/CTLA-4 co-ligation prevented apoptosis under the same conditions where T-cell non-responsiveness was induced. This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death. Our findings therefore define a mechanism by which CTLA-4 can induce anergy (and possibly peripheral tolerance) by preventing the induction of cell death.     

Dysregulation of suppressor of cytokine signaling 3 in keratinocytes causes skin inflammation mediated by interleukin-20 receptor-related cytokines

Homeostatic regulation of epidermal keratinocytes is controlled by the local cytokine milieu. However, a role for suppressor of cytokine signaling (SOCS), a negative feedback regulator of cytokine networks, in skin homeostasis remains unclear. Keratinocyte specific deletion of Socs3 (Socs3 cKO) caused severe skin inflammation with hyper-production of IgE, epidermal hyperplasia, and S100A8/9 expression, although Socs1 deletion caused no inflammation. The inflamed skin showed constitutive STAT3 activation and up-regulation of IL-6 and IL-20 receptor (IL-20R) related cytokines, IL-19, IL-20 and IL-24. Disease development was rescued by deletion of the Il6 gene, but not by the deletion of Il23, Il4r, or Rag1 genes. The expression of IL-6 in Socs3 cKO keratinocytes increased expression of IL-20R-related cytokines that further facilitated STAT3 hyperactivation, epidermal hyperplasia and neutrophilia. These results demonstrate that skin homeostasis is strictly regulated by the IL-6-STAT3-SOCS3 axis. Moreover, the SOCS3-mediated negative feedback loop in keratinocytes has a critical mechanistic role in the prevention of skin inflammation caused by hyperactivation of STAT3.     

Reciprocal modulation of Toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids

Toll-like receptor-4 (TLR4) can be activated by nonbacterial agonists, including saturated fatty acids. However, downstream signaling pathways activated by nonbacterial agonists are not known. Thus, we determined the downstream signaling pathways derived from saturated fatty acid-induced TLR4 activation. Saturated fatty acid (lauric acid)-induced NFkappaB activation was inhibited by a dominant-negative mutant of TLR4, MyD88, IRAK-1, TRAF6, or IkappaBalpha in macrophages (RAW264.7) and 293T cells transfected with TLR4 and MD2. Lauric acid induced the transient phosphorylation of AKT. LY294002, dominant-negative (DN) phosphatidylinositol 3-kinase (PI3K), or AKT(DN) inhibited NFkappaB activation, p65 transactivation, and cyclooxygenase-2 (COX-2) expression induced by lauric acid or constitutively active (CA) TLR4. AKT(DN) blocked MyD88-induced NFkappaB activation, suggesting that AKT is a MyD88-dependent downstream signaling component of TLR4. AKT(CA) was sufficient to induce NFkappaB activation and COX-2 expression. These results demonstrate that NFkappaB activation and COX-2 expression induced by lauric acid are at least partly mediated through the TLR4/PI3K/AKT signaling pathway. In contrast, docosahexaenoic acid (DHA) inhibited the phosphorylation of AKT induced by lipopolysaccharide or lauric acid. DHA also suppressed NFkappaB activation induced by TLR4(CA), but not MyD88(CA) or AKT(CA), suggesting that the molecular targets of DHA are signaling components upstream of MyD88 and AKT. Together, these results suggest that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4 and its downstream signaling pathways involving MyD88/IRAK/TRAF6 and PI3K/AKT and further suggest the possibility that TLR4-mediated target gene expression and cellular responses are also differentially modulated by saturated and unsaturated fatty acids.     

Gemfibrozil, a lipid-lowering drug, upregulates IL-1 receptor antagonist in mouse cortical neurons: implications for neuronal self-defense

Chronic inflammation is becoming a hallmark of several neurodegenerative disorders and accordingly, IL-1β, a proinflammatory cytokine, is implicated in the pathogenesis of neurodegenerative diseases. Although IL-1β binds to its high-affinity receptor, IL-1R, and upregulates proinflammatory signaling pathways, IL-1R antagonist (IL-1Ra) adheres to the same receptor and inhibits proinflammatory cell signaling. Therefore, upregulation of IL-1Ra is considered important in attenuating inflammation. The present study underlines a novel application of gemfibrozil (gem), a Food and Drug Administration-approved lipid-lowering drug, in increasing the expression of IL-1Ra in primary mouse and human neurons. Gem alone induced an early and pronounced increase in the expression of IL-1Ra in primary mouse cortical neurons. Activation of type IA p110α PI3K and Akt by gem and abrogation of gem-induced upregulation of IL-1Ra by inhibitors of PI3K and Akt indicate a role of the PI3K-Akt pathway in the upregulation of IL-1Ra. Gem also induced the activation of CREB via the PI3K-Akt pathway, and small interfering RNA attenuation of CREB abolished the gem-mediated increase in IL-1Ra. Furthermore, gem was able to protect neurons from IL-1β insult. However, small interfering RNA knockdown of neuronal IL-1Ra abrogated the protective effect of gem against IL-1β, suggesting that this drug increases the defense mechanism of cortical neurons via upregulation of IL-1Ra. Taken together, these results highlight the importance of the PI3K-Akt-CREB pathway in mediating gem-induced upregulation of IL-1Ra in neurons and suggest gem as a possible therapeutic treatment for propagating neuronal self-defense in neuroinflammatory and neurodegenerative disorders.     

Immune-mediated signaling in intestinal goblet cells via PI3-kinase- and AKT-dependent pathways

In the intestinal epithelium, activation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways, via growth factor-mediated signaling, has been shown to regulate cell proliferation and inhibit apoptosis. An immune-activated receptor critical for Th2 immune responses, IL-4Ralpha can also activate PI3-kinase via insulin receptor substrate (IRS)-dependent signaling. Here, using the intestinal goblet cell-specific gene RELMbeta, we investigated the effect of PI3-kinase activation via Th2 immune responses on the goblet cell phenotype. IL-13 stimulation activated PI3-kinase and AKT signal transduction in LS174T cells. Not only did pharmacological inhibition of PI3-kinase and AKT1/2 inhibit RELMbeta induction by IL-13, but AKT inhibition also significantly reduced constitutive basal expression of RELMbeta, a response reproduced by the simultaneous pharmacological inhibition of both epidermal growth factor receptor and IGF-I receptor signaling. In vivo, the disruption of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), an inhibitor of PI3-kinase activation, led to the activation of RELMbeta expression in the small intestine. Furthermore, induction of an intestinal Th2 immune response by infection with a small intestinal nematode parasite, Heligmosomoides polygyrus, led to enhanced epithelial cell proliferation, activation of AKT as demonstrated by the loss of Foxo1 nuclear localization, and robust induction of RELMbeta expression in wild-type, but not IL-4Ralpha knockout, mice. These results demonstrate that Th2 immune responses can regulate goblet cell responses by activation of PI3-kinase and AKT pathways via IL-4Ralpha.     

Gemfibrozil,a Lipid-lowering Drug,Increases Myelin Genes in Human Oligodendrocytes via Peroxisome Proliferator-activated Receptor-β

An increase in CNS remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis. Earlier studies have shown that gemfibrozil, a lipid-lowering drug, has anti-inflammatory properties. The current study identified another novel property of gemfibrozil in stimulating the expression of myelin-specific genes (myelin basic protein, myelin oligodendrocyte glycoprotein, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, and proteolipid protein (PLP)) in primary human oligodendrocytes, mixed glial cells, and spinal cord organotypic cultures. Although gemfibrozil is a known activator of peroxisome proliferator-activated receptor-α (PPAR-α), we were unable to detect PPAR-α in either gemfibrozil-treated or untreated human oligodendrocytes, and gemfibrozil increased the expression of myelin genes in oligodendrocytes isolated from both wild type and PPAR-α(−/−) mice. On the other hand, gemfibrozil markedly increased the expression of PPAR-β but not PPAR-γ. Consistently, antisense knockdown of PPAR-β, but not PPAR-γ, abrogated the stimulatory effect of gemfibrozil on myelin genes in human oligodendrocytes. Gemfibrozil also did not up-regulate myelin genes in oligodendroglia isolated from PPAR-β(−/−) mice. Chromatin immunoprecipitation analysis showed that gemfibrozil induced the recruitment of PPAR-β to the promoter of PLP and myelin oligodendrocyte glycoprotein genes in human oligodendrocytes. Furthermore, gemfibrozil treatment also led to the recruitment of PPAR-β to the PLP promoter in vivo in the spinal cord of experimental autoimmune encephalomyelitis mice and suppression of experimental autoimmune encephalomyelitis symptoms in PLP-T cell receptor transgenic mice. These results suggest that gemfibrozil stimulates the expression of myelin genes via PPAR-β and that gemfibrozil, a prescribed drug for humans, may find further therapeutic use in demyelinating diseases.     

PI3-kinase activation by GM-CSF in endothelium is upstream of Jak/Stat pathway: role of alphaGMR

GM-CSF has been identified as a growth factor for endothelial cells. In this study, we investigated the role of PI3-kinase pathway in mediating GM-CSF induced angiogenesis. GM-CSF induced tube formation in human umbilical vein endothelial cells, as examined using Matrigel assay, was inhibited by specific inhibitors of PI3-kinase, wortmannin, and LY294002. The regulatory subunit of PI3-kinase (p85) interacted with alphaGMR via its C-SH2 domain in a GM-CSF-dependent fashion with concomitant phosphorylation of p85 and activation of PI3-kinase pathway. p85 binding site on the alphaGMR was essential to induce GM-CSF receptor-dependent Stat activation. Furthermore, inhibition of PI3-kinase activity also abrogated GM-CSF induced Stat activation. These studies underscore the significance of the GM-CSF mediated PI3-kinase activation and its role in angiogenesis.     

Phosphatidylinositol 3'-kinase, but not S6-kinase, is required for insulin-like growth factor-I and IL-4 to maintain expression of Bcl-2 and promote survival of myeloid progenitors

Phosphatidylinositol 3'-kinase (PI 3-kinase) catalyzes the formation of 3' phosphoinositides and has been implicated in an intracellular signaling pathway that inhibits apoptosis in both neuronal and hemopoietic cells. Here, we investigated two potential downstream mediators of PI 3-kinase, the serine/threonine p70 S6-kinase (S6-kinase) and the antiapoptotic protein B cell lymphoma-2 (Bcl-2). Stimulation of factor-dependent cell progenitor (FDCP) cells with either IL-4 or insulin-like growth factor (IGF)-I induced a 10-fold increase in the activity of both PI 3-kinase and S6-kinase. Rapamycin blocked 90% of the S6-kinase activity but did not affect PI 3-kinase, whereas wortmannin and LY294002 inhibited the activity of both S6-kinase and PI 3-kinase. However, wortmannin and LY294002, but not rapamycin, blocked the ability of IL-4 and IGF-I to promote cell survival. We next established that IL-3, IL-4, and IGF-I increase expression of Bcl-2 by >3-fold. Pretreatment with inhibitors of PI 3-kinase, but not rapamycin, abrogated expression of Bcl-2 caused by IL-4 and IGF-I, but not by IL-3. None of the cytokines affected expression of the proapoptotic protein Bax, suggesting that all three cytokines were specific for Bcl-2. These data establish that inhibition of PI 3-kinase, but not S6-kinase, blocks the ability of IL-4 and IGF-I to increase expression of Bcl-2 and protect promyeloid cells from apoptosis. The requirement for PI 3-kinase to maintain Bcl-2 expression depends upon the ligand that activates the cell survival pathway.