MicroRNA­93 knockdown inhibits acute myeloid leukemia cell growth via inactivating the PI3K/AKT pathway by upregulating DAB2.

Acute myeloid leukemia (AML) is associated with a poor prognosis in elderly adults and currently lacks optimal treatment strategies. MicroRNAs (miRNAs or miRs) have increasingly been reported to be associated with AML progression; however, the mechanisms of action of miR­93 in AML with the involvement of disabled 2 (DAB2) are currently unknown. In the present study, miR­93 expression was assessed in patients with AML and in AML cell lines. The association between miR­93 expression and the pathological characteristics of patients with AML was analyzed. AML cells were then transfected to knockdown or overexpress miR­93 in order to elucidate its function in AML progression. The target gene of miR­93 was assessed using a dual­luciferase reporter gene assay. The expression levels of miR­93, DAB2 and phosphatidylinositol 3­kinase (PI3K)/protein kinase B (AKT) pathway­related proteins were measured and in vivo experiments were conducted to confirm the results. It was observed that miR­93 was highly expressed in patients with AML and in AML cells. The knockdown of miR­93 in HL­60 cells inhibited AML cell proliferation and resistance to apoptosis, while the overexpression of miR­93 in THP­1 cells led to contrasting results. Moreover, miR­93 targeted DAB2 to inactivate the PI3K/AKT pathway, and the overexpression of DAB2 reversed the effects of miR­93 on THP­1 cell growth. Tumor volume, tumor weight, and the positive expression of Ki67, survivin and p53 were increased in THP­1 cells overexpressing miR­93. On the whole, the present study demonstrates that miR­93 is highly expressed in AML cells, and that the suppression of miR­93 inhibits AML cell growth by targeting DAB2 and inhibiting the PI3K/AKT pathway.

Silencing ATG6 and PI3K accelerates petal senescence and reduces flower number and shoot biomass in petunia.

Petal senescence is a form of developmental programmed cell death (PCD) that is regulated by internal and environmental signals. Autophagy, a metabolic pathway that regulates intercellular nutrient recycling, is thought to play an important role in the regulation of petal senescence-associated PCD. To characterize the function of two central autophagy genes in petal senescence, we down-regulated Autophagy Gene 6 (PhATG6) and Phosphoinositide 3-Kinase (PhPI3K) using Virus-Induced Gene Silencing (VIGS) in Petunia × hybrida. The silencing of PhATG6 and PhPI3K accelerated petal senescence, thereby reducing flower longevity. Both PhATG6- and PhPI3K-silenced petunias had reduced flower numbers, flower biomass, and vegetative shoot biomass. These phenotypes were intensified when plants were grown under low nutrient conditions. Additionally, two important regulators of senescence, an ethylene biosynthesis gene (PhACS) and a type I metacaspase gene (PhMC1), were suppressed in senescing petals of PhATG6- and PhPI3K-silenced plants. In conclusion, our study identified PhATG6 and PhPI3K as negative regulators of flower senescence and demonstrated the influence of nutrient limitation on the function of autophagy during petal senescence. Our study also found that autophagy genes potentially influence the transcriptional regulation of metacaspases and ethylene biosynthetic genes during petal senescence. The results of this project will be fundamental for future studies of petal senescence and will provide genetic information for future crop improvement.

Gomisin N Exerts Anti-liver Cancer Effects and Regulates PI3K-Akt and mTOR-ULK1 Pathways in Vitro.

Primary liver cancer is a lethal cancer. The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway has been implicated in the pathogenesis of liver cancer. Gomisin N (GN), a lignan isolated from the dried fruits of Schisandra chinensis (Turca.) Baill., has been reported to reduce viability of, and induce apoptosis in, HepG2 liver cancer cells. In preadipocytes, GN was found to inhibit Akt activity. In the present study, Akt signaling-related anti-liver cancer mechanisms of GN were investigated. We confirmed that GN reduces cell viability of, and triggers apoptosis in, more liver cancer cell lines. Mechanistic studies revealed that GN lowers protein levels of phospho-PI3K (p85 tyrosine (Tyr)458), phospho-Akt (serine (Ser)473), and Akt downstream molecules Mcl-1 in HepG2 and HCCLM3 cells. Meanwhile, GN activates mTOR and inhibits ULK1 (a negative downstream effector of mTOR) activities. Activation of mTOR has been reported to suppress ULK1 activity and repress autophagy. Indeed, we observed that GN inhibits autophagy in liver cancer cells. In summary, we for the first time demonstrated that GN inhibits the PI3K-Akt pathway and regulates the mTOR-ULK1 pathway in liver cancer cells.

Inhibition of phosphatidylinositol kinase-III alpha induces or facilitates lysosome exocytosis from microglia.

Besides degradation, lysosomes can also carry molecules for secretion out of the cell, such as ATP and cytokines, during unconventional secretion. Phosphatidylinositols and their metabolizing enzymes play important roles in the sorting and trafficking of lysosomal materials through the trans-Golgi network. The present study reveals a new function of phosphatidylinositol kinase-III alpha in the 'kiss-and-run' fusion of lysosomes at the plasma membrane to release ATP from microglia.

Involvement of CXCL12/CXCR4 in the motility of human first-trimester endometrial epithelial cells through an autocrine mechanism by activating PI3K/AKT signaling.

BACKGROUND: CXCL12(chemokine ligand 12, CXCL12) and its receptors CXCR4 are widely expressed in maternal-fetal interface and plays an adjust role in materno-fetal dialogue and immune tolerance during early pregnancy. This study aimed to evaluate the role and mechanism of self-derived CXCL12 in modulating the functions of human first-trimester endometrial epithelial cells (EECs) and to identify the potential protein kinase signaling pathways involved in the CXCL12/CXCR4's effect on EECs. METHODS: The expression of CXCL12 and CXCR4 in EECs was measured by using immunohistochemistry, immunofluorescence, real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The effects of EEC-conditioned medium (EEC-CM) and recombinant human CXCL12 (rhCXCL12) on EEC migration and invasion in vitro were evaluated with migration and invasion assays. In-cell western blot analysis was used to examine the phosphorylation of protein kinase B (AKT), extracellular regulated protein kinases (ERKs) and phosphatidylinositol 3-kinase (PI3K) after CXCL12 treatment. RESULTS: CXCL12 and CXCR4 were both expressed in human first-trimester EECs at the mRNA and protein level. Both EEC-CM and rhCXCL12 significantly increased the migration and invasion of EECs (P < 0.05), which could be blocked by neutralizing antibodies against CXCR4 (P < 0.05) or CXCL12 (P < 0.05), respectively. CXCL12 activated both PI3K/AKT and ERK1/2 signaling and CXCR4 neutralizing antibody effectively reduced CXCL12-induced phosphorylation of AKT and ERK1/2. LY294002, a PI3K-AKT inhibitor, was able to reverse the promotive effect of EEC-CM or rhCXCL12 on EEC migration and invasion. CONCLUSIONS: Human first-trimester EECs promoted their own migration and invasion through the autocrine mechanism with CXCL12/CXCR4 axis involvement by activating PI3K/AKT signaling. This study contributes to a better understanding of the epithelium function mediated by chemokine and chemokine receptor during normal pregnancy.

PI3K/Akt/FoxO pathway mediates glycolytic metabolism in HepG2 cells exposed to triclosan (TCS).

Triclosan (TCS) has been widely used as an antibacterial agent for the last several decades in personal care products. The toxicological effect of TCS has attracted more and more attention of researchers. The purpose of this study is to evaluate the cytotoxic effects of TCS in HepG2 cells and to elucidate the molecular mechanism focusing on regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/forkhead box O (FoxO) pathway in the glycolytic metabolism. In this study, we evaluated the adverse effect of TCS exposure on cell viability, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and mitochondrial membrane potential (MMP). In addition, the glycolysis process in HepG2 cells exposed to TCS was examined in terms of glucose consumption, lactate production and ATP generation. Furthermore, Affymetrix Human U133 plus 2.0 gene chips and gene function enrichment analysis were conducted to screen differential expression genes (DEGs) and potential signaling pathway. Expressions of the glycolysis-related proteins were measured and quantified with Western Blotting. The results showed that TCS could suppress the cell viability, induce oxidative stress, and cause mitochondrial damage. In addition, TCS exposure promoted the glycolysis process, as manifested by accelerated conversion of glucose to lactate and increased energy release. Western Blotting results confirmed that the expression levels of glycolysis related proteins were significantly elevated. The PI3K/Akt/FoxO pathway was identified to play a pivot role in TCS-induced glycolysis, which was further confirmed by inhibitor tests using specific inhibitors LY294002 and MK2206. In general, TCS can induce oxidative stress, cause oxidative damages and promote glycolysis in HepG2 cells, which was mediated by the PI3K/Akt/FoxO pathway.

Effects of resveratrol on the expression of molecules related to the mTOR signaling pathway in pathological scar fibroblasts.

BACKGROUND: We observed the effects of resveratrol on the expression of molecules involved in the mTOR signaling pathway in pathological scar fibroblasts, including PI3K, Akt and mTOR. METHODS: We detected the expression of PI3K, Akt and mTOR in pathological scar and normal skin fibroblasts through immunofluorescence. After being treated with different concentrations of resveratrol, the expression of PI3K, Akt and mTOR mRNA and protein were detected by RT-PCR and Western Blot respectively. RESULTS: Results showed that the expression of PI3K, Akt and mTOR were significantly enhanced in pathological scar fibroblasts and mainly expressed in the nucleus, with no expression in normal skin fibroblasts. Results from RT-PCR and Western Blot tests demonstrated that after Res intervention with different concentrations for pathological scar fibroblasts, the relative expression quantity of PI3K mRNA and protein decreased and showed a dose dependent relationship. Compared to the control group, the differences were statistically significant (P<0.05). However, decrease in the expression of PI3K mRNA and protein was not obvious and there were no significant differences in comparison to the control group (P>0.05). CONCLUSIONS: The mechanism of resveratrol in the inhibition of the proliferation of pathological scar fibroblasts may be related to its down-regulation in the expression of Akt and mTOR, which are the key molecules of mTOR signaling pathway.

EGF suppresses the expression of miR-124a in pancreatic ß cell lines via ETS2 activation through the MEK and PI3K signaling pathways.

Diabetes mellitus is characterized by pancreatic ß cell dysfunction. Previous studies have indicated that epidermal growth factor (EGF) and microRNA-124a (miR-124a) play opposite roles in insulin biosynthesis and secretion by beta cells. However, the underlying mechanisms remain poorly understood. In the present study, we demonstrated that EGF could inhibit miR-124a expression in beta cell lines through downstream signaling pathways, including mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) cascades. Further, the transcription factor ETS2, a member of the ETS (E26 transformation-specific) family, was identified to be responsible for the EGF-mediated suppression of miR-124a expression, which was dependent on ETS2 phosphorylation at threonine 72. Activation of ETS2 decreased miR-124a promoter transcriptional activity through the putative conserved binding sites AGGAANA/TN in three miR-124a promoters located in different chromosomes. Of note, ETS2 played a positive role in regulating beta cell function-related genes, including miR-124a targets, Forkhead box a2 (FOXA2) and Neurogenic differentiation 1 (NEUROD1), which may have partly been through the inhibition of miR-124 expression. Knockdown and overexpression of ETS2 led to the prevention and promotion of insulin biosynthesis respectively, while barely affecting the secretion ability. These results suggest that EGF may induce the activation of ETS2 to inhibit miR-124a expression to maintain proper beta cell functions and that ETS2, as a novel regulator of insulin production, is a potential therapeutic target for diabetes mellitus treatment.

The role of PI3K-mediated AMPA receptor changes in post-conditioning of propofol in brain protection.

BACKGROUND: We aimed to study the role of amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) glutamate receptor 2 (GluR2) subunit trafficking, and activity changes in short-term neuroprotection provided by propofol post-conditioning. We also aimed to determine the role of phosphoinositide-3-kinase (PI3K) in the regulation of these processes. METHODS: Rats underwent 1 h of focal cerebral ischemia followed by 23 h of reperfusion were randomly divided into 6 groups (n = 36 per group): sham- operation (S), ischemia-reperfusion (IR), propofol (P group, propofol 20 mg/kg/h at the onset of reperfusion for 2 h after 60 min of occlusion), and LY294002 (PI3K non-selective antagonist) + sham (L + S, LY294002 of 1.5 mg/kg was infused 30 min before sham operation), LY294002+ ischemia-reperfusion (L + IR, LY294002 of 1.5 mg/kg was infused 30 min before middle cerebral artery occlusion), LY294002 + IR + propofol (L + P, LY294002 of 1.5 mg/kg was infused 30 min before middle cerebral artery occlusion and propofol 20 mg/kg/h at the onset of reperfusion for 2 h after 60 min of occlusion). RESULTS: Compared with group IR, rats in group P had significant lower neurologic defect scores and infarct volume. Additionally, consistent with enhanced expression of PI3K-AMPAR GluR2 subunit complex substances in ipsilateral hippocampus, GluR2 subunits showed increased levels in both the plasma and postsynaptic membranes of neurons, while pGluR2 expression was reduced in group P. Furthermore, LY294002, the PI3K non-selective antagonist, blocked those effects. CONCLUSION: These observations demonstrated that propofol post-conditioning revealed acute neuroprotective role against transient MCAO in rats. The short-term neuroprotective effect was contributed by enhanced GluR2 subunits trafficking to membrane and postsynaptic membranes of neurons, as well as down-regulated the expression of pGluR2 in damaged hippocampus. Finally, the above-mentioned protective mechanism might be contributed by increased combination of PI3K to AMPAR GluR2 subunit, thus maintained the expression and activation of AMPAR GluR2 in the ipsilateral hippocampus.

A potential role of toll-like receptors, IFN-γ and the phosphatidylinositol 3-kinase pathway in the pathogenesis of acquired mediastinal lymphatic malformation.

Sarcoidosis is a multisystem disorder with non-caseating granulomas in various organs. The etiology of sarcoid granuloma formation is not clear and likely an antigen-induced process. We came across a previously treated sarcoidosis patient who presented with worsening dyspnea on exertion for several months and several days of difficulty swallowing. On Chest CT imaging, large posterior mediastinal mass was found that subsequently diagnosed as macrocystic lymphatic malformation after surgical resection. Pathophysiology of development of acquired lymphatic malformations in a sarcoidosis patient is currently not clear. We hypothesize there might be a complex interplay of Toll-like receptors, IFN-γ and the phosphatidylinositol 3-kinase pathway in the pathogenesis.

Impact of PI3Kα (Phosphoinositide 3-Kinase Alpha) Inhibition on Hemostasis and Thrombosis.

Objective- PI3Kα (phosphoinositide 3-kinase alpha) is a therapeutic target in oncology, but its role in platelets and thrombosis remains ill characterized. In this study, we have analyzed the role of PI3Kα in vitro, ex vivo, and in vivo in 2 models of arterial thrombosis. Approach and Results- Using mice selectively deficient in p110α in the megakaryocyte lineage and isoform-selective inhibitors, we confirm that PI3Kα is not mandatory but participates to thrombus growth over a collagen matrix at arterial shear rate. Our data uncover a role for PI3Kα in low-level activation of the GP (glycoprotein) VI-collagen receptor by contributing to ADP secretion and in turn full activation of PI3Kß and Akt/PKB (protein kinase B). This effect was no longer observed at high level of GP VI agonist concentration. Our study also reveals that over a vWF (von Willebrand factor) matrix, PI3Kα regulates platelet stationary adhesion contacts under arterial flow through its involvement in the outside-in signaling of vWF-engaged αIIbß3 integrin. In vivo, absence or inhibition of PI3Kα resulted in a modest but significant decrease in thrombus size after superficial injuries of mouse mesenteric arteries and an increased time to arterial occlusion after carotid lesion, without modification in the tail bleeding time. Considering the more discrete and nonredundant role of PI3Kα compared with PI3Kß, selective PI3Kα inhibitors are unlikely to increase the bleeding risk at least in the absence of combination with antiplatelet drugs or thrombopenia. Conclusions- This study provides mechanistic insight into the role of PI3Kα in platelet activation and arterial thrombosis.

Resveratrol promotes in vitro activation of ovine primordial follicles by reducing DNA damage and enhancing granulosa cell proliferation via phosphatidylinositol 3-kinase pathway.

We aimed to study the effects of resveratrol on the morphology, DNA fragmentation, follicular activation and cell proliferation after in vitro culture of ovine ovarian tissue, and to verify if PI3K pathway is involved in resveratrol action in the sheep ovary. Ovaries were collected and divided into fragments. One fragment was fixed for histology (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+ ) alone or with resveratrol (2, 10 or 30 µM). After culture, ovarian tissue was destined to morphological analysis. TUNEL and proliferating cell nuclear antigen (PCNA) analyses were performed in the fresh control, α-MEM+ and 2 µM resveratrol. Inhibition of PI3K activity was performed through pre-treatment with LY294002. The percentage of normal follicles was similar between α-MEM+ and 2 µM resveratrol, and higher than those in other resveratrol treatments. An increase in follicular activation was observed in all treatments compared to fresh control. DNA fragmentation decreased in tissues cultured in 2 µM resveratrol compared to α-MEM+ . Moreover, PCNA-positive cells were higher in 2 µM resveratrol than in α-MEM+ . LY294002 inhibited follicular activation stimulated by α-MEM+ and 2 µM resveratrol. In conclusion, 2 µM resveratrol promotes primordial follicle activation compared to the fresh control by reducing DNA fragmentation and stimulating granulosa cell proliferation through activation of the PI3K pathway.

Neuroprotective effect of zolpidem against glutamate-induced toxicity is mediated via the PI3K/Akt pathway and inhibited by PK11195.

Excitotoxicity is a pathological process in which neuronal dysfunction and death are induced by excessive glutamate stimulation, the major fast excitatory neurotransmitter in the mammalian brain. Excitotoxicity-induced neurodegeneration is a contributing factor in ischemia-induced brain damage, traumatic brain injury, and various neurodegenerative diseases. It is triggered by calcium overload due to prolonged over-activation of ionotropic N-methyl-d-aspartate (NMDA) receptors. Enhanced Ca2+ release results in neuronal vulnerability through several intertwined mechanisms, including activation of proteolytic enzymes, increased production of reactive oxygen species (ROS), mitochondrial dysfunction and modulation of intracellular signalling pathways. We investigated the neuroprotective effect of hypnotic zolpidem, a drug that exerts its central effects at the GABAA receptor complex, against glutamate-induced toxicity in P19 neurons. Zolpidem prevented death of P19 neurons exposed to glutamate, and abolished the glutamate-induced increase in ROS production, p53 and Bax expression, and caspase-3/7 activity. Zolpidem effects were mediated by marked over-activation of Akt kinase. The pro-survival effect, as well as the pAkt induction, were prevented in the presence of wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K) that functions upstream of Akt. The beneficial effect of zolpidem on neuronal survival was not prevented by flumazenil, a GABAA receptor antagonist. PK11195, a drug that modulates the mitochondrial translocator protein 18 kDa (TSPO) and F0F1-ATPase, prevented the beneficial effect of zolpidem, indicating that the mechanism of zolpidem action involves preservation of mitochondrial function and integrity. Zolpidem effects were further mediated by prevention of glutamate-induced increase in the expression of the NR2B subunit of NMDA receptor. The obtained results suggest the promising therapeutic potential of zolpidem against excitotoxic insults and highlight the importance of mitochondria and the Akt pathway as valuable targets for therapeutic interventions in glutamate-mediated neuropathological conditions.

Granulocyte colony-stimulating factor improves neurological function and angiogenesis in intracerebral hemorrhage rats.

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) plays a role in regulating phosphatidylinositol-3-kinase/serine/threonine kinase (PI3K/AKT) pathway, affecting cell proliferation and apoptosis, and inducing vascular endothelial growth factor (VEGF) expression. This study investigated the mechanism of G-CSF on angiogenesis and neural protection after intracerebral hemorrhage (ICH). MATERIALS AND METHODS: The rats were divided into four groups, including sham, ICH, ICH+G-CSF, and ICH+G-CSF+LY294002 (PI3K/AKT signaling pathway specific inhibitor). Cerebral neurological dysfunction was tested by Garcia scoring. Cell apoptosis was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay. Angiogenesis marker CD34 expression, PI3K/AKT signaling pathway, B-cell lymphoma-2 (Bcl-2), and VEGF expressions were compared by IHC. Rat cerebral nerve RN-c cells were divided into four groups, including control, oxygen-glucose deprivation (OGD), OGD+G-CSF, and OGD+G-CSF+LY294002. RESULTS: Neurological dysfunction was more evident; CD34+ cell number, VEGF expression, and cell apoptosis significantly increased; phosphorylated AKT (p-AKT) and Bcl-2 levels markedly reduced in ICH group compared with sham group. G-CSF apparently up-regulated p-AKT and Bcl-2 expressions, attenuated cell apoptosis, and elevated CD34+ cell number. LY294002 significantly decreased p-AKT, Bcl-2, and VEGF expressions, and alleviated the cell apoptosis protective and angiogenesis effect induced by G-CSF. OGD treatment induced RN-c cell apoptosis, down-regulated p-AKT and Bcl-2 expressions, and enhanced the tube capacity of vascular endothelial cells (VEC). G-CSF markedly elevated p-AKT and Bcl-2 contents in RN-c cells, declined cell apoptosis, increased p-AKT and VEGF levels in VEC, and enhanced tube capacity. CONCLUSIONS: G-CSF enhanced PI3K/AKT signaling pathway activity, promoted Bcl-2 and VEGF expression, reduced nerve cell apoptosis, and enhanced tube capacity of VECs, which may be the mechanism of G-CSF in improving neurological function and angiogenesis after ICH.

Cinnamaldehyde accelerates wound healing by promoting angiogenesis via up-regulation of PI3K and MAPK signaling pathways.

The bark of Cinnamomum cassia (C. cassia) has been used for the management of coronary heart disease (CHD) and diabetes mellitus. C. cassia may target the vasculature, as it stimulates angiogenesis, promotes blood circulation and wound healing. However, the active components and working mechanisms of C. cassia are not fully elucidated. The Shexiang Baoxin pill (SBP), which consists of seven medicinal materials, including C. cassia etc., is widely used as a traditional Chinese patent medicine for the treatment of CHD. Here, 22 single effective components of SBP were evaluated against the human umbilical vein endothelial cells (HUVECs). We demonstrated that in HUVECs, cinnamaldehyde (CA) stimulated proliferation, migration, and tube formation. CA also activated the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Furthermore, the secretion of vascular endothelial growth factor (VEGF) from HUVECs was increased by CA. In vivo, CA partially restored intersegmental vessels in zebrafish pretreated with PTK787, which is a selective inhibitor for vascular endothelial growth factor receptor (VEGFR). CA also showed pro-angiogenic efficacy in the Matrigel plug assay. Additionally, CA attenuated wound sizes in a cutaneous wound model, and elevated VEGF protein and CD31-positive vascular density at the margin of these wounds. These results illustrate that CA accelerates wound healing by inducing angiogenesis in the wound area. The potential mechanism involves activation of the PI3K/AKT and MAPK signaling pathways. Such a small non-peptide molecule may have clinical applications for promoting therapeutic angiogenesis in chronic diabetic wounds and myocardial infarction.

PI3K Is a Linker Between L-selectin and PSGL-1 Signaling to IL-18 Transcriptional Activation at the Promoter Level.

L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) are adhesion molecules which induce similar physiological events. Our previous paper showed that phosphatidylinositol 3-kinase (PI3K) played a crucial role in L-selectin- and PSGL-1-mediated F-actin redistribution and assembly during neutrophil rolling on E-selectin. However, it is not clear whether L-selectin and PSGL-1 induce other similar physiology events by PI3K. Here, we investigated the possibility of PI3K linking the signaling pathways of L-selectin and PSGL-1 to IL-18 transcription. We first demonstrated that L-selectin and PSGL-1 stimulation upregulated IL-18 transcription level in Jurkat cells. Then we found that PI3K inhibitor LY294002 reduced L-selectin- and PSGL-1-induced mRNA upregulation of IL-18 in Jurkat cells. Transfection of phosphatase and tensin homolog expressing plasmid inhibited the transcription level of IL-18. Therefore, PI3K is a signal linker between L-selectin and PSGL-1 in IL-18 transcriptional activation at the promoter level. To our knowledge, this is the first time to directly link PI3K to L-selectin- and PSGL-1-mediated IL-18 transcription, providing a foundation for intervention of PI3K-related inflammation.

Role of PKA and PI3K in Leptin and Ghrelin Regulation of Adaptive Subpopulations of Regulatory CD4+ T-Lymphocyte Formation.

The role of phosphatidylinositol-3 kinase (PI3K) and protein kinase A (PKA) in leptin and ghrelin regulation of formation of adaptive (a) subpopulations of CD4+ T-lymphocytes (helper (h) cells producing interleukin-17A) (aTÒ»17) and of T-regulatory lymphocytes (aTreg) in the context of physiological pregnancy is established. It is shown that leptin at a concentration typical for the second half of pregnancy (trimesters II-III) enhances the differentiation of aTÒ»17 with a high level of interleukin-17A (IL-17A) production and the expression of the chemokine receptor CCR6 with the participation of PI3K. Simultaneously, leptin reduces formation of aTreg expressing the suppressor molecule CTLA-4, which determines the function of these cells. Ghrelin at a concentration characteristic of the first half of pregnancy (trimesters I-II), in contrast, enhances aTreg formation and, in parallel, reduces the level aTÒ»17 (that express CCR6) and the IL-17A production by aTh17. PKA, likewise PI3K, participates in regulatory effects of ghrelin on the formation of aTÒ»17 and aTreg. The combined action of leptin and ghrelin (via PKA participation) enhances formation of only aTreg, which determines the priority of this molecular mechanism and explains why the investigated hormones with reciprocal differentiating potential do not come into antagonism at the level of immune system cells during pregnancy.

Inhaled Methane Protects Rats Against Neurological Dysfunction Induced by Cerebral Ischemia and Reperfusion Injury: PI3K/Akt/HO-1 Pathway Involved.

BACKGROUND AND AIMS: Cerebral ischemia and reperfusion (I/R) could produce excess reactive oxygen species (ROS), which in turn induce neurological dysfunction and inflammation in cerebral tissues. This study was designed to study the effect of methane on cerebral I/R injury. METHODS: Fifty Sprague-Dawley (SD) rats were used to induce an animal model of cerebral I/R injury. Methane was mixed with air to achieve a final concentration of 2.2%. Rats started to inhale methane-air mixture after ischemia and continued it during the reperfusion. The neurological deficits, malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α) in the brain tissue were examined. The protein kinase B (Akt) phosphorylation and heme oxygenase-1 (HO-1) expression was measured by Western Blot. The neurological deficits were re-measured after rats were treated with the HO-1 inhibitor Zinc protoporphyrin IX (ZnPP-IX), phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and Akt inhibitor triciribine. RESULTS: Cerebral I/R induced neurological deficit, which was significantly decreased by methane. MDA and TNF-α levels were significantly enhanced by cerebral I/R, while methane caused significant reduction of MDA and TNF-α levels. Methane significantly increased Akt phosphorylation and HO-1 expression. The HO-1 inhibitor ZnPP-IX, PI3K inhibitor LY294002 and Akt inhibitor triciribine all significantly abolished the effect of methane on neurological deficit. CONCLUSIONS: This finding suggests the possible application of methane for cerebral I/R injury and PI3K/Akt/HO-1 dependent antioxidant pathway may be involved.

Phosphatidylinositol 3-Kinase and Protein Kinase C Signaling Pathways Are Involved in Stromal Cell-derived Factor-1α-mediated Transmigration of Stem Cells from Apical Papilla.

INTRODUCTION: Previously, we have shown that stem cells from apical papilla (SCAPs) can be chemoattracted by stromal cell-derived factor-1α (SDF-1α). The purpose of this study was to investigate the intracellular signaling pathways involved in SDF-1α-mediated migration of SCAPs. METHODS: Chemotaxis assays were performed to assess the effect of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) signaling pathways in the SDF-1α-mediated migration of SCAPs using inhibitors of PI3K (LY294002) or PKC (GF109203X). The Cell Counting Kit-8 assay (Dojindo Laboratories, Kumamoto, Japan) was used to evaluate the effect of the inhibitors on the proliferation of SCAPs. The expression of focal adhesion-related proteins was examined by immunofluorescence staining and Western blot analysis. Phosphorylation of PI3K subunit p85 and PKC after SDF-1α induction was evaluated by Western blot. RESULTS: The inhibition of PI3K or PKC signaling pathways significantly reduced SDF-1α-mediated migration of SCAPs. The inhibitors had no effect on the proliferation of SCAPs. Immunofluorescence analysis revealed that SDF-1α stimulated focal adhesion formation and stress fiber assembly in SCAPs, in addition to up-regulation of the expression of focal adhesion molecules, including p-focal adhesion kinase, p-paxillin, and vinculin. Pretreatment with PI3K or PKC inhibitors before SDF-1α induction significantly inhibited focal adhesion molecule expression. Moreover, increased phosphorylation of p85 and PKC were observed after SDF-1α stimulation, whereas these phosphorylations were down-regulated by the inhibition of PI3K or PKC signaling pathways. CONCLUSIONS: PI3K and PKC signaling pathways appear to be required for SDF-1α-mediated transmigration of SCAPs. These findings provide insights into the signaling mechanisms that underlie SDF-1α-mediated migration of SCAPs.