Compound K, a metabolite of ginsenosides, induces cardiac protection mediated nitric oxide via Akt/PI3K pathway

AimsCompound K (C-K; 20-O-d-glucopyranosyl-20(S)-protopanaxadiol) is a novel ginsenoside metabolite formed by intestinal bacteria and does not occur naturally in ginseng. In this study, we investigated whether administration of C-K has protective effects on myocardial ischemia-reperfusion injury and its potential mechanisms.Main methodsWe used in vivo mouse models of ischemia-reperfusion injury and performed biochemical assays in excised hearts.Key findingsC-K reduced infarct size compared with the control group after ischemia-reperfusion. Immunoblot analysis showed that C-K significantly enhanced protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) activity. Wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, blocked cardiac protection in vivo and attenuated phosphorylation of Akt and eNOS. Additionally, the hearts of C-K pretreated mice showed inhibition of mitochondrial swelling induced by Ca²⁺.SignificanceThis study showed that Compound K pretreatment has protective effects on myocardial ischemia-reperfusion injury, partly by mediating the activation of PI3K pathway and phosphorylation of Akt and eNOS.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide

The coupling between neuronal activity and cerebral blood flow (CBF) is essential for normal brain function. The mechanisms behind this neurovascular coupling process remain elusive, mainly because of difficulties in probing dynamically the functional and coordinated interaction between neurons and the vasculature in vivo. Direct and simultaneous measurements of nitric oxide (NO) dynamics and CBF changes in hippocampus in vivo support the notion that during glutamatergic activation nNOS-derived NO induces a time-, space-, and amplitude-coupled increase in the local CBF, later followed by a transient increase in local O₂ tension. These events are dependent on the activation of the NMDA-glutamate receptor and nNOS, without a significant contribution of endothelial-derived NO or astrocyte–neuron signaling pathways. Upon diffusion of NO from active neurons, the vascular response encompasses the activation of soluble guanylate cyclase. Hence, in the hippocampus, neurovascular coupling is mediated by nNOS-derived NO via a diffusional connection between active glutamatergic neurons and blood vessels.     

Mycobacterium tuberculosis-induced expression of Leukotactin-1 is mediated by the PI3-K/PDK1/Akt signaling pathway

Chemokines function in the migration of circulating leukocytes to regions of inflammation, and have been implicated in chronic inflammatory conditions including mycobacterial infection. We investigated whether Leukotactin-1 (Lkn-1), a novel member of the CC-chemokines, is involved in the immune response of macrophages against Mycobacterium tuberculosis (MTB). In PMA-differentiated THP-1 cells, MTB infection increased mRNA expression of Lkn-1 in a dose-dependent manner. Lkn-1 induction peaked 12 h after infection, then declined gradually and returned to its basal level at 72 h. Secretion of Lkn-1 was elevated by MTB infection. The increase in expression and secretion of Lkn-1 caused by MTB was reduced in cells treated with inhibitors of phosphatidylinositol 3-kinase (PI3-K), 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt. MTB-induced Akt phosphorylation was blocked by treatment with a PI3-K inhibitor or a PDK1 inhibitor, implying that PI3-K, PDK1, and Akt are associated with the signaling pathway that up-regulates Lkn-1 in response to MTB. These results suggest that Lkn-1 is novel member of the group of chemokines that is released by macrophages infected with MTB.     

SLC1A3 promotes gastric cancer progression via the PI3K/AKT signalling pathway

Gastric cancer is a major cause of mortality worldwide. The glutamate/aspartate transporter SLC1A3 has been implicated in tumour metabolism and progression, but the roles of SLC1A3 in gastric cancer remain unclear. We used bioinformatics approaches to analyse the expression of SLC1A3 and its role in gastric cancer. The expression levels of SLC1A3 were examined using RT‐qPCR and Western bolting. SLC1A3 overexpressing and knock‐down cell lines were constructed, and the cell viability was evaluated. Glucose consumption, lactate excretion and ATP levels were determined. The roles of SLC1A3 in tumour growth were evaluated using a xenograft tumour growth model. SLC1A3 was found to be overexpressed in gastric cancer, and this overexpression was associated with poor prognosis. In vitro and in vivo assays showed that SLC1A3 affected glucose metabolism and promoted gastric cancer growth. GSEA analysis suggested that SLC1A3 was positively associated with the up‐regulation of the PI3K/AKT pathway. SLC1A3 overexpression activated the PI3K/AKT pathway and up‐regulated GLUT1, HK II and LDHA expression. The PI3K/AKT inhibitor LY294002 prevented SLC1A3‐induced glucose metabolism and cell proliferation. Our findings indicate that SLC1A3 promotes gastric cancer progression via the PI3K/AKT signalling pathway. SLC1A3 is therefore a potential therapeutic target in gastric cancer.     

Isorhamnetin Attenuates Atherosclerosis by Inhibiting Macrophage Apoptosis via PI3K/AKT Activation and HO-1 Induction

Background and Purpose

Isorhamnetin (Iso) is a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L. Previous studies have revealed its anti-cancer, anti-inflammatory, and anti-oxidant activities. This study investigated the ability of Iso to inhibit oxidized low-density lipoprotein (ox-LDL)-induced cell apoptosis in THP-1-derived macrophages. The effects of Iso on atherosclerosis in vivo were also evaluated in apolipoprotein E knockout (ApoE-/-) mice fed a high fat diet.

Methods and Results

Iso showed significant inhibitory effects on ox-LDL-induced THP-1-derived macrophage injuries via decreasing reactive oxygen species levels, lipid deposition, and caspase-3 activation, restoring mitochondrial membrane potential, reducing the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells, and regulating apoptosis-related proteins. We also determined the protective effects of Iso by PI3K/AKT activation and HO-1 induction. Iso reduced the atherosclerotic plaque size in vivo in ApoE-/- mice as assessed by oil red O, Sudan IV staining, and CD68-positive cells, and reduced macrophage apoptosis as assessed by caspase-3 and TUNEL assays in lesions.

Conclusion

In conclusion, our results show that Iso inhibited atherosclerotic plaque development in ApoE-/- mice by PI3K/AKT activation and HO-1 induction.     

OxLDL stimulates Id1 nucleocytoplasmic shuttling in endothelial cell angiogenesis via PI3K pathway

Angiogenesis plays remarkable roles in the development of atherosclerotic rupture plaques. However, its essential mechanism remains unclear. The purpose of the study was to investigate whether inhibitor of DNA binding-1 or inhibitor of differentiation 1 (Id1) promoted angiogenesis when exposed to oxidised low-density lipoprotein (oxLDL), and to determine the molecular mechanism involved. Using aortic ring assay and tube formation assay as a model system, a low concentration of oxLDL was found to induce angiogenic sprouting and capillary lumen formation of endothelial cell. But the Id1 expression was significantly upregulated by oxLDL at low and high concentrations. The Id1 was localised in the nuclei of the human umbilical vein endothelial cells in the control group and in the high-concentration oxLDL group. Id1 was translocated to the cytoplasm at low oxLDL concentrations. The nucleocytoplasmic shuttling at low oxLDL concentration was inhibited by treatment with the nuclear export inhibitor leptomycin B. Protein kinase A (PKA) inhibitor H89 promoted nuclear export of Id1, and phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 reduced the nuclear export of Id1. PI3K inhibition blocked oxLDL-induced angiogenesis. Low concentrations of oxLDL promoted angiogenic sprouting and capillary formation. And this process depends on nuclear export of Id1, which in turn is controlled by the PI3K pathway. This report presents a new link between oxLDL and Id1 localisation, and may provide a new insight into the interactions of ox-LDL and Id1 in the context of atherosclerosis.     

Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway

The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.     

Rapid increase in endothelial nitric oxide production by bradykinin is mediated by protein kinase A signaling pathway

Bradykinin (BK) acutely increases endothelial nitric oxide (NO) production by activating endothelial NO synthase (eNOS), and this increase is in part correlated with enhanced phosphorylation/dephosphorylation of eNOS by several protein kinases and phosphatases. However, the signaling mechanisms producing this increase are still controversial. In an attempt to delineate the acute effect of BK on endothelial NO production, confluent bovine aortic endothelial cells were incubated with BK, and NO production was measured by NO-specific chemiluminescence. Significant increase in NO levels was detected as early as 1 min after BK treatment, with concomitant increase in the phosphorylation of Ser(1179) (bovine sequence) site of eNOS (eNOS-Ser(1179)). This acute effect of BK on both increases was blocked only by treatment of protein kinase A inhibitor H-89, but not by the inhibitors of calmodulin-dependent kinase II and protein kinase B, suggesting that the rapid increase in NO production by BK is mediated by the PKA-dependent phosphorylation of eNOS-Ser(1179).     

Thymus-expressed chemokine promotes survival of PC12 cells via PI3K pathway

We reported previously that CCR9 was neuroprotective in the mouse hippocampal neurons. This study was aimed to investigate if thymus-expressed chemokine (TECK)/CCL25 could promote survival of PC12 cells though its receptor CCR9. pEGFP-N1/CCR9 recombinant was constructed and transfected into PC12 cells. Along with this, 50 nM NGF was used to induce PC12 cells to differentiate into sympathetic-like neurons. We show here that under serum-free conditions and within a concentration range (50-200 nM), TECK rescued pEGFP-N1/CCR9 transfected PC12 cells from undergoing apoptosis in serum-free medium; however, it did not exert a similar effect on the cells in the control. On the other hand, the PC12 cells succumbed to a higher concentration of TECK (≥ 300 nM). Bim expression was up-regulated in PC12 cells cultured in serum-free medium in the absence of factors or with anti-TECK+TECK; however, it was not up-regulated in TECK-treated PC12 cells. p-Akt was detected at 15 min which lasted for at least 60 min when PC12 cells were cultured in serum-free medium with TECK. Additionally, it was shown that such an effect was effectively blocked by PI3K inhibitor, Wortmannin. These data suggest that TECK promotes survival of serum-deprived PC12 cells through its receptor, CCR9, most likely via the PI3K/Akt signaling pathway.     

FGFRL1 affects chemoresistance of small‐cell lung cancer by modulating the PI3K/Akt pathway via ENO1

Fibroblast growth factor receptor‐like 1 (FGFRL1), a member of the FGFR family, has been demonstrated to play important roles in various cancers. However, the role of FGFRL1 in small‐cell lung cancer (SCLC) remains unclear. Our study aimed to investigate the role of FGFRL1 in chemoresistance of SCLC and elucidate the possible molecular mechanism. We found that FGFRL1 levels are significantly up‐regulated in multidrug‐resistant SCLC cells (H69AR and H446DDP) compared with the sensitive parental cells (H69 and H446). In addition, clinical samples showed that FGFRL1 was overexpressed in SCLC tissues, and high FGFRL1 expression was associated with the clinical stage, chemotherapy response and survival time of SCLC patients. Knockdown of FGFRL1 in chemoresistant SCLC cells increased chemosensitivity by increasing cell apoptosis and cell cycle arrest, whereas overexpression of FGFRL1 in chemosensitive SCLC cells produced the opposite results. Mechanistic investigations showed that FGFRL1 interacts with ENO1, and FGFRL1 was found to regulate the expression of ENO1 and its downstream signalling pathway (the PI3K/Akt pathway) in SCLC cells. In brief, our study demonstrated that FGFRL1 modulates chemoresistance of SCLC by regulating the ENO1‐PI3K/Akt pathway. FGFRL1 may be a predictor and a potential therapeutic target for chemoresistance in SCLC.     

Rotavirus-encoded virus-like small RNA triggers autophagy by targeting IGF1R via the PI3K/Akt/mTOR pathway

Rotaviruses are double-stranded RNA viruses that are a major cause of viral diarrhea in infants. Examining virus–host cell interaction is important for elucidating mechanisms of virus proliferation in host cells. Viruses can create an environment that promotes their survival and self-proliferation by encoding miRNAs or miRNA-like molecules that target various host cell. However, it remains unclear whether RNA viruses encode viral miRNAs, and their regulation mechanisms are largely unknown. We previously performed deep sequencing analysis to investigate rotavirus-encoded miRNAs, and identified the small RNA molecule Chr17_1755, which we named RV-vsRNA1755. In our present study, we determined that RV-vsRNA1755 is encoded by the rotavirus NSP4 gene and that it targets the host cell IGF1R, which is part of the PI3K/Akt pathway. We further explored the biological characteristics and functions of RV-vsRNA1755.Our results suggest that rotavirus adapts to manipulate PI3K/Akt signaling at early phases of infection. RV-vsRNA1755 targets IGF1R, blockading the PI3K/Akt pathway and triggering autophagy, but it ultimately inhibits autophagy maturation. A mechanism through which rotavirus encodes a virus-like small RNA (RV-vsRNA1755) that triggers autophagy by targeting the host cell IGF1R gene was revealed. These data provide a theoretical basis for therapeutic drug screening targeting RV-vsRNA1755.     

Regulation of survivin by PI3K/Akt/p70S6K1 pathway

PI3K activation is commonly observed in many human cancer cells. Survivin expression is elevated in cancer cells, and induced by some growth factors through PI3K activation. However, it is not clear whether PI3K activation is sufficient to induce survivin expression. To investigate the role of PI3K pathway in the regulation of survivin, we expressed an active form of PI3K, v-P3k in chicken embryonic fibroblast cells (CEF), and found that overexpression of PI3K-induced survivin mRNA expression. Forced expression of wild-type but not mutant tumor suppressor PTEN in CEF decreased survivin mRNA levels. PI3K regulates survivin expression through Akt activation. To further investigate downstream target of PI3K and Akt in regulating the expression of survivin mRNA, we found that PI3K and Akt-induced p70S6K1 activation and that overexpression of p70S6K1 alone was sufficient to induce survivin expression. The treatment of CEF cells by rapamycin decreased the survivin mRNA expression. This result demonstrated that p70S6K1 is an important target downstream of PI3K and Akt in regulating suvivin mRNA expression. The knockdown of survivin mRNA expression by its specific siRNA induced apoptosis of cancer cells when the cells were treated with LY294002 or taxol. Taken together, these results demonstrated that PI3K/Akt/p70S6K1 pathway is essential for regulating survivin mRNA expression.     

Inhibition of osteocyte apoptosis by fluid flow is mediated by nitric oxide

Bone unloading results in osteocyte apoptosis, which attracts osteoclasts leading to bone loss. Loading of bone drives fluid flow over osteocytes which respond by releasing signaling molecules, like nitric oxide (NO), that inhibit osteocyte apoptosis and alter osteoblast and osteoclast activity thereby preventing bone loss. However, which apoptosis-related genes are modulated by loading is unknown. We studied apoptosis-related gene expression in response to pulsating fluid flow (PFF) in osteocytes, osteoblasts, and fibroblasts, and whether this is mediated by loading-induced NO production. PFF (0.7 ± 0.3 Pa, 5 Hz, 1 h) upregulated Bcl-2 and downregulated caspase-3 expression in osteocytes. l-NAME attenuated this effect. In osteocytes PFF did not affect p53 and c-Jun, but l-NAME upregulated c-Jun expression. In osteoblasts and fibroblasts PFF upregulated c-Jun, but not Bcl-2, caspase-3, and p53 expression. This suggests that PFF inhibits osteocyte apoptosis via alterations in Bcl-2 and caspase-3 gene expression, which is at least partially regulated by NO.     

PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5^Rgsc451 mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.