Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid capable of regulating critical physiological and pathological functions. Here, we report for the first time that S1P stimulates aldosterone secretion in cells of the zona glomerulosa of the adrenal gland. Regulation of aldosterone secretion is important because this hormone controls electrolyte and fluid balance and is implicated in cardiovascular homeostasis. S1P-stimulated aldosterone secretion was dependent upon the protein kinase C (PKC) isoforms alpha and delta and extracellular Ca2+, and it was inhibited by pertussis toxin (PTX). S1P activated phospholipase D (PLD) through a PTX-sensitive mechanism, also involving PKC alpha and delta and extracellular Ca2+. Primary alcohols, which attenuate the formation of phosphatidic acid (the product of PLD), and cell-permeable ceramides, which inhibit PLD activity, blocked S1P-stimulated aldosterone secretion. Furthermore, propranolol, chlorpromazine, and sphingosine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked aldosterone secretion. These data suggest that the PLD/PAP pathway plays a crucial role in the regulation of aldosterone secretion by S1P and that Gi protein-coupled receptors, extracellular Ca2+, and the PKC isoforms alpha and delta are all important components in the cascade of events controlling this process.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

PI3K/AKT通路在红细胞生成素肾保护中的作用

红细胞生成素作为临床上最常用的纠正贫血的药物,近年随着研究的不断深入,其非造血的组织器官保护作用逐渐被认识。PI3K/AKT通路作为介导红细胞生成素生物学作用的通路之一,在红细胞生成素对各种急慢性肾脏疾病的保护过程中占据重要地位。本文就PI3K/AKT通路在红细胞生成素肾保护中的作用方面的研究进展作一综述。     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

Oligomannose-coated liposomes activate ERK via Src kinases and PI3K/Akt in J774A.1 cells

We have previously shown that liposomes coated with a neoglycolipid constructed from mannotriose and dipalmitoylphosphatidylethanolamine (Man3-DPPE) activate peritoneal macrophages to induce enhanced expression of co-stimulatory molecules and MHC class II. In this study, we investigated the signaling pathways activated by the Man3-DPPE-coated liposomes (OMLs) in a murine macrophage cell line, J774A.1. In response to OML stimulation, ERK among MAPKs was clearly and transiently phosphorylated in J774 cells. ERK phosphorylation was also induced by treatment of the cells with Man3-DPPE and Man3-BSA, but not by uncoated liposomes. In addition, rapid and transient phosphorylation of Akt and Src family kinases (SFKs) was observed in response to OMLs. OML-induced ERK phosphorylation was inhibited by specific inhibitors of PI3K and SFKs, and OML-induced Akt phosphorylation was inhibited by a inhibitor of SFKs. Therefore, OMLs may activate the PI3K/Akt pathway through phosphorylation of Src family kinases to induce ERK activation.     

Low-intensity pulsed ultrasound promotes the proliferation of human bone mesenchymal stem cells by activating PI3K/AKt signaling pathways

Low-intensity pulsed ultrasound (LIPUS) is a promising therapy that is widely used in clinical applications and fundamental research. Previous research has shown that LIPUS exposure has a positive effect on stem cell proliferation. However, the impact of LIPUS exposure on human bone marrow mesenchymal stem cells (hBMSCs) remains unknown. In our study, the effect and mechanism of LIPUS exposure on the proliferation of hBMSCs were investigated, and the optimal parameters of LIPUS were determined. hBMSCs were obtained and identified by flow cytometry, and the proliferation of hBMSCs was measured using the Cell Counting Kit-8 assay to determine cell cycle and cell count. Expression levels of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKt) pathway proteins and cyclin D1 were determined by western blot analysis. Next, hBMSCs were successfully cultured and identified as multipotent mesenchymal stem cells. We found that LIPUS could promote the proliferation of hBMSCs when the exposure time was 5 or 10 minutes per day. Furthermore, 50 or 60 mW/cm² LIPUS had a more significant effect on cell proliferation, but if cells were irradiated by LIPUS for 20 minutes once a day, an intensity of at least 50 mW/cm² could markedly inhibit cell growth. Cell cycle analysis demonstrated that LIPUS treatment drives cells to enter S and G2/M phases from the G0/G1 phase. LIPUS exposure increased phosphorylation of PI3K/AKt and significantly upregulated expression of cyclin D1. However, these effects were inhibited when cells were treated with PI3K inhibitor (LY294002), which in turn reduced LIPUS-mediated proliferation of hBMSCs. These results suggest that LIPUS exposure may be involved in the proliferation of hBMSCs via activation of the PI3K/AKt signaling pathway and high expression of cyclin D1, and the intensity of 50 or 60 mW/cm² and exposure time of 5 minutes were determined to be the optimal parameters for LIPUS exposure.     

Interference with the contractile machinery of the fibroblastic chondrocyte cytoskeleton induces re-expression of the cartilage phenotype through involvement of PI3K,PKC and MAPKs

Chondrocytes rapidly lose their phenotypic expression of collagen II and aggrecan when grown on 2D substrates. It has generally been observed that a fibroblastic morphology with strong actin–myosin contractility inhibits chondrogenesis, whereas chondrogenesis may be promoted by depolymerization of the stress fibers and/or disruption of the physical link between the actin stress fibers and the ECM, as is the case in 3D hydrogels. Here we studied the relationship between the actin–myosin cytoskeleton and expression of chondrogenic markers by culturing fibroblastic chondrocytes in the presence of cytochalasin D and staurosporine. Both drugs induced collagen II re-expression; however, renewed glycosaminoglycan synthesis could only be observed upon treatment with staurosporine. The chondrogenic effect of staurosporine was augmented when blebbistatin, an inhibitor of myosin/actin contractility, was added to the staurosporine-stimulated cultures. Furthermore, in 3D alginate cultures, the amount of staurosporine required to induce chondrogenesis was much lower compared to 2D cultures (0.625 nM vs. 2.5 nM). Using a selection of specific signaling pathway inhibitors, it was found that PI3K-, PKC- and p38-MAPK pathways positively regulated chondrogenesis while the ERK-pathway was found to be a negative regulator in staurosporine-induced re-differentiation, whereas down-regulation of ILK by siRNA indicated that ILK is not determining for chondrocyte re-differentiation. Furthermore, staurosporine analog midostaurin displayed only a limited chondrogenic effect, suggesting that activation/deactivation of a specific set of key signaling molecules can control the expression of the chondrogenic phenotype. This study demonstrates the critical importance of mechanobiological factors in chondrogenesis suggesting that the architecture of the actin cytoskeleton and its contractility control key signaling molecules that determine whether the chondrocyte phenotype will be directed along a fibroblastic or chondrogenic path.     

Noradrenaline enhances the expression of the neuronal monocarboxylate transporter MCT2 by translational activation via stimulation of PI3K/Akt and the mTOR/S6K pathway

Monocarboxylate transporter 2 (MCT2) expression is up-regulated by noradrenaline (NA) in cultured cortical neurons via a putative but undetermined translational mechanism. Western blot analysis showed that p44/p42 mitogen-activated protein kinase (MAPK) was rapidly and strongly phosphorylated by NA treatment. NA also rapidly induced serine/threonine protein kinase from AKT virus (Akt) phosphorylation but to a lesser extent than p44/p42 MAPK. However, Akt activation persisted over a longer period. Similarly, NA induced a rapid and persistent phosphorylation of mammalian target of rapamycin (mTOR), a kinase implicated in the regulation of translation in the central nervous system. Consistent with activation of the mTOR/S6 kinase pathway, phosphorylation of the ribosomal S6 protein, a component of the translation machinery, could be observed upon treatment with NA. In parallel, it was found that the NA-induced increase in MCT2 protein was almost completely blocked by LY294002 (phosphoinositide 3-kinase inhibitor) as well as by rapamycin (mTOR inhibitor), while mitogen-activated protein kinase kinase and p38 MAPK inhibitors had much smaller effects. Taken together, these data reveal that NA induces an increase in neuronal MCT2 protein expression by a mechanism involving stimulation of phosphoinositide 3-kinase/Akt and translational activation via the mTOR/S6 kinase pathway. Moreover, considering the role of NA in synaptic plasticity, alterations in MCT2 expression as described in this study might represent an adaptation to face energy demands associated with enhanced synaptic transmission.     

Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways

Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum.     

急、慢性运动对2型糖尿病大鼠脂肪PI3K/AKT/GLUT4通路的影响

目的:探讨急性和慢性运动对2型糖尿病(T2DM)大鼠脂肪组织明磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)/葡萄糖运载体4(GLUT4)信号通路的影响。方法:15月龄SD雄性大鼠52只随机分为正常对照组(n=13)和高脂组(n=39),分别喂养普通和高脂饲料。8周后,高脂组体重>正常对照组20%,注射小剂量STZ后,血糖>16.7 mmol/l,造模成功。将糖尿病模型组随机分为糖尿病对照组(DC,n=13),糖尿病慢性运动组(DCE,n=13),糖尿病急性运动组(DAE,n=13)。DCE组进行8周的游泳运动,DAE组进行一次性游泳运动。测定血脂,血糖和血清胰岛素,Western blot法测定脂肪PI3K、AKT和GLUT4蛋白含量。结果:糖尿病组体重、血脂、血糖、胰岛素显著高于正常对照组(P均<0.01);高密度脂蛋白胆固醇(HDL-C)水平降低(P<0.05),脂肪组织中PI3K、AKT和GLUT4蛋白表达下降(P均<0.01)。糖尿病慢性运动组体重、血脂、血糖、胰岛素均出现显著性下降(P均<0.01);HDL-C升高(P<0.05...     

Ghrelin stimulates angiogenesis via GHSR1a-dependent MEK/ERK and PI3K/Akt signal pathways in rat cardiac microvascular endothelial cells

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), is thought to exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function such as cell proliferation, migration, survival and angiogenesis. However, the effect of ghrelin on angiogenesis and the corresponding mechanisms have not yet been extensively studied in cardiac microvascular endothelial cells (CMECs) isolated from left ventricular myocardium of adult Sprague-Dawley (SD) rats. In our study, we found that ghrelin and GHSR are constitutively expressed in CMECs. Ghrelin significantly increases CMECs proliferation, migration, and in vitro angiogenesis. The ghrelin-induced angiogenic process was accompanied by phosphorylation of ERK and Akt. MEK inhibitor PD98059 abolished ghrelin-induced phosphorylation of ERK, but had no effect on Akt phosphorylation. PI3K inhibitor LY294002 abolished ghrelin-induced phosphorylation of Akt, but had no effect on ERK phosphorylation. Ghrelin-induced angiogenesis was partially blocked by treatment with PD98059 or LY294002. In addition, this angiogenic effect was almost completely inhibited by PD98059+LY294002. Pretreatment with GHSR1a blocker [D-Lys3]-GHRP-6 abolished ghrelin-induced phosphorylation of ERK, Akt and in vitro angiogenesis. In conclusion, this is the first demonstration that ghrelin stimulates CMECs angiogenesis through GHSR1a-mediated MEK/ERK and PI3K/Akt signal pathways, indicating that two pathways are required for full angiogenic activity of ghrelin. This study suggests that ghrelin may play an important role in myocardial angiogenesis.     

Immunomodulatory activity of Ganoderma lucidum immunomodulatory protein via PI3K/Akt and MAPK signaling pathways in RAW264.7 cells

Ganoderma lucidum immunomodulatory protein (FIP-glu) is an active ingredient with potential immunoregulatory functions. The study was conducted to explore the immunomodulatory activities of recombinant FIP-glu (rFIP-glu) and its possible mechanism in macrophage RAW264.7 cells. In vitro assays of biological activity indicated that rFIP-glu significantly activated RAW264.7 cells and possessed proinflammatory and anti-inflammatory abilities. RNA sequencing analysis and Western blot analysis showed that macrophage activation involved PI3K/Akt and MAPK pathways. Furthermore, real-time quantitative polymerase chain reaction indicated that the PI3K inhibitor LY294002 blocked the messenger RNA (mRNA) levels of MCP-1 (CCL-2), the MEK1/2 inhibitor U0126 reduced the mRNA levels of TNF-α and MCP-1 (CCL-2), and the JNK1/2/3 inhibitor SP600125 prevented the upregulation of inducible nitric oxide synthase mRNA in rFIP-glu-induced cells. rFIP-glu did not mediate these inflammatory effects through a general pathway but rather through a different pathway for a different inflammatory mediator. These data imply that rFIP-glu possessed immunomodulatory activity in macrophages, which was mediated through PI3K/Akt and MAPK pathways.     

Phosphorylation of cold shock domain/Y-box proteins by ERK2 and GSK3beta and repression of the human VEGF promoter

The hypoxia responsive region (HRR) of the VEGF promoter plays a key role in regulating VEGF expression. We found that the cold shock domain (Y-box) repressor proteins, dbpA and dbpB/YB-1, bind distinct strands of the human VEGF HRR. We find both dbpA and dbpB are phosphorylated by ERK2 and GSK3beta in vitro, and the binding of dbpB to single-strand VEGF HRR DNA is regulated by this phosphorylation. These findings suggest the ERK/MAPK and PI3K pathways may regulate VEGF expression in part through regulating the action of these repressor proteins.     

The hyaluronan-binding protease upregulates ERK1/2 and PI3K/Akt signalling pathways in fibroblasts and stimulates cell proliferation and migration

The hyaluronan-binding protease (HABP) is a serine protease in human plasma which is structurally related to plasminogen activators, coagulation factor XII and hepathocyte growth factor activator. It can in vitro activate the coagulation factor FVII, kininogen and plasminogen activators. The present study was initiated to gain a more complete picture of the cell-associated activities of this fibrinolysis-related protease. Treatment of lung fibroblasts with HABP lead to a rapid activation of signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway with c-Raf, MEK and ERK1/2. Additionally the activation of the PI3K/Akt pathway and of several translation-related proteins was found. Proliferation assays confirmed the assumption of a strong growth-stimulating effect of HABP on human lung and skin fibroblasts. Intracellular signalling and growth stimulation were strongly dependent on the proteolytic activity of HABP. Stimulation of signalling and proliferation by HABP involved the fibroblast growth factor receptor 1 (FGFR-1). HABP-stimulated proliferation of lung fibroblasts MRC-5 was accompanied by a significant intracellular increase in basic fibroblast growth factor (bFGF), the major ligand of FGFR-1; bFGF could however not be identified in the supernatant of HABP-treated cells. Though, the conditioned medium from HABP-treated cells showed a strong growth-promoting activity on quiescent fibroblasts, indicating the release of a yet unknown growth factor amplifying the initial growth stimulus. In a two-dimensional wound model HABP stimulated the invasion of fibroblasts into a scratch area, adding a strong pro-migratory activity to this plasma protease. In summary, HABP exhibits a significant growth factor-like activity on quiescent human lung and dermal fibroblasts. Our findings suggest that this fibrinolysis-related plasma protease may participate in physiologic or pathologic processes where cell proliferation and migration are pivotal, like tissue repair, vascular remodelling, wound healing or tumor development.