High glucose induces alternative activation of macrophages via PI3K/Akt signaling pathway

Objective: It has been proved that lactate-4.25% dialysate could result in peritoneal fibrosis by inducing alternative activation of macrophages in our previous study, but the mechanism of high glucose-induced alternative activation has not been elucidated. This study was, therefore, to investigate the mechanism by high glucose stimuli.

Methods: In this study, Raw264.7 (murine macrophage cell line) cells were cultured and stimulated by 4.25% glucose medium, and mannitol medium was used as osmotic pressure control. Cells were harvested at 0?h, 4?h, 8?h, and 12?h to examine the expression of Arg-1, CD206, and p-Akt. After blocking PI3K by LY294002, the expression of Arg-1, CD206, and p-Akt was examined again.

Results: The expression of Arg-1 and CD206 was increased in a time-dependent manner induced by high glucose medium. On the contrary, there was mainly no Agr-1 or CD206 expressed in cells cultured in the mannitol medium with the same osmotic pressure. What’s more, Akt was phosphorylated at the eighth hour stimulated by high glucose medium, and LY294002 inhibited the expression of Arg-1 and CD206 by blocking the phosphorylation of Akt.

Conclusions: Our study indicated that high glucose rather than high osmotic pressure induced M2 phenotype via PI3K/Akt signaling pathway.     

PI3K in lymphocyte development,differentiation and activation

Phosphoinositide 3-kinases (PI3Ks) regulate numerous biological processes, including cell growth, differentiation, survival, proliferation, migration and metabolism. In the immune system, impaired PI3K signalling leads to immunodeficiency, whereas unrestrained PI3K signalling contributes to autoimmunity and leukaemia. New insights into the role of PI3Ks in lymphocyte biology have been derived from gene-targeting studies, which have identified the PI3K subunits that are involved in B-cell and T-cell signalling. In particular, the catalytic subunit p110delta seems to be adapted to transmit antigen-receptor signalling in B and T cells. Additional recent work has provided new insights into the molecular interactions that lead to PI3K activation and the signalling pathways that are regulated by PI3K.     

CpG-ODN,the TLR9 agonist,attenuates myocardial ischemia/reperfusion injury: Involving activation of PI3K/Akt signaling

BackgroundToll-like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. The TLR9 ligand, CpG-ODN has been reported to improve cell survival. We examined effect of CpG-ODN on myocardial I/R injury.MethodsMale C57BL/6 mice were treated with either CpG-ODN, control-ODN, or inhibitory CpG-ODN (iCpG-ODN) 1 h prior to myocardial ischemia (60 min) followed by reperfusion. Untreated mice served as I/R control (n = 10/each group). Infarct size was determined by TTC straining. Cardiac function was examined by echocardiography before and after myocardial I/R up to 14 days.ResultsCpG-ODN administration significantly decreased infarct size by 31.4% and improved cardiac function after myocardial I/R up to 14 days. Neither control-ODN nor iCpG-ODN altered I/R-induced myocardial infarction and cardiac dysfunction. CpG-ODN attenuated I/R-induced myocardial apoptosis and prevented I/R-induced decrease in Bcl2 and increase in Bax levels in the myocardium. CpG-ODN increased Akt and GSK-3β phosphorylation in the myocardium. In vitro data suggested that CpG-ODN treatment induced TLR9 tyrosine phosphorylation and promoted an association between TLR9 and the p85 subunit of PI3K. Importantly, PI3K/Akt inhibition and Akt kinase deficiency abolished CpG-ODN-induced cardioprotection.ConclusionCpG-ODN, the TLR9 ligand, induces protection against myocardial I/R injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.     

SARS-CoV-2 spike promotes inflammation and apoptosis through autophagy by ROS-suppressed PI3K/AKT/mTOR signaling

BackgroundSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection-induced inflammatory responses are largely responsible for the death of novel coronavirus disease 2019 (COVID-19) patients. However, the mechanism by which SARS-CoV-2 triggers inflammatory responses remains unclear. Here, we aimed to explore the regulatory role of SARS-CoV-2 spike protein in infected cells and attempted to elucidate the molecular mechanism of SARS-CoV-2-induced inflammation.MethodsSARS-CoV-2 spike pseudovirions (SCV-2-S) were generated using the spike-expressing virus packaging system. Western blot, mCherry-GFP-LC3 labeling, immunofluorescence, and RNA-seq were performed to examine the regulatory mechanism of SCV-2-S in autophagic response. The effects of SCV-2-S on apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Western blot, and flow cytometry analysis. Enzyme-linked immunosorbent assay (ELISA) was carried out to examine the mechanism of SCV-2-S in inflammatory responses.ResultsAngiotensin-converting enzyme 2 (ACE2)-mediated SCV-2-S infection induced autophagy and apoptosis in human bronchial epithelial and microvascular endothelial cells. Mechanistically, SCV-2-S inhibited the PI3K/AKT/mTOR pathway by upregulating intracellular reactive oxygen species (ROS) levels, thus promoting the autophagic response. Ultimately, SCV-2-S-induced autophagy triggered inflammatory responses and apoptosis in infected cells. These findings not only improve our understanding of the mechanism underlying SARS-CoV-2 infection-induced pathogenic inflammation but also have important implications for developing anti-inflammatory therapies, such as ROS and autophagy inhibitors, for COVID-19 patients.     

Src family kinases regulate renal epithelial dedifferentiation through activation of EGFR/PI3K signaling

Dedifferentiation, a process by which differentiated cells become mesenchymal‐like proliferating cells, is the first step in renal epithelium repair and occurs in vivo after acute kidney injury and in vitro in primary culture. However, the underlying mechanism remains poorly understood. In this report, we studied the signaling events that mediate dedifferentiation of proximal renal tubular cells (RPTC) in primary culture. RPTC dedifferentiation characterized by increased expression of vimentin concurrent with decreased expression of cytokeratin‐18 was observed at 24 h after the initial plating of freshly isolated proximal tubules and persisted for 72 h. At 96 h, RPTC started to redifferentiate as revealed by reciprocal expression of cytokeratin‐18 and vimentin and completed at 120 h. Phosphorylation levels of Src, epidermal growth factor receptor (EGFR), AKT (a target of phosphoinositide‐3‐kinase (PI3K)), and ERK1/2 were increased in the early time course of culture (<72 h). Inhibition of Src family kinases (SFKs) with PP1 blocked EGFR, AKT, and ERK1/2 phosphorylation, as well as RPTC dedifferentiation. Inhibition of EGFR with AG1478 also blocked AKT and ERK1/2 phosphorylation and RPTC dedifferentiation. Although inactivation of the PI3K/AKT pathway with LY294002 inhibited RPTC dedifferentiation, blocking the ERK1/2 pathway with U0126 did not show such an effect. Moreover, inhibition of SFKs, EGFR, PI3K/AKT, but not ERK1/2 pathways abrogated RPTC outgrowth and SFK inhibition decreased RPTC proliferation and migration. These findings demonstrate a critical role of SFKs in mediating RPTC dedifferentiation through activation of the EGFR/PI3K signaling pathway. J. Cell. Physiol. 227: 2138–2144, 2012. © 2011 Wiley Periodicals, Inc.     

Absence of Appl2 sensitizes endotoxin shock through activation of PI3K/Akt pathway

Background

The adapter proteins Appl1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif 1) and Appl2 are highly homologous and involved in several signaling pathways. While previous studies have shown that Appl1 plays a pivotal role in adiponectin signaling and insulin secretion, the physiological functions of Appl2 are largely unknown.

Results

In the present study, the role of Appl2 in sepsis shock was investigated by using Appl2 knockout (KO) mice. When challenged with lipopolysaccharides (LPS), Appl2 KO mice exhibited more severe symptoms of endotoxin shock, accompanied by increased production of proinflammatory cytokines. In comparison with the wild-type control, deletion of Appl2 led to higher levels of TNF-α and IL-1β in primary macrophages. In addition, phosphorylation of Akt and its downstream effector NF-κB was significantly enhanced. By co-immunoprecipitation, we found that Appl2 and Appl1 interacted with each other and formed a complex with PI3K regulatory subunit p85α, which is an upstream regulator of Akt. Consistent with these results, deletion of Appl1 in macrophages exhibited characteristics of reduced Akt activation and decreased the production of TNFα and IL-1β when challenged by LPS.

Conclusions

Results of the present study demonstrated that Appl2 is a critical negative regulator of innate immune response via inhibition of PI3K/Akt/NF-κB signaling pathway by forming a complex with Appl1 and PI3K.

     

Prolactin induces chitotriosidase expression in human macrophages through PTK,PI3‐K,and MAPK pathways

We previously reported that prolactin (PRL) induces chitotriosidase (CHIT‐1) mRNA expression in human macrophages. In this investigation we determined the signaling pathways involved in CHIT‐1 induction in response to PRL. The CHIT‐1 induction PRL‐mediated was reduced by wortmannin and LY‐294002, inhibitors of phosphatidylinositol 3‐kinase (PI3‐K) and by genistein an inhibitor of protein tyrosine kinase (PTK). Pre‐treatment of macrophages with SB203580, a specific inhibitor of the mitogen‐activated kinases (MAPK) p38, or with U0126, an inhibitor of MAPK p44/42, prevented both basal and exogenous PRL‐mediated CHIT‐1 expression. No significant effects on CHIT‐1 induction PRL‐mediated were observed with a protein kinase C inhibitor (PKC), rottlerin, or with an Src inhibitor, PP2, or with JAK2 inhibitor, AG490. In addition, PRL induced a phosphorylation of AKT that was prevented both by the two MAPK inhibitors SB203580 and U0126 and by the PI3‐K inhibitors wortmannin and LY‐294002. In conclusion, our results indicate that PRL up‐regulated CHIT‐1 expression via PTK, PI3‐K, MAPK, and signaling transduction components. J. Cell. Biochem. 107: 881–889, 2009. © 2009 Wiley‐Liss, Inc.     

High glucose promotes retinal endothelial cell migration through activation of Src, PI3K/Akt1/eNOS, and ERKs

Hyperglycemia impacts retinal vascular function and promotes the development and progression of diabetic retinopathy, which ultimately results in growth of new blood vessels and loss of vision. How high glucose affects retinal endothelial cell (EC) properties requires further investigation. Here we determined the impact of high glucose on mouse retinal EC function in vitro. High glucose significantly enhanced the migration of retinal EC without impacting their proliferation, apoptosis, adhesion, and capillary morphogenesis. The enhanced migration of retinal EC under high glucose was reversed in the presence of the antioxidant N-acetylcysteine, suggesting increased oxidative stress under high-glucose conditions. Retinal EC under high-glucose conditions also expressed increased levels of fibronectin, osteopontin, and alpha(v)beta(3)-integrin, and reduced levels of thrombospondin-1. These changes were concomitant with sustained activation of the downstream prosurvival and promigratory signaling pathways, including Src kinase, phosphatidylinositol 3-kinase/Akt1/endothelial nitric oxide synthase, and ERKs. The sustained activation of these signaling pathways was essential for enhanced migration of retinal EC under high-glucose conditions. Together, our results indicate the exposure of retinal EC to high glucose promotes a promigratory phenotype. Thus alterations in the proangiogenic properties of retinal EC during diabetes may contribute to the development and pathogenesis of diabetic retinopathy.     

Overexpression of PEMT2 downregulates the PI3K/Akt signaling pathway in rat hepatoma cells

Phosphatidylethanolamine N-methyltransferase 2 (PEMT2) is an isoform of PEMT that converts phosphatidylethanolamine to phosphatidylcholine in mammalian liver. Overexpression of PEMT2 led to inhibition of proliferation of hepatoma cells [J. Biol. Chem. 269 (1994) 24531]. The present study aims to unravel the molecular mechanism of the reduced proliferation, especially the signaling transducer proteins involved in this process. Thus, we chose PI3K/Akt pathway that is initiated by growth factors and leads to cell survival and proliferation. Rat hepatoma CBRH-7919 cells transfected with pemt2-cDNA showed that: (1) signaling proteins including c-Met, PDGF receptor, PI3K, Akt and Bcl-2 all had reduced expression as shown by Western blotting studies; (2) flow cytometric and DNA ladder assays showed that 22.9% of the pemt2-transfected cells were undergoing apoptosis; (3) the activity of Akt was decreased as shown by Western blotting using antibody directed against p-Akt (Thr308); (4) wortmannin and PD98059, inhibitors of PI3K and MEK, respectively, both inhibited Akt activity, indicating that PI3K and MAPK pathways were merging at Akt in CBRH-7919 cells. The above results suggest that overexpression of PEMT2 strongly downregulated the PI3K/Akt signaling pathway at multiple sites and induced apoptosis. This, at least partly, explains the molecular mechanism of impaired proliferation induced by pemt2 transfection.     

Bilobalide prevents apoptosis through activation of the PI3K/Akt pathway in SH-SY5Y cells

Bilobalide, a sesquiterpene trilactone constituent of Ginkgo biloba leaf extracts, has been proposed to exert protective and trophic effects on neurons. However, mechanisms underlying the protective effects of bilobalide remain unclear. Using human SH-SY5Y neuroblastoma cells and primary hippocampal neurons, this study investigated the neuroprotective effects of bilobalide. We mimicked aging-associated neuronal impairments by applying external factors (beta amyloid protein (Aβ) 1-42, H₂O₂ and serum deprivation) consequently inducing cell apoptosis. As markers for apoptosis, cell viability, DNA fragmentation, mitochondrial membrane potential and levels of cleaved caspase 3 were measured. We found that, bilobalide prevented Aβ 1-42-, H₂O₂- and serum deprivation-induced apoptosis. To better understand the neuroprotective effects of bilobalide, we also tested the ability of bilobalide to modulate pro-survival signaling pathways such as protein kinase C (PKC), extracellular-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. It was found that, bilobalide dose-dependently increased PI3K activity and levels of phosphorylated Akt (p-Akt Ser473 and Thr308), which could be maintained up to at least 2 h after bilobalide withdrawal in cells treated with or without Aβ 1-42, H₂O₂ or serum-free medium. In addition, application of PI3K/Akt inhibitor LY294002 could abrogate both the protective effects of bilobalide against Aβ 1-42-, H₂O₂- and serum deprivation-induced apoptotic cell damage and bilobalide-induced increase in PI3K activity and levels of p-Akt (Ser473 and Thr308). In contrast, application of PKC inhibitor staurosporine (STS) did not affect the protective effects of bilobalide. Moreover, no change in levels of phosphorylated ERK1/2 (p-ERK1/2) was observed in bilobalide-treated cells. These results further suggested that the PI3K/Akt pathway might be involved in the protective effects of bilobalide. Since modern technology allows production of purified bilobalide with high bioavailability, bilobalide may be useful in developing therapy for diseases involving age-associated neurodegeneration.     

Angiotensin AT2 receptor agonist stimulates high stretch induced- ANP secretion via PI3K/NO/sGC/PKG/pathway

Angiotensin II (Ang II) type 1 receptor (AT₁R) mediates the major cardiovascular effects of Ang II. However, the effects mediated via AT₂R are still controversial. The aim of the present study is to define the effect of AT₂R agonist CGP42112A (CGP) on high stretch-induced ANP secretion and its mechanism using in vitro and in vivo experiments. CGP (0.01, 0.1 and 1 μM) stimulated high stretch-induced ANP secretion and concentration from isolated perfused rat atria. However, atrial contractility and the translocation of extracellular fluid did not change. The augmented effect of CGP (0.1 μM) on high stretch-induced ANP secretion was attenuated by the pretreatment with AT₂R antagonist or inhibitor for phosphoinositol 3-kinase (PI3K), nitric oxide (NO), soluble guanylyl cyclase (sGC), or protein kinase G (PKG). However, antagonist for AT₁R or Mas receptor did not influence CGP-induced ANP secretion. In vivo study, acute infusion of CGP for 10 min increased plasma ANP level without blood pressure change. In renal hypertensive rat atria, AT₂R mRNA and protein levels were up-regulated and the response of plasma ANP level to CGP infusion in renal hypertensive rats augmented. The pretreatment with AT₂R antagonist for 10 min followed by CGP infusion attenuated an increased plasma ANP level induced by CGP. However, pretreatment with AT₁R or Mas receptor antagonist unaffected CGP-induced increase in plasma ANP level. Therefore, we suggest that AT₂R agonist CGP stimulates high stretch-induced ANP secretion through PI3K/NO/sGC/PKG pathway and these effects are augmented in renal hypertensive rats.     

Insulin-induced NADPH oxidase activation promotes proliferation and matrix metalloproteinase activation in monocytes/macrophages

Insulin stimulates superoxide (O₂^?) production in monocytes and macrophages. However, the mechanisms through which insulin induces O₂^? production are not completely understood. In this study, we (a) characterized the enzyme and the pathways involved in insulin-stimulated O₂^? production in human monocytes and murine macrophages, and (b) analyzed the consequences of insulin-stimulated O₂^? production on the cellular phenotype in these cells. We showed that insulin stimulated O₂^? production, and promoted p47^phox translocation to the plasma membrane. Insulin-induced O₂^? production and p47^phox translocation were prevented in the presence of specific inhibitors of PI3K and PKC. Insulin-mediated NADPH oxidase activation stimulated MMP-9 activation in monocytes and cell proliferation in macrophages. The effect of insulin on these phenotypic responses was mediated through NFκB, p38MAPK, and ERK 1/2 activation. Small-interfering RNA-specific gene silencing targeted specifically against Nox2 reduced the cognate protein expression, decreased insulin-induced O₂^? production, inhibited the turn on of NFκB, p38MAPK, and ERK 1/2, and reduced cell proliferation in macrophages. These findings suggest a pivotal role for NADPH oxidase in insulin-induced proliferation and proteolytic activation in monocytes and macrophages, respectively, and identify a pathway that may play a pathological role in hyperinsulinemic states.     

Pure lipopolysaccharide or synthetic lipid A induces activation of p21Ras in primary macrophages through a pathway dependent on Src family kinases and PI3K

Recognition of bacterial LPS by macrophages plays a critical role in host defense against infection by Gram-negative bacteria. However, when not tightly regulated, the macrophage's response to LPS can induce severe disease and septic shock. Although LPS triggers the activation of multiple signaling pathways in macrophages, it was unclear whether these include activation of the p21Ras GTPases. We report that p21Ras is rapidly and transiently activated in murine primary macrophages stimulated with an ultra-pure preparation of LPS or with synthetic lipid A. The molecular basis of this activation was investigated using a pharmacological approach. LPS-induced activation of p21Ras was inhibited in the presence of PP2, LY294002, or wortmannin, suggesting that it depends on the activity of one or more members of the Src kinase family and the subsequent activation of PI3K. In that pharmacological inhibitors of PI3K inhibited LPS-induced activation of p21Ras, but not activation of ERK, we concluded that LPS-induced activation of ERK occurs through a pathway that is not dependent on the activation of p21Ras.     

PI3K/mTORC1 activation in hamartoma syndromes: Therapeutic prospects

Dysregulated activity of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) is characteristic feature of hamartoma syndromes. Hamartoma syndromes, dominantly inherited cancer predisposition disorders, affect multiple organs and are manifested by benign tumors consisting of various cell types native to the tissues in which they arise. In the past few years, three inherited hamartoma syndromes, Cowden syndrome (CS), tuberous sclerosis complex (TSC) syndrome, and Peutz-Jeghens syndrome (PJS), have all been linked to a common biochemical pathway: the hyperactivation of PI3K/mTORC1 intracellular signaling. Three tumor suppressors, PTEN (phosphatases and tensin homolog), tuberous sclerosis complex TSC1/TSC2, and LKB1, are negative regulators of PI3K/mTORC1 signaling; disease-related inactivation of these tumor suppressors results in the development of PTEN-associated hamartoma syndromes, TSC, and PJS, respectively. The goal of this review is to provide a roadmap for navigating the inherently complex regulation of PI3K/mTORC1 signaling while highlighting the progress that has been made in elucidating the cellular and molecular mechanisms of hamartoma syndromes and identificating potential therapeutic targets for their treatment. Importantly, because the PI3K/mTORC1 pathway is activated in the majority of common human cancers, the identification of novel molecular target(s) for the treatment of hamartoma syndromes may have a broader translational potential, and is critically important not only for therapeutic intervention in hamartoma disorders, but also for the treatment of cancers.     

PI3K regulates the activation of NLRP3 inflammasome in atherosclerosis through part-dependent AKT signaling pathway

PI3K is a downstream target of multiple cell-surface receptors, which acts as a crucial modulator of both cell polarization and survival. PI3K/AKT signaling pathway is commonly involved in cancer, atherosclerosis, and other diseases. However, its role in cardiovascular diseases, especially in atherosclerosis, remains to be further investigated. To determine the effect of PI3K/AKT signaling pathway on cellular inflammatory response and oxidative stress, PI3K inhibitor (GDC0941) and AKT inhibitor (MK2206) were used. First, THP-1 cells were incubated with ox-LDL (100 µg/ml) to establish an in vitro atherosclerosis model. The inflammatory factors and foam cell formation were then evaluated to ascertain and compare the effects of PI3K and AKT inhibition. ApoE^−/− mice fed a high-fat diet were used to assess the roles of PI3K and AKT in aortic plaque formation. Our results showed that the inhibition of PI3K or AKT could suppress the activation of NLRP3, decreased the expression levels of p-p65/p65 and reduced the production of mitochondrial reaction oxygen species (mitoROS) in THP-1 cells. Inhibition of PI3K or AKT could also reduced atherosclerosis lesion and plaque area, and decreased the levels of NLRP3 and IL-1β in ApoE^−/− mice. The effect of PI3K inhibition was more significant than AKT. Therefore, PI3K inhibition can retard the progress of atherosclerosis. Besides, there may be other AKT-independent pathways that regulate the formation of atherosclerosis.