Direct stimulation of adult neural stem/progenitor cells in vitro and neurogenesis in vivo by salvianolic acid B

Background

Small molecules have been shown to modulate the neurogenesis processes. In search for new therapeutic drugs, the herbs used in traditional medicines for neurogenesis are promising candidates.

Methodology and Principal Findings

We selected a total of 45 natural compounds from Traditional Chinese herbal medicines which are extensively used in China to treat stroke clinically, and tested their proliferation-inducing activities on neural stem/progenitor cells (NSPCs). The screening results showed that salvianolic acid B (Sal B) displayed marked effects on the induction of proliferation of NSPCs. We further demonstrated that Sal B promoted NSPCs proliferation in dose- and time-dependent manners. To explore the molecular mechanism, PI3K/Akt, MEK/ERK and Notch signaling pathways were investigated. Cell proliferation assay demonstrated that {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 (PI3K/Akt inhibitor), but neither U0126 (ERK inhibitor) nor DAPT (Notch inhibitor) inhibited the Sal B-induced proliferation of cells. Western Blotting results showed that stimulation of NSPCs with Sal B enhanced the phosphorylation of Akt, and {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 abolished this effect, confirming the role of Akt in Sal B mediated proliferation of NSPCs. Rats exposed to transient cerebral ischemia were treated for 4 weeks with Sal B from the 7th day after stroke. BrdU incorporation assay results showed that exposure Sal B could maintain the proliferation of NSPCs after cerebral ischemia. Morris water maze test showed that delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats.

Significance

Sal B could maintain the NSPCs self-renew and promote proliferation, which was mediated by PI3K/Akt signal pathway. And delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats. These findings suggested that Sal B may act as a potential drug in treatment of brain injury or neurodegenerative diseases.     

PAK4 confers cisplatin resistance in gastric cancer cells via PI3K/Akt- and MEK/ERK-dependent pathways

CDDP [cisplatin or cis-diamminedichloroplatinum(II)] and CDDP-based combination chemotherapy have been confirmed effective against gastric cancer. However, CDDP efficiency is limited because of development of drug resistance. In this study, we found that PAK4 (p21-activated kinase 4) expression and activity were elevated in gastric cancer cells with acquired CDDP resistance (AGS/CDDP and MKN-45/CDDP) compared with their parental cells. Inhibition of PAK4 or knockdown of PAK4 expression by specific siRNA (small interfering RNA)-sensitized CDDP-resistant cells to CDDP and overcome CDDP resistance. Combination treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [the inhibitor of PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B or PKB) pathway] or PD98509 {the inhibitor of MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] pathway} with PF-3758309 (the PAK4 inhibitor) resulted in increased CDDP efficacy compared with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or PD98509 alone. However, after the concomitant treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98509, PF-3758309 administration exerted no additional enhancement of CDDP cytotoxicity in CDDP-resistant cells. Inhibition of PAK4 by PF-3758309 could significantly suppress MEK/ERK and PI3K/Akt signalling in CDDP-resistant cells. Furthermore, inhibition of PI3K/Akt pathway while not MEK/ERK pathway could inhibit PAK4 activity in these cells. The in vivo results were similar with those of in vitro. In conclusion, these results indicate that PAK4 confers CDDP resistance via the activation of MEK/ERK and PI3K/Akt pathways. PAK4 and PI3K/Akt pathways can reciprocally activate each other. Therefore, PAK4 may be a potential target for overcoming CDDP resistance in gastric cancer.     

Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

Background

Traumatic brain injury (TBI) induces a complex sequence of apopototic cascades that contribute to secondary tissue damage. The aim of this study was to investigate the effects of salidroside, a phenolic glycoside with potent anti-apoptotic properties, on behavioral and histological outcomes, brain edema, and apoptosis following experimental TBI and the possible involvement of the phosphoinositide 3-kinase/protein kinase B (PI3K)/Akt signaling pathway.

Methodology/Principal Findings

Mice subjected to controlled cortical impact injury received intraperitoneal salidroside (20, or 50 mg/kg) or vehicle injection 10 min after injury. Behavioral studies, histology analysis and brain water content assessment were performed. Levels of PI3K/Akt signaling-related molecules, apoptosis-related proteins, cytochrome C (CytoC), and Smac/DIABLO were also analyzed. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, was administered to examine the mechanism of protection. The protective effect of salidroside was also investigated in primary cultured neurons subjected to stretch injury. Treatment with 20 mg/kg salidroside_significantly improved functional recovery and reduced brain tissue damage up to post-injury day 28. Salidroside_also significantly reduced neuronal death, apoptosis, and brain edema at day 1. These changes were associated with significant decreases in cleaved caspase-3, CytoC, and Smac/DIABLO at days 1 and 3. Salidroside increased phosphorylation of Akt on Ser473 and the mitochondrial Bcl-2/Bax ratio at day 1, and enhanced phosphorylation of Akt on Thr308 at day 3. This beneficial effect was abolished by pre-injection of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Moreover, delayed administration of salidroside at 3 or 6 h post-injury reduced neuronal damage at day 1. Salidroside treatment also decreased neuronal vulnerability to stretch-induced injury in vitro.

Conclusions/Significance

Post-injury salidroside improved long-term behavioral and histological outcomes and reduced brain edema and apoptosis following TBI, at least partially via the PI3K/Akt signaling pathway.     

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice

Background

Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model.

Results

The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 μg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-μg LPS injection in Tlr4^-/- mice. When the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4^-/- mice was 30%. In the Tlr4^-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 μg LPS led to a significant induction in O₂^- detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4^-/- mice. Addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 only significantly increased the O₂^- level in the lung and liver of the Tlr4^-/- mice but not in the C57BL/6 mice following 500-μg LPS injection. In addition, the serum IL-1β and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4^-/- mice. Notably, IL-1β and IL-2 were significantly increased in Tlr4^-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 prior to LPS injection.

Conclusions

In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4^-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O₂^- and inflammatory cytokines.     

Lysophosphatidic acid induces MDA-MB-231 breast cancer cells migration through activation of PI3K/PAK1/ERK signaling

Background

Enhanced motility of cancer cells is a critical step in promoting tumor metastasis. Lysophosphatidic acid (LPA), representing the major mitogenic activity in serum, stimulates migration in various types of cancer cells. However, the underlying signaling mechanisms for LPA-induced motility of cancer cells remain to be elucidated.

Methodology/Principal Findings

In this study, we found that LPA dose-dependently stimulated migration of MDA-MB-231 breast cancer cells, with 10 µM being the most effective. LPA also increased ERK activity and the MEK inhibitor U0126 could block LPA-induced ERK activity and cell migration. In addition, LPA induced PAK1 activation while ERK activation and cell migration were inhibited by ectopic expression of an inactive mutant form of PAK1 in MDA-MB-231 cells. Furthermore, LPA increased PI3K activity, and the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 inhibited both LPA-induced PAK1/ERK activation and cell migration. Moreover, in the breast cancer cell, LPA treatment resulted in remarkable production of reactive oxygen species (ROS), while LPA-induced ROS generation, PI3K/PAK1/ERK activation and cell migration could be inhibited by N-acetyl-L-Cysteine, a scavenger of ROS.

Conclusions/Significance

Taken together, this study identifies a PI3K/PAK1/ERK signaling pathway for LPA-stimulated breast cancer cell migration. These data also suggest that ROS generation plays an essential role in the activation of LPA-stimulated PI3K/PAK1/ERK signaling and breast cancer cell migration. These findings may provide a basis for designing future therapeutic strategy for blocking breast cancer metastasis.     

Nitrogen-containing bisphosphonates inhibit RANKL- and M-CSF-induced osteoclast formation through the inhibition of ERK1/2 and Akt activation

Background

Bisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Recent studies have shown that nitrogen-containing bisphosphonates induced apoptosis in rabbit osteoclasts and prevented prenylated small GTPase. However, whether bisphosphonates inhibit osteoclast formation has not been determined. In the present study, we investigated the inhibitory effect of minodronate and alendronate on the osteoclast formation and clarified the mechanism involved in a mouse macrophage-like cell lines C7 and RAW264.7.

Results

It was found that minodronate and alendronate inhibited the osteoclast formation of C7 cells induced by receptor activator of NF-κB ligand and macrophage colony stimulating factor, which are inhibited by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. It was also found that minodronate and alendronate inhibited the osteoclast formation of RAW264.7 cells induced by receptor activator of NF-κB ligand. Furthermore, minodronate and alendornate decreased phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; similarly, U0126, a mitogen protein kinase kinase 1/2 (MEK1/2) inhibitor, and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited osteoclast formation.

Conclusions

This indicates that minodronate and alendronate inhibit GGPP biosynthesis in the mevalonate pathway and then signal transduction in the MEK/ERK and PI3K/Akt pathways, thereby inhibiting osteoclast formation. These results suggest a novel effect of bisphosphonates that could be effective in the treatment of bone metabolic diseases, such as osteoporosis.     

Multi-level targeting of the phosphatidylinositol-3-kinase pathway in non-small cell lung cancer cells

Introduction

We assessed expression of p85 and p110α PI3K subunits in non-small cell lung cancer (NSCLC) specimens and the association with mTOR expression, and studied effects of targeting the PI3K/AKT/mTOR pathway in NSCLC cell lines.

Methods

Using Automated Quantitative Analysis we quantified expression of PI3K subunits in two cohorts of 190 and 168 NSCLC specimens and correlated it with mTOR expression. We studied effects of two PI3K inhibitors, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and NVP-BKM120, alone and in combination with rapamycin in 6 NSCLC cell lines. We assessed activity of a dual PI3K/mTOR inhibitor, NVP-BEZ235 alone and with an EGFR inhibitor.

Results

p85 and p110α tend to be co-expressed (p<0.001); p85 expression was higher in adenocarcinomas than squamous cell carcinomas. High p85 expression was associated with advanced stage and poor survival. p110α expression correlated with mTOR (ρ = 0.276). In six NSCLC cell lines, addition of rapamycin to {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or NVP-BKM120 was synergistic. Even very low rapamycin concentrations (1 nM) resulted in sensitization to PI3K inhibitors. NVP-BEZ235 was highly active in NSCLC cell lines with IC₅₀s in the nanomolar range and resultant down-regulation of pAKT and pP70S6K. Adding Erlotinib to NVP-BEZ235 resulted in synergistic growth inhibition.

Conclusions

The association between PI3K expression, advanced stage and survival in NSCLC suggests that it might be a valuable drug target. Concurrent inhibition of PI3K and mTOR is synergistic in vitro, and a dual PI3K/mTOR inhibitor was highly active. Adding EGFR inhibition resulted in further growth inhibition. Targeting the PI3K/AKT/mTOR pathway at multiple levels should be tested in clinical trials for NSCLC.     

Aquaporin3 Is Required for FGF-2-Induced Migration of Human Breast Cancers

Purpose

The aquaporin (AQP) family consists of a number of small integral membrane proteins that transport water and glycerol. AQPs are critical for trans-epithelial fluid transport. Recent reports demonstrated that AQPs, particularly AQP1 and AQP5, are expressed in high grade tumor cells of a variety of tissue origins, and that AQPs are involved in cell migration and metastasis. Based on this background, we examined whether AQP3, another important member of the AQP family, could facilitate cell migration in human breast cancers.

Methods

Potential role of AQP3 was examined using two representative breast cancer cell lines (MDA-MB-231 and Bcap-37). Briefly, AQP3 expression was inhibited with a lentivirus construct that stably expressed shRNA against the AQP3 mRNA. AQP3 expression inhibition was verified with Western blot. Cell migration was examined using a wound scratch assay in the presence of fibroblast growth factor-2 (FGF-2). In additional experiments, AQP3 was inhibited by CuSO₄. Fibroblast growth factor receptor (FGFR) kinase inhibitor PD173074, PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, and MEK1/2 inhibitor PD98059 were used to dissect the molecular mechanism of FGF-2 induced AQP3 expression.

Results

FGF-2 treatment increased AQP3 expression and induced cell migration in a dose dependent manner. Silencing AQP3 expression by a lentiviral shRNA inhibited FGF-2 induced cell migration. CuSO₄, a water transport inhibitor selective for AQP3, also suppressed FGF-2-induced cell migration. The FGFR kinase inhibitor PD173074, significantly inhibited FGF-2-induced AQP3 expression and cell migration. The PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and MEK1/2 inhibitor PD98059 inhibited, but not fully blocked, FGF-2-induced AQP3 expression and cell migration.

Conclusions

AQP3 is required for FGF-2-induced cell migration in cultured human breast cancer cells. Our findings also suggest the importance of FGFR-PI3K and FGFR-ERK signaling in FGF-2-induced AQP3 expression. In summary, our findings suggest a novel function of AQP3 in cell migration and metastasis of breast cancers.     

Neurotrophic effects of a cyanine dye via the PI3K-Akt pathway: attenuation of motor discoordination and neurodegeneration in an ataxic animal model

Background

Neurotrophic factors may be future therapeutic agents for neurodegenerative disease. In the screening of biologically active molecules for neurotrophic potency, we found that a photosensitizing cyanine dye, NK-4, had remarkable neurotrophic activities and was a potent radical scavenger.

Methodology/Principal Findings

In this study, we evaluated the effect of NK-4 on the protection of neurons against oxidative damage and investigated the associated intracellular signaling pathways. Subsequently, we evaluated the effect of NK-4 in an animal model of neurodegeneration. In vitro, NK-4 showed dose-dependent protection of PC12 cells from toxicity induced by oxidative stress caused by hydrogen peroxide (H₂O₂) or 6-hydroxydopamine (6-OHDA). Comparison of extracellular signal-regulated kinase signaling pathways between treatment with NK-4 and nerve growth factor (NGF) using K252a, an inhibitor of the NGF receptor TrkA, revealed that NK-4 activity occurs independently of NGF receptors. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the protective effect of NK-4, and NK-4 caused activation of Akt/protein kinase B, a downstream effector of PI3K. These results suggest that the neuroprotective effects of NK-4 are mediated by the PI3K-Akt signaling pathway. NK-4 treatment also attenuated stress-induced activation of SAPK/JNK, which suggests that NK-4 activates a survival signaling pathway and inhibits stress-activated apoptotic pathways independently of the TrkA receptor in neuronal cells. In vivo, administration of NK-4 improved motor coordination in genetic ataxic hamsters, as assessed by rota-rod testing. Histological analysis showed that cerebellar atrophy was significantly attenuated by NK-4 treatment. Notably, the Purkinje cell count in the treated group was threefold higher than that in the vehicle group.

Conclusions/Significance

These results suggest that NK-4 is a potential agent for therapy for neurodegenerative disorders based on the activation of survival signaling pathways.     

Supernatant from bifidobacterium differentially modulates transduction signaling pathways for biological functions of human dendritic cells

Background

Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, the signaling pathways engaged by probiotics are poorly understood. We have previously reported that a fermentation product from Bifidobacterium breve C50 (BbC50sn) could induce maturation, high IL-10 production and prolonged survival of DCs via a TLR2 pathway. We therefore studied the roles of mitogen-activated protein kinases (MAPK), glycogen synthase kinase-3 (GSK3) and phosphatidylinositol 3-kinase (PI3K) pathways on biological functions of human monocyte-derived DCs treated with BbC50sn.

Methodology/Principal Findings

DCs were differentiated from human monocytes with IL-4 and GM-CSF for 5 days and cultured with BbC50sn, lipopolysaccharide (LPS) or Zymosan, with or without specific inhibitors of p38MAPK (SB203580), ERK (PD98059), PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and GSK3 (SB216763). We found that 1) the PI3K pathway was positively involved in the prolonged DC survival induced by BbC50sn, LPS and Zymosan in contrast to p38MAPK and GSK3 which negatively regulated DC survival; 2) p38MAPK and PI3K were positively involved in DC maturation, in contrast to ERK and GSK3 which negatively regulated DC maturation; 3) ERK and PI3K were positively involved in DC-IL-10 production, in contrast to GSK3 that was positively involved in DC-IL-12 production whereas p38MAPK was positively involved in both; 4) BbC50sn induced a PI3K/Akt phosphorylation similar to Zymosan and a p38MAPK phosphorylation similar to LPS.

Conclusion/Significance

We report for the first time that a fermentation product of a bifidobacteria can differentially activate MAPK, GSK3 and PI3K in order to modulate DC biological functions. These results give new insights on the fine-tuned balance between the maintenance of normal mucosal homeostasis to commensal and probiotic bacteria and the specific inflammatory immune responses to pathogen bacteria.     

Non-opsonic phagocytosis of Legionella pneumophila by macrophages is mediated by phosphatidylinositol 3-kinase

Background

Legionella pneumophila, is an intracellular pathogen that causes Legionnaires'' disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis.

Methodology/Principal Findings

Phagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by ³²P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3), a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K) inhibitors, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. In contrast, PI3K and protein kinase B (PKB/Akt) activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85α subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells.

Conclusion/Significance

Entry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires'' disease.     

PI3K and ERK-induced Rac1 activation mediates hypoxia-induced HIF-1α expression in MCF-7 breast cancer cells

Background

Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown.

Methodology/Principal Findings

In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression.

Conclusions/Significance

Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1.     

Macrophage-Activating Lipopeptide-2 Requires Mal and PI3K for Efficient Induction of Heme Oxygenase-1

Aims

This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes.

Methods

Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay.

Results

MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions

These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.     

Anthraquinone-2-sulfonic acid (AQ2S) is A Novel Neurotherapeutic Agent

Anthraquinone derivatives such as emodin have recently been shown to protect in models of beta amyloid β (Aβ) and tau aggregation-induced cell death. The mechanisms of action possibly involve preconditioning effects, anti-aggregation properties, and/or enhancing the phosphatidylinositol-3-kinase (PI3K)/AKT survival mechanism. We studied several natural (emodin, rhein, and aloin) and synthetic (AQ2S) anthraquinones, to screen for post-treatment therapeutic benefit in two models of neuronal death, namely hydrogen peroxide (H₂O₂) and staurosporine (STS)-induced injury. Treatment with emodin, rhein, or aloin failed to reduce H₂O₂ injury. Moreover, consistent with emodin behaving like a mild toxin, it exacerbated oxidative injury at the highest concentration used (50 μM) in our post-treatment paradigm, and potently inhibited AKT. In contrast, AQ2S was neuroprotective. It reduced H₂O₂ injury at 50 and 75 μM. In addition, AQ2S potently inhibited staurosporine (STS)-induced injury. The mechanisms of action involve caspase inhibition and AKT activation. However, blockade of AKT signaling with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 failed to abolish AQ2S-mediated protection on the STS assay. This is the first study to report that AQ2S is a new neuroprotective compound and a novel caspase inhibitor.     

Ursolic Acid Attenuates Diabetic Mesangial Cell Injury through the Up-Regulation of Autophagy via miRNA-21/PTEN/Akt/mTOR Suppression

ObjectiveTo investigate the effect of ursolic acid on autophagy mediated through the miRNA-21-targeted phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in rat mesangial cells cultured under high glucose (HG) conditions.MethodsRat glomerular mesangial cells were cultured under normal glucose, HG, HG with the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or HG with ursolic acid conditions. Cell proliferation and hypertrophy were assayed using an MTT assay and the ratio of total protein to cell number, respectively. The miRNA-21 expression was detected using RT-qPCR. The expression of phosphatase and tensin homolog (PTEN)/AKT/mTOR signaling signatures, autophagy-associated protein and collagen I was detected by western blotting and RT-qPCR. Autophagosomes were observed using electron microscopy.ResultsCompared with mesangial cells cultured under normal glucose conditions, the cells exposed to HG showed up-regulated miRNA-21 expression, down-regulated PTEN protein and mRNA expression, up-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and down-regulated LC3II expression. Ursolic acid and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation, down-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and up-regulated LC3II expression. However, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not affect the expression of miRNA-21 and PTEN. Ursolic acid down-regulated miRNA-21 expression and up-regulated PTEN protein and mRNA expression.ConclusionsUrsolic acid inhibits the glucose-induced up-regulation of mesangial cell miRNA-21 expression, up-regulates PTEN expression, inhibits the activation of PI3K/Akt/mTOR signaling pathway, and enhances autophagy to reduce the accumulation of the extracellular matrix and ameliorate cell hypertrophy and proliferation.     

CCN4 induces IL-6 production through αvβ5 receptor,PI3K,Akt, and NF-κB singling pathway in human synovial fibroblasts

Introduction

Osteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells.

Methods

CCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvβ5 but not α5β1 and αvβ3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation.

Conclusions

Our results suggest that CCN4 activates αvβ5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.     

Effects of Bone Marrow-Derived Mesenchymal Stem Cells on the Axonal Outgrowth through Activation of PI3K/AKT Signaling in Primary Cortical Neurons Followed Oxygen-Glucose Deprivation Injury

Background

Transplantation with bone marrow-derived mesenchymal stem cells (BMSCs) improves the survival of neurons and axonal outgrowth after stroke remains undetermined. Here, we investigated whether PI3K/AKT signaling pathway is involved in these therapeutic effects of BMSCs.

Methodology/Principal Findings

(1) BMSCs and cortical neurons were derived from Sprague-Dawley rats. The injured neurons were induced by Oxygen–Glucose Deprivation (OGD), and then were respectively co-cultured for 48 hours with BMSCs at different densities (5×10³, 5×10⁵/ml) in transwell co-culture system. The average length of axon and expression of GAP-43 were examined to assess the effect of BMSCs on axonal outgrowth after the damage of neurons induced by OGD. (2) The injured neurons were cultured with a conditioned medium (CM) of BMSCs cultured for 24 hours in neurobasal medium. During the process, we further identified whether PI3K/AKT signaling pathway is involved through the adjunction of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a specific phosphatidylinositide-3-kinase (PI3K) inhibitor). Two hours later, the expression of pAKT (phosphorylated AKT) and AKT were analyzed by Western blotting. The length of axons, the expression of GAP-43 and the survival of neurons were measured at 48 hours.

Results

Both BMSCs and CM from BMSCs inreased the axonal length and GAP-43 expression in OGD-injured cortical neurons. There was no difference between the effects of BMSCs of 5×10⁵/ml and of 5×10³/ml on axonal outgrowth. Expression of pAKT enhanced significantly at 2 hours and the neuron survival increased at 48 hours after the injured neurons cultured with the CM, respectively. These effects of CM were prevented by inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions/Significance

BMSCs promote axonal outgrowth and the survival of neurons against the damage from OGD in vitro by the paracrine effects through PI3K/AKT signaling pathway.     

Varicella-Zoster Virus ORF12 Protein Activates the Phosphatidylinositol 3-Kinase/Akt Pathway To Regulate Cell Cycle Progression

Varicella-zoster virus (VZV) activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and alters cell cycle progression, but the viral protein(s) responsible for these activities is unknown. We previously reported that the VZV open reading frame 12 (ORF12) protein triggers phosphorylation of ERK. Here, we demonstrate that the VZV ORF12 protein also activates the PI3K/Akt pathway to regulate cell cycle progression. Transfection of cells with a plasmid expressing the ORF12 protein induced phosphorylation of Akt, which was dependent on PI3K. Infection of cells with wild-type VZV triggered phosphorylation of Akt, while infection with an ORF12 deletion mutant induced less phosphorylated Akt. The activation of Akt by ORF12 protein was associated with its binding to the p85 subunit of PI3K. Infection of cells with wild-type VZV resulted in increased levels of cyclin B1, cyclin D3, and phosphorylated glycogen synthase kinase 3β (GSK-3β), while infection with the ORF12 deletion mutant induced lower levels of these proteins. Wild-type VZV infection reduced the G₁ phase cell population and increased the M phase cell population, while infection with the ORF12 deletion mutant had a reduced effect on the G₁ and M phase populations. Inhibition of Akt activity with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 reduced the G₁ and M phase differences observed in cells infected with wild-type and ORF12 mutant viruses. In conclusion, we have found that the VZV ORF12 protein activates the PI3K/Akt pathway to regulate cell cycle progression. Since VZV replicates in both dividing (e.g., keratinocytes) and nondividing (neurons) cells, the ability of the VZV ORF12 protein to regulate the cell cycle is likely important for VZV replication in various cell types in the body.     

PI3Kγ contributes to MEK1/2 activation in oxidative glutamate toxicity via PDK1

The role of phosphoinositide 3‐kinase (PI3K) in oxidative glutamate toxicity is not clear. Here, we investigate its role in HT22 mouse hippocampal cells and primary cortical neuronal cultures, showing that inhibitors of PI3K, LY294002, and wortmannin suppress extracellular hydrogen peroxide (H₂O₂) generation and increase cell survival during glutamate toxicity in HT22 cells. The mitogen‐activated protein kinase kinase (MEK) inhibitor U0126 also reduced glutamate‐induced H₂O₂ generation and inhibited phosphorylation of extracellular signal‐regulated kinase (ERK) 1/2. LY294002 was seen to abolish phosphorylation of both ERK1/2 and Akt. A small interfering RNA (siRNA) study showed that PI3Kβ and PI3Kγ, rather than PI3Kα and PI3Kδ, contribute to glutamate‐induced H₂O₂ generation and cell death. PI3Kγ knockdown also inhibited glutamate‐induced ERK1/2 phosphorylation, whereas transfection with the constitutively active form of human PI3Kγ (PI3Kγ‐CAAX) triggered MEK1/2 and ERK1/2 phosphorylation and H₂O₂ generation without glutamate exposure. This H₂O₂ generation was reduced by inhibition of MEK. Transfection with kinase‐dead 3‐phosphoinositide‐dependent protein kinase 1 (PDK1‐KD) reduced glutamate‐induced ERK1/2 phosphorylation and H₂O₂ generation. Accordingly, cotransfection of cells with PDK1‐KD and PI3Kγ‐CAAX suppressed PI3Kγ‐CAAX‐triggered ERK1/2 phosphorylation and H₂O₂ generation. These results suggest that activation of PI3Kγ induces ERK1/2 phosphorylation, leading to extracellular H₂O₂ generation via PDK1 in oxidative glutamate toxicity.