Andrographolide acts through inhibition of ERK1/2 and Akt phosphorylation to suppress chemotactic migration

We now evaluated the anti-inflammatory mechanisms of andrographolide on complement 5a (C5a)-induced macrophage recruitment in vitro. Andrographolide concentration dependently inhibited cell migration toward C5a with an IC50 of 5.6+/-0.7 microM. With relatively specific kinase inhibitors (PD98059, SB203580, SP600125, wortmannin and LY294002, respectively) the results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol-3-kinase (PI3K) were necessary for C5a-induced migration, whereas c-Jun N-terminal kinase (JNK) was nonessential. Andrographolide significantly attenuated C5a-stimulated phosphorylation of ERK1/2, and of its upstream activator, MAP kinase-ERK kinase (MEK1/2). C5a-activated ERK1/2 phosphorylation was 86+/-9% inhibited by 30 microM andrographolide. Under the same conditions, however, andrographolide failed to affect C5a-stimulated p38 MAPK and JNK phosphorylation. Andrographolide also strongly abolished C5a-stimulated Akt phosphorylation, a downstream target protein for PI3K. These results indicate that inhibition of cell migration by interfering with ERK1/2 and PI3K/Akt signal pathways may contribute to the anti-inflammatory activity of andrographolide.     

C5a differentially stimulates the ERK1/2 and p38 MAPK phosphorylation through independent signaling pathways to induced chemotactic migration in RAW264.7 macrophages

We elucidate the roles of various protein kinases involved in complement 5a (C5a)-induced cell migration. Results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (P13K) were necessary for C5a-induced migration, whereas protein kinase C and c-Jun N-terminal kinase (JNK) were nonessential. C5a-induced migration was also suppresses by phospholipase C (PLC) inhibitor U73122 and pertussis toxin (PTX). We found that C5a-induced, time-dependent (1) ERK1/2 phosphorylation was markedly diminished by PTX, U73122, P13K inhibitors wortmannin and LY294002 and ERK1/2 inhibitor PD98059; (2) Akt phosphorylation was also attenuated by the above inhibitors except PD98059; (3) p38 MAPK phosphorylation was only affected by PTX. Furthermore, C5a also stimulated PLCbeta(2) membrane translocation in a time-dependent manner that occurred early prior to Akt phosphorylation and could be abolished only by PTX and U73122. These results suggest that C5a, through the activation of PTX-sensitive G protein, to differentially stimulate ERK1/2 and p38 MAPK phosphorylation and evoke cell migration. That is, ERK1/2 but not p38 MAPK phosphorylation is down stream of P13K/Akt and modulated by PLC. Additionally, beta(2) isoform may be one of the participates in C5a signal and acts more upstream of P13K/Akt.     

Cardiotoxin III suppresses MDA-MB-231 cell metastasis through the inhibition of EGF/EGFR-mediated signaling pathway

Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. Epidermal growth factor (EGF) and its receptor, EGFR, play roles in cancer metastasis in various tumors. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of CTX III on cell migration. CTX III inhibited the EGF-induced activation of matrix metalloproteinase-9 (MMP-9), and further suppressed cell invasion and migration without obvious cellular cytotoxicity. CTX III suppressed EGF-induced nuclear factor-kappaB (NF-κB) nuclear translocation and also abrogated the EGF-induced phosphorylation of EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt, and extracellular regulated kinase (ERK)1/2. In addition, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an up-stream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by EGF. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that CTX III inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt, ERK1/2, and NF-κB signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of CTX III as a potent anti-metastatic agent in MDA-MB-231 cells.     

Water extract of Korean red ginseng stimulates angiogenesis by activating the PI3K/Akt-dependent ERK1/2 and eNOS pathways in human umbilical vein endothelial cells

Angiogenesis is important for promoting cardiovascular disease, wound healing, and tissue regeneration. We investigated the effects of Korean red ginseng water extract (KRGE) on angiogenesis and its underlying signal mechanism. KRGE increased in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells, as well as stimulated in vivo angiogenesis without increasing VEGF expression. KRGE-induced angiogenesis was accompanied by phosphorylation of ERK1/2, phosphatidylinositol 3-kinase (Akt), and endothelial nitric oxide synthase (eNOS) as well as an increase in NO production. Inhibition of PI3K activity by wortmannin completely inhibited KRGE-induced angiogenesis and phosphorylation of Akt, ERK1/2, and eNOS, indicating that PI3K/Akt activation is an upstream event of the KRGE-mediated angiogenic pathway. The MEK inhibitor PD98059 blocked KRGE-induced ERK1/2 phosphorylation without affecting Akt and eNOS activation. However, the eNOS inhibitor N(G)-monomethyl-L-arginine effectively inhibited tube formation, but partially blocked proliferation and migration as well as ERK phosphorylation, without altering Akt and eNOS activation, revealing that the eNOS/NO pathway is partially involved in ERK1/2 activation. This study demonstrated that KRGE stimulates in vitro and in vivo angiogenesis through the activation of the PI3K/Akt-dependent ERK1/2 and eNOS signal pathways and their cross talk.     

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.     

磷脂酰肌醇-3-激酶在胰岛素与表皮生长因子激活MAPK信号通路中的不同作用

目的　研究磷脂酰肌醇 3 激酶 (phosphatidylinositol3 kinase, PI3K)在胰岛素和表皮生长因子 (epidermalgrowthfactor, EGF)刺激丝裂原激活的蛋白激酶 (mitogen activatedproteinkinase,MAPK)信号通路中的作用。方法　主要应用免疫印迹方法分析MAPK的磷酸化水平,利用PI3K特异性抑制剂wortmannin了解PI3K在其中所起的作用。结果　胰岛素和EGF均能有效、快速地刺激MAPK的磷酸化;wort mannin抑制胰岛素刺激的MAPK磷酸化,但对EGF刺激的MAPK磷酸化却没有明显影响。与此相反,胰岛素和EGF刺激的蛋白激酶B的磷酸化,均可被wortmannin同样有效地抑制。另外,wortmannin抑制胰岛素刺激的MAPK磷酸化具浓度依赖性;其不能抑制EGF刺激的MAPK磷酸化的性质并不随EGF刺激浓度的改变而改变。结论　PI3K在胰岛素和EGF刺激的MAPK磷酸化信号通路中起不同的作用。     

Honokiol-induced neurite outgrowth promotion depends on activation of extracellular signal-regulated kinases (ERK1/2)

We have found that honokiol [4-allyl-2-(3-allyl-4-hydroxy-phenyl)-phenol] can promote neurite outgrowth and mobilize intracellular Ca2+ store in primary cultured rat cortical neurons. In this study, we examined the effects of honokiol on extracellular signal-regulated kinases (ERK1/2) and Akt, and their possible relationship to neurite outgrowth and Ca2+ mobilization. Honokiol-induced neurite outgrowth in the cultured rat cortical neurons was significantly reduced by PD98059, a mitogen-activated protein kinase kinase (MAPKK, MAPK/ERK kinase MEK, direct upstream of ERK1/2) inhibitor, but not by LY294002, a phosphoinositide 3-kinase (PI3K, upstream of Akt) inhibitor. Honokiol also significantly enhanced the phosphorylation of ERK1/2 in a concentration-dependent manner, whereas the effect of honokiol on Akt phosphorylation was characterized by transient enhancement in 10 min and lasting inhibition after 30 min. The phosphorylation of ERK1/2 enhanced by honokiol was inhibited by PD98059 as well as by KN93, a Ca2+/calmodulin-dependent kinase II (CaMK II) inhibitor. Moreover, the products of the phosphoinositide specific phospholipase C (PLC)-derived inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG) were measured after honokiol treatment. Together with our previous findings, these results suggest that the signal transduction from PLC, IP3, Ca2+, and CaMK II to ERK1/2 is involved in honokiol-induced neurite outgrowth.     

TGF-beta1 increases motility and alphavbeta3 integrin up-regulation via PI3K, Akt and NF-kappaB-dependent pathway in human chondrosarcoma cells

Transforming growth factor-beta1 (TGF-beta1) plays an essential role in tumor progression and metastasis. Integrins are the major adhesive molecules in mammalian cells. Here we found that TGF-beta1 increased the migration and cell surface expression of alphavbeta3 integrin in human chondrosarcoma cells (JJ012 cells). Phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002) or Akt inhibitor inhibited the TGF-beta1-induced increase the migration of chondrosarcoma cells. TGF-beta1 stimulation increased the phosphorylation of p85 subunit of PI3K, and serine 473 of Akt. In addition, treatment of JJ102 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) inhibited TGF-beta1-induced cells migration and integrins expression. Treatment of JJ012 cells with TGF-beta1-induced IkappaB kinase alpha/beta (IKKalpha/beta) phosphorylation, IkappaBalpha phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The TGF-beta1-mediated increases in IKKalpha/beta phosphorylation and p65 Ser(536) phosphorylation were inhibited by Ly294002 and Akt inhibitor. Cotransfection with p85 and Akt mutants also reduced the TGF-beta1-induced kappaB-luciferase activity. Taken together, these results suggest that the TGF-beta1 acts through PI3K/Akt, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of alphavbeta3 integrins and contributing the migration of chondrosarcoma cells.     

Extracellular taurine induces angiogenesis by activating ERK-, Akt-, and FAK-dependent signal pathways

Taurine, a non essential sulfur-containing amino acid, plays a critical role in cardiovascular functions. We here examined the effect of taurine on angiogenesis and its underlying signal pathway. Taurine treatment increased angiogenesis in vitro and in vivo, which was followed by activation of the phosphatidylinositol 3-kinase (PI3K)/Akt, MEK/ERK, and Src/FAK signaling pathways. Further, taurine promoted endothelial cell cycle progression to the S and G2/M phases by up-regulating the positive cell cycle proteins, particularly cyclins D1 and B, as well as down-regulating the negative cell cycle proteins, p53 and p21(WAF1/CIP1), resulting in Rb phosphorylation. This angiogenic event was inhibited by inhibitors of PI3K and MEK. In addition, a PI3K inhibitor blocked the activation of Akt and ERK, while Akt knockdown did not affect taurine-induced ERK activation, indicating that PI3K is an upstream mediator of both MEK and Akt. Taurine-induced endothelial cell migration was suppressed by Src inhibitor, but not by other inhibitors, suggesting that the increase in cell migration is regulated by Src-dependent pathway. Moreover, inhibition of cellular taurine uptake by β-alanine and taurine transporter knockdown promoted taurine-induced cell proliferation, ERK and Akt activation, and in vivo angiogenesis, suggesting that extracellular taurine induces angiogenesis. However, taurine did not induce vascular inflammation and permeability in vitro and in vivo. These data demonstrate that extracellular taurine promotes angiogenesis by Akt- and ERK-dependent cell cycle progression and Src/FAK-mediated cell migration without inducing vascular inflammation, indicating that it is potential use for the treatment of vascular dysfunction-associated human diseases.     

Phosphatidylinositol 3-kinase mediates pain behaviors induced by activation of peripheral ephrinBs/EphBs signaling in mice

EphBs receptors and their ephrinBs ligands are present in the adult brain and peripheral tissue and play a critical role in modulating multiple aspects of physiology and pathophysiology. Our recent evidence has shown that ephrinBs acted as a sensitizer to participate in peripheral sensitization and hyperalgesia induced by activation of peripheral ephrinBs/EphBs signaling. In the present study, we explored the role of phosphatidylinositol 3-kinase (PI3K) in ephrinB1-Fc-induced pain behaviors. Intraplantar injection of ephrinB1-Fc produced a time- and dose-dependent increase of PI3K-p110γ expression and of phosphorylation of AKT in skin of injection site. Pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented activation of peripheral AKT by ephrinB1-Fc. The activated AKT expressed in peripheral nerve terminals and DRG peptide-containing and small non-peptide-containing neurons. Inhibition of peripheral PI3K signaling dose-dependently prevented and reversed pain behaviors and spinal Fos protein expression induced by intraplantar injection of ephrinB1-Fc. Furthermore, pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented ephrinB1-Fc-induced ERK activation in a dose-dependent manner. These data demonstrated that PI3K and PI3K crosstalk to ERK signaling mediated pain behaviors induced by activation of peripheral ephrinBs/EphBs signaling in mice.     

CCL5 increases lung cancer migration via PI3K, Akt and NF-κB pathways

CCL5 (previously called RANTES) is in the CC-chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. Besides, integrins are the major adhesive molecules in mammalian cells. Here we found CCL5 increased the migration and cell surface expression of αvβ3 integrin in human lung cancer cells (A549 cells). CCL5 stimulation increased phosphorylation of the p85α subunit of phosphatidylinositol 3-kinase (PI3K) and serine 473 of Akt. Also, we found that PI3K inhibitor (Ly294002) or Akt inhibitor suppressed CCL5-induced migration activities and integrin expression of A549 cells. Transfection of cells with p85 or Akt mutant also reduced CCL5-mediated cancer migration. In addition, treatment of A549 cells with CCL5 induced IκB kinase α/β (IKK α/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB-luciferase activity. Furthermore, the CCL5-mediated increases in p65 Ser⁵³⁶ phosphorylation were inhibited by Ly294002 and Akt inhibitor. Taken together, our results suggest that CCL5 acts through PI3K/Akt, which in turn activates IKKα/β and NF-κB, resulting in the activation of αvβ3 integrin and contributing to the migration of human lung cancer cells.     

Red wine polyphenols induce EDHF-mediated relaxations in porcine coronary arteries through the redox-sensitive activation of the PI3-kinase/Akt pathway

Red wine polyphenolic compounds (RWPCs) are potent inducers of endothelium-dependent relaxations of coronary arteries, which involve both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF). The EDHF-mediated relaxation to RWPCs is critically dependent on the formation of reactive oxygen species by a flavin-dependent enzyme. The aim of the present study was to determine the role of redox-sensitive protein kinases including p38 MAPK, ERK1/2 and PI3-kinase/Akt in RWPCs-induced EDHF-mediated relaxation. Porcine coronary artery rings were suspended in organ chambers for measurement of changes in isometric tension. Confluent cultures of porcine coronary artery endothelial cells were used to determine the phosphorylation level of p38 MAPK, ERK1/2 and Akt by Western blot analysis. All experiments were performed in the presence of indomethacin and Nomega-nitro-L-arginine. RWPCs caused pronounced endothelium-dependent relaxations, which were significantly reduced by wortmannin and LY294002, two inhibitors of PI3-kinase, and not affected by PD98059 (an inhibitor of ERK1/2 kinase kinase) and SB203580 (an inhibitor of p38 MAPK). In contrast, wortmannin did not affect relaxations to bradykinin or levcromakalim. RWPCs elicited within minutes a sustained and concentration-dependent phosphorylation of p38 MAPK, ERK1/2 and Akt in endothelial cells. The phosphorylation of Akt in response to RWPCs was abolished by wortmannin and LY294002, and by the membrane-permeant analogue of superoxide dismutase Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin. The present findings demonstrate that RWPCs cause EDHF-mediated relaxations of coronary arteries; these responses are critically dependent on the redox-sensitive activation of the PI3-kinase/Akt pathway in endothelial cells.     

Phosphatidylinositol inhibits vascular endothelial growth factor-A--induced migration of human umbilical vein endothelial cells

Phosphatidylinositol (PI), a phospholipid in component of cell membranes, is widely distributed in animals, plants, and microorganisms. Here, we examined in vitro whether PI inhibits the angiogenesis induced by vascular endothelial growth factor-A (VEGF-A). PI concentration-relatedly and significantly (at 10 and 30 microg/ml) inhibited VEGF-A-induced tube formation in a co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. PI also inhibited the migration, but not proliferation, induced in HUVECs by VEGF-A. Furthermore, PI at 30 microg/ml inhibited the VEGF-A-induced phosphorylation of serine/threonine protein kinase family protein kinase B (Akt) and p38 mitogen activate kinase (p38MAPK), key molecules in cell migration, but not phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a key molecule in cell proliferation. These findings indicate that PI inhibits VEGF-induced angiogenesis by inhibiting HUVECs migration and that inhibition of phosphorylated-Akt and -p38MAPK may be involved in the mechanism. Therefore, PI may be expected to prevent some diseases caused by angiogenesis.     

Activation of phosphoinositide 3-kinase, protein kinase C, and extracellular signal-regulated kinase is required for oridonin-enhanced phagocytosis of apoptotic bodies in human macrophage-like U937 cells

Our previous study showed that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic cells by macrophage-like U937 cells through tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta release. In this study, we further investigated signaling events involved in oridonin-augmented phagocytosis. Phagocytic stimulation was significantly suppressed by inhibitors, including a phosphoinositide 3-kinases (PI3K) inhibitor (wortmannin), a protein kinase C (PKC) inhibitor (stauroporine), and a phospholipase C (PLC) inhibitor (U73122). Exposure of U937 cells to oridonin caused an increase in PKC activity time- dependently, which was prevented by pretreatment with inhibitors of PI3K and PLC. Simultaneously, the activation of protein kinase B (PKB/Akt) and the increased expression of PLCgamma2 were also blocked by wortmannin. In addition, an extracellular signal-regulated kinase (ERK) MAPK inhibitor, PD98059, suppressed oridonin-augmented phagocytosis, whereas the p38 MAPK inhibitor (SB203580) and c-Jun N-terminal kinase (JNK) MAPK inhibitor (SP98059) had no inhibitory effect. Furthermore, pretreatment of U937 cells with anti-TNFalpha and anti-IL-1beta antibodies blocked oridonin-induced phagocytic stimulation as well as phosphorylation of ERK, but did not block the activation of PKC, indicating that these signaling events are triggered by oridonin, whereas secreted TNFalpha or IL-1beta only activate the ERK-dependent pathway. Taken together, oridonin is suggested to enhance phagocytosis of apoptotic bodies by activating PI3K, PKC, and ERK-dependent pathways.     

Major role of the PI3K/Akt pathway in ischemic tolerance induced by sublethal oxygen-glucose deprivation in cortical neurons in vitro

Ischemic preconditioning can provide protection to neurons from subsequent lethal ischemia. The molecular mechanisms of neuronal ischemic tolerance, however, are still not well-known. The present study, therefore, examined the role of MAPK and PI3K/Akt pathways in ischemic tolerance induced by preconditioning with sublethal oxygen-glucose deprivation (OGD) in cultured rat cortical neurons. Ischemic tolerance was simulated by preconditioning of the neurons with sublethal 1-h OGD imposed 12 h before lethal 3-h OGD. The time-course studies of relative phosphorylation and expression levels of ERK1/2, JNK and p38 MAPK showed lack of their involvement in ischemic tolerance. However, there were significant increases in Akt phosphorylation levels during the reperfusion period following preconditioned lethal OGD. In addition, Bcl-2 associated death promoter (Bad) and GSK-3β were also found to be inactivated during that reperfusion period. Finally, treatment with an inhibitor of PI3K, wortmannin, applied from 15 min before and during lethal OGD abolished not only the preconditioning-induced neuroprotection but also the Akt activation. Concomitant with blockade of the Akt activation, PI3K inhibition also resulted in activation of Bad and GSK-3β. The results suggest that ischemic tolerance induced by sublethal OGD preconditioning is primarily mediated through activation of the PI3K/Akt pathway, but not the MAPK pathway, in rat cortical neurons.     

Angiotensin-induced EGF receptor transactivation inhibits insulin signaling in C9 hepatic cells

To investigate the potential interactions between the angiotensin II (Ang II) and insulin signaling systems, regulation of IRS-1 phosphorylation and insulin-induced Akt activation by Ang II were examined in clone 9 (C9) hepatocytes. In these cells, Ang II specifically inhibited activation of insulin-induced Akt Thr³⁰⁸ and its immediate downstream substrate GSK-3α/β in a time-dependent fashion, with ∼70% reduction at 15 min. These inhibitory actions were associated with increased IRS-1 phosphorylation of Ser⁶³⁶/Ser⁶³⁹ that was prevented by selective blockade of EGFR tyrosine kinase activity with AG1478. Previous studies have shown that insulin-induced phosphorylation of IRS-1 on Ser⁶³⁶/Ser⁶³⁹ is mediated mainly by the PI3K/mTOR/S6K-1 sequence. Studies with specific inhibitors of PI3K (wortmannin) and mTOR (rapamycin) revealed that Ang II stimulates IRS-1 phosphorylation of Ser⁶³⁶/Ser⁶³⁹ via the PI3K/mTOR/S6K-1 pathway. Both inhibitors blocked the effect of Ang II on insulin-induced activation of Akt. Studies using the specific MEK inhibitor, PD98059, revealed that ERK1/2 activation also mediates Ang II-induced S6K-1 and IRS-1 phosphorylation, and the impairment of Akt Thr³⁰⁸ and GSK-3α/β phosphorylation. Further studies with selective inhibitors showed that PI3K activation was upstream of ERK, suggesting a new mechanism for Ang II-induced impairment of insulin signaling. These findings indicate that Ang II has a significant role in the development of insulin resistance by a mechanism that involves EGFR transactivation and the PI3K/ERK1/2/mTOR-S6K-1 pathway.     

Beta-sitosterol-induced-apoptosis is mediated by the activation of ERK and the downregulation of Akt in MCA-102 murine fibrosarcoma cells

Beta-sitosterol (SITO) is a potential candidate for cancer chemotherapy, however, little is known about the cellular and molecular mechanisms in cancer cells. We herein identified how SITO induces anti-proliferation and cell death in MCA-102 fibrosarcoma cells. SITO exposure induced-apoptosis and the cell death resulted from a significant loss of the Bcl-2 and the inhibitor of apoptosis protein (IAP) family (XIAP, cIAP-1 and cIAP-2), and increased Bax with an alteration of p53 and p21. SITO-induced cell death significantly also increased caspase activity and poly(ADP-ribose) polymerase (PARP) cleavage, and caspase-3 inhibitor z-DEVD-fmk significantly inhibited SITO-induced cell death. These data suggest that the activation of caspase-3 is associated with SITO-induced-apoptosis. Treatment with SITO also induced phosphorylation of extracellular-signal regulating kinase (ERK) and p38 mitogen-activated protein kinase (MARK), but not c-Jun N-terminal kinase (JNK). A specific ERK inhibitor PD98059 significantly blocks SITO-induced-apoptosis, whereas a JNK inhibitor SP600125 has no affect. A p38 MAPK inhibitor SB203580 very slightly suppressed cell death. The induction of apoptosis was also accompanied by an inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt, and PI3K inhibitor LY29004 significantly increases SITO-induced cell death. These findings provide evidence demonstrating that the proapoptotic effect of SITO is mediated through the activation of ERK and the block of the PI3K/Akt signal pathway in MCA-102 cells. Therefore, SITO has a strong potential as a therapeutic agent for preventing cancers such as fibrosarcoma.     

Effect of bradykinin on bradykinin-B2 receptor in rat aortic vascular smooth muscle cells and the involved signal transduction pathways

The aim of this paper is to study the effect of bradykinin (BK) on bradykinin-B2 receptor as well as the possible involved signal transduction pathways in cultured rat aortic vascular smooth muscle cells (VSMCs). Rat aortic VSMCs were cultured. Cells after 4–6 passages were used in the experiment. VSMCs were incubated with BK, BK + B2 receptor inhibitor (HOE-140), BK + MEK inhibitor (PD98059), BK + mitogen-activated protein kinase (MAPK) inhibitor (apigenin), BK + phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), and BK + Akt inhibitor to evaluate the expression of B2 receptor and phosphorylation of signaling molecules MAPK, Akt, and PI3K by Western blot. (1) BK markedly up-regulated the expression of B2 receptor in VSMC. (2) Apigenin, PD98059, Akt inhibitor, and LY294002 inhibited upregulation of B2 receptor induced by BK. (3) Signal transduction pathways of MAPK and PI3K were involved in the up-regulation of B2 receptor by BK mediation. Results suggest that bradykinin can up-regulate the expression of B2 receptor in VSMCs.     

Carboxymethylated chitosan stimulates proliferation of Schwann cells in vitro via the activation of the ERK and Akt signaling pathways

Proliferation of Schwann cell in the injured peripheral nerve supports axonal regeneration and also is critical for the regeneration of injured nerves. In this publication, carboxymethylated chitosan (CMCS) was studied to determine its capacity (i) to induce proliferation and synthesis of proliferating cell nuclear antigen (PCNA) and (ii) to activate mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) and phosphatidylinositil-3 kinase (PI3K)/Akt signaling pathways in rat Schwann cells. CMCS was found to induce proliferation and PCNA synthesis in Schwann cells in a dose and time dependent manner. CMCS was shown to phosphorylate ERK1/2 and Akt in Schwann cell proliferation. The phosphorylation of ERK1/2 and Akt in Schwann cells was blocked by the MEK inhibitor PD98059 and the PI3K inhibitor wortmannin. In addition, inhibition of the MEK/ERK or the PI3K/Akt signaling pathways significantly decreased the proliferative effects of CMCS in Schwann cells. Overall, the above results indicate that CMCS stimulates proliferation of Schwann cells by activating the intracellular signaling cascades of ERK1/2 and PI3K/Akt.