TRAIL promotes the survival,migration and proliferation of vascular smooth muscle cells

Human and rat primary sub-cultured vascular smooth muscle cells (VSMCs) showed clear expression of the death receptors TRAIL-R1 and TRAIL-R2; however, recombinant soluble TRAIL did not induce cell death when added to these cells. TRAIL tended to protect rat VSMCs from apoptosis induced either by inflammatory cytokines tumor necrosis factor- + interleukin-1 + interferon- or by prolonged serum withdrawal, and promoted a significant increase in VSMC proliferation and migration. Of note, all the biological effects induced by TRAIL were significantly inhibited by pharmacological inhibitors of the ERK pathway. Western blot analysis consistently showed that TRAIL induced a significant activation of ERK1/2, and a much weaker phosphorylation of Akt, while it did not affect the p38/MAPK pathway. Taken together, these data strengthen the notion that the TRAIL/TRAIL-R system likely plays a role in the biology of the vascular system by affecting the survival, migration and proliferation of VSMCs.Received 6 May 2004; received after revision 7 June 2004; accepted 8 June 2004     

Impaired apoptosis in lymphoblasts from Alzheimer’s disease patients: Cross-talk of Ca<Superscript>2+</Superscript>/calmodulin and ERK1/2 signaling pathways

We have analyzed the intracellular signals that allow lymphoblasts from Alzheimer’s disease (AD) patients to escape from serum deprivation-induced apoptosis. The following observations suggested that modulation of ERK1/2 activity by Ca²⁺/calmodulin (CaM) is involved in preventing apoptosis: (i) ERK1/2 activity seems to support lethality in control cells, as PD98059, the inhibitor of the activating MEK prevented cell death; (ii) control cells show a persistent and higher stimulation of ERK1/2 than that of AD cells in the absence of serum; (iii) CaM antagonists have no effects on control cells, but sensitize AD cells to death induced by serum withdrawal and increased ERK1/2 phosphorylation, and (iv) no apoptotic effects of CaM antagonists were observed in AD cells treated with PD98059. These results suggest the existence of an activation threshold of the ERK1/2 pathway setting by Ca²⁺/CaM-dependent mechanisms, which appears to be the critical factor controlling cell survival or death decision under trophic factor withdrawal. F. Bartolomé, N. de las Cuevas: These authors contributed equally to this work. Received 14 February 2007; received after revision 16 April 2007; accepted 23 April 2007     

Aloe emodin decreases the ERK-dependent anticancer activity of cisplatin

The present study describes the ability of an anthraquinone derivative aloe emodin (AE) to reduce the cytotoxic activity of the platinum(II)-based anticancer agent cisplatin toward murine L929 fibrosarcoma and C6 glioma cell lines. The protective effect of AE was demonstrated by MTT and crystal violet assays for cell viability, and involved supression of cisplatin-induced apoptosis and necrosis, as assessed by lactate dehydrogenase release and flow cytometric analysis of DNA fragmentation or phosphatidylserine exposure. Cell-based ELISA and Western blot analysis revealed that AE abolished cisplatin-triggered activation of extracellular signal-regulated kinase (ERK) in tumor cells, while activation of c-Jun N-terminal kinase was not significantly altered. A selective blockade of ERK activation with PD98059 mimicked the protective effect of AE treatment in both tumor cell lines. Moreover, AE failed to protect tumor cells against the ERK-independent toxicity of the Pt(IV)-based complex tetrachloro(O,O-dibutyl-ethylenediamine-N,N′-di-3-propanoate)platinum(IV). Taken together, these data indicate that herbal anthraquinone AE can downregulate the anticancer activity of cisplatin by blocking the activation of ERK in tumor cells.Received 30 January 2005; received after revision 21 March 2005; accepted 31 March 2005     

Calcium-mediated activation of PI3K and p53 leads to apoptosis in thyroid carcinoma cells

The molecular mechanism responsible for cadmium-induced cell death in thyroid cancer cells (FRO) is unknown. We demonstrated that apoptosis of FRO cells induced by cadmium was concentration and time dependent. Cadmium caused the rapid elevation of intracellular calcium and induced phosphorylation of Akt, p53, JNK, ERK and p38. Inhibition of PI3K/Akt attenuated the cadmium-induced apoptosis, but the inhibition of JNK inhibitor, ERK or p38 aggravated it, indicating that activation of PI3K/Akt was a pro-apoptosis signal in response to cadmium treatment, whereas the activation of stress-activated protein kinase JNK, ERK and p38 functioned as survival signals to counteract the cadmium-induced apoptosis. Buffering of the calcium response attenuated mitochondrial impairment, recovered the cadmium-activated Akt, p53, JNK, ERK and p38, and subsequently blocked the apoptosis. These results suggested that apoptosis induced by cadmium in FRO cells was initiated by the rapid elevation of intracellular calcium, followed by calcium-mediated activation of PI3K/Akt and mitochondrial impairment. Received 28 February 2007; received after revision 2 April 2007; accepted 23 April 2007     

Combining naturally occurring polyphenols with TNF-related apoptosis-inducing ligand: a promising approach to kill resistant cancer cells?

TNF-related apoptosis-inducing ligand (TRAIL) and its receptors are attractive targets for anticancer therapy owing to their ability to trigger apoptosis selectively in cancer cells but not in normal cells. To date, many combinatorial strategies, such as chemotherapy or radiotherapy, have given encouraging results for overcoming TRAIL resistance in preclinical models. In this review, we provide an overview of the molecular mechanisms underlying sensitization to TRAIL-induced apoptosis by polyphenols. These naturally occurring compounds can restore tumor cell sensitivity to TRAIL-induced cell death with no apparent toxicity towards normal cells. Both extrinsic and intrinsic pathways can be modulated by polyphenols, the activation of which largely depends on the cell type, the particular polyphenolic compound, and the conditions of treatment. The large variety of polyphenol cellular targets could prove useful in circumventing TRAIL resistance. The relevance of these combined treatments for cancer therapy is discussed in the light of recent preclinical studies.     

The protease-activated receptor-3 (PAR-3) can signal autonomously to induce interleukin-8 release

Protease-activated receptors (PARs) play a clear role in the burst of inflammatory reactions and immune responses. However, for PAR-3, the most elusive member of the PAR family, the functional role is still largely unclear. It has been claimed that PAR-3 does not signal autonomously, although the wide expression of human PAR-3 indicates its important physiological roles. We demonstrate that in HEK-293 cells, stably transfected with human PAR-3, thrombin induced calcium signaling, IL-8 gene expression and IL-8 release. We confirmed this finding using human lung epithelial and human astrocytoma cells that express endogenous PAR-3. Moreover, thrombin exposure of HEK-293 cells resulted in ERK1/2 activation coinciding with IL-8 release. The effects of thrombin were not dependent on PAR-1 activation, as confirmed by PAR-1 gene silencing. Thus, we propose that PAR-3 is able to signal autonomously to induce IL-8 release mediated by ERK1/2 phosphorylation, which contributes actively to inflammatory responses. Received 9 December 2007; received after revision 16 January 2008; accepted 18 January 2008     

Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G₀/G₁ phase without inducing significant cell death. In confluent C6 cultures, on the other hand, metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP-activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251, while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. Received 14 February 2007; received after revision 26 March 2007; accepted 3 April 2007     

MAPK/ERK1/2 signaling mediates endothelial-like differentiation of immature DCs in the microenvironment of esophageal squamous cell carcinoma

Endothelial-like differentiation of dendritic cells (DCs) is a new phenomenon, and the mechanism is still elusive. Here, we show that the tumor microenvironment derived from the human esophageal squamous cell carcinoma (ESCC) cell line EC9706 can induce immature DCs (iDCs) differentiate toward endothelial cells, and become endothelial-like cells, but it has no obvious influence on mature DCs. During the course of endothelial-like differentiation of iDCs, a sustained activation of mitogen-activated protein kinase/extracelluar signal-regulated kinase1/2 (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) was detected. Incubation of iDCs with MEK phosphorylation inhibitor PD98059 blocked the MAPK/ERK1/2 and CREB phosphorylation as well as the endothelial-like differentiation of iDCs. Inhibition of vascular endothelial growth factor-A (VEGF-A) in the microenvironment with its antibody blocked the endothelial-like differentiation and the phosphorylation of MAPK/ERK1/2 and CREB. These data suggest that MAPK/ERK1/2 signaling pathway activated by VEGF-A could mediate endothelial-like differentiation of iDCs in the ESCC microenvironment.     

ADP receptor P2Y13 induce apoptosis in pancreatic β-cells

Pancreatic β-cell loss represents a key factor in the pathogenesis of diabetes. Since the influence of purinergic signaling in β-cell apoptosis has not been much investigated, we examined the role of the ADP receptor P2Y₁₃ using the pancreatic insulinoma-cell line MIN6c4 as a model system. Real time-PCR revealed high expression of the ADP receptors P2Y₁ and P2Y₁₃. Adding the ADP analogue, 2MeSADP, to MIN6c4 cells induced calcium influx/mobilization and inhibition of cAMP production by activation of P2Y₁ and P2Y₁₃, respectively. 2MeSADP reduced cell proliferation and increased Caspase-3 activity; both these effects could be fully reversed by the P2Y₁₃ receptor antagonist MRS2211. We further discovered that blocking the P2Y₁₃ receptor results in enhanced ERK1/2, Akt/PKB and CREB phosphorylation mechanisms involved in β-cell survival. These results indicate that P2Y₁₃ is a proapoptotic receptor in β-cells as the P2Y₁₃ receptor antagonist MRS2211 is able to protect the cells from ADP induced apoptosis.     

ERK activation by Ca2+ ionophores depends on Ca2+ entry in lymphocytes but not in platelets, and does not conduct membrane scrambling

Rapid Ca²⁺-dependent phospholipid (PL) reorganization (scrambling) at the plasma membrane is a mechanism common to hematopoietic cells exposing procoagulant phosphatidylserine (PS). The aim of this research was to determine whether activation of the extracellular signal-regulated kinase (ERK) pathway was required for PL scrambling, based on a single report analyzing both responses induced by Ca²⁺ ionophores in megakaryoblastic HEL cells. Ca²⁺ ionophore-stimulated ERK phosphorylation was induced in platelets without external Ca²⁺, whereas exogenous Ca²⁺ entry was crucial for ERK activation in Jurkat T cells. In both cells, membrane scrambling only occurred following Ca²⁺ entry and was not blocked by inhibiting ERK phosphorylation. Furthermore, ERK proteins are strongly phosphorylated in transformed B lymphoblastic cell lines, which do not expose PS in their resting state. Overall, the data demonstrated that ERK activation and membrane scrambling are independent mechanisms. A. Arachiche, I. Badirou: These authors contributed equally to this work. Received 18 June 2008; received after revision 24 September 2008; accepted 1 October 2008     

刚地弓形虫培养上清抑制THP-1细胞增殖及诱导凋亡的研究

摘要：目的提取弓形虫体外细胞共培养上清,并研究上清对人急性单核细胞白血病细胞THP-1增殖及凋亡的影响。方法收集对数生长期的THP-1细胞以5X10^7／ml细胞浓度接种于不同培养瓶中,对照组加入含10％胎牛血清的RPMll640,实验组加入相同体积不同数量（2×10^7／ml、4X10^7／ml、8×10^7／m1）弓形虫速殖子培养上清,采用四甲基氮噻唑蓝（MTY）法检测吸光度（A490值）并计算THP-1细胞增殖抑制率;倒置显微镜下观察细胞形态变化;Annexin-V-FITC／PI染色细胞后上流式细胞仪检测各个时间点细胞凋亡率变化,以Western印迹方法分析凋亡相关蛋白Bax、Bcl-2的表达或活性。结果MTY法检测结果弓形虫培养上清呈时间剂量依赖性抑制THP-1细胞株增殖,倒置显微镜下观察处理组细胞有发泡现象和凋亡小体出现。流式细胞仪检测弓形虫感染后的THP-1细胞凋亡率较对照组有升高趋势（P〈0．05）,呈量效依赖性,Westernblot检测刚地弓形虫培养上清作用于THP-l细胞48h后实验组的Bax、Bcl-2蛋白表达较对照组的比值分别有明显的升高与降低（P〈0．05）。结论刚地弓形虫速殖子培养上清对体外培养THP-l细胞增殖有明显的抑制作用,并可诱导THP-1细胞凋亡。     

Culture in low levels of oxygen enhances in vitro proliferation potential of satellite cells from old skeletal muscles

The proliferation ability of satellite cells (considered the 'stem cells' of mature myofibers) declines with increasing age when cultured under standard cell culture conditions of 21% oxygen. However, actual oxygen levels in the intact myofiber in vivo are an order of magnitude lower. No studies to date have addressed the issue of whether culturing satellite cells from old muscles under more 'physiologic' conditions would enhance their proliferation and/or differentiation ability. Therefore, we analyzed satellite cells derived from 31-month-old rats in standard cultures with 21% O₂ and in lowered (∼3%) O₂. Under the lowered O₂ conditions, we noted a remarkable increase in the percentage of large-sized colonies, activation of cell cycle progression factors, phosphorylation of Akt, and downregulation of the cell cycle inhibitor p27^Kip1. These data suggest that lower O₂ levels provide a milieu that stimulates proliferation by allowing continued cell cycle progression, to result ultimately in the enhanced in vitro replicative life span of the old satellite cells. Such a method therefore provides an improved means for the ex vivo generation of progenitor satellite cell populations for potential therapeutic stem cell transplantation. Received 20 April 2001; received after revision 28 May 2001; accepted 31 May 2001     

Cell migration to the chemokine CXCL8: Paxillin is activated and regulates adhesion and cell motility

The chemokine CXCL8 is a powerful inducer of directional cell motility, primarily during inflammation. In this study, we found that CXCL8 stimulation led to paxillin phosphorylation in normal neutrophils, and that both CXCL8 receptors (CXCR1 and CXCR2) mediated CXCL8-induced paxillin phosphorylation. In CXCR2-transfected cells, the process depended on G_αi and G_αs coupling to CXCR2. Dominant negative (DN) paxillin increased CXCL8-induced adhesion and migration, indicating that endogenous paxillin keeps migration at submaximal levels. Furthermore, using activating antibodies to β1 integrins, analyses with focal adhesion kinase (FAK) DN variant (FRNK) and co-immunoprecipitations of FAK and paxillin, we found that β1 integrin ligation cooperates with CXCL8-induced stimulation, leading to FAK activation and thereafter to FAK-mediated paxillin phosphorylation. Our findings indicate that paxillin keeps directional motility at a restrained magnitude, and suggest that perturbations in its activation may lead to chemotactic imbalance and to pathological conditions associated with excessive or reduced leukocyte migration. R. Mintz, T. Meshel: These authors contributed equally to this work. Received 31 July 2008; received after revision 14 December 2008; accepted 16 December 2008     

Cathepsin X-mediated β2 integrin activation results in nanotube outgrowth

Membrane nanotubes were recently described as a new principle of cell–cell communication enabling complex and specific messaging to distant cells. Calcium fluxes, vesicles, and cell-surface components can all traffic between cells connected by nanotubes. Here we report for the first time the mechanism of membrane nanotube formation in T cells through LFA-1 (CD11a/CD18; α_Lβ₂) integrin activation by the cysteine protease cathepsin X. Cathepsin X is shown to induce persistent LFA-1 activation. Cathepsin X-upregulated T cells exhibit increased homotypic aggregation and polarized, migration-associated morphology in 2D and 3D models, respectively. In these cells, extended uropods are frequently formed, which subsequently elongate to nanotubes connecting T lymphocytes. Our results demonstrate that LFA-1 activation with subsequent cytoskeletal reorganization induces signal transmission through a physically connected network of T lymphocytes for better coordination of their action at various stages of the immune response. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 26 December 2008; received after revision 26 January 2009; accepted 27 January 2009 N. Obermajer, Z. Jevnikar: These authors contributed equally to the present work.     

Interaction between PGE2 and EGF receptor through MAPKs in mouse embryonic stem cell proliferation

Identifying the small molecules that permit precise regulation of embryonic stem (ES) cell proliferation should further support our understanding of the underlying molecular mechanisms of self renewal. In the present study, we showed that PGE₂ increased [³H]-thymidine incorporation in a time and dose dependent manner. In addition, PGE₂ increased the expression of cell cycle regulatory proteins, the percentage of cells in S phase and the total number of cells. PGE₂ obviously increased E-type prostaglandin (EP) receptor 1 mRNA expression level compare to 2, 3, 4 subtypes. EP1 antagonist also blocked PGE₂-induced cell cycle regulatory protein expression and thymidine incorporation. PGE₂ caused phosphorylation of protine kinase C, Src, epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation, and p44/42 mitogen-activated protein kinase (MAPK), which were blocked by each inhibitors. In conclusion, PGE₂-stimulated proliferation is mediated by MAPK via EP1 receptor-dependent PKC and EGF receptor-dependent PI3K/Akt signaling pathways in mouse ES cells. Received 30 January 2009; received after revision 03 March 2009; accepted 10 March 2009     

MSAP enhances migration of C6 glioma cells through phosphorylation of the myosin regulatory light chain

A key regulatory mechanism in cell motility is the control of myosin activity, which in non-muscle cells is determined by phosphorylation of the myosin regulatory light chain (MRLC). Here we show that MRLC-interacting protein (MIR)-interacting saposin-like protein (MSAP) enhances cell spreading in fibroblasts and migration of rat C6 glioma cells through increases in MRLC phosphorylation. Overexpression of MSAP enhanced the motility of glioma cells measured in matrigel invasion chambers and using a scratch assay. Downregulation of MSAP by RNA interference significantly decreased glioma cell migration and phosphorylation of MRLC. Inhibition of the corresponding MRLC kinase by ML-7 did not affect migration of MSAP-overexpressing cells. The present results show that MSAP controls glioma cell migration via enhancement of MRLC phosphorylation. This effect is independent of the activity of MRLC kinase. Thus, MSAP is a novel modulator of cell motility that influences migration of glioma cells and possibly other tumors.Received 9 February 2005; received after revision 2 March 2005; accepted 21 March 2005