Hypoxia-Induced Modulation of Apoptosis and BCL-2 Family Proteins in Different Cancer Cell Types

Hypoxia plays an important role in the resistance of tumour cells to chemotherapy. However, the exact mechanisms underlying this process are not well understood. Moreover, according to the cell lines, hypoxia differently influences cell death. The study of the effects of hypoxia on the apoptosis induced by 5 chemotherapeutic drugs in 7 cancer cell types showed that hypoxia generally inhibited the drug-induced apoptosis. In most cases, the effect of hypoxia was the same for all the drugs in one cell type. The expression profile of 93 genes involved in apoptosis as well as the protein level of BCL-2 family proteins were then investigated. In HepG2 cells that are strongly protected against cell death by hypoxia, hypoxia decreased the abundance of nearly all the pro-apoptotic BCL-2 family proteins while none of them are decreased in A549 cells that are not protected against cell death by hypoxia. In HepG2 cells, hypoxia decreased NOXA and BAD abundance and modified the electrophoretic mobility of BIM_EL. BIM and NOXA are important mediators of etoposide-induced cell death in HepG2 cells and the hypoxia-induced modification of these proteins abundance or post-translational modifications partly account for chemoresistance. Finally, the modulation of the abundance and/or of the post-translational modifications of most proteins of the BCL-2 family by hypoxia involves p53-dependent and –independent pathways and is cell type-dependent. A better understanding of these cell-to-cell variations is crucial in order to overcome hypoxia-induced resistance and to ameliorate cancer therapy.     

Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas RhoA is activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors, it is unknown how LPA receptors may signal to Rac. Here we report that the prototypic LPA(1) receptor (previously named Edg2), when expressed in B103 neuroblastoma cells, mediates transient activation of RhoA and robust, prolonged activation of Rac leading to cell spreading, lamellipodia formation, and stimulation of cell migration. LPA-induced Rac activation is inhibited by pertussis toxin and requires phosphoinositide 3-kinase activity. Strikingly, LPA fails to activate Rac in cell types that lack the Rac-specific exchange factor Tiam1; however, enforced expression of Tiam1 restores LPA-induced Rac activation in those cells. Tiam1-deficient cells show enhanced RhoA activation, stress fiber formation, and cell rounding in response to LPA, consistent with Tiam1/Rac counteracting RhoA. We conclude that LPA(1) receptors couple to a G(i)-phosphoinositide 3-kinase-Tiam1 pathway to activate Rac, with consequent suppression of RhoA activity, and thereby stimulate cell spreading and motility.     

RAFTK/Pyk2 mediates LPA-induced PC12 cell migration

The phospholipid lysophosphatidic acid (LPA) is a normal constituent of serum that functions as a lipid growth factor and intracellular signaling molecule. In this report, we have investigated the signaling mechanism and function of the tyrosine kinase RAFTK/Pyk2 in LPA-induced cell migration. Analysis of tyrosine phosphorylation upon LPA stimulation in neuroendocrine PC12 cells revealed 6 major tyrosine-phosphorylated proteins with estimated sizes of 180, 120, 115, 68, 44, and 42 kDa. These proteins were identified as epidermal growth factor receptor (EGFR), focal adhesion kinase, RAFTK/Pyk2, paxillin, Erk 1, and Erk 2, respectively. Using specific pharmacological inhibitors, we found that the tyrosine phosphorylation of RAFTK/Pyk2 was intracellular Ca2+-dependent, but not EGFR-dependent, during LPA stimulation of these cells. Moreover, the cytoskeletal and signal scaffolding protein, paxillin, associated with and was regulated by RAFTK/Pyk2 in a Ca2+-dependent manner. Characterization of LPA receptors showed that LPA1 (Edg2) and LPA2 (Edg4) are major receptors for LPA, while LPA3 receptor (Edg7) expression was limited. Upon using the LPA1/LPA3 receptor-specific antagonist VPC 32179, we observed that inhibition of the LPA1/LPA3 receptors had no effect on the LPA-induced phosphorylation of RAFTK, strongly suggesting that the LPA2 receptor is a key mediator of RAFTK phosphorylation. Furthermore, LPA induced PC12 cell migration, which was subsequently blocked by the dominant-negative form of FAK, FRNK. Expression of a dominant-negative form of the small GTPase Ras also blocked LPA-induced cell migration and RAFTK phosphorylation. Taken together, these results indicate that RAFTK is a key signaling molecule that mediates LPA-induced PC12 cell migration in a Ras-dependent manner.     

TRIP6 enhances lysophosphatidic acid-induced cell migration by interacting with the lysophosphatidic acid 2 receptor

Lysophosphatidic acid (LPA) induces actin rearrangement, focal adhesion assembly, and cell migration through the activation of small G protein Rho and its downstream effectors. These diverse cellular responses are mediated by its associated G protein-coupled receptors. However, the mechanisms and specificity by which these LPA receptors mediate LPA actions are still poorly understood. Here we show that LPA stimulation promotes the interaction of the LPA(2) receptor with a focal adhesion molecule, TRIP6 (thyroid receptor interacting protein 6)/ZRP-1 (zyxin-related protein 1). TRIP6 directly binds to the carboxyl-terminal tail of the LPA(2) receptor through its LIM domains. LPA-dependent recruitment of TRIP6 to the plasma membrane promotes its targeting to focal adhesions and co-localization with actin stress fibers. In addition, TRIP6 associates with the components of focal complexes including paxillin, focal adhesion kinase, c-Src, and p130(cas) in an agonist-dependent manner. Overexpression of TRIP6 augments LPA-induced cell migration; in contrast, suppression of endogenous TRIP6 expression by a TRIP6-specific small interfering RNA reduces it in SKOV3 ovarian cancer cells. Strikingly, the association with TRIP6 is specific to the LPA(2) receptor but not LPA(1) or LPA(3) receptor, indicating a specific role for TRIP6 in regulating LPA(2) receptor-mediated signaling. Taken together, our results suggest that TRIP6 functions at a point of convergence between the activated LPA(2) receptor and downstream signals involved in cell adhesion and migration.     

The role of lysophosphatidic acid receptors in phenotypic modulation of vascular smooth muscle cells

Lysophosphatidic acid (LPA) is a bioactive lipid with diverse physiological effects via activation of G protein-coupled receptors (GPCRs). It has been implicated as a specific dedifferentiation factors that can promote phenotypic modulation of cultured vascular smooth muscle cells (VSMCs) which is critically involved in various vascular disease. However, the role of LPA receptors and details of their signaling in LPA induced phenotypic modulation are largely unexplored. In this study we detect the expression of LPA1 and LPA3 in rat aortic smooth muscle cells (RASMCs). LPA promoted RASMCs phenotypic modulation in a dose-dependent manner and coordinated induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell phenotypic modulation was significantly inhibited by specific LPA1/LPA3-receptor antagonist dioctyl-glycerol pyrophosphate (DGPP8:0) at concentration, but this inhibitive effect was lost when the antagonist was coadministered with a highly selective LPA3 agonist,1-oleoyl-2-Omethyl-rac-glycero-phosphothionate (OMPT). In addition, pertussis toxin (PTX), a Gi protein inhibitor had little affect on the LPA-induced phenotypic modulation in RASMC. These data suggest that LPA-induced phenotypic modulation is mediated through the PTX-insensitive G-protein(s), possibly Gq-coupled LPA3 receptor.     

Lysophosphatidic acid as a novel cell survival/apoptotic factor

Lysophosphatidic acid (LPA) activates its cognate G protein-coupled receptors (GPCRs) LPA(1-3) to exert diverse cellular effects, including cell survival and apoptosis. The potent survival effect of LPA on Schwann cells (SCs) is mediated through the pertussis toxin (PTX)-sensitive G(i/o)/phosphoinositide 3-kinase (PI3K)/Akt signaling pathways and possibly enhanced by the activation of PTX-insensitive Rho-dependent pathways. LPA promotes survival of many other cell types mainly through PTX-sensitive G(i/o) proteins. Paradoxically, LPA also induces apoptosis in certain cells, such as myeloid progenitor cells, hippocampal neurons, and PC12 cells, in which the activation of the Rho-dependent pathways and caspase cascades has been implicated. The effects of LPA on both cell survival and apoptosis underscore important roles for this lipid in normal development and pathological processes.     

Results and prospects of the yeast three-hybrid system

The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53, p300 (a major component of histone acetyl transferase complexes), and p65(RelA) subunit of NF-κB. In this study, we investigated the cellular behaviors of HepG2 cells with exogenous ING4. Interestingly, the overexpression of ING4 negatively regulated the cell growth with significant G2/M arrest of cell cycle, and moreover, enhanced the cell apoptosis triggered by serum starvation in HepG2 cells. Furthermore, the exogenous ING4 could upregulate endogenous p21 and Bax in HepG2 cells, not in p53-deficient Saos-2 cells, suggesting that G2/M arrest induced by ING4 could be mediated by the increased p21 expression in a p53-dependent manner, although there is no significant increase of p53 expression in HepG2 cells. Moreover, HepG2 cells with exogenous ING4 could significantly increase cell death, as exposed to some DNA-damage agents, such as etoposide and doxorubicin, implying that ING4 could enhance chemosensitivity to certain DNA-damage agents in HepG2 cells.     

Physical and functional interactions of the lysophosphatidic acid receptors with PDZ domain-containing Rho guanine nucleotide exchange factors (RhoGEFs)

Lysophosphatidic acid (LPA) is a serum-derived phospholipid that induces a variety of biological responses in various cells via heterotrimeric G protein-coupled receptors (GPCRs) including LPA1, LPA2, and LPA3. LPA-induced cytoskeletal changes are mediated by Rho family small GTPases, such as RhoA, Rac1, and Cdc42. One of these small GTPases, RhoA, may be activated via Galpha(12/13)-linked Rho-specific guanine nucleotide exchange factors (RhoGEFs) under LPA stimulation although the detailed mechanisms are poorly understood. Here, we show that the C terminus of LPA1 and LPA2 but not LPA3 interact with the PDZ domains of PDZ domain-containing RhoGEFs, PDZ-RhoGEF, and LARG, which are comprised of PDZ, RGS, Dbl homology (DH), and pleckstrin homology (PH) domains. In LPA1- and LPA2-transfected HEK293 cells, LPA-induced RhoA activation was observed although the C terminus of LPA1 and LPA2 mutants, which failed to interact with the PDZ domains, did not cause LPA-induced RhoA activation. Furthermore, overexpression of the PDZ domains of PDZ domain-containing RhoGEFs served as dominant negative mutants for LPA-induced RhoA activation. Taken together, these results indicate that formation of the LPA receptor/PDZ domain-containing RhoGEF complex plays a pivotal role in LPA-induced RhoA activation.     

The lysophosphatidic acid 2 receptor mediates down-regulation of Siva-1 to promote cell survival

Lysophosphatidic acid (LPA) promotes cell survival through the activation of G protein-coupled LPA receptors. However, whether different LPA receptors activate distinct anti-apoptotic signaling pathways is not yet clear. Here we report a novel mechanism by which the LPA(2) receptor targets the proapoptotic Siva-1 protein for LPA-dependent degradation, thereby attenuating Siva-1 function in DNA damage response. The carboxyl-terminal tail of the LPA(2) receptor, but not LPA(1) or LPA(3) receptor, specifically associates with the carboxyl cysteine-rich domain of Siva-1. Prolonged LPA stimulation promotes the association of Siva-1 with the LPA(2) receptor and targets both proteins for ubiquitination and degradation. As a result, adriamycin-induced Siva-1 protein stabilization is attenuated by LPA in an LPA(2)-dependent manner, and the function of Siva-1 in promoting DNA damage-induced apoptosis is inhibited by LPA pretreatment. Consistent with this result, inhibition of the LPA(2) receptor expression increases Siva-1 protein levels and augments adriamycin-induced caspase-3 cleavage and apoptosis. Together, these findings reveal a critical and specific role for the LPA(2) receptor through which LPA directly inactivates a critical component of the death machinery to promote cell survival.     

PTPL1/FAP-1 negatively regulates TRIP6 function in lysophosphatidic acid-induced cell migration

The LIM domain-containing TRIP6 (Thyroid Hormone Receptor-interacting Protein 6) is a focal adhesion molecule known to regulate lysophosphatidic acid (LPA)-induced cell migration through interaction with the LPA2 receptor. LPA stimulation targets TRIP6 to the focal adhesion complexes and promotes c-Src-dependent phosphorylation of TRIP6 at Tyr-55, which creates a docking site for the Crk Src homology 2 domain, thereby promoting LPA-induced morphological changes and cell migration. Here we further demonstrate that a switch from c-Src-mediated phosphorylation to PTPL1/Fas-associated phosphatase-1-dependent dephosphorylation serves as an inhibitory feedback control mechanism of TRIP6 function in LPA-induced cell migration. PTPL1 dephosphorylates phosphotyrosine 55 of TRIP6 in vitro and inhibits LPA-induced tyrosine phosphorylation of TRIP6 in cells. This negative regulation requires a direct protein-protein interaction between these two molecules and the phosphatase activity of PTPL1. In contrast to c-Src, PTPL1 prevents TRIP6 turnover at the sites of adhesions. As a result, LPA-induced association of TRIP6 with Crk and the function of TRIP6 to promote LPA-induced morphological changes and cell migration are inhibited by PTPL1. Together, these results reveal a novel mechanism by which PTPL1 phosphatase plays a counteracting role in regulating TRIP6 function in LPA-induced cell migration.     

HBx抑制IGFBP3转录的机制

【目的】在细胞水平上研究乙型肝炎病毒X蛋白(HBx)对胰岛素样生长因子结合蛋白3(IGFBP3)转录的影响并对具体机制进行初步探索。【方法】首先采用RNA-Deep-Sequencing技术分析HepG2和乙型肝炎病毒(HBV)转基因细胞HepG2-4D14中表达差异的基因,然后通过实时定量PCR对相关基因进行验证;利用启动子报告基因分析,研究HBx对相关基因IGFBP3转录的调控;通过染色质免疫共沉淀方法,分析HBx抑制IGFBP3启动子活性的机制。【结果】RNA-Deep-Sequencing的结果表明IGFBP3在HBV转基因细胞HepG2-4D14中水平显著下调,实时定量PCR结果与RNA-Deep-Sequencing一致。进一步研究表明HBx能明显抑制IGFBP3的转录,通过实时定量PCR发现HBx对IGFBP3转录的抑制作用依赖于p53;染色质免疫共沉淀实验结果表明HBx能够通过抑制p53与IGFBP3启动子的结合,从而抑制IGFBP3的转录。IGFBP3是一种细胞周期负调控蛋白,我们推测HBx对IGFBP3水平的下调是其促进细胞增殖的途径之一。【结论】HBV能显著下调IGFBP3的转录,机制研究揭示HBV HBx通过干扰p53与IGFBP3启动子的结合进而抑制IGFBP3的转录。     

Triad 1 induces apoptosis by p53 activation

Triad 1 (2 RING [really interesting new gene] fingers and DRIL [double RING finger linked] 1) is an E3 ligase that induces apoptosis and clonogenic inhibition in myeloid cells through Gfi-1 stabilization. Here we demonstrate that Triad 1 induces apoptosis in several cancer cell lines including MCF7, A549, U2OS, and HCT 116 p53^+/+ cells via its RING ligase activity. Interestingly, in these cancer cells, Triad 1-induced apoptosis is not mediated by Gfi-1 stabilization but is instead p53-dependent. Moreover, Triad 1 promotes transactivation of p53. These results suggest that Triad 1 can induce apoptosis through its ligase activity via p53 activation.     

MAGI-3 regulates LPA-induced activation of Erk and RhoA

Lysophosphatidic acids (LPA) exert multiple biological effects through specific G protein-coupled receptors. The LPA-activated receptor subtype LPA(2) contains a carboxyl-terminal motif that allows interaction with PDZ domain-containing proteins, such as NHERF2 and PDZ-RhoGEF. To identify additional interacting partners of LPA(2), the LPA(2) carboxyl-terminus was used to screen a proteomic array of PDZ domains. In addition to the previously identified NHERF2, several additional LPA(2)-interacting PDZ domains were found. These included MAGI-2, MAGI-3 and neurabin. In the present work, we demonstrate the specific interaction between LPA(2) and MAGI-3, and the effects of MAGI-3 in colon cancer cells using SW480 as a cell model. MAGI-3 specifically bound to LPA(2), but not to LPA(1) and LPA(3). This interaction was mediated via the fifth PDZ domain of MAGI-3 interacting with the carboxyl-terminal 4 amino acids of LPA(2), and mutational alteration of the carboxyl-terminal sequences of LPA(2) severely attenuated its ability to bind MAGI-3. LPA(2) also associated with MAGI-3 in cells as determined by co-affinity purification. Overexpression of MAGI-3 in SW480 cells showed no apparent effect on LPA-induced activation of Erk and Akt. In contrast, silencing of MAGI-3 expression by siRNA drastically inhibited LPA-induced Erk activation, suggesting that the lack of an effect by overexpression was due to the high endogenous MAGI-3 level in these cells. Previous studies have shown that the cellular signaling elicited by LPA results in activation of the small GTPase RhoA by Galpha(12/13) - as well as Galpha(q)-dependent pathways. Overexpression of MAGI-3 stimulated LPA-induced RhoA activation, whereas silencing of MAGI-3 by siRNA resulted in a small but statistically significant decrease in RhoA activation. These results demonstrate that MAGI-3 interacts directly with LPA(2) and regulates the ability of LPA(2) to activate Erk and RhoA.