17β-雌二醇通过PI3K/Akt/mTOR通路抑制白介素1β诱导的大鼠椎间盘髓核细胞的凋亡

髓核细胞(nucleus pulposus cells,NPCs)的异常凋亡是导致椎间盘退变(intervertebral disc degeneration,IVDD)的主要原因。本研究组前期研究显示,17β-雌二醇(17β-estradiol,E2)能够通过PI3K/Akt信号通路抑制白介素1β(interleukin-1β,IL-1β)诱导的大鼠椎间盘NPCs凋亡。本研究旨在探讨PI3K/Akt途径的下游蛋白是否参与E2对NPCs凋亡的抑制作用。用胰蛋白酶消化法分离原代大鼠NPCs,采用E2和PI3K/Akt信号通路下游蛋白的不同抑制剂预处理后用IL-1β处理,用Annexin V/PI染色法检测凋亡率,用CCK-8法检测细胞活力,用细胞黏附试验检测NPCs与Ⅱ型胶原的黏附能力,用Western blot检测哺乳动物雷帕霉素靶蛋白(mammalian target of Rapamycin,mTOR)、糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)和核因子κB(nuclear factor kappaB,NF-κB)磷酸化水平。结果显示,E2显著抑制IL-1β诱导的NPCs凋亡,逆转由IL-1β引起的细胞活力和黏附能力的降低,抑制IL-1β对mTOR磷酸化水平的下调作用,而雷帕霉素可以阻断E2的这些保护作用。以上结果提示,E2可能通过PI3K/Akt/mTOR信号通路抑制IL-1β诱导的NPCs凋亡。     

The anti-apoptotic effect of Notch-1 requires p56lck-dependent, Akt/PKB-mediated signaling in T cells

The Notch family of transmembrane receptors have been implicated in a variety of cellular decisions in different cell types. Here we investigate the mechanism underlying Notch-1-mediated anti-apoptotic function in T cells using model cell lines as the experimental system. Ectopic expression of the intracellular domain of Notch-1/activated Notch (AcN1) increases expression of anti-apoptotic proteins of the inhibitors of apoptosis (IAP) family, the Bcl-2 family, and the FLICE-like inhibitor protein (FLIP) and inhibits death triggered by multiple stimuli that activate intrinsic or extrinsic pathways of apoptosis in human and murine T cell lines. Numb inhibited the AcN1-dependent induction of anti-apoptotic proteins and anti-apoptotic function. Using pharmacological inhibitors and dominant-negative approaches, we describe a functional role for phosphatidylinositol 3-kinase (PI3K)-dependent activation of the serine-threonine kinase Akt/PKB in the regulation of AcN1-mediated anti-apoptotic function and the expression of FLIP and IAP family proteins. Using a cell line deficient for the T cell-specific, Src family protein, the tyrosine kinase p56(lck) and by reconstitution approaches we demonstrate that p56(lck) is required for the Notch-1-mediated activation of Akt/PKB function. Furthermore, the Src tyrosine kinase inhibitor, PP2, abrogated ectopically expressed AcN1-mediated anti-apoptotic function and phosphorylation of p56(lck). We present evidence that endogenous Notch-1 associates with p56(lck) and PI3K but that Akt/PKB does not co-immunoprecipitate with the Notch1.p56(lck).PI3K complex. Finally, we demonstrate that the Notch1.p56(lck).PI3K complex is present in primary T cells that have been activated in vitro and sustained in culture with the cytokine interleukin-2.     

Phospholipase D elevates the level of MDM2 and suppresses DNA damage-induced increases in p53

Phospholipase D (PLD) has been reported to generate survival signals that prevent apoptosis induced by serum withdrawal. We have now found that elevated expression of PLD also suppresses DNA damage-induced apoptosis. Since DNA damage-induced apoptosis is often mediated by p53, we examined the effect of elevated PLD expression on the regulation of p53 stabilization. We report here that PLD suppresses DNA damage-induced increases in p53 stabilization in cells where PLD has been shown to provide a survival signal. Elevated expression of PLD also led to increased expression of the p53 E3 ubiquitin ligase MDM2 and increased turnover of p53. PLD1-stimulated increases in MDM2 expression and suppression of p53 activation were blocked by inhibition of mTOR and the mitogen-activated protein kinase pathway. Although PLD did not activate the phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway activate the basal levels of PI3K activity were partially required for PLD1-induced increases in MDM2. These data provide evidence that survival signals generated by PLD involve suppression of the p53 response pathway.     

Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent senescence and apoptosis in HeLa cervical carcinoma cells

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine the role of p53 signaling in senescence or apoptosis after repression of HPV oncogenes, we introduced a dominant-negative allele of p53 into HeLa cells. Dominant-negative p53 prevented senescence and apoptosis when E6 alone was repressed but did not inhibit senescence when both E6 and E7 were repressed. To determine whether reduced telomerase activity was involved in senescence or apoptosis after E6 repression, we generated HeLa cells stably expressing an exogenous hTERT gene, which encodes the catalytic subunit of telomerase. Although these cells contained markedly elevated telomerase activity and elongated telomeres, hTERT expression did not prevent senescence and apoptosis when E6 alone was repressed. These results demonstrate that when the Rb tumor suppressor pathway is inactivated by the E7 protein, E6 repression activates p53 signaling, which in turn is required for growth inhibition, senescence, and apoptosis. Thus, sustained inactivation of the p53 pathway by the E6 protein is required for maintenance of the proliferative phenotype of HeLa cervical carcinoma cells.     

Mdm2 inhibition induces apoptosis in p53 deficient human colon cancer cells by activating p73- and E2F1-mediated expression of PUMA and Siva-1

Camptothecin (CPT) and Nutlin-3 caused apoptosis by increasing p53 protein and its activation in intestinal epithelial cells (IEC-6). We studied the effectiveness of these inducers on apoptosis in human colon cancer cells (Caco2) lacking p53 expression. CPT failed to activate caspase-3 and cause apoptosis in these cells. The absence of p53 expression, higher basal Bcl-xL and lower Bax proteins prevented CPT-induced apoptosis. However, the Mdm2 antagonist Nutlin-3 induced apoptosis in a dose dependent manner by activating caspases-9 and -3. Nutlin-3 prevented the activation of AKT via PTEN-mediated inhibition of the PI3K pathway. Nutlin-3 increased the phosphorylation of retinoblastoma protein causing E2F1 release leading to induction of Siva-1. Nutlin-3-mediated degradation of Mdm2 caused the accumulation of p73, which induced the expression of p53 up-regulated modulator of apoptosis (PUMA). E2F1 and p73 knockdown decreased the expression of Siva and PUMA, respectively and abolished Nutlin-3-induced caspase-3 activation. Cycloheximide (CHX) inhibited Nutlin-3-induced Siva, Noxa, and PUMA expression and inhibited apoptosis in IEC-6 and Caco2 cells. These results indicate that translation of mRNAs induced by Nutlin-3 is critical for apoptosis. In summary, apoptosis in Caco2 cells lacking functional p53 occurred following the disruption of Mdm2 binding with p73 and Rb leading to the expression of pro-apoptotic proteins, PUMA, Noxa, and Siva-1.     

Activated H-ras rescues E1A-induced apoptosis and cooperates with E1A to overcome p53-dependent growth arrest.

The adenovirus E1A oncogene products stimulate DNA synthesis and cell proliferation but fail to transform primary baby rat kidney (BRK) cells because of the induction of p53-mediated programmed cell death (apoptosis). Overexpression of dominant mutant p53 (to abrogate wild-type p53 function) or introduction of apoptosis inhibitors, such as adenovirus E1B 19K or Bcl-2 oncoproteins, prevents E1A-induced apoptosis and permits transformation of BRK cells. The ability of activated Harvey-ras (H-ras) to cooperate with E1A to transform BRK cells suggests that H-ras is capable of overcoming the E1A-induced, p53-dependent apoptosis. We demonstrate here that activated H-ras was capable of suppressing apoptosis induced by E1A and wild-type p53. However, unlike Bcl-2 and the E1B 19K proteins, which completely block apoptosis but not p53-dependent growth arrest, H-ras expression permitted DNA synthesis and cell proliferation in the presence of high levels of wild-type p53. The mechanism by which H-ras regulates apoptosis and cell cycle progression is thereby strikingly different from that of the E1B 19K and Bcl-2 proteins. BRK cells transformed with H-ras and the temperature sensitive murine mutant p53(val 135), which lack E1A, underwent growth arrest at the permissive temperature for wild-type p53. p53-dependent growth arrest, however, could be relieved by E1A expression. Thus, H-ras alone was insufficient and cooperation of H-ras and E1A was required to override growth suppression by p53. Our data further suggest that two complementary growth signals from E1A plus H-ras can rescue cell death and thus permit transformation.     

Class I phosphatidylinositol 3-kinase inhibitor LY294002 activates autophagy and induces apoptosis through p53 pathway in gastric cancer cell line SGC7901

We aimed to study the effects of LY294002, an inhibitor of class I phosphatidylinositol 3-kinase (PI3K), on proliferation, apoptosis, and autophagy in gastric cancer cell line SGC7901. In this study, we showed that LY294002 inhibited the viability of gastric cancer SGC7901 cells. We also showed that LY294002 increased the expression of microtubule-associated protein 1 light chain 3 (LC3), and increased monodansylcadaverine (MDC)-labeled vesicles. LY294002 activated autophagy by activating p53 and caspase-3, and induced apoptosis by up-regulatingp53 and p53-up-regulated modulator of apoptosis ( PUMA ). Therefore, LY294002 might induce cytotoxicity in SGC7901 cells through activation of p53 and the downstream point PUMA . These findings suggest that inhibition of the class I PI3K signaling pathway is a potential strategy for managing gastric cancers.     

Phosphorylation of MNAR promotes estrogen activation of phosphatidylinositol 3-kinase

Estrogen actions are mediated by a complex interface of direct control of gene expression (the so-called "genomic action") and by regulation of cell signaling/phosphorylation cascades, referred to as the "nongenomic," or extranuclear, action. We have previously described the identification of MNAR (modulator of nongenomic action of estrogen receptor) as a novel scaffold protein that regulates estrogen receptor alpha (ERalpha) activation of cSrc. In this study, we have investigated the role of MNAR in 17beta-estradiol (E2)-induced activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Consistent with our previous results, a direct correlation was established between MNAR expression levels and E2-induced activation of PI3 and Akt kinases. Endogenous MNAR, ERalpha, cSrc, and p85, the regulatory subunit of PI3 kinase, interacted in MCF7 cells treated with E2. The interaction between p85 and MNAR required activation of cSrc and MNAR phosphorylation on Tyr 920. Consequently, the mutation of this tyrosine to alanine (Y920A) abrogated the interaction between MNAR and p85 and the E2-induced activation of the PI3K/Akt pathway, which was required for the E2-induced protection of MCF7 cells from apoptosis. Nonetheless, the Y920A mutant potentiated the E2-induced activation of the Src/MAPK pathway and MCF7 cell proliferation, as observed with the wild-type MNAR. These results provide new and important insights into the molecular mechanisms of E2-induced regulation of cell proliferation and apoptosis.     

Notch signaling protects retina from nuclear factor-κB- and poly-ADP-ribose-polymerase-mediated apoptosis under high-glucose stimulation

Proliferative diabetic retinopathy, the primary cause of vision loss in adults, is one of serious microvascular complications caused by diabetes. Both poly-ADP-ribose-polymerase (PARP) and nuclear factor (NF)-κB signaling are involved in the injury process. Injury activates PARP, which in turn potentiates NF-κB activation and causes cell apoptosis. Like the NF-κB pathway, Notch1 signaling plays a key role in the regulation of cell proliferation, differentiation, and apoptosis. However, the connections between these signaling pathways are not well understood. In this study, we used both streptozotocin (STZ)-induced diabetic mice and human retinal vascular endothelial cells (HRVECs) cultured in high glucose to detect these relationships. We found that apoptosis was increased in both STZ-induced diabetic mice and high-glucose-treated HRVECs, which was due to increased activation of PARP, cleaved caspase3, and reduced expression of Notch1 and p-Akt. The results of Notch1 overexpression and knockdown indicated that Notch1 signaling participated in the interaction of PARP and p50, and inhibited PARP- and p50-mediated apoptosis directly. These phenomena could be blocked by pretreatment with the PI3K inhibitor wortmannin via reducing p-Akt levels. Thus, our study demonstrated that Notch1 signaling protects cells from PARP- and NF-κB-induced apoptosis under high glucose through the activation of Akt.     

Higenamine mitigates interleukin-1β-induced human nucleus pulposus cell apoptosis by ROS-mediated PI3K/Akt signaling

Intervertebral disc degeneration (IDD) is a natural problem linked to the inflammation. Higenamine exerts multiple pharmacological properties in inflammation-related disorders. Our study aimed to explore the function of higenamine on interleukin (IL)-1β-caused apoptosis of human nucleus pulposus cells (HNPCs). Cell apoptosis was investigated by TUNEL and flow cytometry. Apoptosis-related biomarkers were determined by qRT-PCR or Western blotting. The protein in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling was measured by Western blotting. We found that higenamine showed little effect on cell apoptosis, but mitigated IL-1β-caused apoptosis in a dose-dependent pattern. Higenamine attenuated IL-1β-induced decrease of Bcl-2 and increase of Bax and cleaved caspase-3. Higenamine did not affect the reactive oxygen species (ROS) level and the PI3K/Akt signaling, but attenuated IL-1β-induced ROS production and inhibition of the PI3K/Akt signaling. IL-1β repressed the activation of the PI3K/Akt pathway, but ROS inhibition using N-acetylcysteine (NAC) rescued this pathway. The PI3K/Akt signaling suppression using LY294002 reversed the inhibitive effect of higenamine on IL-1β-caused apoptosis, and this effect was weakened by ROS inhibition. In conclusion, higenamine attenuates IL-1β-caused apoptosis of HNPCs via ROS-mediated PI3K/Akt pathway.

     

Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis.

The phosphoinositide 3-OH kinase (PI3K)-PKB/Akt signaling pathway has been shown to mediate both Ras- and cytokine-induced protection from apoptosis. In addition, apoptosis induced by the p53 tumor suppressor protein can be inhibited by Ras- and cytokine-mediated signaling pathways. It was therefore of interest to determine if the PI3K-PKB/Akt signaling pathway was capable of conferring protection from apoptosis induced by p53. We demonstrate in this report that constitutively active PI3K and PKB/Akt are capable of significantly delaying the onset of p53-mediated apoptosis. This was manifested as a delay in the kinetics of DNA degradation and cell death as well as a profound attenuation in the accumulation of cells with a sub-G(1) DNA content. Moreover, we found that this effect is mediated in the absence of changes in expression of Bcl-2, Bcl-Xl, and the pro-apoptotic protein Bax. Our results provide the first direct and unambiguous link between p53-mediated apoptosis and the PI3K-PKB/Akt signaling pathway.     

Bcl-xL and E1B-19K proteins inhibit p53-induced irreversible growth arrest and senescence by preventing reactive oxygen species-dependent p38 activation

In this study, we describe novel functions of the anti-apoptotic Bcl-2 family proteins. Bcl-x(L) and E1B-19K were found to inhibit p53-induced irreversible growth arrest and senescence, but not to inhibit transient growth arrest, implying that Bcl-x(L) and E1B-19K are specifically involved in senescence without participating in growth arrest. We provide several lines of evidences showing that the functions of Bcl-x(L) and E1B-19K to prevent generation of reactive oxygen species (ROS) are important to inhibit senescence induction. First, we found that that ROS are increased during p53-induced senescence. Moreover, Bcl-x(L) and E1B-19K inhibit this p53-induced ROS generation. Second, antioxidants prevent the induction of senescence and ROS by p53, but not the persistence of the senescence phenotype. Third, the anti-senescence functions of Bcl-x(L) and E1B-19K were suppressed by adding exogenous ROS. These results suggest that Bcl-x(L) and E1B-19K inhibit senescence induction by preventing ROS generation. Furthermore, p38 kinase was found to be activated during p53-induced senescence, but not in cells expressing Bcl-x(L) or E1B-19K, or in cells treated with anti-oxidants. Consistently, a chemical inhibitor of p38 kinase, SB203580, was found to inhibit p53-induced senescence, but only when treated before the cellular commitment to senescence, implying that p38 kinase is necessary for senescence induction. Therefore, Bcl-x(L) and E1B-19K inhibit p53-induced senescence by preventing ROS generation, which in turn leads to the activation of p38 kinase. These results also suggest that the oncogenic potential of Bcl-2 is due to its ability to inhibit senescence as well as apoptosis.     

PI3K inhibitors changed the p53-induced response of Saos-2 cells from growth arrest to apoptosis

p53 is activated by stress leading to oncogenic alteration, which induces either cell cycle arrest or apoptosis, although the mechanism involved in this decision has not been fully clarified as yet. This work was undertaken to change the cellular response by inducing apoptosis with PI3K inhibitors to Saos-2 cells that had been growth-arrested in both G1 and G2/M by the wild-type activity of temperature-sensitive (ts) p53. We found that the PI3K/Akt inhibitors LY294002 and wortmannin, but not the MEK inhibitor U0126, were capable of inducing apoptosis in growth-arrested Saos-2 cells, as assessed by an increase in the sub-G1 population, pyknotic nuclei, and DNA ladder formation. We detected the cleavage of caspases 9 and 3, and PARP after LY294002 addition, accompanied by a loss of cytochrome c from the mitochondria, and observed Bax translocation to the mitochondria and down-regulation of phospho-Akt, suggesting that blocking of survival signals triggered the apoptotic signal through the mitochondrial apoptotic pathway. It is thus suggested that the PI3K/Akt pathway played an important role in determining cell fate between growth arrest and apoptosis.     

Cross-talk between phosphatidylinositol 3-kinase and sphingomyelinase pathways as a mechanism for cell survival/death decisions

Peptide hormones act to regulate apoptosis through activation of multiple pro- and anti-apoptotic signaling cascades of which lipid signaling events represent an important facet of the cellular rheostat that determines survival and death decisions. Activation of sphingomyelinase, which generates ceramide, is an intermediate in cellular stress responses and induction of apoptosis in many systems. Conversely, phosphatidylinositol 3-kinase (PI3K) is a critical signaling molecule involved in regulating cell survival and proliferation pathways. In the present study, we investigate cross-talk between the PI3K and sphingomyelinase pathways as a mechanism for regulation of cell survival/death decisions. We show that phorbol ester, insulin-like growth factor 1, and a constitutively active PI3K suppress both tumor necrosis factor-induced apoptosis and ceramide generation. Conversely, inhibition of the PI3K pathway with expression of a kinase-dead PI3K both prevented survival signaling and enhanced tumor necrosis factor-induced ceramide generation. The ability of exogenous sphingomyelinase to induce ceramide generation was partially suppressed by expression of constitutively active PI3K and enhanced by inhibition of PI3K suggesting that cross-talk between PI3K and ceramide generation within cells is regulated subsequent to activation of sphingomyelinase.