Proteolytic control of neurite outgrowth inhibitor NOGO-A by the cAMP/PKA pathway

Protein kinase A (PKA) controls major aspects of neurite outgrowth and morphogenesis and plays an essential role in synaptic plasticity and memory. However, the molecular mechanism(s) of PKA action on neurite sprouting and activity are still unknown. Here, we report that in response to neurotrophin or cAMP stimulation the RING ligase praja2 ubiquitinates and degrades NOGO-A, a major inhibitor of neurite outgrowth in mammalian brain. Genetic silencing of praja2 severely inhibited neurite extension of differentiating neuroblastoma cells and mesencephalic neurons and axon outgrowth and sprouting of striatal terminals in developing rat brain. This phenotype was rescued when both praja2 and NOGO-A were depleted, suggesting that NOGO-A is, indeed, a biologically relevant target of praja2 in neuronal cells. Our findings unveil a novel mechanism that functionally couples cAMP signaling with the proteolytic turnover of NOGO-A, positively impacting on neurite outgrowth in mammalian brain.Neurite outgrowth plays an essential role in embryonic development, neuronal differentiation, and central nervous system (CNS) plasticity. Outgrowth can be also altered in several neurological disorders, as well as by neuronal injury and degeneration (1, 2). Extracellular signals, such as neurotrophins (NTFs) and neurotransmitters, regulate neurite outgrowth, dendritic arborization, and synaptic activity, establishing a dynamic neuronal network in developing and adult CNS. NTFs and neurotransmitters act at the cell membrane by generating intracellular second messengers that, in turn, reversibly modulate the activity of signaling proteins and effector enzymes (3, 4).cAMP is an ancient second messenger that controls a variety of biological cues. In neurons, essential functions such as neurite outgrowth and morphogenesis, synaptic transmission, and plasticity require tightly regulated responses to cAMP/protein kinase A (PKA) stimulation (5). PKA holoenzyme localizes in subcellular microdomains through interactions with A-Kinase-Anchor-Proteins (AKAPs). AKAP forms a local transduction unit, which includes signaling/metabolic enzymes, receptors, ion channels, adaptor molecules, and mRNAs (6, 7). Space-restricted activation of PKA provides a control mechanism to direct, integrate, and locally attenuate the cAMP cascade (8). praja2 belongs to a growing family of mammalian RING ligases abundantly expressed in the brain that finely tune the stability of intracellular substrates and play an essential role in critical aspects of cell signaling. In response to cAMP stimulation, praja2 couples ubiquitination and proteolysis of the inhibitory PKA regulatory (R) subunits by the proteasome to a sustained cAMP/PKA signaling, significantly impacting on synaptic plasticity and long-term memory (9). In addition to enhancing cAMP signaling, the role of praja2 in neuronal differentiation and dendritic network in the CNS and the molecular targets involved are unknown.NOGO-A is a member of the reticulon (RTN) family of integral membrane proteins with a conserved C terminus reticulon homology domain (RHD) and abundantly expressed in oligodendrocytes and in distinct neuronal subpopulations (10, 11). NOGO-A was originally identified as a potent inhibitor of neurite outgrowth (1, 10). In the adult CNS and in injured neurons, NOGO-A restricts the capacity of an axon to grow and regenerate. Genetic ablation of NOGO-A promotes neuritogenesis and fasciculation of oligodendrocytes and culture of dorsal root ganglion neurons, functionally improving neuronal plasticity and recovery of postischemic adult rat brain (12).Although the role of NOGO-A in neurite outgrowth is well established, regulation of NOGO-A levels in differentiating neurons and the mechanism(s) involved have been, to date, unknown. Here, we report a novel mechanism of neuritogenesis based on proteolytic turnover of NOGO-A (13). In response to cAMP or neurothrophin stimulation, RING ligase praja2 ubiquitinates and degrades NOGO-A. Proteolysis of NOGO-A by praja2 is functionally linked to neurite outgrowth in both differentiating neurons and developing rat brain.     

ERK1/2通路在TIMP-1抑制大鼠肾小球系膜细胞凋亡中的作用

目的探讨细胞外信号调节激酶(ERK1/2)通路是否参与基质金属蛋白酶抑制剂(TIMP-1)抑制大鼠肾小球系膜细胞的凋亡。方法选择大连医科大学附属第一医院2005年8月至2006年2月应用人正、反义TIMP-1转染大鼠肾小球系膜细胞,分别用高糖(25mmol/L)和MEK1特异性抑制剂PD98059(50μmol/L)刺激24h,采用流式细胞仪检测细胞凋亡情况;逆转录聚合酶链式扩增(RT-PCR)观察细胞外信号调节激酶信使RNA(ERK1 messenger RNA)的表达;酶联免疫吸附实验(ELISA)方法检测胞浆中p-ERK1/2的表达情况。结果(1)未加PD98059的未转染组、空载体组、正义转染组及反义转染组各组细胞的凋亡率分别为(12.10±2.21)%、(11.90±3.34)%、(5.50±0.50)%和(20.70±3.41)%;正义转染组的凋亡率明显低于未转染组(P<0.01);反义转染组凋亡率高于未转染组(P<0.05)。加入PD98059后各组细胞的凋亡率明显增加。(2)加入PD98059后,各组细胞ERK1mRNA的表达较相应未加PD98059各组明显上调;以正义转染组上调最为明显。(3)ELISA结果显示:加入PD98059后,p-ERK1/2较相应未加PD98059各组明显下调(P<0.05),以正义组下调最为明显(P<0.01)。结论ERK1/2信号传导通路是TIMP-1抑制高糖诱导的大鼠肾小球系膜细胞凋亡的主要途径之一。     

cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins

Although rates of protein degradation by the ubiquitin-proteasome pathway (UPS) are determined by their rates of ubiquitination, we show here that the proteasome’s capacity to degrade ubiquitinated proteins is also tightly regulated. We studied the effects of cAMP-dependent protein kinase (PKA) on proteolysis by the UPS in several mammalian cell lines. Various agents that raise intracellular cAMP and activate PKA (activators of adenylate cyclase or inhibitors of phosphodiesterase 4) promoted degradation of short-lived (but not long-lived) cell proteins generally, model UPS substrates having different degrons, and aggregation-prone proteins associated with major neurodegenerative diseases, including mutant FUS (Fused in sarcoma), SOD1 (superoxide dismutase 1), TDP43 (TAR DNA-binding protein 43), and tau. 26S proteasomes purified from these treated cells or from control cells and treated with PKA degraded ubiquitinated proteins, small peptides, and ATP more rapidly than controls, but not when treated with protein phosphatase. Raising cAMP levels also increased amounts of doubly capped 26S proteasomes. Activated PKA phosphorylates the 19S subunit, Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11) at Ser14. Overexpression of a phosphomimetic Rpn6 mutant activated proteasomes similarly, whereas a nonphosphorylatable mutant decreased activity. Thus, proteasome function and protein degradation are regulated by cAMP through PKA and Rpn6, and activation of proteasomes by this mechanism may be useful in treating proteotoxic diseases.In mammalian cells, the bulk of cell proteins are degraded by the ubiquitin-proteasome system (UPS) (1, 2). Misfolded proteins, which arise from mutations or postsynthetic damage, and normal proteins with regulatory functions tend to be degraded more rapidly than average cell proteins (2). To be degraded by the UPS, proteins are first modified by ubiquitination (2). In this highly selective process, ubiquitin moieties are conjugated to individual proteins by one of the cell’s many ubiquitin ligases (E3s) (3). Protein ubiquitination is generally assumed to be the rate-limiting step in the degradation pathway, and once ubiquitinated, proteins are believed to be efficiently hydrolyzed by the 26S proteasome. This 2.5-MDa proteolytic complex is composed of about 60 subunits (3). Proteins are digested within the core 20S proteasome, a hollow cylindrical particle containing three types of peptidase activities: chymotrypsin-like, trypsin-like, and caspase-like (3). This particle can be associated with one or two 19S regulatory particles forming a 26S proteasome (3). The 19S complex serves multiple key functions: it binds the ubiquitinated substrate, removes the ubiquitin chain, unfolds the protein substrate, and translocates it through a narrow gated channel into the 20S particle (3). This multistep process is coupled to ATP hydrolysis by the hexameric ATPase ring at the base of the 19S complex adjacent to the core particle (3, 5). These various steps are tightly coordinated; for example, gate opening into the 20S particle and ATP hydrolysis are activated upon binding of the ubiquitin (Ub) chain to the deubiquitinating enzymes, Usp14 or Uch37 (5, 6).The development of inhibitors of proteasome function have advanced our knowledge of cell regulation and proven very valuable in treating hematological cancers (7). In principle, agents that enhance proteasome function could be valuable in combating the various diseases resulting from the toxic accumulation of misfolded proteins. In the major neurodegenerative diseases [amyotrophic lateral sclerosis (ALS), Alzheimer’s, Parkinson’s, and Huntington’s diseases (8, 9)], aggregation-prone proteins build up, often as protein inclusions that contain Ub and proteasomes (10). One factor that may contribute to the pathogenesis of these diseases is the progressive impairment of the capacity of the UPS to degrade misfolded proteins (11). In fact, several studies of neurodegenerative disease models have suggested that proteasome function is impaired when these misfolded proteins (e.g., huntingtin aggregates, mutant tau, or PrP^Sc prions) accumulate in cells (11–14).A number of postsynthetic modifications of 26S proteasome subunits have been reported, including O-GlcNAc modification (15), ADP ribosylation (16), and especially phosphorylation (17–19). The subunit phosphorylation may influence the localization (20), activity (17), and formation (18, 21) of the 26S proteasome. For example, phosphorylation of one of the 19S ATPases, Rpt6, in neurons by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), has been reported to cause proteasome entry into dendrites and promote synaptic plasticity (22, 23). In addition, phosphorylation of Rpt6 by cAMP-dependent proteins kinase (PKA) was reported to increase proteasome activity against small peptides (17, 24, 25). However, the effects of this modification on the proteasome’s capacity to degrade ubiquitin conjugates and on protein degradation in cells were not examined. Although raising cAMP levels and phosphorylation by PKA alter many cellular functions, effects on protein breakdown by the UPS have not been reported, aside from a suppression of overall proteolysis in skeletal muscle (26). Here we demonstrate that PKA directly phosphorylates the 19S subunit Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11), and that this modification stimulates several key proteasomal processes and enhances its capacity to degrade ubiquitinated proteins. As a result, pharmacological agents that raise cAMP levels and activate PKA promote the breakdown of short-lived cell proteins by the ubiquitin proteasome pathway, and can accelerate the degradation of aggregation-prone proteins that cause major neurodegenerative diseases.     

Aspirin attenuates monocrotaline-induced pulmonary arterial hypertension in rats by suppressing the ERK/MAPK pathway

This study aimed to investigate the therapeutic effects of aspirin (ASA) and its potential mechanisms of action in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats. PAH was induced in a rat model by a single intraperitoneal (IP) injection of MCT. Saline was injected in a control group. Two weeks following MCT injection, right ventricular systolic pressure (RVSP) and systolic blood pressure (SBP) were measured in six rats from each group to confirm establishment of a PAH model. The remaining MCT-treated rats were randomly allocated to receive IP injection of saline, ASA, or ERK1/2 inhibitor PD98059. Four weeks following treatment, RVSP was measured and all rats were sacrificed for histological study. There was no significant difference in SBP in any group two weeks following MCT administration. Nonetheless RVSP was significantly increased in the MCT group compared with the control group. At 6 weeks, ASA treatment remarkably attenuated MCT-induced increased RVSP, RV hypertrophy, and pulmonary artery remodeling compared with the MCT group. The density of pulmonary capillaries in ASA-treated rats was also dramatically increased. Treatment with ASA significantly inhibited the increased p-ERK1/2 and restored the impaired endothelial nitric oxide synthase (eNOS) in MCT-treated rats. This study demonstrated that ASA distinctively attenuates MCT-induced PAH by inhibition of the ERK1/2 signaling pathway.     

Phosphorylation of Arl4A/D promotes their binding by the HYPK chaperone for their stable recruitment to the plasma membrane

The Arl4 small GTPases participate in a variety of cellular events, including cytoskeleton remodeling, vesicle trafficking, cell migration, and neuronal development. Whereas small GTPases are typically regulated by their GTPase cycle, Arl4 proteins have been found to act independent of this canonical regulatory mechanism. Here, we show that Arl4A and Arl4D (Arl4A/D) are unstable due to proteasomal degradation, but stimulation of cells by fibronectin (FN) inhibits this degradation to promote Arl4A/D stability. Proteomic analysis reveals that FN stimulation induces phosphorylation at S143 of Arl4A and at S144 of Arl4D. We identify Pak1 as the responsible kinase for these phosphorylations. Moreover, these phosphorylations promote the chaperone protein HYPK to bind Arl4A/D, which stabilizes their recruitment to the plasma membrane to promote cell migration. These findings not only advance a major mechanistic understanding of how Arl4 proteins act in cell migration but also achieve a fundamental understanding of how these small GTPases are modulated by revealing that protein stability, rather than the GTPase cycle, acts as a key regulatory mechanism.

The Arl4 [ADP ribosylation factor (Arf)-like 4 proteins] small GTPases, comprising Arl4A, Arl4C, and Arl4D, are developmentally regulated. In adults, they have restricted tissue expression and have been found to act in a variety of cellular events. Arl4A recruits ELMO-Dock180, a conserved Rac regulator, to membranes for actin remodeling (1) and also directs cell migration through Robo1 and Pak1 effectors (2, 3). Several studies have reported that Arl4C functions in filopodia formation (4), cancer cell invasion (5, 6), and tumorigenesis (7–9). Arl4D has been found to activate Arf6 by recruiting cytohesin-2/ARNO for actin remodeling to promote cell migration (10, 11). How the Arl4 proteins are regulated in these cellular roles has been enigmatic. Small GTPases are typically activated by guanine nucleotide exchange factors and deactivated by GTPase-activating proteins (12, 13). However, neither class of key regulators has been identified for the Arl4 proteins. Consistent with this current state of knowledge, these small GTPases have been found to undergo rapid nucleotide exchange and appear structurally unable to bind GDP (13, 14). Thus, a major goal has been to elucidate how Arl4 proteins are regulated independent of the canonical mechanism that involves the GTPase cycle.The p21-activated kinases (Paks) stand at the hub of several signaling pathways for cell proliferation, migration, and survival (15). In cell migration, Pak1 phosphorylates paxillin at the leading edge of migrating cells for the rapid turnover of focal adhesion (16). Pak1 also phosphorylates LIMK1 and myosin light chain for actin reorganization to promote cell migration (17, 18). Pak activation requires cell adhesion through the interaction between integrins and the extracellular matrix (19). A well-characterized example has been the stimulation of cells by fibronectin (FN), which recruits Pak1 to the plasma membrane, where it then interacts with other molecules to coordinate downstream events (20, 21). In this role, FN acts in a variety of physiologic and pathologic circumstances, with wound healing being an example of the former (22) and cancer cell invasion being an example of the latter (23). With respect to the latter, alternatively spliced forms of FN have been found to act in collective tumor migration and are predictive of adverse outcomes for patients with cancer (24–26).We have recently found that FN promotes the cooperative recruitment of Arl4A and Pak1 to the plasma membrane, which contributes to sustained Pak1 activation needed for cell migration (3). In this study, we find that Arl4A and Arl4D (Arl4A/D) undergo rapid proteasomal degradation. FN stimulation activates Pak1 to phosphorylate these small GTPases, which leads to their binding by the chaperone-like protein HYPK (Huntingtin-interacting protein K). This binding stabilizes the targeting of Arl4A/D to the plasma membrane to promote cell migration.     

FTY720通过miR-494/MST1抑制结肠癌细胞并增加吉西他滨敏感性的分子机制

背景结肠癌在我国恶性肿瘤中发病率和死亡率均居第5位,化疗是其主要治疗方式,有研究表明免疫抑制剂FT Y720对癌症细胞增殖起一定的抑制作用,抑制剂联合化疗药物可提高癌症治疗效果.本研究使用免疫抑制剂FTY720与吉西他滨联合处理结肠癌细胞,并探索miR-494/哺乳动物Ste20样激酶1 (mammalian Ste20-like kinase 1,MST1)对在此过程中对结肠癌细胞增殖和凋亡的影响,以期为结肠癌的治疗提供新的思路.目的研究FT Y720和吉西他滨对结肠癌细胞存活率和凋亡的影响和潜在的分子机制.方法用0.0001μg/mL、0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL的吉西他滨和2.5μmol/L、5μmol/L、7.5μmol/L、10μmol/L、12.5μmol/L的FTY720处理结肠癌SW1116细胞,CCK8法和流式细胞术检测SW1116细胞存活率和凋亡率,实时定量聚合酶链式反应检测miR-494和MST1 mRNA的含量,Western blot检测MST1、p21和Caspase-3蛋白表达水平,双荧光素酶报告系统验证miR-494与MST1的调控关系.结果吉西他滨(0.0001μg/mL、0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL)和FTY720 (2.5μmol/L、5μmol/L、7.5μmol/L、10μmol/L、12.5μmol/L)均可降低结肠癌SW1116细胞的存活率,且具有浓度依赖性,根据结果选取抑制率约为50%的0.1μg/mL吉西他滨和10μmol/L的FTY720进行后续实验,吉西他滨和FT Y720均可抑制细胞存活并促进细胞凋亡,且联合使用比单独使用效果更好;过表达miR-494可逆转FTY720、吉西他滨对SW1116细胞存活率和凋亡的作用;miR-494靶向调控MST1;抑制MST1可逆转FTY720和吉西他滨对SW1116细胞存活率和凋亡的影响.结论FTY720和吉西他滨通过miR-494/MST1抑制SW1116细胞存活,促进细胞凋亡.FTY720和吉西他滨对结肠癌SW1116具有抑制作用,且联合使用效果更佳.     

PGE1 abolishes the mitochondrial-independent cell death pathway induced by D-galactosamine in primary culture of rat hepatocytes

Background and Aim:  PGE₁ reduces in vivo and in vitro D-galactosamine (D-GalN)-induced cell death in hepatocytes. The present study was undertaken to elucidate the intracellular pathway by which D-GalN induces cell death in cultured hepatocytes. In addition, we evaluated if PGE₁ was able to modulate different parameters related to D-GalN-induced apoptosis in cultured rat hepatocytes.
Methods:  Hepatocytes were isolated from male Wistar rats (225–275 g) by the classical collagenase procedure. PGE₁ (1 µM) was administered 2 h before D-GalN (5 mM) in primary culture of rat hepatocytes. Apoptosis was determined by DNA fragmentation and caspase-3, -6, -8 and -9 activation in hepatocytes. Caspase activation was evaluated by the detection of the related cleaved product and its associated activity. Cell necrosis was determined by the measurement of lactate dehydrogenase (LDH) activity in culture medium. To elucidate the role of mitochondria, we measured neutral (nSMase) and acid (aSMase) sphingomyelinase, as well as the expression of cytochrome c in mitochondria and cytoplasm fractions from D-GalN treated hepatocytes.
Results:  D-GalN induced caspase-3 activation and DNA fragmentation in hepatocytes. This apoptotic response was not associated with the activation of caspase-6, -8 or -9. The use of specific inhibitors confirmed that only caspase-3 was involved in D-GalN-induced apoptosis. D-GalN did not modify nSMase and aSMase activities, nor mitochondrial cytochrome c release in hepatocytes.
Conclusions:  D-GalN induced apoptosis through caspase-3 activation but without modification of the activity of caspase-6, -8, -9, SMases or cytochrome c release. PGE₁ appears to prevent D-GalN-induced apoptosis by a mitochondria-independent mechanism.     

Epigenetic dysregulation of the Jak/STAT pathway by frequent aberrant methylation of SHP1 but not SOCS1 in acute leukaemias

SOCS1 and SHP1 are negative regulators of the Jak/STAT signalling pathway that is implicated in leukaemogenesis. We studied if aberrant methylation of SOCS1 and SHP1 might be involved in the pathogenesis and prognostication of acute leukaemias by methylation-specific polymerase chain reaction (MSP). At diagnosis, methylation of SHP1 occurred more frequently in acute myeloid leukaemia (AML) (n=26, 52%) than acute lymphoblastic leukaemia (ALL) (n=6, 24%) (p=0.02). Methylation of SOCS1 was absent in both AML and ALL patients. SHP1 methylation was not associated with specific clinicopathologic features and had no prognostic impact on AML patients. Frequent methylation of SHP1, but not SOCS1, may be important in the pathogenesis, but not prognosis, of acute leukaemias.     

Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway

BACKGROUND Hepatic stellate cell(HSC)hyperactivation is a central link in liver fibrosis development.HSCs perform aerobic glycolysis to provide energy for their activation.Focal adhesion kinase(FAK)promotes aerobic glycolysis in cancer cells or fibroblasts,while FAK-related non-kinase(FRNK)inhibits FAK phosphorylation and biological functions.AIM To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODS Mouse liver fibrosis models were established by administering CCl4,and the effect of FRNK on the degree of liver fibrosis in the model was evaluated.Transforming growth factor-β1 was used to activate LX-2 cells.Tyrosine phosphorylation at position 397(pY397-FAK)was detected to identify activated FAK,and the expression of the glycolysis-related proteins monocarboxylate transporter 1(MCT-1)and enolase1(ENO1)was assessed.Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region,which were validated with chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays.RESULTS The pY397-FAK level was increased in human fibrotic liver tissue.FRNK knockout promoted liver fibrosis in mouse models.It also increased the activation,migration,proliferation and aerobic glycolysis of primary hepatic stellate cells(pHSCs)but inhibited pHSC apoptosis.Nevertheless,opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells.Mechanistically,the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis,which was inhibited by exogenous FRNK.CONCLUSION FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway,thereby improving liver fibrosis.FRNK might be a potential target for liver fibrosis treatment.     

Ursodeoxycholic acid ameliorates hepatic lipid metabolism in LO2 cells by regulating the AKT/mTOR/SREBP-1 signaling pathway

BACKGROUND Nonalcoholic fatty liver disease(NAFLD), the most common chronic liver disease, can progress into nonalcoholic steatohepatitis(NASH), cirrhosis, and even hepatocellular carcinoma. Bile acids such as ursodeoxycholic acid(UDCA)play an essential role in the pathogenesis of NAFLD by regulating the level of sterol regulatory element-binding protein(SREBP) 1 c, but the underlying regulatory mechanism remains elusive. Increased evidence indicates that the AKT/mTOR/SREBP-1 signaling pathway is a key pathway to regulate hepatic cellular lipid metabolism. UDCA may regulate the AKT/mTOR/SREBP-1 signaling pathway to ameliorate hepatic lipid metabolism.AIM To investigate the functional mechanism of UDCA in an oleic acid(OA)-induced cellular model of NAFLD.METHODS The cellular model of NAFLD was established using OA and treated with UDCA.First, the best concentration of UDCA was selected. For the best time-dependent assay, cells were stimulated with OA only or co-treated with OA and 2 mmol/L UDCA for 24 h, 48 h, and 72 h. Oil red O staining was used to observe the accumulation of intracellular lipids, while the intracellular contents of triglyceride, alanine aminotransferase(ALT), gamma-glutamyl transpeptidase(GGT), and aspartate aminotransferase(AST) were detected by enzymatic methods. Meanwhile, the expression levels of AKT/mTOR/SREBP-1 signaling pathway-related proteins were detected by real-time PCR and Western blot.RESULTS In the NAFLD cell model established with LO2 cells induced using OA, lipid accumulation was obvious. UDCA significantly inhibited lipid accumulation at different concentrations(especially 2 mmol/L) and decreased cell growth ability at different time points. The biochemical parameters like ALT, AST, and GGT were significant improved by UDCA. UDCA treatment vividly repressed the activation of AKT, mTOR, and CRTC2 and the expression of nSREBP-1 in LO2 cells induced with OA.CONCLUSION Our findings demonstrate the effect of UDCA in improving NAFLD. UDCA attenuates OA-induced hepatic steatosis mainly by regulation of AKT/mTOR/SREBP-1 signal transduction.     

ERK1/2信号途径与弓形虫侵入细胞及胞内增殖关系的研究

目的探讨阻断细胞外蛋白调节激酶(ERK1/2)信号途径对弓形虫速殖子侵入宿主细胞及在胞内增殖的影响。方法姬氏染色法检测ERK1/2途径不同时间及不同剂量的抑制剂U0126或PD98059作用下宿主细胞感染弓形虫速殖子的百分率,根据细胞感染率和感染细胞内虫荷量分析ERK1/2途径抑制剂对速殖子在细胞中增殖的影响。结果速殖子在细胞培养系统中以1、10和100μmol/L U0126或PD98059作用3 h、6 h和9 h后,前者细胞培养孔中的细胞感染率分别较对照组平均下降34.62%(P<0.01)、53.55%(P<0.01)和67.76%(P<0.01),后者分别较对照组平均下降22.67%(P<0.01)、52.21%(P<0.01)和58.99%(P<0.01);感染细胞感染速殖子率与对照组比较差异无统计学意义(P>0.05)。结论阻断ERK1/2途径的不同信号位点对弓形虫速殖子侵入宿主细胞影响不同,ERK1/2途径在速殖子侵入宿主细胞起主要作用,但对侵入后的增殖无明显影响。     

阿芬太尼调节SphK1/S1P信号通路保护心肌缺血再灌注损伤大鼠

[目的]探究阿芬太尼对心肌缺血再灌注损伤(MIRI)大鼠的作用及在该过程中对鞘氨醇激酶1(SphK1)/鞘氨醇-1-磷酸(S1P)信号通路的调节机制。[方法]将SPF级SD雄性大鼠随机分为假手术组、模型组、阳性药物组(复方丹参组)和阿芬太尼低剂量组、阿芬太尼高剂量组、阿芬太尼高剂量+SphK1激动剂组(阿芬太尼+PMA组),每组20只。除假手术组,其余组均利用结扎左前降支冠状动脉后再灌注复制MIRI模型。全自动生物化学分析仪检测血清乳酸脱氢酶(LDH)、肌酸激酶(CK)和谷草转氨酶(AST)的活性;TTC检测大鼠心肌梗死面积;HE染色观察大鼠心肌组织形态学特征;TUNEL染色检测大鼠心肌细胞凋亡;ELISA检测血清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)及S1P的水平;试剂盒检测心肌组织中丙二醛(MDA)含量和超氧化物歧化酶(SOD)的活性;Western blot检测心肌组织SphK1蛋白表达。[结果]相较于假手术组,模型组大鼠心肌组织病理损伤严重,血清中心肌损伤标志物LDH、CK和AST的活性,心肌梗死面积和心肌细胞凋亡率,TNF-α、I...     

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage,while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4^CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4^CDT2 ubiquitin ligase.

Schlafen-11 (SLFN11) is an emergent restriction factor against genomic instability acting by eliminating cells with replicative damage (1–6) and potentially acting as a tumor suppressor (6, 7). SLFN11-expressing cancer cells are consistently hypersensitive to a broad range of chemotherapeutic drugs targeting DNA replication, including topoisomerase inhibitors, alkylating agents, DNA synthesis, and poly(ADP-ribose) polymerase (PARP) inhibitors compared to SLFN11-deficient cancer cells, which are chemoresistant (1, 2, 4, 8–17). Profiling SLFN11 expression is being explored for patients to predict survival and guide therapeutic choice (8, 13, 18–24).The Cancer Genome Atlas (TCGA) and cancer cell databases demonstrate that SLFN11 mRNA expression is suppressed in a broad fraction of common cancer tissues and in ∼50% of all established cancer cell lines across multiple histologies (1, 2, 5, 8, 13, 25, 26). Silencing of the SLFN11 gene, like known tumor suppressor genes, is under epigenetic mechanisms through hypermethylation of its promoter region and activation of histone deacetylases (HDACs) (21, 23, 25, 26). A recent study in small-cell lung cancer patient-derived xenograft models also showed that SLFN11 gene silencing is caused by local chromatin condensation related to deposition of H3K27me3 in the gene body of SLFN11 by EZH2, a histone methyltransferase (11). Targeting epigenetic regulators is therefore an attractive combination strategy to overcome chemoresistance of SLFN11-deficient cancers (10, 25, 26). An alternative approach is to attack SLFN11-negative cancer cells by targeting the essential pathways that cells use to overcome replicative damage and replication stress. Along these lines, a prior study showed that inhibition of ATR (Ataxia Telangiectasia- and Rad3-related) kinase reverses the resistance of SLFN11-deficient cancer cells to PARP inhibitors (4). However, targeting the ATR pathway in SLFN11-deficient cells has not yet been fully explored.SLFN11 consists of two functional domains: A conserved nuclease motif in its N terminus and an ATPase motif (putative helicase) in its C terminus (2, 6). The N terminus nuclease has been implicated in the selective degradation of type II tRNAs (including those coding for ATR) and its nuclease structure can be derived from crystallographic analysis of SLFN13 whose N terminus domain is conserved with SLFN11 (27, 28). The C terminus is only present in the group III Schlafen family (24, 29). Its potential ATPase activity and relationship to chemosensitivity to DNA-damaging agents (3–5) imply that the ATPase/helicase of SLFN11 is involved specifically in DNA damage response (DDR) to replication stress. Indeed, inactivation of the Walker B motif of SLFN11 by the mutation E669Q suppresses SLFN11-mediated replication block (5, 30). In addition, SLFN11 contains a binding site for the single-stranded DNA binding protein RPA1 (replication protein A1) at its C terminus (3, 31) and is recruited to replication damage sites by RPA (3, 5). The putative ATPase activity of SLFN11 is not required for this recruitment (5) but is required for blocking the replication helicase complex (CMG-CDC45) and inducing chromatin accessibility at replication origins and promoter sites (5, 30). Based on these studies, our current model is that SLFN11 is recruited to “stressed” replication forks by RPA filaments formed on single-stranded DNA (ssDNA), and that the ATPase/helicase activity of SLFN11 is required for blocking replication progression and remodeling chromatin (5, 30). However, underlying mechanisms of how SLFN11 irreversibly blocks replication in DNA damage are still unclear.Increased RPA-coated ssDNA caused by DNA damage and replication fork stalling also triggers ATR kinase activation, promoting subsequent phosphorylation of CHK1, which transiently halts cell cycle progression and enables DNA repair (32). ATR inhibitors are currently in clinical development in combination with DNA replication damaging drugs (33, 34), such as topoisomerase I (TOP1) inhibitors, which are highly synergistic with ATR inhibitors in preclinical models (35). ATR inhibitors not only inhibit DNA repair, but also lead to unscheduled replication origin firing (36), which kills cancer cells (37, 38) by inducing genomic alterations due to faulty replication and mitotic catastrophe (33).The replication licensing factor CDT1 orchestrates the initiation of replication by assembling prereplication complexes (pre-RC) in G1-phase before cells enter S-phase (39). Once replication is started by loading and activation of the MCM helicase, CDT1 is degraded by the ubiquitin proteasomal pathway to prevent additional replication initiation and ensure precise genome duplication and the firing of each origin only once per cell cycle (39, 40). At the end of G2 and during mitosis, CDT1 levels rise again to control kinetochore-microtubule attachment for accurate chromosome segregation (41). Deregulated overexpression of CDT1 results in rereplication, genome instability, and tumorigenesis (42). The cellular CDT1 levels are tightly regulated by the damage-specific DNA binding protein 1 (DDB1)–CUL4^CDT2 E3 ubiquitin ligase complex in G1-phase (43) and in response to DNA damage (44, 45). How CDT1 is recognized by CUL4^CDT2 in response to DNA damage remains incompletely known.In the present study, starting with a human genome-wide RNAi screen, bioinformatics analyses, and mechanistic validations, we explored synthetic lethal interactions that overcome the chemoresistance of SLFN11-deficient cells to the TOP1 inhibitor camptothecin (CPT). The strongest synergistic interaction was between depletion of the ATR/CHK1-mediated DNA damage response pathways and DNA-damaging agents in SLFN11-deficient cells. We validated and expanded our molecular understanding of combinatorial strategies in SLFN11-deficient cells with the ATR (M4344 and M6620) and CHK1 (SRA737) inhibitors in clinical development (33, 46, 47) and found that ATR inhibition leads to CDT1 stabilization and hyperphosphorylation with mitotic catastrophe. Our study also establishes that SLFN11 promotes the degradation of CDT1 by binding to DDB1, an adaptor molecule of the CUL4^CDT2 E3 ubiquitin ligase complex, leading to an irreversible replication block in response to replicative DNA damage.     

TBL1XR1 induces cell proliferation and inhibit cell apoptosis by the PI3K/AKT pathway in pancreatic ductal adenocarcinoma

BACKGROUND Pancreatic ductal adenocarcinoma(PDAC) is one of the deadliest solid tumors. Identification of diagnostic and therapeutic biomarkers for PDAC is urgently needed. Transducin(β)-like 1 X-linked receptor 1(TBL1 XR1) has been linked to the progression of various human cancers. Nevertheless, the function and role of TBL1 XR1 in pancreatic cancers are unclear.AIM To elucidate the function and potential mechanism of TBL1 XR1 in the development of PDAC.METHODS Ninety patients with histologically-confirmed PDAC were included in this study. PDAC tumor samples and cell lines were used to determine the expression of TBL1 XR1. CCK-8 assays and colony formation assays were carried out to assess PDAC cell viability. Flow cytometry was performed to measure the changes in the cell cycle and cell apoptosis. Changes in related protein expression were measured by western blot analysis. Animal analysis was conducted to confirm the impact of TBL1 XR1 in vivo.RESULTS Patients with TBL1 XR1-positive tumors had worse overall survival than those with TBL1 XR1-negative tumors. Moreover, we found that TBL1 XR1 strongly promoted PDAC cell proliferation and inhibited PDAC cell apoptosis. Moreover, knockdown of TBL1 XR1 induced G0/G1 phase arrest. In vivo animal studies confirmed that TBL1 XR1 accelerated tumor cell growth. The results of western blot analysis showed that TBL1 XR1 might play a key role in regulating PDAC cell proliferation and apoptosis via the PI3 K/AKT pathway.CONCLUSION TBL1 XR1 promoted PDAC cell progression and might be an effective diagnostic and therapeutic marker for pancreatic cancer.