Identification of the Serine 307 of LKB1 as a Novel Phosphorylation Site Essential for Its Nucleocytoplasmic Transport and Endothelial Cell Angiogenesis

LKB1, a master kinase that controls at least 13 downstream protein kinases including the AMP-activated protein kinase (AMPK), resides mainly in the nucleus. A key step in LKB1 activation is its export from the nucleus to the cytoplasm. Here, we identified S307 of LKB1 as a putative novel phosphorylation site which is essential for its nucleocytoplasmic transport. In a cell-free system, recombinant PKC-ζ phosphorylates LKB1 at S307. AMPK-activating agents stimulate PKC-ζ activity and LKB1 phosphorylation at S307 in endothelial cells, hepatocytes, skeletal muscle cells, and vascular smooth muscle cells. Like the kinase-dead LKB1 D194A mutant (mutation of Asp194 to Ala), the constitutively nucleus-localized LKB1 SL26 mutant and the LKB1 S307A mutant (Ser307 to Ala) exhibit a decreased association with STRADα. Interestingly, the PKC-ζ consensus sequence surrounding LKB1 S307 is disrupted in the LKB1 SL26 mutant, thus providing a likely molecular explanation for this mutation causing LKB1 dysfunction. In addition, LKB1 nucleocytoplasmic transport and AMPK activation in response to peroxynitrite are markedly reduced by pharmacological inhibition of CRM1, which normally facilitates nuclear export of LKB1-STRAD complexes. In comparison to the LKB1 wild type, the S307A mutant complexes show reduced association with CRM1. Finally, adenoviral overexpression of wild-type LKB1 suppresses, while the LKB1 S307A mutant increases, tube formation and hydrogen peroxide-enhanced apoptosis in cultured endothelial cells. Taken together, our results suggest that, in multiple cell types the signaling pathways engaged by several physiological stimuli converge upon PKC-ζ-dependent LKB1 phosphorylation at S307, which directs the nucleocytoplasmic transport of LKB1 and consequent AMPK activation.LKB1 is a tumor suppressor (3, 25, 33, 42, 59) that is mutated in Peutz-Jeghers cancer syndrome (20, 24). This serine/threonine protein kinase phosphorylates and activates at least 13 downstream kinases, which in turn regulate multiple cellular processes, including the cell cycle, cellular proliferation, apoptosis, and energy metabolism (1, 30). One of the key downstream kinases of LKB1 is the 5′-AMP-activated protein kinase (AMPK), a serine/threonine kinase that serves as a master regulator of energy metabolism (18, 19, 28). LKB1 is ubiquitously expressed in adult and fetal tissue, particularly pancreatic, liver, testicular, cardiac, and skeletal muscle tissue (21, 25, 43, 60). In humans, LKB1 comprises 433 amino acids (436 residues in mouse LKB1) and is located predominantly in the nucleus due to its nuclear localization signal in the N-terminal noncatalytic region (residues 38 to 43) (36, 53). Paradoxically, LKB1 activation takes place predominantly in the cytoplasm, after it complexes with STRAD (STE-related adapter) and MO25 (mouse protein 25). As a result, the nucleocytoplasmic transport and subsequent association of LKB1 with STRAD and MO25 in the cytoplasm are required for full activation of LKB1 (2, 5) and its downstream kinases, including AMPK. Consistent with this theory, 12 mutants of LKB1 (including the SL26 mutants) found in patients with Peutz-Jeghers cancer syndrome are constitutively nuclear (5, 6). Further, a recent study from Macara''s group (13) shows that STRAD regulates LKB1 localization by blocking access to importin and by association with CRM1 and exportin-7, two nuclear protein exportins.LKB1 is phosphorylated at S325, T366, and S431 by upstream kinases. In addition, LKB1 autophosphorylates at S31, T185, T189, T336, and S404 (1). Mutation of any of these phosphorylation sites to Ala (to abolish phosphorylation) or Glu (to mimic phosphorylation) does not significantly affect the in vitro catalytic activity of LKB1 or its intracellular localization (5, 44, 45). Recently, we demonstrated that phosphorylation of LKB1 S428 is required for metformin-enhanced AMPK activation (56). Nevertheless, several questions such as the precise mechanism(s) underlying LKB1 activation, the relevant phosphorylation sites, and the upstream activating kinase(s) remain unclear. While it has been shown that LKB1 S428 phosphorylation is required for nucleocytoplasmic transport of LKB1, the translocation of LKB1 to the cytosol could be further regulated by unknown mechanisms. Here, we have identified S307 as a novel phosphorylation site in LKB1 and provide evidence that, in multiple cell types, phosphorylation of this site by protein kinase C ζ (PKC-ζ) induces nucleocytoplasmic transport of LKB1 and subsequent activation of AMPK and suppression of angiogenesis and apoptosis. Importantly, we provide a molecular explanation for the constitutive nuclear localization of the LKB1 SL26 mutant. Taken together, our results suggest that the phosphorylation of LKB1 S307 by PKC-ζ is essential for LKB1 regulation of cell cycle progression, proliferation, angiogenesis, and apoptosis.     

The Phosphate Transporter PiT1 (Slc20a1) Revealed As a New Essential Gene for Mouse Liver Development

Background

PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na⁺-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.

Methodology/Principal Findings

To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and null PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and null alleles in heterozygous compounds results in late embryonic lethality (E14.5–E16.5) with phenotypic features intermediate between null and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.

Conclusion/Significance

This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.     

A Novel Tumor Suppressor Function of Glycine N-Methyltransferase Is Independent of Its Catalytic Activity but Requires Nuclear Localization

Glycine N-methyltransferase (GNMT), an abundant cytosolic enzyme, catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine generating S-adenosylhomocysteine and sarcosine (N-methylglycine). This reaction is regulated by 5-methyltetrahydrofolate, which inhibits the enzyme catalysis. In the present study, we observed that GNMT is strongly down regulated in human cancers and is undetectable in cancer cell lines while the transient expression of the protein in cancer cells induces apoptosis and results in the activation of ERK1/2 as an early pro-survival response. The antiproliferative effect of GNMT can be partially reversed by treatment with the pan-caspase inhibitor zVAD-fmk but not by supplementation with high folate or SAM. GNMT exerts the suppressor effect primarily in cells originated from malignant tumors: transformed cell line of non-cancer origin, HEK293, was insensitive to GNMT. Of note, high levels of GNMT, detected in regenerating liver and in NIH3T3 mouse fibroblasts, do not produce cytotoxic effects. Importantly, GNMT, a predominantly cytoplasmic protein, was translocated into nuclei upon transfection of cancer cells. The presence of GNMT in the nuclei was also observed in normal human tissues by immunohistochemical staining. We further demonstrated that the induction of apoptosis is associated with the GNMT nuclear localization but is independent of its catalytic activity or folate binding. GNMT targeted to nuclei, through the fusion with nuclear localization signal, still exerts strong antiproliferative effects while its restriction to cytoplasm, through the fusion with nuclear export signal, prevents these effects (in each case the protein was excluded from cytosol or nuclei, respectively). Overall, our study indicates that GNMT has a secondary function, as a regulator of cellular proliferation, which is independent of its catalytic role.     

A Novel Role for the Hypoxia Inducible Transcription Factor HIF-1alpha: Critical Regulation of Inflammatory Cell Function

No abstract available.     

吡啶锰配合物诱导肿瘤细胞死亡的作用及对线粒体功能的影响

研究新合成的小分子吡啶锰配合物Adpa-Mn（III）（[（Adpa）Mn（μ2-O）2Mn（Adpa）]PF6.8H2O（Adpa=bis（2-pyridylmethyl）amino-2-propionic acid））的抗肿瘤作用,初步探索其抗肿瘤的机制。MTT分析Adpa-Mn（III）对细胞活性的影响;活细胞工作站观察GFP荧光标记组蛋白HeLa细胞的细胞核形态,MDC染色以及GFP-LC3质粒转染,探讨细胞死亡的方式;JC-1染色检测线粒体膜电位;Fluo-3-AM和DCFH-DA荧光探针分别检测细胞中Ca^2＋和ROS的含量。结果发现,Adpa-Mn（III）剂量依赖性地抑制细胞活性;给药后细胞核出现固缩、片段化;自噬小泡增多,GFP-LC3荧光强度增强;线粒体膜电位下降;细胞内Ca^2＋发生超载,ROS含量升高。由此,Adpa-Mn（III）可抑制肿瘤细胞活性,其机制与引起线粒体膜电位下降、增加ROS生成及诱导细胞的死亡有关,同时胞内Ca^2＋超载也参与了该作用。这些数据显示,Adpa-Mn（III）具有成为抗肿瘤先导金属配合物的潜在可能性。     

Identification of a Novel Role for Dematin in Regulating Red Cell Membrane Function by Modulating Spectrin-Actin Interaction

The membrane skeleton plays a central role in maintaining the elasticity and stability of the erythrocyte membrane, two biophysical features critical for optimal functioning and survival of red cells. Many constituent proteins of the membrane skeleton are phosphorylated by various kinases, and phosphorylation of β-spectrin by casein kinase and of protein 4.1R by PKC has been documented to modulate erythrocyte membrane mechanical stability. In this study, we show that activation of endogenous PKA by cAMP decreases membrane mechanical stability and that this effect is mediated primarily by phosphorylation of dematin. Co-sedimentation assay showed that dematin facilitated interaction between spectrin and F-actin, and phosphorylation of dematin by PKA markedly diminished this activity. Quartz crystal microbalance measurement revealed that purified dematin specifically bound the tail region of the spectrin dimer in a saturable manner with a submicromolar affinity. Pulldown assay using recombinant spectrin fragments showed that dematin, but not phospho-dematin, bound to the tail region of the spectrin dimer. These findings imply that dematin contributes to the maintenance of erythrocyte membrane mechanical stability by facilitating spectrin-actin interaction and that phosphorylation of dematin by PKA can modulate these effects. In this study, we have uncovered a novel functional role for dematin in regulating erythrocyte membrane function.     

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2

The general phosphate need in mammalian cells is accommodated by members of the P_i transport (PiT) family (SLC20), which use either Na⁺ or H⁺ to mediate inorganic phosphate (P_i) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na⁺-dependent P_i (NaP_i) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with ³²P_i as a traceable P_i source. For PiT1, the Michaelis-Menten constant for P_i was determined as 322.5 ± 124.5 µM. PiT2 was analyzed for the first time and showed positive cooperativity in P_i uptake with a half-maximal activity constant for P_i of 163.5 ± 39.8 µM. PiT1- and PiT2-mediated Na⁺-dependent P_i uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na⁺ dependency patterns. However, only PiT2 was capable of Na⁺-independent P_i transport at acidic pH. Study of the impact of divalent cations Ca²⁺ and Mg²⁺ revealed that Ca²⁺ was important, but not critical, for NaP_i transport function of PiT proteins. To gain insight into the NaP_i cotransport function, we analyzed PiT2 and a PiT2 P_i transport knockout mutant using ²²Na⁺ as a traceable Na⁺ source. Na⁺ was transported by PiT2 even without P_i in the uptake medium and also when P_i transport function was knocked out. This is the first time decoupling of P_i from Na⁺ transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E⁵⁵ and E⁵⁷⁵ are responsible for linking P_i import to Na⁺ transport in PiT2. inorganic phosphate transport; retroviral receptor; SLC20     

Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast

Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Δ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Δ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Δ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.Boron has been proposed as an important micronutrient in plants and animals. Studies have shown the presence of several genes associated with boron transport and tolerance in plants (18, 25, 27); however, boron transport mechanisms in other organisms, including animals, remain unclear. In plants, boron functions as a cross-linker for rhammogalacturanon II in the cell membrane (9, 14, 21) and also as a structural component in cytoskeleton assembly (1). Arabidopsis thaliana BOR1 was the first gene shown to play a role in boron tolerance (28). Homologs of BOR1 were found in many organisms, including yeasts, plants, and mammals (22, 25, 29). A high level of boron leads to degradation of its own exporter, BOR1, in A. thaliana (27), and A. thaliana BOR1 cannot be used to produce genetically modified plants that grow in soil with high boron levels. However, transgenic plants expressing BOR4, one of six paralogs of BOR1, showed high tolerance to toxic levels of boron (18). Multicopy expression of BOT1, a BOR1 ortholog, provided boron tolerance to barley (25).The yeast Saccharomyces cerevisiae has been used as a model organism for characterization of plant boron tolerance genes (19, 20, 25, 26, 29). While 10 mM boric acid is lethal to Arabidopsis (18), yeast can grow in the presence of 80 mM boron and is considered a boron-tolerant organism (19, 20). Yeast Bor1 was characterized in detail (10). This protein is localized to the plasma membrane and functions as a boric acid exporter (26). The bor1Δ yeast strain overaccumulates boron (20, 28), and cells that overexpress BOR1 have less intracellular boron and show resistance to boron treatment (20). In addition to Bor1, two other proteins, Dur3 and Fps1, have been implicated in boron tolerance in yeast, but their functions are not clear (20). Dur3 is a plasma membrane transporter that plays a role in urea and polyamine transport (5, 31), and Fps1 is a member of the major intrinsic protein family and plays a role in glycerol, acetic acid, arsenite, and antimonite transport (16, 30, 33). Overexpression of FPS1 and DUR3 showed controversial effects on cellular boron levels. While FPS1 expression lowered the protoplasmic boron concentration, DUR3 expression led to a small increase in boron (20).The objective of this study was to identify proteins that are primarily responsible for boron transport in yeast. ATR1 was identified as a boron tolerance gene by screening a yeast DNA expression library. Yeast Atr1 is a member of the DHA2 family of drug-H⁺ antiporters with 14 predicted membrane-spanning segments (7). It was first characterized in a genetic screen as a high-copy-number suppressor of the 3-amino-1,2,4-triazole sensitivity of gcn4Δ mutants (11). It also conferred resistance to the DNA-damaging agent 4-nitroquinoline-N-oxide in a separate genetic screen (17). In this study, we demonstrated that high-copy-number expression of ATR1 conferred extreme resistance to boron and reduced intracellular levels of the element, whereas cells lacking the ATR1 gene were hypersensitive to boron and increased its intracellular levels. We analyzed changes in the global gene expression profile in response to boron and found that ATR1 is the most induced transporter gene. The Atr1-green fluorescent protein (GFP) fusion protein localized to the plasma membrane and vacuole. Taken together, our data show that Atr1 functions as a major boron efflux pump and provides tolerance of the element by pumping boron out of cells.     

A Novel Bipartite Nuclear Localization Signal Guides BPM1 Protein to Nucleolus Suggesting Its Cullin3 Independent Function

BPM1 belongs to the MATH-BTB family of proteins, which act as substrate-binding adaptors for the Cullin3-based E3 ubiquitin ligase. MATH-BTB proteins associate with Cullin3 via the BTB domain and with the substrate protein via the MATH domain. Few BPM1-interacting proteins with different functions are recognized, however, specific roles of BPM1, depending on its cellular localization have not been studied so far. Here, we found a novel bipartite nuclear localization signal at the C-terminus of the BPM1 protein, responsible for its nuclear and nucleolar localization and sufficient to drive the green fluorescent protein and cytoplasmic BPM4 protein into the nucleus. Co-localization analysis in live Nicotiana tabacum BY2 cells indicates a Cullin3 independent function since BPM1 localization is predominantly nucleolar and thus devoid of Cullin3. Treatment of BY2 cells with the proteasome inhibitor MG132 blocks BPM1 and Cullin3 degradation, suggesting turnover of both proteins through the ubiquitin–proteasome pathway. Possible roles of BPM1 in relation to its in vivo localization are discussed.     

Upregulation of Cleavage and Polyadenylation Specific Factor 4 in Lung Adenocarcinoma and Its Critical Role for Cancer Cell Survival and Proliferation

Cleavage and polyadenylation specific factor 4 (CPSF4), a member of CPSF complex, plays a key role in mRNA polyadenylation and mRNA 3′ ends maturation. However, its possible role in lung cancer pathogenesis is unknown. In this study, we investigated the biological role and clinical significance of CPSF4 in lung cancer growth and survival and elucidated its underlying molecular mechanisms. We found that CPSF4 was highly expressed in lung adenocarcinoma cell lines and tumor tissue but was undetectable in 8 normal human tissues. We also found that CPSF4 overexpression was correlated with poor overall survival in patients with lung adenocarcinomas (P<0.001). Multivariate survival analyses revealed that higher CPSF4 expression was an independent prognostic factor for overall survival of the patients with lung adenocarcinomas. Suppression of CPSF4 by siRNA inhibited lung cancer cells proliferation, colony formation, and induced apoptosis. Mechanism studies revealed that these effects were achieved through simultaneous modulation of multiple signaling pathways. Knockdown of CPSF4 expression by siRNA markedly inhibited the phosphorylation of PI3K, AKT and ERK1/2 and JNK proteins. In contrast, the ectopic expression of CPSF4 had the opposite effects. Moreover, CPSF4 knockdown also induced the cleavage of caspase-3 and caspse-9 proteins. Collectively, these results demonstrate that CPSF4 plays a critical role in regulating lung cancer cell proliferation and survival and may be a potential prognostic biomarker and therapeutic target for lung adenocarcinoma.     

Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis

Rickettsia prowazekii is an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion to sn-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway is R. prowazekii open reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3P in vitro. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of the R. prowazekii gpsA gene functioned to complement an Escherichia coli gpsA mutant. Furthermore, gpsA mRNA was detected in R. prowazekii purified from hen egg yolk sacs, and G3PDH activity was assayable in R. prowazekii lysed-cell extracts. Together, these data strongly suggested that R. prowazekii encodes and synthesizes a functional GpsA enzyme, yet R. prowazekii is unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned that R. prowazekii transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purified R. prowazekii transported and incorporated DHAP into phospholipids, thus implicating a role for GpsA in vivo as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.Rickettsia prowazekii is an obligate intracellular pathogen, a select agent, and the etiologic cause of epidemic typhus fever in humans. R. prowazekii is transmitted by the human body louse, Pediculus humanus, and is prevalent in areas of low socioeconomic conditions where hygiene and sanitation are lacking (3, 12, 35). Known reservoirs include humans with recrudescent Brill-Zinsser disease and flying squirrels (17, 29). If they are misdiagnosed and/or improperly treated, typhus infections result in substantial morbidity and mortality (36).Upon entering a eukaryotic host cell, R. prowazekii quickly escapes the endosomal vesicle and replicates directly within the cytoplasm (21, 45, 48). As an obligate intracellular pathogen, R. prowazekii has evolved to exploit the nutrient-rich cytoplasmic growth niche by transporting the end products of various host cell metabolic pathways (7, 10, 39, 42, 50, 52). As a presumed consequence of its reliance on the transport of host cell metabolites, R. prowazekii has lost many of its biosynthetic pathway genes through the process of reductive evolution (2, 4-6). In short, essential metabolites that cannot be synthesized by the rickettsiae de novo are transported from the eukaryotic host cell cytosol. The ongoing loss of R. prowazekii biochemical pathways from the genome is evidenced by the identification of pseudogenes, biochemical pathway remnants that have acquired mutations and that encode nonfunctional proteins (2, 4, 5, 22).Interestingly, there exists another class of rickettsial biochemical pathway remnants that may be mistaken as pseudogenes. We have termed members of this class “functional orphaned enzymes” because the purified enzyme, unlike the product of a pseudogene, exhibits its annotated biochemical activity. However, the other enzymes in the corresponding pathway are absent from the R. prowazekii genome; thus, there is no endogenous enzymatic synthesis of substrate for the functional orphaned enzyme. We hypothesize that rickettsial functional orphaned enzymes are fed their substrate from the host cell cytosol via the activity of novel rickettsial transport systems. Together, functional orphaned enzymes and their cognate transport systems serve as streamlined biosynthetic pathways for required metabolites.Here we present the characterization of a R. prowazekii functional orphaned enzyme, an sn-glycerol-3-phosphate (G3P) dehydrogenase (G3PDH) enzyme, RP442 (or GpsA), that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to G3P for phospholipid biosynthesis. GpsA appears to be a pathway remnant because R. prowazekii lacks glycolytic and gluconeogenic pathways and thus does not possess the capacity to synthesize DHAP (6, 30, 56). Evidence is presented to suggest that GpsA is a functional enzyme and that R. prowazekii transports and incorporates DHAP into phospholipid, indicating that DHAP transport and GpsA are part of a novel G3P acquisition pathway. This is the first report of DHAP transport by a bacterium.     

Identification and Antioxidant Activity of a Novel Peptide from Baijiu

International Journal of Peptide Research and Therapeutics - A novel peptide, Cys-Trp-Cys (CWC), which firstly isolated from Guojing Baijiu was qualitative and quantitative studied by...     

靶向IRE1a干扰质粒构建及对ERS时细胞增殖及凋亡的影响

目的构建靶向人IRE1a的shRNA干扰质粒（pSUPER-IRE1a）并观察其对人HeLa细胞和HepG2细胞增殖及凋亡的影响。方法设计并合成靶向IRE1a基因的两条shRNA,分别克隆至真核表达载体pSUPER构建重组质粒pS1、pS2,依次转染入HeLa细胞和HepG2细胞中。采用RT—PCR检测pS1、pS2转染前后IRE1a在HeLa细胞和HepG2细胞中的mRNA水平,免疫印迹检测pS1、pS2转染前后IRE1a蛋白的表达;MTT比色法、BrdU／DAPI双免疫荧光法及流式细胞仪分别检测各重组质粒对HeLa细胞和HepG2细胞增殖及凋亡的影响。结果干扰质粒（pSUPER-IRE1a）能有效抑制HeLa细胞和HepG2细胞中IRE1a基因的表达;成功转染后,细胞处于内质网应激（ER stress）状态时,各实验组细胞增殖率及凋亡率与对照组比较,差异均具有统计学意义P〈0．05）。结论成功构建靶向人IRE1a的shRNA真核表达载体pS1、pS2,有效抑制了HeLa细胞和HepG2细胞中IRE1a的表达;细胞处于ERS状态时,IRE1a-shRNA有效促进HeLa、HepG2两种肿瘤细胞的增殖;抑制HeLa和HepG2细胞的凋亡。     

Functional Identification of the Glycerol Transport Activity of Chlamydomonas reinhardtii CrMIP1

The above article appeared in Plant and Cell Physiology 45(9):1313–1319 (2004). Fig. 2 in the     

PDE8 Regulates Rapid Teff Cell Adhesion and Proliferation Independent of ICER

Background

Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions.

Methodology/Principal Findings

We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem ^−/− mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection.

Conclusion/Significance

Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.