P53-dependent expression of the stress-induced protein (SIP)

The mouse stress-induced protein (SIP) mRNA is activated in the pancreas with acute pancreatitis and in several cell lines in response to various stress agents. The SIP gene is alternatively spliced, generating two proteins (SIP'8 and SIP27). Both proteins, located mainly in the nucleus, promote cell death when overexpressed in vitro. We show that induction by stress agents of the expression of SIP18 and SIP27 mRNAs, observed in human- and mouse-derived cell lines, is absent from cells with deleted, mutated or inactive p53, suggesting that regulation of SIP gene expression is dependent on p53. That hypothesis is consistent with the presence of a functional p53-response element within the promoter region of the mouse SIP gene and confirmed by the induction of SIP mRNA expression in mouse embryo fibroblasts upon activation of a p53-dependent pathway by transfection with rasV12 or rasV12/E1A. In conclusion, SIP being a proapoptotic gene induced through p53 activation could be a stress-induced gene with antitumour properties.     

Interaction of CacyBP/SIP with NPM1 and its influence on NPM1 localization and function in oxidative stress

Calcyclin (S100A6) binding protein/Siah‐1 interacting protein (CacyBP/SIP) is mainly a cytoplasmic protein; however, some literature data suggested its presence in the nucleus. In this work we examined more precisely the nuclear localization and function of CacyBP/SIP. By applying mass spectrometry, we have identified several nuclear proteins, among them is nucleophosmin (NPM1), that may interact with CacyBP/SIP. Subsequent assays revealed that CacyBP/SIP forms complexes with NPM1 in the cell and that the interaction between these two proteins is direct. Interestingly, although CacyBP/SIP exhibits phosphatase activity, we have found that its overexpression favors phosphorylation of NPM1 on S125. In turn, the RNA immunoprecipitation assay indicated that the altered CacyBP/SIP level has an impact on the amount of 28S and 18S rRNA bound to NPM1. The overexpression of CacyBP/SIP resulted in a significant increase in the binding of 28S and 18S rRNA to NPM1, whereas silencing of CacyBP/SIP expression decreased 28S rRNA binding and had no effect on the binding of 18S rRNA. Further studies have shown that under oxidative stress, CacyBP/SIP overexpression alters NPM1 distribution in cell nuclei. In addition, staining for a nucleolar marker, fibrillarin, revealed that CacyBP/SIP is indispensable for maintaining the nucleolar structure. These results are in agreement with data obtained by western blot analysis, which show that upon oxidative stress the NPM1 level decreases but that CacyBP/SIP overexpression counteracts the effect of stress. Altogether, our results show for the first time that CacyBP/SIP binds to and affects the properties of a nuclear protein, NPM1, and that it is indispensable for preserving the structure of nucleoli under oxidative stress.     

SIP蛋白调节p65核转位的作用机制

核因子κB（nuclear factor-κB, NF-κB）参与转录调控许多与细胞生长、凋亡、肿瘤形成和转移、胚胎发育及炎症反应相关的基因.它的二聚体与抑制蛋白结合,如抑制蛋白κB（IκBα,β或γ）,而被滞留在细胞质中处于失活状态.然而NF-κB是否还有其它的抑制因子目前还不清楚.本研究结果表明,SIP（steroid receptor coactivator, SRC,SRC-interacting protein）是NF-κB家族的一个新抑制因子,它通过PEST结构域与NF-κB家族的p65蛋白相互作用.当细胞处于静息状态时,SIP将p65蛋白隔离于细胞质中;当有刺激因子TNFα或IL-1作用时,SIP与p65解离继而使p65进入细胞核启动下游靶基因转录激活.该研究为进一步认识NF-κB介导基因转录调控机制和相关疾病的发生发展提供了重要的理论依据.     

糖皮质激素与炎症因子对应急时相反应蛋白SIP24/24p3的协同调控作用及其意义

小鼠应急时相反应蛋白SIP24／24p3有抗炎症和特异性诱导白细胞凋亡的功能,其在体内的表达是高度特异性的。为研究SIP24／24p3的调控因子及机制,我们在小鼠Balb／c3T3和BNL细胞培养中通过灵敏的弱S代谢标记方法检测SIP24／24p3蛋白的表达水平,定量观测分析了糖皮质激素化合物dexamethasone对SIP24／24p3的诱导作用及其与炎症因子白介素6(IL-6)和肿瘤坏死因子α(TNF-α)的协同调控作用。结果显示：(1)在Balb／c3T3和BNL细胞中,dexamethasone对SIP24／24p3都有明显诱导作用,这种诱导作用在BNL细胞中尤其显著;(2)在Balb／c3T3和BNL细胞中dexamethasone与IL-6协同诱导SIP24／24p3;(3)在Balb／c 3T3细胞中dexamethasone与TNF-α对SIP24／24p3有协同诱导效应,而在BNL细胞中dexamethasone与TNF-α对SIP24／24p3的诱导表现为相加效应;(4)在Balb／c3T3和BNL细胞中dexamethasone与IL-6／TNF-α对SIP24／24p3的诱导分别表现出协同和相加效应。多种因子对SIP24／24p3的协同诱导调控有助于阐明其在体内的高度特异表达及机制,SIP24／24p3在不同细胞中的不同表达格局也对体内应急时相反应蛋白在肝脏外和肝脏内的表达方式及诱导机制有提示作用。SIP24／24p3能同时被炎症因子和抗炎症因子诱导的事实显示了其在炎症全过程中的重要作用。     

The mechanism whereby heat shock induces apoptosis depends on the innate sensitivity of cells to stress

The cellular response to heat shock (HS) is a paradigm for many human diseases collectively known as “protein conformation diseases” in which the accumulation of misfolded proteins induces cell death. Here, we analyzed how cells having a different apoptotic threshold die subsequent to a treatment with HS. Cells with a low apoptotic threshold mainly induced apoptosis through activation of conventional stress kinase signaling pathways. By contrast, cells with a high apoptotic threshold also died by apoptosis but likely after the accumulation of heat-aggregated proteins as revealed by the formation of aggresomes in these cells, which were associated with the generation of atypical nuclear deformations. Inhibition of the proteasome or expression of an aggregation prone protein produced similar nuclear alterations. Furthermore, elevated levels of chaperones markedly suppressed both HS-induced nuclear deformations and apoptosis induced upon protein aggregation whereas they had little effect on stress kinase-mediated apoptosis. We conclude that the relative contribution of stress signaling pathways and the accumulation of protein aggregates to cell death by apoptosis is related to the innate sensitivity of cells to deadly insults.     

Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis in pancreatic acinar AR42J cells

Cell death linked to oxidative DNA damage has been implicated in acute pancreatitis. The severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. It has been hypothesized that oxidative stress may induce a decrease in the Ku70 and Ku80 levels and apoptosis in pancreatic acinar cells. In this study, it was found that oxidative stress caused by glucose oxidase (GO) acting on beta-d-glucose, glucose/glucose oxidase (G/GO), induced slight changes in cytoplasmic Ku70 and Ku80 but drastically induced a decrease in nuclear Ku70 and Ku80 both time- and concentration-dependently in AR42J cells. G/GO induced apoptosis determined by poly(ADP-ribose) polymerase cleavage, an increase in expression of p53 and Bax, and a decrease in Bcl-2 expression. G/GO-induced apoptosis was in parallel with the loss of nuclear Ku proteins in AR42J cells. Caspase-3 inhibitor prevented G/GO-induced nuclear Ku loss and cell death. G/GO did not induce apoptosis in the cells transfected with either the Ku70 or Ku80 expression gene but increased apoptosis in those transfected with the Ku dominant negative mutant. Pulse and pulse-chase results show that G/GO induced Ku70 and Ku80 syntheses, even though Ku70 and Ku80 were degraded both in cytoplasm and nucleus. G/GO-induced decrease in Ku binding to importin alpha and importin beta reflects possible modification of nuclear import of Ku proteins. The importin beta level was not changed by G/GO. These results demonstrate that nuclear decrease in Ku70 and Ku80 may result from the decrease in Ku binding to nuclear transporter importins and the degradation of Ku proteins. The nuclear loss of Ku proteins may underlie the mechanism of apoptosis in pancreatic acinar cells after oxidative stress.     

The CacyBP/SIP Protein is Sumoylated in Neuroblastoma NB2a Cells

The Calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein is highly expressed in mammalian brain as well as in neuroblastoma NB2a cells and pheochromocytoma PC12 cells. This protein interacts with several targets such as cytoskeletal proteins or ERK1/2 kinase and seems to be involved in many cellular processes. In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function. Since theoretical analysis of the amino acid sequence of CacyBP/SIP indicated several lysine residues which could potentially be sumoylated we checked experimentally whether this protein might be modified by SUMO attachment. We have shown that indeed CacyBP/SIP bound the E2 SUMO ligase, Ubc9, in neuroblastoma NB2a cell extract and was sumoylated in these cells. By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction. We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.     

Ataxin-1 poly(Q)-induced proteotoxic stress and apoptosis are attenuated in neural cells by docosahexaenoic acid-derived neuroprotectin D1

Neurodegenerative diseases share two common features: enhanced oxidative stress and cellular inability to scavenge structurally damaged abnormal proteins. Pathogenesis of polyglutamine (poly(Q)) diseases involves increased protein misfolding, along with ubiquitin and chaperon protein-containing nuclear aggregates. In spinocerebellar ataxia, the brain and retina undergo degeneration. Neuroprotectin D1 (NPD1) is made on-demand in the nervous system and retinal pigment epithelial (RPE) cells in response to oxidative stress, which activates prosurvival signaling via regulation of gene expression and other processes. We hypothesized that protein misfolding-induced proteotoxic stress triggers NPD1 synthesis. We used ARPE-19 cells as a cellular model to assess stress due to ataxin-1 82Q protein expression and determine whether NPD1 prevents apoptosis. Ectopic ataxin-1 expression induced RPE cell apoptosis, which was abrogated by 100 nm docosahexaenoic acid, 10 ng/ml pigment epithelium-derived factor, or NPD1. Similarly, NPD1 was protective in neurons and primary human RPE cells. Furthermore, when ataxin-1 82Q was expressed in 15-lipoxygenase-1-deficient cells, apoptosis was greatly enhanced, and only NPD1 (50 nm) rescued cells from death. NPD1 reduced misfolded ataxin-1-induced accumulation of proapoptotic Bax in the cytoplasm, suggesting that NPD1 acts by preventing proapoptotic signaling pathways from occurring. Finally, NPD1 signaling interfered with ataxin-1/capicua repression of gene expression and decreased phosphorylated ataxin-1 in an Akt-independent manner, suggesting that NPD1 signaling modulates formation or stabilization of ataxin-1 complexes. These data suggest that 1) NPD1 synthesis is an early response induced by proteotoxic stress due to abnormally folded ataxin-1, and 2) NPD1 promotes cell survival through modulating stabilization of ataxin-1 functional complexes and pro-/antiapoptotic and inflammatory pathways.     

SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase.

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.     

Alteration in inflammatory/apoptotic pathway and histone modifications by nordihydroguaiaretic acid prevents acute pancreatitis in swiss albino mice

Reactive oxygen radicals, pro-inflammatory mediators and cytokines have been implicated in caerulein induced acute pancreatitis. Nordihydroguaiaretic acid (NDGA), a plant lignin, has marked anti-inflammatory properties. The present study aimed to investigate the possible protective effect of NDGA against caerulein induced pancreatitis. Acute pancreatitis was induced by intraperitoneal administration of eight doses of caerulein in male swiss albino mice. NDGA was administered after 9 h of acute pancreatitis induction. Pancreatic damage and the protective effect of NDGA were assessed by oxidative stress parameters and histopathology of pancreas. The mRNA expression of heat shock proteins (DNAJ C15 and HSPD1) was examined by real-time RT-PCR analysis. Expression of HSP 27, NF-κB, TNF-α, p-p38, Bcl-2, p-PP2A, procaspase-3, caspase-3 and histone modifications were examined by western blotting. NDGA attenuated the oxidative stress, led to increased plasma α-amylase and decreased IGF-1 in AP mice. It modulated the mRNA and protein levels of heat shock proteins and reduced the expression of NF-κB, TNF-α and p-p38. It increased the number of TUNEL positive apoptotic cells in the pancreas of AP mice. In addition, NDGA prevented the changes in modifications of histone H3 in acute pancreatitis. To best of our knowledge, this is the first report which suggests that NDGA prevents the progression of acute pancreatitis by involving alteration of histone H3 modifications and modulating the expression of genes involved in inflammatory/apoptotic cascade, which may be responsible for decreased necrosis and increased apoptosis in this model of acute pancreatitis.     

Ezrin and HNRNP expression correlate with increased virus release rate and early onset of virus‐induced apoptosis of MDCK suspension cells

With the aim to adapt high‐yield adherent cell lines to suspension growth, Madin Darby canine kidney (MDCK) suspension cells were developed recently that achieved comparable influenza virus yields despite an early induction of apoptosis compared to the parental adherent cell line. For both cell lines, a comprehensive study under comparable infection conditions is performed comprising information on: time course of viral infection, antiviral state of cells, virus‐induced apoptosis, and virus‐induced cellular protein expression for early and late infection with influenza A/PuertoRico/8/34 H1N1. The proteomic analysis is performed with 2D differential gel electrophoreses followed by mass spectrometry. Based on flow cytometric data and on the differential expression of various stress and apoptosis‐related proteins, the earlier onset of virus‐induced apoptosis is confirmed for suspension cells. Surprisingly, the data indicated an increased virus release rate for suspension cells. These observations correlate with an increased expression of the apical marker protein ezrin, known to play a role in influenza‐induced cytoskeletal rearrangement, and the differential expression of heterogeneous nuclear ribonucleoproteins, known to bind actively influenza viral proteins and play a central role in regulating gene expression. Based on these findings, additional studies towards the design of MDCK suspension cells with further increase in influenza virus yields will be performed.     

Protective up-regulation of CK2 by mutant huntingtin in cells co-expressing NMDA receptors

Huntington's disease is caused by a polyglutamine expansion in the huntingtin (htt) protein, and previous data indicate that over-activation of NMDA receptors (NMDARs) may be involved in the selective degeneration of cells expressing NR1/NR2B NMDARs. We used Kinetworks™ multi-immunoblotting screens to examine expression of 76 protein kinases, 18 protein phosphatases, 25 heat shock/stress proteins, and 27 apoptosis proteins in human embryonic kidney 293 cells transfected with NR1/NR2B and htt containing 15 (htt-15Q; wild-type) or 138 (htt-138Q; mutant) glutamine repeats. Follow-up experiments revealed several proteins involved in the heat-shock response pathway to be up-regulated in the soluble fraction from cells expressing htt-138Q, including protein phosphatase 5 and cyclin-dependent kinase 5. Increased expression in the soluble fraction of htt-138Q-expressing cells was also noted for the stress- and calcium-activated protein-serine/threonine kinase casein kinase 2, a change which was confirmed in striatal tissue of yeast artificial chromosome transgenic mice expressing full-length mutant htt. Inhibition of casein kinase 2 activity in cultured striatal neurons from these mice significantly exacerbated NMDAR-mediated toxicity, as assessed by labeling of apoptotic nuclei. Our findings are consistent with up-regulation of components of the stress response pathway in the presence of polyglutamine-expanded htt and NR1/NR2B which may reflect an attempt at the cellular level to ameliorate the detrimental effects of mutant htt expression.