Presynaptic protein synaptotagmin1 regulates the activity‐induced remodeling of synaptic structures in cultured hippocampal neurons

Activity‐dependent reorganizations of central neuronal synapses are thought to play important roles in learning and memory. Although the precise mechanisms of how neuronal activities modify synaptic connections in neurons remain to be clarified, the activity‐induced neuronal presynaptic proteins such as synaptotagmin1 may contribute to the onset of synaptic remodeling. To understand better the physiological roles of synaptotagmin1, we first examined the prolonged effects of neuronal stimulation capable of inducing synaptotagmin1 on the distribution of a postsynaptic proteins (PSD) protein Homer1c by immunostaining. Previously we found that glutamate stimulation induced other postsynaptic proteins, such as postsynaptic density‐95 (PSD95), a biphasic change with an initially diffuse distribution after 30 min to 1 hr, followed by reassembly to more than the original level after 4–8 hr, suggesting that glutamate stimulation induces a global biphasic alteration in synaptic structures. To dissect further the functions of synaptotagmin1 in the activity‐induced synaptic remodeling, short hairpin RNA (shRNA) vectors that specifically block the expression of endogenous synaptotagmin1 were constructed. When the shRNA of synaptotagmin1 was introduced to the neurons, the activity‐induced changes were almost completely suppressed. We found that synaptotagmin1 contributes to the postsynaptic remodeling in a retrograde manner. Our data indicate that synaptotagmin1 regulates the activity‐induced biphasic changes of post‐ and presynaptic sites. © 2013 Wiley Periodicals, Inc.     

E2F1 is not essential for apoptosis induced by potassium deprivation in cerebellar granule neurons

Cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing concentration of potassium. A key regulator of cell cycle, E2F1, was believed to play a role in CGN apoptosis induced by potassium deprivation. However, here we demonstrated that although E2F1 was upregulated in wild type CGNs following potassium deprivation, CGNs that derived from E2F1 knockout mice underwent apoptosis at a similar rate as the wild type. Analysis of the apoptotic neurons revealed no difference in the activation of caspase-3 in E2F1 null and wild type CGNs. Furthermore, knockdown of E2F1 expression by RNA interference failed to attenuate the apoptosis of CGNs induced by potassium deprivation. Taken together, our results suggested that E2F1 is not essential for apoptosis induced by potassium deprivation in CGNs.     

Involvement of an Orphan Transporter,SLC22A18, in Cell Growth and Drug Resistance of Human Breast Cancer MCF7 Cells

The SLC22A18 gene, which encodes an orphan transporter, is located at the 11p15.5 imprinted region, an important tumor suppressor gene region. However, the role of SLC22A18 in tumor suppression remains unclear. Here, we investigated the involvement of SLC22A18 in cell growth, invasion, and drug resistance of MCF7 human breast cancer cell line. Western blot analysis indicated that SLC22A18 is predominantly expressed at intracellular organelle membranes. Quantitative proteomics showed that knockdown of SLC22A18 significantly altered the expression of 578 (31.0%) of 1867 proteins identified, including proteins related to malignancy and poor prognosis of breast cancer. SLC22A18 knockdown (1) increased MCF7 cell growth concomitantly with a >7-fold increase of annexin A8 (involved in cell growth and migration; a predictor of poor prognosis), (2) induced spherical morphology of MCF7 cells concomitantly with a nearly 3-fold increase of CD44 (involved in regulation of malignant phenotypes), and (3) increased chemosensitivity to vinca alkaloids concomitantly with a >80% reduction of doublecortin-like kinase 1 (involved in regulation of microtubule polymerization). Our results suggest that SLC22A18 may act as a tumor suppressor by regulating the expression levels of cell growth–related proteins, and vinca alkaloids might show therapeutic efficacy against low-SLC22A18–expressing breast cancer.     

Protein kinase C isoforms in atherosclerosis: Pro- or anti-inflammatory?

Atherosclerosis is a pathologic condition caused by chronic inflammation in response to lipid deposition in the arterial wall. There are many known contributing factors such as long-term abnormal glucose levels, smoking, hypertension, and hyperlipidemia. Under the influence of such factors, immune and non-immune effectors cells are activated and participate during the progression of atherosclerosis. Protein kinase C (PKC) family isoforms are key players in the signal transduction pathways of cellular activation and have been associated with several aspects of the atherosclerotic vascular disease. This review article summarizes the current knowledge of PKC isoforms functions during atherogenesis, and addresses differential roles and disputable observations of PKC isoforms. Among PKC isoforms, both PKCβ and PKCδ are the most attractive and potential therapeutic targets. This commentary discusses in detail the outcomes and current status of clinical trials on PKCβ and PKCδ inhibitors in atherosclerosis-associated disorders like diabetes and myocardial infarction. The risk and benefit of these inhibitors for clinical purposes will be also discussed. This review summarizes what is already being done and what else needs to be done in further targeting PKC isoforms, especially PKCβ and PKCδ, for therapy of atherosclerosis and atherosclerosis-associated vasculopathies in the future.     

Lysophosphatidic acid-induced IL-8 secretion involves MSK1 and MSK2 mediated activation of CREB1 in human fibroblast-like synoviocytes

Lysophosphatidic acid (LPA) is a pleiotropic lipid mediator that promotes motility, survival, and the synthesis of chemokines/cytokines such as interleukin-8 (IL-8) and interleukin-6 by human fibroblast-like synoviocytes from patients with rheumatoid arthritis (RAFLS). In those cells LPA was reported to induce IL-8 secretion through activation of various signaling pathways including p38 mitogen-activated protein kinase (p38 MAPK), p42/44 MAPK, and Rho kinase. In addition to those pathways we report that mitogen- and stress-activated protein kinases (MSKs) known to be activated downstream of the ERK1/2 and p38 MAPK cascades and CREB are phosphorylated in response to LPA. The silencing of MSKs with small-interfering RNAs and the pharmacological inhibitor of MSKs SB747651A shows a role for both MSK1 and MSK2 in LPA-mediated phosphorylation of CREB at Ser-133 and secretion of IL-8 and MCP-1. Whereas CREB inhibitors have off target effects and increased LPA-mediated IL-8 secretion, the silencing of CREB1 with short hairpin RNA significantly reduced LPA-induced chemokine production in RAFLS. Taken together the data clearly suggest that MSK1 and MSK2 are the major CREB kinases in RAFLS stimulated with LPA and that phosphorylation of CREB1 at Ser-133 downstream of MSKs plays a significant role in chemokine production.     

Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

RNA interference (RNAi) is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4), which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA) to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3) were designed to target the coding sequence (CDS) of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55%) in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.KEY WORDS: RNAi, Cytochrome P450, CYP3A4, shRNA, Chemosensitivity     

抑制MDR1基因表达shRNA RNAi系统的构建

目的构建抑制多药耐药基因(MDR1)表达的短发夹RNA(sh RNA)真核表达载体系统.方法根据MDR1基因已知序列,设计两段21个碱基的MDR1特异性靶序列,合成两对62nt 并含有编码shRNA序列的寡核苷酸,双链退火后,克隆到经过BamH I和Xba I双酶切后线性 PG E-1载体的U6启动子下游,重组构建RNA干扰(RNAi)质粒.结果对重新构建的pshRNA-MDR1载体经P CR扩增后行电泳分析,并对含有插入基因片段行测序分析.结果表明,62个碱基均成功插入到预计位点,并且序列完全一致.结论载体的成功构建为研究其对MDR1 基因表达的抑制作用打下基础,同时使我们发展了在体内合成siRNA的方法.