Lipofectamine LTX Research Articles

Gas7 Functions with N-WASP to Regulate the Neurite Outgrowth of Hippocampal Neurons

Neuritogenesis, or neurite outgrowth, is a critical process for neuronal differentiation and maturation in which growth cones are formed from highly dynamic actin structures. Gas7 (growth arrest-specific gene 7), a new member of the PCH (Pombe Cdc15 homology) protein family, is predominantly expressed in neurons and is required for the maturation of primary cultured Purkinje neurons as well as the neuron-like differentiation of PC12 cells upon nerve growth factor stimulation. We report that Gas7 co-localizes and physically interacts with N-WASP, a key regulator of Arp2/3 complex-mediated actin polymerization, in the cortical region of Gas7-transfected Neuro-2a cells and growth cones of hippocampal neurons. The interaction between Gas7 and N-WASP is mediated by WW-Pro domains, which is unique in the PCH protein family, where most interactions are of the SH3-Pro kind. The interaction contributes to the formation of membrane protrusions and processes by recruiting the Arp2/3 complex in a Cdc42-independent manner. Importantly, specific interaction between Gas7 and N-WASP is required for regular neurite outgrowth of hippocampal neurons. The data demonstrate an essential role of Gas7 through its interaction with N-WASP during neuronal maturation/differentiation.

EKLF/KLF1 Controls Cell Cycle Entry via Direct Regulation of E2f2

Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF(-/-) fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.

Expression of Cyclophilin B is Associated with Malignant Progression and Regulation of Genes Implicated in the Pathogenesis of Breast Cancer

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor alpha, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility.

Functional Interactions between Mldp (LSDP5) and Abhd5 in the Control of Intracellular Lipid Accumulation

Cellular lipid metabolism is regulated in part by protein-protein interactions near the surface of intracellular lipid droplets. This work investigated functional interactions between Abhd5, a protein activator of the lipase Atgl, and Mldp, a lipid droplet scaffold protein that is highly expressed in oxidative tissues. Abhd5 was highly targeted to individual lipid droplets containing Mldp in microdissected cardiac muscle fibers. Mldp bound Abhd5 in transfected fibroblasts and directed it to lipid droplets in proportion to Mldp concentration. Analysis of protein-protein interactions in situ demonstrated that the interaction of Abhd5 and Mldp occurs mainly, if not exclusively, on the surface of lipid droplets. Oleic acid treatment rapidly increased the interaction between Abhd5 and Mldp, and this effect was suppressed by pharmacological inhibition of triglyceride synthesis. The functional role of the Abhd5-Mldp interaction was explored using a mutant of mouse Abhd5 (E262K) that has greatly reduced binding to Mldp. Mldp promoted the subcellular colocalization and interaction of Atgl with wild type, but not mutant, Abhd5. This differential interaction was reflected in cellular assays of Atgl activity. In the absence of Mldp, wild type and mutant Abhd5 were equally effective in reducing lipid droplet formation. In contrast, mutant Abhd5 was unable to prevent lipid droplet accumulation in cells expressing Mldp despite considerable targeting of Atgl to lipid droplets containing Mldp. These results indicate that the interaction between Abhd5 and Mldp is dynamic and essential for regulating the activity of Atgl at lipid droplets containing Mldp.

Hepatitis B Virus-induced hFGL2 Transcription Is Dependent on c-Ets-2 and MAPK Signal Pathway

Fibrinogen-like protein 2 (FGL2)/fibroleukin plays a pivotal role in the pathogenesis of experimental and human fulminant and chronic viral hepatitis. To define the transcription factor(s) and upstream signal transduction pathways involved in the transcription of human FGL2 (hFGL2) in response to hepatitis B (HB) virus, hepatitis B core (HBc), hepatitis B virus S protein (HBs), or hepatitis B virus X protein (HBx) protein, expression plasmids were cotransfected with an hFGL2 promoter luciferase reporter construct into Chinese hamster ovary and HepG2 cells, respectively. HBc and HBx proteins, but not HBs protein, enhanced hFGL2 transcription in both cell lines. A strong regulatory region from -712 to -568 (relative to the transcriptional starting site) was shown to be responsible for hFGL2 gene transcription in response to both HBc and HBx proteins. c-Ets-2 was shown to be translocated to the nucleus in association with hFGL2 expression in response to both HBc and HBx proteins. Short hairpin RNA (shRNA) interference of c-Ets-2 expression inhibited hFGL2 gene transcription by 64.8 and 60.0% in response to HBc and HBx, respectively. c-Ets-2 protein was highly expressed in peripheral blood mononuclear cells from patients with severe chronic hepatitis B (CHB) in contrast to patients with mild CHB. Increased phosphorylation of ERK and JNK was detected in peripheral blood mononuclear cells from patients with severe CHB. ERK inhibitor PD098059 or ERK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBc, whereas JNK inhibitor SP600125 or JNK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBx. In conclusion, HBc and HBx proteins enhance transcription of hFGL2 through c-Ets-2 dependent on MAPK signal pathways.

Glucocorticoid Receptor-mediated Expression of Caldesmon Regulates Cell Migration via the Reorganization of the Actin Cytoskeleton

Glucocorticoids (GCs) play important roles in numerous cellular processes, including growth, development, homeostasis, inhibition of inflammation, and immunosuppression. Here we found that GC-treated human lung carcinoma A549 cells exhibited the enhanced formation of the thick stress fibers and focal adhesions, resulting in suppression of cell migration. In a screen for GC-responsive genes encoding actin-interacting proteins, we identified caldesmon (CaD), which is specifically up-regulated in response to GCs. CaD is a regulatory protein involved in actomyosin-based contraction and the stability of actin filaments. We further demonstrated that the up-regulation of CaD expression was controlled by glucocorticoid receptor (GR). An activated form of GR directly bound to the two glucocorticoid-response element-like sequences in the human CALD1 promoter and transactivated the CALD1 gene, thereby up-regulating the CaD protein. Forced expression of CaD, without GC treatment, also enhanced the formation of thick stress fibers and focal adhesions and suppressed cell migration. Conversely, depletion of CaD abrogated the GC-induced phenotypes. The results of this study suggest that the GR-dependent up-regulation of CaD plays a pivotal role in regulating cell migration via the reorganization of the actin cytoskeleton.

Characterization of β-N-Acetylglucosaminidase Cleavage by Caspase-3 during Apoptosis

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.

DNA Internalized via Caveolae Requires Microtubule-dependent, Rab7-independent Transport to the Late Endocytic Pathway for Delivery to the Nucleus

Using cationic liposomes to mediate gene delivery by transfection has the advantages of improved safety and simplicity of use over viral gene therapy. Understanding the mechanism by which cationic liposome:DNA complexes are internalized and delivered to the nucleus should help identify which transport steps might be manipulated in order to improve transfection efficiencies. We therefore examined the endocytosis and trafficking of two cationic liposomes, DMRIE-C and Lipofectamine LTX, in CHO cells. We found that DMRIE-C-transfected DNA is internalized via caveolae, while LTX-transfected DNA is internalized by clathrin-mediated endocytosis, with both pathways converging at the late endosome or lysosome. Inhibition of microtubule-dependent transport with nocodazole revealed that DMRIE-C:DNA complexes cannot enter the cytosol directly from caveosomes. Lysosomal degradation of transfected DNA has been proposed to be a major reason for poor transfection efficiency. However, in our system dominant negatives of both Rab7 and its effector RILP inhibited late endosome to lysosome transport of DNA complexes and LDL, but did not affect DNA delivery to the nucleus. This suggests that DNA is able to escape from late endosomes without traversing lysosomes and that caveosome to late endosome transport does not require Rab7 function. Lysosomal inhibition with chloroquine likewise had no effect on transfection product titers. These data suggest that DMRIE-C and LTX transfection complexes are endocytosed by separate pathways that converge at the late endosome or lysosome, but that blocking lysosomal traffic does not improve transfection product yields, identifying late endosome/lysosome to nuclear delivery as a step for future study.

Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes

This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormonesensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.

Low-Molecular-Weight Polyethylenimine Enhanced Gene Transfer by Cationic Cholesterol-Based Nanoparticle Vector

Both polyethylenimine (PEI) polymers and cationic nanoparticles have been widely used for non-viral DNA transfection. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA (pDNA) with high transfection efficiency. To increase the transfection activity of NP-OH, we investigated the potential synergism of PEI and NP-OH for the transfection of DNA into human prostate tumor PC-3, human cervices tumor Hela, and human lung adenocarcinoma A549 cells. The transfection efficiency with low-molecular PEI (MW 600) was low, but that with a combination of NP-OH and PEI was higher than with NP-OH alone, being comparable to commercially available lipofectamine 2,000 and lipofectamine LTX, with very low cytotoxicity. Low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex and release pDNA from the endosome to cytoplasm by the proton sponge effect. Therefore, the combination of cationic cholesterol-based nanoparticles and a low-molecular PEI has potential as a non-viral DNA vector for gene delivery.