swinholide-a and jasplakinolide

Cancer progression depends on an accumulation of metastasis-supporting physiological changes which are regulated by cell signaling molecules. One such molecule, osteopontin (OPN), is a secreted phosphoprotein which mediates increased cellular migratory and invasive behavior, increased metastasis, protection from apoptosis, promotion of colony formation and 3D growth ability, induction of tumor-associated inflammatory cells, and induction of expression of angiogenic factors. Studies show that OPN expression is controlled by complex regulatory pathways at the transcriptional level in several cancers, but the molecular mechanisms which determine expression of OPN in HCC are largely unknown. In HepG2 and Hep3B tumor cell lines that differentially express OPN mRNA and protein, we identify elongation translation factor-1A1 (EF1A1) to be the trans-acting factor regulating differential OPN mRNA stability between HepG2 and Hep3B cell lines and characterize its interactions with G- and F-actin. EF1A1 binds to the OPN 5'-UTR to regulate OPN mRNA half-life. EF1A1 binds to actin in Hep3B cells. Pharmacologic manipulation to increase the G:F actin ratio in Hep3B increases OPN mRNA half-life and protein expression with simultaneous decrease in EF1A1 binding to OPN 5'-UTR. The converse findings were noted in HepG2 cells. Overall, our results suggest that EF1A1 regulation of OPN mRNA stability is actin dependent. EF1A1 has not been previously identified as a regulatory factor in OPN expression in cancer.

Topics: 5' Untranslated Regions; Actin Cytoskeleton; Actins; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytotoxins; Depsipeptides; Electrophoretic Mobility Shift Assay; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Marine Toxins; Osteopontin; Peptide Elongation Factor 1; Protein Binding; Regulatory Elements, Transcriptional; RNA Stability; RNA, Small Interfering; Sequence Deletion; Transfection

Insulin-increased prolactin gene transcription in GH4 cells was enhanced by binding on fibronectin. This was mediated by receptor-like protein tyrosine phosphatase alpha, which activated Src, Rho, and phosphatidylinositol 3-kinase. It suggested that insulin signaling to gene transcription was partly dependent on actin rearrangement. This was confirmed through studies using inhibitors of actin treadmilling. Cytochalasin D, jasplakinolide, latrunculin B, and swinholide A altered the actin cytoskeleton of GH4 cells, as assessed by Alexa Fluor phalloidin staining, and inhibited insulin-increased prolactin gene transcription. These reagents did not affect the controls. Nor was it due to a gross defect of insulin signaling because activation/translocation of glycogen synthase kinase 3beta and mammalian target of rapamycin were not affected. Expression of wild-type and mutant actin treadmilling agents, Cdc42, TC10, neuronal Wiskott-Aldrich syndrome protein, and Nck, indicated that they were essential to insulin-increased prolactin gene expression, and suggested that activation of p21 associated kinase (PAK) might also be essential to this process. PAK expression also increased and PAK mutants decreased prolactin promoter activity in insulin-treated cells. The activation of PAK in the presence of inhibitors was also consistent with a role in activation of insulin-increased prolactin gene expression. Finally, small interfering RNA-mediated reduction of PAK decreased the effect of insulin on prolactin gene expression. Thus, it is likely that insulin activation of actin treadmilling through Cdc42/TC10 and neuronal Wiskott-Aldrich syndrome protein activates PAK and prolactin gene transcription.

Topics: Actins; Animals; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; cdc42 GTP-Binding Protein; Cytochalasin D; Depsipeptides; Electrophoresis, Polyacrylamide Gel; Gene Expression; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Insulin; Marine Toxins; Microscopy, Fluorescence; Mutation; p21-Activated Kinases; PC12 Cells; Phosphorylation; Prolactin; Protein Kinases; Rats; rho GTP-Binding Proteins; RNA, Small Interfering; Signal Transduction; Thiazolidines; TOR Serine-Threonine Kinases; Transcription, Genetic; Transfection

Posttranscriptional regulation of endothelial nitric oxide synthase (eNOS) expression is an important mechanism by which endothelial cells respond to various physiological and pathophysiological stimuli. Previously, we showed that eNOS expression was dramatically altered by the state of cell growth and that the mechanism responsible for this regulation was entirely posttranscriptional, occurring via changes in eNOS mRNA stability. The present study identifies a role for actin cytoskeleton organization in the posttranscriptional regulation of eNOS during cell growth and examines the relationship between the state of actin polymerization and eNOS expression. We identified monomeric actin (globular [G]-actin) as the major component of a 51-kDa ribonucleoprotein that binds to the eNOS mRNA 3' untranslated region in UV-crosslinking analysis. Binding activity of the ribonucleoprotein complex correlated with the relative concentration of G-actin versus filamentous actin (F-actin). ENOS transcripts colocalized with cytoplasmic G-actin in cells subjected to fluorescence in situ hybridization and G-actin fluorescence staining. In subcellular fractionation studies, eNOS transcripts were enriched in the free polysomal fraction of nonproliferating cells and enriched in the cell matrix-associated polysomal fraction of proliferating cells. Furthermore, an inverse relationship between the concentration of G-actin and eNOS expression was observed in endothelial cells subjected to pharmacological alteration of their cytoskeleton; lower G/F-actin ratios correlated with increased eNOS expression. Our findings provide some insight into how endothelial cells may use the dynamic organization of the actin cytoskeleton to regulate expression of an enzyme that is crucial to vascular homeostasis.

Topics: 3' Untranslated Regions; Actin Cytoskeleton; Actins; Animals; Binding Sites; Cattle; Cell Proliferation; Cells, Cultured; Depsipeptides; Endothelium, Vascular; Gene Expression Regulation, Enzymologic; Marine Toxins; Microfilament Proteins; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Ribonucleoproteins; RNA Stability; RNA, Messenger

The microvilli of the apical membrane of proximal tubule (PT) cells are supported by the underlying actin cytoskeleton. Ischaemic or anoxic ATP-depletion leads to the disruption of the actin cytoskeleton, resulting in microvillar retraction and loss of membrane polarity. Using isolated PT cells, we have previously demonstrated that actin filaments (F-actin) are likely severed during ATP-depletion. A sequential extraction protocol revealed a decrease in actin solubility, resulting in the sequestration of a distinct F-actin pool with the insoluble cellular complex in ATP-depleted PT cells. We demonstrate here that decreased actin solubility is not only a reliable end-marker of ATP-depletion induced injury in freshly isolated PT cells, but also serves as a biochemical marker in the cultured proximal tubular cell line LLC-PK1. In the present studies, we also investigated specific actin-binding drugs to determine if they mimic the effects observed during energy depletion. Jasplakinolide (JP), a compound which binds F-actin and prevents depolymerization, did not effect actin solubility during ATP-depletion. Furthermore, swinholide A (SA), an F-actin severing agent, resulted in decreased actin solubility, mimicking the effects of ATP-depletion. Interestingly, latrunculin A (LA), an agent which depolymerizes F-actin, did not reduce actin solubility, but rather resulted in an increase in digitonin-soluble actin. Taken collectively, our results support previous work and suggest that disruption of the actin cytoskeleton during ATP-depletion is mediated by F-actin severing/fragmentation and not depolymerization. The differential effects of F-actin disrupting agents and the consistencies observed in both models of ischaemic injury will provide a basis for a more detailed understanding of the pathological events of PT-cell dysfunction.

Topics: Actins; Adenosine Triphosphate; Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Depsipeptides; Female; Kidney Tubules, Proximal; LLC-PK1 Cells; Marine Toxins; Microscopy, Fluorescence; Peptides, Cyclic; Polymers; Rabbits; Solubility; Swine; Thiazoles; Thiazolidines

We investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton, L-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca(2+)/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50-100 nM, 2 h treatment) induced a reversible activation of L-[(3)H]arginine uptake by PAEC, whereas Swinh (10-50 nM) decreased L-[(3)H]arginine uptake. The two drugs could abrogate the effect of each other on L-[(3)H]arginine uptake. The effects of both drugs on L-[(3)H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular L-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC.

Topics: Actins; Animals; Arginine; Cells, Cultured; Cytoskeleton; Depsipeptides; Endothelium, Vascular; Marine Toxins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Peptides, Cyclic; Pulmonary Artery; Swine

To determine effects of the marine macrolides swinholide A (Swin A) and jasplakinolide (Jas), alone or in conjunction with latrunculin B (Lat B) on outflow facility in monkeys.. Total outflow facility was measured by two-level constant-pressure perfusion of the anterior chamber before and after exchange with Swin A, Jas, or vehicles followed by continuous anterior chamber infusion of drug or vehicle, in opposite eyes of cynomolgus monkeys. The effect of a facility-ineffective dose of Jas plus a threshold or submaximal facility-effective dose of the actin depolymerizer Lat B on outflow facility was also determined.. Ten or 100 nM Swin A or 20, 100, or 500 nM Jas had no significant effect on outflow facility. However, 500 nM Swin A and 2.5 microM Jas significantly increased facility by 80% +/- 21% and 157% +/- 57% (mean +/- SEM) respectively, adjusted for corresponding baselines and resistance washout in contralateral control eyes. The facility increase in the eye treated with 500 nM Jas with 60 or 200 nM Lat B was similar to that in the eye treated with 60 or 200 nM Lat B only.. Swin A (which severs actin filaments and sequesters actin dimers) and Lat B (which sequesters actin monomers) similarly increase outflow facility. The potent inducer of actin polymerization Jas (500 nM) neither inhibits nor potentiates the facility increase induced by Lat B (60 or 200 nM). A higher dose of Jas increases rather than decreases outflow facility.

Topics: Animals; Anterior Chamber; Bridged Bicyclo Compounds, Heterocyclic; Depsipeptides; Dose-Response Relationship, Drug; Drug Combinations; Macaca fascicularis; Marine Toxins; Peptides, Cyclic; Thiazoles; Thiazolidines

Depolymerization of actin by latrunculin A transiently promotes neurotransmitter release. The mean rate of mEPSCs increases by a Ca2+-independent process, without a concomitant change in the mean amplitude. The readily releasable vesicle pool size and the rate of refilling of the readily releasable pool remain unaltered by latrunculin treatment. Evoked neurotransmitter release also increases in a manner consistent with an increase in vesicle release probability. The observed enhancement of neurotransmitter release is specific to actin depolymerization mediated by latrunculin A and is not caused by cytochalasin D. Our findings indicate that actin participates in a regulatory mechanism that restrains fusion of synaptic vesicles at the active zone.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cells, Cultured; Cytochalasin D; Depsipeptides; Egtazic Acid; Excitatory Postsynaptic Potentials; Fluorescent Antibody Technique; Green Fluorescent Proteins; Hippocampus; Luminescent Proteins; Marine Toxins; Mice; Nerve Tissue Proteins; Neurotransmitter Agents; Peptides, Cyclic; Presynaptic Terminals; Rats; Recombinant Fusion Proteins; Synapses; Synaptic Transmission; Synaptic Vesicles; Thiazoles; Thiazolidines