Focal adhesion assembly and disassembly are essential for cell migration and cancer invasion, but the detailed molecular mechanisms regulating these processes remain to be elucidated. ...Phosphatidylinositol phosphate kinase type Iγ (PIPKIγ) binds talin and is required for focal adhesion formation in EGF-stimulated cells, but its role in regulating focal adhesion dynamics and cancer invasion is poorly understood. We show here that overexpression of PIPKIγ promoted focal adhesion formation, whereas cells expressing either PIPKIγ(K188,200R) or PIPKIγ(D316K), two kinase-dead mutants, had much fewer focal adhesions than those expressing WT PIPKIγ in CHO-K1 cells and HCT116 colon cancer cells. Furthermore, overexpression of PIPKIγ, but not PIPKIγ(K188,200R), resulted in an increase in both focal adhesion assembly and disassembly rates. Depletion of PIPKIγ by using shRNA strongly inhibited formation of focal adhesions in HCT116 cells. Overexpression of PIPKIγ(K188,200R) or depletion of PIPKIγ reduced the strength of HCT116 cell adhesion to fibronection and inhibited the invasive capacities of HCT116 cells. PIPKIγ depletion reduced PIP₂ levels to ∼40% of control and PIP₃ to undetectable levels, and inhibited vinculin localizing to focal adhesions. Taken together, PIPKIγ positively regulates focal adhesion dynamics and cancer invasion, most probably through PIP₂-mediated vinculin activation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Coplanar polychlorinated biphenyls (PCBs) may facilitate development of atherosclerosis by stimulating pro-inflammatory pathways in the vascular endothelium. Nutrition, including fish oil-derived ...long-chain omega-3 fatty acids, such as docosahexaenoic acid (DHA, 22:6ω-3), can reduce inflammation and thus the risk of atherosclerosis. We tested the hypothesis that cyclopentenone metabolites produced by oxidation of DHA can protect against PCB-induced endothelial cell dysfunction. Oxidized DHA (oxDHA) was prepared by incubation of the fatty acid with the free radical generator 2,2-azo-bis(2-amidinopropane) dihydrochloride (AAPH). Cellular pretreatment with oxDHA prevented production of superoxide induced by PCB77, and subsequent activation of nuclear factor-κB (NF-κB). A
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4-neuroprostanes (NPs) were identified and quantitated using HPLC ESI tandem mass spectrometry. Levels of these NPs were markedly increased after DHA oxidation with AAPH. The protective actions of oxDHA were reversed by treatment with sodium borohydride (NaBH
4), which concurrently abrogated A
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4-NP formation. Up-regulation of monocyte chemoattractant protein-1 (MCP-1) by PCB77 was markedly reduced by oxDHA, but not by un-oxidized DHA. These protective effects were proportional to the abundance of A
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4 NPs in the oxidized DHA sample. Treatment of cells with oxidized eicosapentaenoic acid (EPA, 20:5ω-3) also reduced MCP-1 expression, but less than oxDHA. Treatment with DHA-derived cyclopentenones also increased DNA binding of NF-E2-related factor-2 (Nrf2) and downstream expression of NAD(P)H:quinone oxidoreductase (NQO1), similarly to the Nrf-2 activator sulforaphane. Furthermore, sulforaphane prevented PCB77-induced MCP-1 expression, suggesting that activation of Nrf-2 mediates the observed protection against PCB77 toxicity. Our data implicate A
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4-NPs as mediators of omega-3 fatty acid-mediated protection against the endothelial toxicity of coplanar PCBs.
In hepatocytes, aging-associated decline in GSH has been linked to activation of neutral SMase (nSMase), accumulation of bioactive ceramide, and inflammation. In this study, we seek to test whether ...dietary supplementation with the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC), would correct the aging-associated differences in hepatic GSH, nSMase, and ceramide. Young and aged mice were placed on a diet that either lacked sulfur-containing amino acids (SAAs) or had 0.5% OTC for 4 weeks. Mice fed standard chow were used as an additional control. SAA-deficient mice exhibited significant aging-associated differences in hepatic GSH, GSH/GSSG, ceramide, and nSMase. C24:1 ceramide, the major ceramide species in liver, was affected the most by aging, followed by the less abundant C16:0 ceramide. OTC supplementation eliminated the aging-associated differences in hepatic GSH and GSH/GSSG ratio. Surprisingly, however, instead of decreasing, the nSMase activity and ceramide increased in the OTC-fed mice irrespective of their age. These effects were due to elevated nSMase-2 mRNA and protein and appeared to be direct. Similar increases were seen in HepG2 cells following treatment with OTC. The OTC-fed aged mice also exhibited hepatic steatosis and triacylglyceride accumulation. These results suggest that OTC is a potent stimulant of nSMase-2 expression and that there may be unanticipated complications of OTC supplementation.
Methionine adenosyltransferase (MAT) is a family of enzymes that utilizes ATP and methionine to produce S‐adenosylmethionine (AdoMet), the most crucial methyl donor in the biological methylation of ...biomolecules and bioactive natural products. Here, we report that the MAT from Sulfolobus solfataricus (sMAT), an enzyme from a poorly explored class of the MAT family, has the ability to produce a range of differentially alkylated AdoMet analogs in the presence of non‐native methionine analogs and ATP. To investigate the molecular basis for AdoMet analog production, we have crystallized the sMAT in the AdoMet bound, S‐adenosylethionine (AdoEth) bound and unbound forms. Notably, among these structures, the AdoEth bound form offers the first MAT structure containing a non‐native product, and cumulatively these structures add new structural insight into the MAT family and allow for detailed active site comparison with its homologs in Escherichia coli and human. As a thermostable MAT structure from archaea, the structures herein also provide a basis for future engineering to potentially broaden AdoMet analog production as reagents for methyltransferase‐catalyzed ‘alkylrandomization’ and/or the study of methylation in the context of biological processes. DATABASES: PDB IDs: 4HPV, 4L7I, 4K0B and 4L2Z. EC 2.5.1.6 STRUCTURED DIGITAL ABSTRACT: • sMAT and sMAT bind by x-ray crystallography (View interaction)
An efficient diastereoselective oxa-Pictet–Spengler reaction strategy was developed to construct benzoisochroman diastereomers. The utility of the reaction was demonstrated in the context of both the ...total synthesis of naturally occurring pyranonaphthoquinones (+)-frenolicin B and epi-(+)-frenolicin B as well as a range of frenolicin precursor analogs. The method is versatile and offers exquisite stereocontrol and, as such, offers a synthetic advance for the synthesis of pyranonaphthoquinone analogs.
Sphingosine 1-phosphate (S1P), a potent phospholipid growth and trophic factor, is synthesized in vivo by two sphingosine kinases. Thus these kinases have been proposed as important drug targets for ...treatment of hyperproliferative diseases and inflammation. We report here a new class of amidine-based sphingosine analogues that are competitive inhibitors of sphingosine kinases exhibiting varying degrees of enzyme selectivity. These inhibitors display K I values in the submicromolar range for both sphingosine kinases and, in cultured vascular smooth muscle cells, decrease S1P levels and initiate growth arrest.
A novel crosslinker 4,4′-dihydroxybiphenyl diacrylate (44BDA) was developed, and a series of temperature-responsive hydrogels were synthesized through free radical polymerization of ...N-isopropylacrylamide (NIPAAm) with 44BDA. The temperature-responsive behavior of the resulting gels was characterized by swelling studies, and the lower critical solution temperature (LCST) of the hydrogels was characterized through differential scanning calorimetry. Increased content of 44BDA led to a decreased swelling ratio and shifted the LCST to lower temperatures. These novel hydrogels also displayed resiliency through multiple swelling–deswelling cycles, and their temperature responsiveness was reversible. The successful synthesis of NIPAAm-based hydrogels crosslinked with 44BDA has led to a new class of temperature-responsive hydrogel systems with a variety of potential applications.
► DGKα contributes to cell-associated alkyl-LPA production in SKOV-3 cells. ► Alkyl amino alcohols stimulate DGKα, increasing SKOV-3 cell-associated alkyl-LPA. ► Results suggest cross-talk between ...sphingolipid and glycerol lipid pathways.
Lysophosphatidic acid (LPA) designates a family of bioactive phosphoglycerides that differ in the length and degree of saturation of their radyl chain. Additional diversity is provided by the linkage of the radyl chain to glycerol: acyl, alkyl, or alk-1-enyl. Acyl-LPAs are the predominate species in tissues and biological fluids. Alkyl-LPAs exhibit distinct pharmacodynamics at LPA receptors, potently drive platelet aggregation, and contribute to ovarian cancer aggressiveness. Multiple biosynthetic pathways exist for alkyl-LPA production. Herein we report that diacylglycerol kinases (DGKs) contribute to cell-associated alkyl-LPA production involving phosphorylation of 1-alkyl-2-acetyl glycerol and document the biosynthesis of alkyl-LPA by DGKs in SKOV-3 ovarian cancer cells, specifically identifying the contribution of DGKα. Concurrently, we discovered that treating SKOV-3 ovarian cancer cell with a sphingosine analog stimulates conversion of exogenous 1-alkyl-2-acetyl glycerol to alkyl-LPA, indicating that DGKα contributes significantly to the production of alkyl-LPA in SKOV-3 cells and identifying cross-talk between the sphingolipid and glycerol lipid pathways.
Venturicidin C (1), a new 20-membered macrolide along with the known venturicidins A (2) and B (3) were isolated from the crude extract of the Appalachian bacterial strain Streptomyces sp. TS-2-2. ...Additionally, nine other known compounds namely nocardamine, dehydroxynocardamine, desmethylenylnocardamine, ferrioxamine E, adenosine, riboflavin, cyclo(D)-trans-4-OH-Pro-(D)-Phe, cyclo(D)-Pro-(D)-Phe and N-(2-phenylethyl)-acetamide were also isolated and identified. The structure of the new macrolide 1 was elucidated by the cumulative analyses of NMR spectroscopy and HR-MS data. Complete NMR assignments for the known venturicidins A (2) and B (3) are also provided, for the first time, in this report. Venturicidins A-C did not inhibit the proliferation of A549 lung cancer cell line but all displayed potent antifungal activity.
The findings have demonstrated that early transient sphingosine‐1 phosphate lyase inhibition after acute myocardial infarction (AMI) correlates with increased stem cell mobilization and homing to the ...infarct border zones. These data indicate that pharmacological elevation of bioactive lipid levels can be beneficial in the early phase after cardiac ischemic injury and provide the first evidence that carefully timed transient pharmacological upregulation of bioactive lipids after AMI could be therapeutic.
Acute myocardial infarction (AMI) triggers mobilization of bone marrow (BM)‐derived stem/progenitor cells (BMSPCs) through poorly understood processes. Recently, we postulated a major role for bioactive lipids such as sphingosine‐1 phosphate (S1P) in mobilization of BMSPCs into the peripheral blood (PB). We hypothesized that elevating S1P levels after AMI could augment BMSPC mobilization and enhance cardiac recovery after AMI. After AMI, elevating bioactive lipid levels was achieved by treating mice with the S1P lyase inhibitor tetrahydroxybutylimidazole (THI) for 3 days (starting at day 4 after AMI) to differentiate between stem cell mobilization and the known effects of S1P on myocardial ischemic pre‐ and postconditioning. Cardiac function was assessed using echocardiography, and myocardial scar size evolution was examined using cardiac magnetic resonance imaging. PB S1P and BMSPCs peaked at 5 days after AMI and returned to baseline levels within 10 days (p < .05 for 5 days vs. baseline). Elevated S1P paralleled a significant increase in circulating BMSPCs (p < .05 vs. controls). We observed a greater than twofold increase in plasma S1P and circulating BMSPCs after THI treatment. Mechanistically, enhanced BMSPC mobilization was associated with significant increases in angiogenesis, BM cell homing, cardiomyocytes, and c‐Kit cell proliferation in THI‐treated mice. Mice treated with THI demonstrated better recovery of cardiac functional parameters and a reduction in scar size. Pharmacological elevation of plasma bioactive lipids after AMI could contribute to BMSPC mobilization and could represent an attractive strategy for enhancing myocardial recovery and improving BMSC targeting.
Significance
Acute myocardial infarction (AMI) initiates innate immune and reparatory mechanisms through which bone marrow‐derived stem/progenitor cells (BMSPCs) are mobilized toward the ischemic myocardium and contribute to myocardial regeneration. Although it is clear that the magnitude of BMSPC mobilization after AMI correlates with cardiac recovery, the molecular events driving BMSPC mobilization and homing are poorly understood. The present study confirms the role of bioactive lipids in BMSPC mobilization after AMI and proposes a new strategy that improves cardiac recovery. Inhibiting sphingosine‐1 phosphate (S1P) lyase (SPL) allows for the augmentation of the plasma levels of S1P and stem cell mobilization. These findings demonstrate that early transient SPL inhibition after MI correlates with increased stem cell mobilization and their homing to the infarct border zones. Augmenting BMSPC mobilization correlated with the formation of new blood vessels and cardiomyocytes and c‐Kit cell proliferation. These novel findings on the cellular level were associated with functional cardiac recovery, reduced adverse remodeling, and a decrease in scar size. Taken together, these data indicate that pharmacological elevation of bioactive lipid levels can be beneficial in the early phase after cardiac ischemic injury. These findings provide the first evidence that a carefully timed transient pharmacological upregulation of bioactive lipids after AMI could be therapeutic, because it results in significant cardiac structural and functional improvements.
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