ABSTRACT
Spontaneously Diabetic Torii (SDT) fatty rats, a new obese diabetic model, reportedly presented with features of non-alcoholic steatohepatitis (NASH) after 32 weeks of age. We tried to accelerate the onset of NASH in SDT fatty rats using dietary cholesterol loading and noticed changes in the blood choline level which is expected to be a NASH biomarker. Body weight and biochemical parameters were measured from 8 to 24 weeks of age. At 16, 20, 24 weeks, pathophysiological analysis of the livers were performed. Hepatic lipids, lipid peroxides, and the expression of mRNA related to triglyceride (TG) synthesis, inflammation, and fibrosis were evaluated at 24 weeks. Hepatic fibrosis was observed in SDT fatty rats fed cholesterol-enriched diets (SDT fatty-Cho) from 16 weeks. Furthermore, hepatic lipids and lipid peroxide were significantly higher in SDT fatty-Cho than SDT fatty rats fed normal diets at 24 weeks. Hepatic mRNA expression related to TG secretion decreased in SDT fatty-Cho, and the mRNA expression related to inflammation and fibrosis increased in SDT fatty-Cho at 24 weeks. Furthermore, SDT fatty-Cho presented with increased plasma choline, similar to human NASH. There were no significant changes in the effects of feeding a cholesterol-enriched diet in Sprague-Dawley rats. SDT fatty-Cho has the potential to become a valuable animal model for NASH associated with type 2 diabetes and obesity.
Subject(s)
Cholesterol, Dietary/adverse effects , Diabetes Mellitus, Type 2/physiopathology , Non-alcoholic Fatty Liver Disease/etiology , Non-alcoholic Fatty Liver Disease/physiopathology , Animals , Cholesterol, Dietary/administration & dosage , Diabetes Mellitus, Type 2/blood , Female , Non-alcoholic Fatty Liver Disease/blood , Rats , Rats, Sprague-DawleyABSTRACT
The protein p38 mitogen-activated protein kinase (MAPK) delta isoform (p38δ) is a poorly studied member of the MAPK family. Data analysis from The Cancer Genome Atlas database revealed that p38δ is highly expressed in all types of human breast cancers. Using a human breast cancer tissue array, we confirmed elevation in cancer tissue. The breast cancer mouse model, MMTV-PyMT (PyMT), developed breast tumors with lung metastasis; however, mice deleted in p38δ (PyMT/p38δ-/-) exhibited delayed primary tumor formation and highly reduced lung metastatic burden. At the cellular level, we demonstrate that targeting of p38δ in breast cancer cells, MCF-7 and MDA-MB-231 resulted in a reduced rate of cell proliferation. In addition, cells lacking p38δ also displayed an increased cell-matrix adhesion and reduced cell detachment. This effect on cell adhesion was molecularly supported by the regulation of the focal adhesion kinase by p38δ in the human breast cell lines. These studies define a previously unappreciated role for p38δ in breast cancer development and evolution by regulating tumor growth and altering metastatic properties. This study proposes MAPK p38δ protein as a key factor in breast cancer. Lack of p38δ resulted in reduced primary tumor size and blocked the metastatic potential to the lungs.
Subject(s)
Breast Neoplasms/pathology , Cell Adhesion , Cell Proliferation , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/pathology , Mitogen-Activated Protein Kinase 13/metabolism , Animals , Breast/pathology , Disease Progression , Female , Humans , MCF-7 Cells , Mammary Neoplasms, Experimental/genetics , Mice , Mice, Transgenic , Mitogen-Activated Protein Kinase 13/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Tissue Array AnalysisABSTRACT
Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2ß-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2ß and affect its compartmentalization, thereby suppressing PI3KC2ß activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2ß knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2ß in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2ß-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.
Subject(s)
Cell Movement/drug effects , Ceramides/pharmacology , Ovarian Neoplasms/drug therapy , Phosphatidylinositol 3-Kinase/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Mice , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/pathologyABSTRACT
Fallopian tubal epithelium is a candidate for the origin of high-grade serous ovarian cancer. Transferrin-containing follicular fluid and/or retrograde menstrual blood are possible risk factors for carcinogenesis. Accumulation of DNA double-strand breaks (DNA-DSBs) in the fallopian tubal epithelium is considered to play an important role in the development of cancer. However, the mechanisms by which DNA-DSBs accumulate have not yet been fully elucidated. The hydroxyl radical, which is produced in a Fenton reaction catalyzed by an iron ion, serves as a potent DNA-DSB-inducing molecule, raising the potential of an iron ion transporter of transferrin in the formation of DNA-DSBs. We studied the potential involvement of transferrin in DNA damage and the development of ovarian cancer. Treatment with transferrin facilitated the formation of histone 2AX phosphorylated at Serine 139 (γH2AX), which is known as a DNA-DSB marker, in human fallopian tube secretory epithelial cells and A2780 ovarian cancer cells. Knockdown of transferrin receptor 1 (TfR1), but not transferrin receptor 2, suppressed the transferrin uptake and consequent formation of γH2AX. As hydroxyl radicals in reactive oxygen species (ROS) are involved in DNA-DSBs, the formation of ROS was determined. Treatment with TfR1-specific small interference RNAs significantly diminished transferrin-induced formation of ROS. Moreover, TfR1-dependent uptake of transferrin was revealed to augment the formation of DNA-DSBs in the presence of hydrogen peroxide, which served as a substrate for the Fenton reaction. An ex vivo study with murine fallopian tubes further demonstrated that transferrin treatment introduced DNA-DSBs in the fallopian tubal epithelium. Collectively, these data suggested that the transferrin-TfR1 axis accounts for the induction of DNA-DSBs that potentially lead to DNA damage/genome instability. These findings also suggested that exposure to transferrin initiates and promotes the development of ovarian cancer by aiding the accumulation of DNA-DSBs in the fallopian tubal epithelium.
Subject(s)
Carcinogenesis/drug effects , Cystadenocarcinoma, Serous/metabolism , DNA Breaks, Double-Stranded/drug effects , Ovarian Neoplasms/metabolism , Receptors, Transferrin/metabolism , Transferrin/pharmacology , Animals , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Line, Tumor , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/pathology , Epithelium/drug effects , Epithelium/metabolism , Fallopian Tubes/drug effects , Fallopian Tubes/metabolism , Female , Histones/metabolism , Humans , Hydrogen Peroxide/pharmacology , Immunoblotting , Mice, Inbred C57BL , Microscopy, Confocal , Neoplasm Grading , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Oxidants/pharmacology , RNA Interference , Reactive Oxygen Species/metabolism , Receptors, Transferrin/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We previously reported that IL-2 deprivation induced acid sphingomyelinase-mediated (ASM-mediated) ceramide elevation and apoptosis in an NK/T lymphoma cell line KHYG-1. However, the molecular mechanism of ASM-ceramide-mediated apoptosis during IL-2 deprivation is poorly understood. Here, we showed that IL-2 deprivation induces caspase-dependent apoptosis characterized by phosphatidylserine externalization, caspase-8, -9, and -3 cleavage, and degradation of X-linked inhibitor of apoptosis protein (XIAP). IL-2 re-supplementation rescued apoptosis via inhibition of XIAP degradation without affecting caspase cleavage. However, IL-2 deprivation induced ceramide elevation via ASM in lysosomes and activated lysosomal cathepsin B (CTSB) but not cathepsin D. A CTSB inhibitor CA-074 Me and knockdown of CTSB inhibited ceramide-mediated XIAP degradation and apoptosis. Inhibition of ceramide accumulation in lysosomes using an ASM inhibitor, desipramine, decreased cytosolic activation of CTSB by inhibiting its transfer into cytosol from the lysosome. Knockdown of ASM also inhibited XIAP degradation and apoptosis. Furthermore, cell permeable N-acetyl sphingosine (C2-ceramide), which increases mainly endogenous d18:1/16:0 and d18:1/24:1 ceramide-like IL-2 deprivation, induced caspase-dependent apoptosis with XIAP degradation through CTSB. These findings suggest that lysosomal ceramide produced by ASM mediates XIAP degradation by activation of cytosolic CTSB and caspase-dependent apoptosis. The ASM-ceramide-CTSB signaling axis is a novel pathway of ceramide-mediated apoptosis in IL-2-deprived NK/T lymphoma cells.
Subject(s)
Cathepsin B/metabolism , Ceramides/biosynthesis , Killer Cells, Natural/pathology , Lymphoma, T-Cell/pathology , Sphingomyelin Phosphodiesterase/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolism , Apoptosis/physiology , Caspase 3/metabolism , Cell Line, Tumor , Ceramides/metabolism , Cytosol/metabolism , Humans , Interleukin-2/deficiency , Interleukin-2/metabolism , Killer Cells, Natural/metabolism , Lymphoma, T-Cell/metabolism , Lysosomes/metabolismABSTRACT
AIM: Vascular calcification, which significantly increases cardiovascular and other causes of mortality, is highly prevalent in hemodialysis patients. The aim of the present study was to examine the association between serum magnesium levels and vascular calcification in hemodialysis patients. METHODS: 390 nondiabetic patients on maintenance hemodialysis (226 males and 164 females, 59 +/- 13 years) were examined. Hand roentgenography was performed in each patient, and visible vascular calcification of the hand arteries was evaluated. Blood was drawn to measure serum calcium, phosphate, magnesium and intact parathyroid hormone levels. RESULTS: There were 52 patients (38 males and 14 females) with vascular calcification, and 338 (188 males and 150 females) without. Serum phosphate was significantly higher in the former compared with the latter group (p < 0.005); serum intact parathyroid hormone was significantly higher (p < 0.05), whereas serum calcium was not statistically different between the two groups. Serum magnesium was significantly lower in patients with vascular calcification than in those without (2.69 +/- 0.28 vs. 2.78 +/- 0.33 mg/dl, p < 0.05). Multivariate logistic regression analysis revealed that serum magnesium concentration was a significant independent factor associated with the presence of vascular calcification in hemodialysis patients (odds ratio 0.28, 95% CI 0.09 - 0.92/1 mg/dl increase in serum magnesium, p = 0.036) after adjustment for age, gender, duration of hemodialysis, calcium, phosphate and intact parathyroid hormone concentrations. CONCLUSION: Hypomagnesemia is significantly associated with the presence of vascular calcification of the hand arteries, independent of serum calcium and phosphate levels. These results suggest that higher serum magnesium concentrations may play an important protective role in the development of vascular calcification in hemodialysis patients, and that magnesium concentration of dialysis fluid may be reconsidered in view of preventing vascular calcification in hemodialysis patients.
Subject(s)
Calcinosis/physiopathology , Magnesium/blood , Peripheral Vascular Diseases/physiopathology , Renal Dialysis , Aged , Calcinosis/diagnostic imaging , Female , Hand/blood supply , Hand/diagnostic imaging , Humans , Male , Middle Aged , Peripheral Vascular Diseases/diagnostic imaging , RadiographyABSTRACT
Although labile, assay of intact osteocalcin (iOC) is established as the standard assay for evaluating osteoblastic function. The present study examines the clinical usefulness of the newly developed immunoradiometric assay for osteocalcin (OC), which identifies the stable N-terminal midfragment of OC (N-MID OC assay), in hemodialysis (HD) patients. The performance of N-MID OC assay was compared to that of iOC assay in the sera obtained from 137 male HD patients, by comparing these assays with those of other bone metabolic markers and bone loss during a 1-year period before determination of serum markers. Serum N-MID OC values did not decrease significantly during 24 h incubation at room temperature, whereas serum iOC decreased significantly after 1 h incubation. Serum N-MID and iOC in the 137 male HD patients were 197.3 +/- 57.8 and 34.6 +/- 30.0 ng/ml, respectively, or 3.9 +/- 3.1 and 2.8 +/- 2.4 times above the respective reported normal upper limits. Serum N-MID OC correlated significantly in a positive manner with serum iOC (r = 0.934, P < 0.0001). Serum N-MID OC correlated no less significantly in a positive manner with serum levels of bone alkaline phosphatase, deoxypyridinoline, and intact parathyroid hormone compared to serum iOC. Of interest was the fact that serum N-MID OC, but not iOC, correlated significantly with both the amount and the rate of bone loss at the distal radius 1/3. In summary, the findings suggest that N-MID OC immunoreactivity is much more stable than iOC immunoreactivity and that N-MID OC assay may be less susceptible to the OC fragments reported to accumulate in uremic serum. It may, therefore, prove more reliable than iOC assay for evaluating bone turnover, and thus for reflecting bone loss, in HD patients.
Subject(s)
Bone Resorption/blood , Osteocalcin/blood , Peptide Fragments/blood , Renal Dialysis/adverse effects , Adult , Aged , Aged, 80 and over , Alkaline Phosphatase/blood , Biomarkers , Bone Density , Bone Resorption/diagnosis , Bone and Bones/metabolism , Humans , Immunoradiometric Assay/methods , Male , Middle Aged , Osteocalcin/chemistry , Time Factors , Uremia/therapyABSTRACT
AIM/HYPOTHESIS: Although derangements of calcium and phosphate control have been emphasized as important risk factors for vascular calcification in non-diabetic haemodialysis patients, similar risk factors for diabetic haemodialysis patients are not known. We compared factors affecting peripheral vascular calcification between haemodialysis patients with and without diabetes. METHODS: We examined 421 patients on maintenance haemodialysis. There were 89 patients with Type II (non-insulin-dependent) diabetes mellitus (53 men and 36 women, 62+/-10 years old) and 332 patients without diabetes (192 men and 140 women, 59+/-13 years old). Hand roentgenography was carried out, and visible vascular calcification of the hand arteries was evaluated. RESULTS: There were 42 diabetic patients and 45 non-diabetic patients with vascular calcification. The prevalence of vascular calcification in diabetic patients (47.1%) was higher than in non-diabetic patients (13.6%) ( p<0.001). In multivariate logistic regression, the main factors affecting vascular calcification in non-diabetic patients were advanced age, longer duration of haemodialysis, increased phosphate concentrations, male gender, and lower predialysis diastolic pressure. In diabetic patients, predictors for vascular calcification were higher values of HbA(1C) and longer duration of haemodialysis. In diabetic patients, a 1% increase in HbA(1C) increased the risk of calcification by 2.1-fold (95% CI 1.282-3.575, p=0.0029). CONCLUSION/INTERPRETATION: We have shown that poor glycaemic control, rather than calcium and phosphate concentrations, is a predictor of peripheral vascular calcification in diabetic patients on haemodialysis. This study emphasizes that glycaemic control remains critical even in diabetic patients with end-stage renal disease.
Subject(s)
Calcinosis/epidemiology , Diabetes Mellitus, Type 2/physiopathology , Diabetic Nephropathies/complications , Kidney Failure, Chronic/therapy , Peripheral Vascular Diseases/epidemiology , Renal Dialysis , Blood Glucose/metabolism , Blood Pressure , Diabetes Mellitus, Type 2/blood , Diabetic Nephropathies/blood , Diabetic Nephropathies/mortality , Diabetic Nephropathies/therapy , Female , Humans , Kidney Failure, Chronic/mortality , Male , Middle Aged , Parathyroid Hormone/blood , Phosphates/blood , Predictive Value of Tests , Risk Factors , SmokingABSTRACT
In the present study, the effect of ceramide on antigen-stimulated phosphorylation of extracellular signal-regulated kinase (ERK) in the mechanism responsible for regulating production of prostaglandin (PG) D(2) was investigated in the mast cell line, RBL-2H3 cells. Cell-permeable C(6)-ceramide (N-hexanoylsphingosine) suppressed antigen-stimulated phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase. Ceramide also inhibited production of PGD(2) and an increase in the activity of cytosolic phospholipase A(2) (cPLA(2)), whereas it did not influence the tyrosine phosphorylation of major cellular proteins in response to antigen. The ceramide-induced inhibition of ERK1/2 phosphorylation and of cPLA(2) activation was suppressed by orthovanadate, a tyrosine phosphatase inhibitor, but not by okadaic acid, a serine/threonine phosphatase inhibitor. Addition of ceramide to the lysate prepared from antigen-stimulated cells reduced the phosphorylated ERK1/2, and orthovanadate effectively prevented the reduction. These results suggest that ceramide accelerates the dephosphorylation of phosphorylated ERK1/2 via activation of a protein tyrosine phosphatase, thus preventing activation of cPLA(2) and production of PGD(2).
Subject(s)
Ceramides/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Prostaglandin D2/biosynthesis , Animals , Antigens/biosynthesis , Arachidonic Acid/metabolism , Cell Line , Ceramides/metabolism , Dose-Response Relationship, Drug , Immunoblotting , Mast Cells/metabolism , Mitogen-Activated Protein Kinase 3 , Okadaic Acid/pharmacology , Phospholipase D/metabolism , Phospholipases A/metabolism , Phosphorylation , Protein Tyrosine Phosphatases/metabolism , Rats , Tumor Cells, Cultured , Tyrosine/metabolism , Vanadates/pharmacology , p38 Mitogen-Activated Protein KinasesABSTRACT
Transforming growth factor (TGF)-alpha and interleukin (IL)-1beta are responsible for the healing of gastric lesions through, in part, prostaglandin (PG) generation. We examined the contribution of cytosolic and secretory phospholipase A(2)s (cPLA(2) and sPLA(2)) to the PG generation by rat gastric epithelial cells in response to both stimuli. Stimulation with TGF-alpha for 24 h increased cPLA(2) and cyclooxygenase (COX)-2 markedly, PGE(2) slightly, and type IIA sPLA(2) and COX-1 not at all, whereas IL-1beta increased sPLA(2) only. Both stimuli synergistically increased PGE(2), sPLA(2), and the two COXs but not cPLA(2). The onset of the PGE(2) generation paralleled the sPLA(2) release but was apparently preceded by increases in cPLA(2) and the two COXs. The increase in PGE(2) was impaired by inhibitors for sPLA(2) and COX-2 but not COX-1. cPLA(2) inhibitors suppressed PGE(2) generation by TGF-alpha alone but not augmentation of PGE(2) generation or sPLA(2) release by IL-1beta in combination with TGF-alpha. Furthermore, despite an increase in cPLA(2) including its phosphorylated form (phosphoserine), -induced arachidonic acid liberation was impaired in the TGF-alpha/IL-1beta-stimulated cells, in which p11, a putative cPLA(2) inhibitory molecule, was also increased and co-immunoprecipitated with cPLA(2). These results suggest that synergistic stimulation of sPLA(2) and COX-2 expression by TGF-alpha and IL-1beta results in an increase in PGE(2). Presumably, the preceding cPLA(2) expression is not involved in the PGE(2) generation, because of impairment of its hydrolytic activity in the stimulated cells.
Subject(s)
Gastric Mucosa/enzymology , Interleukin-1/pharmacology , Isoenzymes/metabolism , Phospholipases A/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Recombinant Proteins/pharmacology , Transforming Growth Factor alpha/pharmacology , Animals , Arachidonic Acid/metabolism , Cell Line, Transformed , Cyclooxygenase 1 , Cyclooxygenase 2 , Dinoprostone/metabolism , Gastric Mucosa/cytology , Gene Expression Regulation, Enzymologic/genetics , Humans , Isoenzymes/genetics , Kinetics , Membrane Proteins , Phospholipases A/genetics , Phospholipases A2 , Prostaglandin-Endoperoxide Synthases/genetics , RatsABSTRACT
The effects of docosapentaenoic acid (DPA) on platelet aggregation and arachidonic acid metabolism were studied in comparison to those of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Collagen- or arachidonic acid-stimulated platelet aggregation was inhibited dose-dependently by n-3 fatty acids, among which DPA was the most potent inhibitor. These fatty acids inhibited U46619-induced aggregation but to almost the same extent. No effect of the acids on thrombin-induced aggregation was observed. Furthermore, these fatty acids suppressed thromboxane A2 formation by platelets which were exposed to collagen or thrombin, or by platelets to which arachidonic acid was added. In these experiments also, DPA was the most potent inhibitor, whereas DHA was the most effective inhibitor of cyclooxygenase-1 activity. DPA enhanced formation of 12-hydroxyeicosatetraenoic acid in response to collagen or from arachidonic acid by intact platelets, while the other two acids had less of an effect. These results suggest that DPA possesses potent activity for interfering with the cyclooxygenase pathway and accelerating the lipoxygenase pathway, thus inhibiting platelet aggregation most effectively.
Subject(s)
Fatty Acids, Unsaturated/pharmacology , Lipoxygenase/metabolism , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/blood , Animals , Collagen/pharmacology , Cyclooxygenase 1 , Eicosapentaenoic Acid/pharmacology , In Vitro Techniques , Isoenzymes/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Rabbits , Thromboxane B2/bloodABSTRACT
Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.
Subject(s)
Adipocytes/enzymology , Leucyl Aminopeptidase/chemistry , Amino Acid Sequence , Angiotensin II/metabolism , Animals , CHO Cells , COS Cells , Cricetinae , Humans , Kidney/enzymology , Kinetics , Leucyl Aminopeptidase/genetics , Molecular Sequence Data , TransfectionABSTRACT
The effect of ceramide on Ca2+-dependent translocation of cytosolic phospholipase A2 (cPLA2) to membranes was studied. Pretreatment of platelets with sphingomyelinase or C6-ceramide (N-hexanoylsphingosine) led to apparent enhancement of Ca2+-ionophore A23187-stimulated arachidonic acid release but did not affect the cytosolic phospholipase A2 (cPLA2) activity. Under these conditions, the cPLA2 proteins in membranes increased significantly, compared with those by A23187 alone. Sphingomyelinase and C6-ceramide, but not C6-dihydroceramide, a control analog of C6-ceramide, also facilitated the Ca2+-dependent increase in the cPLA2 protein, as well as the activity, in membranes induced by addition of Ca2+ into platelet lysate. Protein kinase Calpha, which possesses a Ca2+-dependent lipid binding domain, was increased in membranes in a Ca2+-dependent manner, but the increase was not accelerated by sphingomyelinase or C6-ceramide. These findings suggest that ceramide in membranes potentiates Ca2+-dependent cPLA2 translocation from cytosol to membranes, probably through modification of membrane phospholipid organization.
Subject(s)
Blood Platelets/drug effects , Blood Platelets/metabolism , Calcium/blood , Ceramides/pharmacology , Phospholipases A/blood , Animals , Arachidonic Acid/blood , Biological Transport, Active/drug effects , Cytosol/metabolism , In Vitro Techniques , Isoenzymes/blood , Membrane Lipids/blood , Membranes/metabolism , Phospholipases A2 , Phospholipids/blood , Protein Kinase C/blood , Protein Kinase C-alpha , Rabbits , Sphingomyelin Phosphodiesterase/pharmacologyABSTRACT
The implications of phospholipase D (PLD) in cytosolic phospholipase A2 (cPLA2) activation were studied in a mast cell line, RBL-2H3, upon stimulation with antigen. Antigen-stimulated prostaglandin D2 generation was apparently suppressed by ethanol with a concomitant decrease in phosphatidic acid (PA) formation. The prostaglandin D2 generation was also inhibited almost completely by methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of cPLA2, but not by diacylglycerol lipase inhibitor. Furthermore, stimulation with antigen resulted in an increase in lysophosphatidic acid formation, which was suppressed by MAFP in parallel with an increase in PA formation. These results suggest that PA formed by the catalytic action of PLD is used as a substrate for cPLA2, thus PLD regulates cPLA2 activation in antigen-stimulated RBL-2H3 cells.
Subject(s)
Phosphatidic Acids/metabolism , Phospholipase D/metabolism , Phospholipases A/metabolism , Animals , Antigens/pharmacology , Arachidonic Acids/pharmacology , Cytosol/enzymology , Dinitrophenols/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Leukemia, Basophilic, Acute/enzymology , Leukemia, Basophilic, Acute/metabolism , Organophosphonates , Phospholipases A/antagonists & inhibitors , Phospholipases A2 , Prostaglandin D2/biosynthesis , Rats , Serum Albumin, Bovine/pharmacology , Tumor Cells, CulturedABSTRACT
To study the involvement of sphingolipids in glycerophospholipid metabolism, the contribution of ceramide to the activation of group IV cytosolic phospholipase A2 (cPLA2) was investigated in platelets using cell-permeable C6-ceramide (N-hexanoylsphingosine). The addition of ceramide led to potentiation of thrombin-induced activation of cPLA2 and mitogen-activated protein kinase (MAPK) as well as arachidonic acid release and lysophosphatidylcholine formation. However, ceramide by itself did not induce any response. The arachidonic acid release due to the synergistic action of ceramide and thrombin was inhibited by PD98059, a MAPK kinase inhibitor. Ceramide also stimulated thrombin-induced protein kinase C (PKC) activation, but ceramide by itself failed to do so. Furthermore, ceramide synergistically enhanced diacylglycerol (DAG) formation and Ca2+ mobilization with thrombin, and also DAG formation with Ca2+-ionophore A23187. The DAG formation in response to ceramide with thrombin or A23187, as well as arachidonic acid release with thrombin were completely inhibited by U73122, a phospholipase C (PLC) inhibitor. These results suggest that ceramide triggers PLC activation through its synergistic action with thrombin, and subsequently potentiates the sequential PKC-MAPK cascade-cPLA2 pathway, thus resulting in enhancement of arachidonic acid release.
Subject(s)
Blood Platelets/metabolism , Ceramides/metabolism , Phospholipases A/metabolism , Type C Phospholipases/metabolism , Animals , Blood Platelets/drug effects , Calcimycin/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Ceramides/pharmacology , Drug Synergism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Ionophores/pharmacology , Phospholipases A/drug effects , Phospholipases A2 , Rabbits , Signal Transduction , Sphingomyelin Phosphodiesterase/metabolism , Thrombin/metabolism , Thrombin/pharmacology , Type C Phospholipases/drug effectsABSTRACT
The effects of ceramide on agonist-stimulated phospholipase A2 (PLA2) activity were studied in platelets. Cell-permeable C6-ceramide (N-hexanoylsphingosine) exogenously added to platelet suspension enhanced U46619-stimulated arachidonic acid release and lysophosphatidylcholine production. Treatment of platelets with sphingomyelinase also led to an enhancement of the release. The enhanced arachidonic acid release by exogenous ceramide was completely inhibited by methyl arachidonyl fluorophosphonate, a cytosolic PLA2 inhibitor. However, U46619-stimulated PLA2 activity was not significantly potentiated by ceramide. These results suggest that enrichment of ceramide in membranes causes modification of intermolecular organization, leading to increased susceptibility of substrate phospholipids to PLA2.
Subject(s)
Blood Platelets/drug effects , Ceramides/pharmacology , Phospholipases A/metabolism , Phospholipids/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Animals , Arachidonic Acid/metabolism , Arachidonic Acids/pharmacology , Blood Platelets/enzymology , Cell Membrane/drug effects , Cell Membrane/metabolism , Cytosol/drug effects , Cytosol/enzymology , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Lysophosphatidylcholines/biosynthesis , Organophosphonates/pharmacology , Phospholipases A2 , Rabbits , Signal Transduction , Thromboxane A2/analogs & derivativesABSTRACT
Ipriflavone (7-isopropoxyisoflavone), a non-hormonal isoflavone derivative, is currently used in several countries for prevention and treatment of postmenopausal osteoporosis. This compound is devoid of estrogenic activity in humans, but increases the activity of estrogens. Ipriflavone has been shown to be effective in reducing bone turnover rate mainly through an inhibition of bone resorption, and has been effect to stimulate of bone formation. Inhibitory effect of ipriflavone on bone resorption has been demonstrated both directly by the activation of mature osteoclast and the formation of new osteoclasts by stimulating estrogen-induced calcitonin secretion by thyroids in vivo. There are some evidence that ipriflavone has direct effect on bone formation. Several clinical studies have demonstrated that bone mineral density (BMD) was increased or maintained in patients treated with ipriflavone. Recently, a large multicentral study, Ipriflavone Multicenter European Fracture Study (IMEFS), was designed in order to investigate the efficacy of ipriflavone on the prevention of vertebral and the effect on BMD in women with postmenopausal osteoporosis.
Subject(s)
Isoflavones/therapeutic use , Osteoporosis, Postmenopausal/drug therapy , Bone Remodeling/drug effects , Female , HumansABSTRACT
PIP: At the Population and Natural Resources Workshop of the World Conservation Union (IUCN) General Assembly in Perth, Australia, December 1990, population and quality of life issues were stressed as one of the central items to be placed on the 1992 Agenda of the UN Conference on Environment. The pace of environmental degradation is quickening, the causes are becoming more entrenched, and indecision will narrow our options. Poverty and population growth are making development unsustainable. Technological miracles will not appear to restore balance. Deforestation, soil erosion, decertification and loss of water resources are fueling urbanization. Therefore the World Commission on Environment and Development, known as the Brundtland Commission, ranks human resources development as a top priority in sustainable development and quality of life. Human resources can be improved by providing maternal and child care, family planning and improving the status of women. Successful family planning programs as seen in Thailand and Malaysia can show results very quickly once national population policies, institutions and capacity are in place.^ieng