Cellular concentrations of plasmalogen species containing a polyunsaturated fatty acid significantly increase under hypoxia in human colorectal cancer, Caco2 cells.

Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2ß and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.

IL-6 promotes cell adhesion in human endothelial cells via microRNA-126-3p suppression.

Atherosclerosis is an important underlying cause of cardiovascular diseases; vascular endothelial cells play a vital role in inflammatory responses in the initial steps of atherosclerosis. High levels of the pro-inflammatory cytokine interleukin-6 (IL-6) long have been considered a risk factor in the development and complications of atherosclerotic disease. However, it is still controversial whether IL-6 is atherogenic or atheroprotective. Recently, miR-126-3p, an endothelial cell-specific microRNA, has been proposed as an atheroprotective molecule. Therefore, we investigated whether IL-6 accelerates endothelial cell responses through the suppression of miR-126-3p expression in human endothelial cell line EA.hy926. IL-6 yielded concentration-dependent decreases in miRNA-126-3p accumulation in EA.hy926 cells, leading in turn to increased expression of genes targeted by miRNA-126-3p. In addition, adhesion of the human monocyte cell line THP-1 was enhanced by the exposure of EA.hy926 cells to IL-6, with associated increases in the levels of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1). Suppression of miR-126-3p expression resulted in upregulation of miRNA-126-3p-regulated genes, enhanced adhesion of THP-1 cells, and increased ICAM-1 accumulation in EA.hy926 cells. In contrast, miR-126-3p overproduction had the opposite effects. The regulation of miRNA-126-3p by IL-6 may have important implications for the development of novel protective therapies targeting atherosclerosis.

CERS6 required for cell migration and metastasis in lung cancer.

Sphingolipids constitute a class of bio-reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non-small-cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR-101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1-positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.

Necrostatin-7 suppresses RANK-NFATc1 signaling and attenuates macrophage to osteoclast differentiation.

Osteoclasts play a crucial role in osteolytic bone diseases, such as osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease of bone and bone metastatic tumors. Therefore, controlling osteoclast differentiation and function has been considered a promising therapeutic strategy. Here, we show that necrostatin (Nec)-7, an inhibitor of programmed necrosis, strongly suppressed receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption, without compromising macrophage colony-stimulating factor (M-CSF)-supported survival and growth of osteoclast precursor cells. Accordingly, Nec-7 significantly decreased the levels of RANKL-induced osteoclastogenic marker genes, such as cathepsin K. Mechanistically, Nec-7 neither affected MAPK nor NF-κB activation; however, it strongly inhibited the RANKL receptor (RANK) to nuclear factor of activated T cells c1 (NFATc1) signaling. Lentiviral expression of RANK in bone marrow-derived macrophages significantly restored osteoclastogenesis and NFATc1 amplification in Nec-7-treated cells. In this study, we revealed that Nec-7-sensitive pathways are crucially involved in osteoclast formation and function. Investigation of the molecular mechanism(s) through which Nec-7 inhibits RANK-NFATc1 signaling axis may lead to the development of new therapeutic strategies for bone disease.

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by sphingosine 1-phosphate in HepG2 liver cells.

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a major physiologic inhibitor of fibrinolysis, is implicated in the progression of atherosclerosis. Sphingosine 1-phosphate (S1P) regulates expression of diverse genes and alters expression of PAI-1 in several types of cells. However, the nature of posttranscriptional regulation of expression of PAI-1 by S1P has not yet been thoroughly elucidated. The present study was undertaken to determine whether S1P has important effects on the posttranscriptional regulation of PAI-1 expression. To evaluate this possibility, we determined promoter activity, mRNA levels, 3'-untranslated region (UTR) activity, and protein levels of PAI-1 in HepG2 cells. S1P increased PAI-1 promoter activity and the expression of PAI-1 mRNA within 4h of exposure. It decreased the expression of PAI-1 mRNA and the accumulation of PAI-1 protein into the media in 24h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2kb). S1P decreased the baseline luciferase activity of the 1kb fragment of the 3' terminus (+2177 to 3176nt) of the 3'-UTR of the 3.2kb PAI-1 mRNA [3'-UTR (+2177-3176)]. S1P decreased expression of PAI-1 protein, presumably by regulating PAI-1 expression at the posttranscriptional level thereby affecting mRNA stability. SERPINE1 mRNA binding protein (SERBP1) and ARE3 in the 3'-UTR were involved in the posttranscriptional regulation by S1P. Our data suggest that S1P can destabilize 3.2kb PAI-1 mRNA through specific effects on the 3'-UTR. These effects appear to involve SERBP1 leading to decreased expression of PAI-1 protein.

Hypoxia increases cellular levels of phosphatidic acid and lysophospholipids in undifferentiated Caco-2 cells.

Cancer cells are known to survive in a hypoxic microenvironment by altering their lipid metabolism as well as their energy metabolism. In this study, Caco-2 cells derived from human colon cancer, were found to have elevated intracellular levels of phosphatidic acid and its lysoform, lysophosphatidic acid (LPA), under hypoxic conditions. Our results suggested that the elevation of LPA in Caco-2 cells was mainly due to the combined increases in cellular levels of lysophosphatidylcholine and lysophosphatidylethanolamine by phospholipase A2 and subsequent hydrolysis to LPA by lysophospholipase D. We detected the Ca2+ -stimulated choline-producing activities toward exogenous lysophosphatidylcholines in whole Caco-2 cell homogenates, indicating their involvement in the LPA production in intact Caco-2 cells.

Circulating microRNA-126 in patients with coronary artery disease: correlation with LDL cholesterol.

BACKGROUND: Coronary artery disease (CAD) is a major problem worldwide. Atherosclerosis and thrombosis underlying CAD involve multiple cell types. New and useful diagnostic markers are required. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the gene expressions involved in various cellular processes. Endothelial dysfunction is implicated in early processes of athero-thrombosis. Thus, it was hypothesized that the level of vascular endothelium-enriched miRNAs would be altered in plasma samples of CAD patients. METHODS: Vascular endothelium-enriched miRNA (miR-126) level was analyzed in plasma from 31 patients with CAD and 36 patients without CAD (qRT-PCR analysis). RESULTS: MiR-126 was not significantly down-regulated or up-regulated in CAD patients. Interestingly, the level of miR-126 was significantly decreased in patients with CAD and high low-density lipoprotein (LDL) cholesterol level. In contrast, the level of miR-126 was significantly increased when LDL cholesterol was high in patients who had risk factors for CAD but did not have angiographically significant CAD. CONCLUSION: MiR-126 was not significantly down-regulated or up-regulated in CAD patients and was not suitable for discriminating CAD patients from patients without CAD. The oppositely-directed relationship between miR-126 and LDL cholesterol in patients with or without CAD may have significant implications for identifying a potential role of miR-126 in cholesterol metabolism.

Long-chain base kinase Lcb4 Is anchored to the membrane through its palmitoylation by Akr1.

Sphingoid long-chain base kinase Lcb4 catalyzes the production of the bioactive lipid molecules the long-chain base 1-phosphates. Although Lcb4 has no apparent transmembrane-spanning domain, it is tightly associated with the membrane. Here, we demonstrate that Lcb4 is modified by palmitoylation. This modification was greatly reduced in mutants for AKR1, which was recently identified as encoding a protein acyltransferase. In vitro experiments revealed that Akr1 indeed acts as a protein acyltransferase for Lcb4. Studies using site-directed mutagenesis indicated that Cys-43 and Cys-46 are palmitoylated. The loss of palmitoylation on Lcb4 caused several effects, including mislocalization of the protein to the cytosol, reduced phosphorylation, and loss of downregulation during the stationary phase. Although Akr2 is highly homologous to Akr1, the deletion of AKR2 did not result in any remarkable phenotypes. However, overproduction of Akr2 resulted in reduced amounts of Lcb4. We demonstrated that Akr2 is an unstable protein and is degraded in the vacuole. Akr2 exhibits high affinity for Lcb4, and in Akr2-overproducing cells this interaction caused unusual delivery of Lcb4 to the vacuole and degradation.

Investigation into Differences in Level of Knowledge about Hypertension between High School Students and Elderly People.

As a major chronic non-communicable disease, hypertension is the most important risk factor for cardiovascular disease, chronic kidney disease, stroke and, if not treated appropriately, premature death. A population-based approach aimed at decreasing high blood pressure among the general population is an important component of any comprehensive plan to prevent hypertension. However, few studies have investigated generational differences in knowledge about, and consciousness of, hypertension. Thus, we conducted a questionnaire survey about hypertension, with the aim of clarifying differences of understanding about hypertension between high school students and elderly people. The results of this investigation suggested that there is indeed a generational difference: knowledge about hypertension, and awareness of its relationship with salt intake, was higher in elderly people than in high school students. Furthermore, our study showed that among high school students, salt intake consciousness correlated with a family history of hypertension. By contrast, in elderly people, salt intake consciousness is related to age and to an awareness of recommended daily salt intake. This study strongly showed that knowledge and consciousness of hypertension varied among generations, with the elderly being more aware and conscientious about salt intake. Acknowledgement of this generational diversity is critical to developing an effective overall preventive strategy for hypertension.

Sphingosine 1­phosphate induced by hypoxia increases the expression of PAI­1 in HepG2 cells via HIF­1α.

Our group has recently reported that in the immortal human HepG2 liver cell line, sphingosine 1­phosphate (S1P) increases transcription of plasminogen activator inhibitor type­1 (PAI­1), the major physiological inhibitor of fibrinolysis, within 4 h. The present study aimed to elucidate the molecular mechanisms underlying this effect. PAI­1 expression was measured by reverse transcription­quantitative polymerase chain reaction and immunoblotting. It was demonstrated that S1P increased PAI­1 promoter activity but did not increase the activity of promoters lacking the hypoxia responsive element (HRE) 2. In addition, S1P transiently increased the concentration of hypoxia inducible factor (HIF)­1α, a transcription factor capable of binding to HRE. When HIF­1α was knocked down, the induction of transcription of PAI­1 by S1P was no longer observed. Sphingosine kinase (SPHK) activity is increased by hypoxia. It was demonstrated that increases in the concentration of the HIF­1α protein induced by hypoxia were prevented by treatment with SPHK inhibitor or S1P receptor antagonists. Thus, modification of the induction of HIF­1α by S1P, leading to increased transcription of PAI­1, may be an attractive therapeutic target for thrombosis and consequent inhibition of fibrinolysis associated with hypoxia.

Targeting ceramide synthase 6-dependent metastasis-prone phenotype in lung cancer cells.

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.

Circulating level of microRNA-126 may be a potential biomarker for recovery from smoking-related vascular damage in middle-aged habitual smokers.

BACKGROUND: Cigarette smoking promotes vascular endothelial damage and accelerates progression of atherosclerosis. The purpose of this study was to examine whether the circulating level of vascular endothelium-enriched microRNA-126 (miR-126), which is highlighted as a regulator of gene expression, would serve as a novel biomarker for recovery from smoking-related vascular damage. METHODS: Middle-aged male smokers (n = 30) were enrolled and instructed to stop smoking. Their clinical profiles and laboratory findings including expression of miR-126 were investigated before and after 8 weeks of smoking cessation. Serum levels of cotinine, metabolites of nicotine, were measured to confirm smoking cessation. Endothelial function for peripheral small vessels was assessed and expressed as reactive hyperemia peripheral arterial tonometry (RH-PAT) index. The expression of miR-126 in plasma was analyzed by quantitative real-time PCR. RESULTS: At baseline, serum cotinine levels were inversely correlated with RH-PAT index (r = - 0.48, P < 0.01) and positively correlated with levels of metabolic parameters such as non-HDL cholesterol (r = 0.53, P < 0.01) and HOMA-IR (r = 0.52, P < 0.01). The RH-PAT index was not significantly changed after 8 weeks in all subjects, because only 13 subjects could attain smoking cessation. However, changes in the RH-PAT index showed a significant correlation with those in systolic blood pressure (r = - 0.54, P < 0.01). In smokers who completely attained smoking cessation (n = 13), RH-PAT index and plasma levels of miR-126 were significantly increased (P < 0.05, respectively). CONCLUSIONS: Endothelial damage was improved and plasma levels of circulating miR-126 were increased after 8 weeks of smoking cessation. These findings suggested a potential use of miR-126 as a biomarker for recovery from smoking-induced vascular damage.

Increased acid ceramidase expression depends on upregulation of androgen-dependent deubiquitinases, USP2, in a human prostate cancer cell line, LNCaP.

Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression.

TNF-α production in NKT cell hybridoma is regulated by sphingosine-1-phosphate: implications for inflammation in atherosclerosis.

OBJECTIVES: Natural killer T (NKT) cells are unique T lymphocytes that recognize glycolipid antigen and produce various cytokines. NKT cells accelerate atherosclerosis in mice. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid and regulates T-lymphocyte trafficking. We aimed to determine the effects of S1P on the production of proinflammatory cytokine, tumor necrosis factor (TNF)-α, in NKT cell hybridomas and mouse NKT cells. MATERIALS AND METHODS: NKT cell hybridomas and sorted mouse NKT cells were stimulated with S1P and α-galactosylceramide (α-GalCer), the major ligand to produce cytokines in NKT cells. TNF-α mRNA expression and protein production were determined by real-time PCR and ELISA, respectively. Cell migration was assayed using chemotaxicell. Plasma S1P was measured using HPLC. RESULTS: Hybridomas expressed S1P receptors, S1P1, S1P2, and S1P4. S1P and α-GalCer increased TNF-α mRNA expression and protein production. S1P enhanced TNF-α induction by α-GalCer. S1P receptor antagonists decreased the TNF-α mRNA expression induced by S1P. FTY720, an immunosuppressive S1P receptor modulator, also decreased the TNF-α mRNA expression. The migration of NKT cell hybridomas was increased by S1P. FTY720 reduced the migration induced by S1P. S1P also increased the TNF-α mRNA expression in mouse NKT cells. Plasma TNF-α levels in patients with high plasma S1P (≥500 nmol/l) were higher than those in patients with low S1P (<500 nmol/l). CONCLUSION: S1P binds to S1P receptors in NKT cells and enhances TNF-α production. TNF-α overproduction may induce atherogenic inflammatory responses. S1P may serve as a novel therapeutic target for amelioration of vascular inflammatory diseases.

Increased plasma sphingosine-1-phosphate in obese individuals and its capacity to increase the expression of plasminogen activator inhibitor-1 in adipocytes.

OBJECTIVES: Concentrations of plasminogen activator inhibitor-1 (PAI-1) are increased in obese individuals. One source of PAI-1 is adipocytes. Hypoxia develops within adipose tissue as it expands, presumably contributing to increased levels of sphingosine-1-phosphate (S1P). S1P is a breakdown product of sphingosine, ubiquitous in cell membranes. We have shown previously that S1P increases the expression of PAI-1 in human liver-derived cell line. In the present study, we aimed to determine whether hypoxia induces S1P in adipocytes, thereby potentially contributing to an increase in PAI-1 and hence constraints on fibrinolysis associated with obesity. MATERIALS AND METHODS: Mouse 3T3-L1 adipocytes were exposed to CoCl2 to simulate hypoxia. Assays were performed for PAI-1 mRNA (quantitative PCR) and S1P (high-performance liquid chromatography). RESULTS: The physiologic concentration of S1P increased PAI-1 mRNA expression. The S1P2 receptor antagonist attenuated the increase in PAI-1. Adipocytes expressed sphingosine kinase 1/2 (SPHK1/2) and S1P lyase, key enzymes involved in S1P production and degradation. Hypoxia increased SPHK activity and decreased S1P lyase mRNA. Hypoxia reduced cytosolic sphingosine and increased S1P release into conditioned medium. Inhibitors of ABCA1 and ABCC1 reduced the release of S1P into conditioned media. In obese patients with uncomplicated dyslipidemia and hypertension, plasma S1P was increased compared with that in nonobese and lean individuals. CONCLUSION: Hypoxia in adipose tissue of obesity can promote elaboration of S1P that binds to S1P2 receptors in an autocrine or a paracrine manner. S1P potentially contributes toward increased expression of PAI-1 and consequent constraints on fibrinolysis. S1P production and extracellular transport provide an attractive target for therapy to attenuate impaired fibrinolysis associated with obesity.

Analytical evaluation of plasma serotonin and sphingosine 1-phosphate and their clinical assessment in early atherosclerosis.

OBJECTIVES: Serotonin stored in platelets is released into plasma on aggregation and activation in atherosclerotic diseases. Sphingosine 1-phosphate (S1P) in plasma is mainly derived from red blood cells and is responsible for the production of nitric oxide in endothelial cells and protects vasculature. The purpose of this study was to investigate the plasma levels of serotonin, S1P, and their clinical relationships with vascular endothelial function in patients with early atherosclerosis. METHODS: Blood was withdrawn from patients with low-to-moderate risks of atherosclerotic diseases (n=49, 39 ± 7 years). Platelet-poor plasma was immediately centrifuged. Serotonin levels in plasma were measured with high-performance liquid chromatography. S1P levels in plasma were measured by high-performance liquid chromatography after fluorescent derivatization with o-phthaldialdehyde. Endothelial function was assessed by endothelium-dependent flow-mediated dilation (FMD) and endothelium-independent dilation was measured by glycerol trinitrate-induced dilation using an ultrasound system. RESULTS: Plasma serotonin was inversely correlated with the FMD value (r=-0.287, P<0.05). Fourteen patients with dyslipidemia, who had not shown improvements after lifestyle modifications, were subsequently treated with rosuvastatin (2.5 mg/day). After 4 weeks of treatment, rosuvastatin improved lipid profiles. Rosuvastatin increased FMD, whereas glycerol trinitrate-induced dilation was unchanged. Notably, percentage decrease in plasma serotonin was inversely correlated with percentage increase in plasma S1P (r=-0.557, P<0.05). CONCLUSION: Plasma serotonin was inversely correlated with FMD and a decrease in plasma serotonin was inversely correlated with an increase in plasma S1P after statin treatment. The results suggested that plasma levels of serotonin and S1P may be useful for the assessment of endothelial function of patients with low-to-moderate risks of atherosclerotic diseases.

Role of down-regulated neutral ceramidase during all-trans retinoic acid-induced neuronal differentiation in SH-SY5Y neuroblastoma cells.

Neutral ceramidase (NCDase) is considered to be a critical enzyme for controlling the turnover of ceramide, an important bioactive lipid, which determines cell's fate. All-trans retinoic acid (ATRA) has been reported to induce neuronal differentiation and cell-cycle arrest [Lopez-Carballo, Moreno, Masia, Perez, and Barettino (Activation of the phosphatidylinositol 3-kinase/Akt signalling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 2002:277:25297-304.)]. In this study, we observed that ATRA-induced cellular ceramide accumulation, cell-growth arrest and differentiation accompanied with down-regulation of NCDase in SH-SY5Y cells, without a decrease in sphingosine or sphingosine 1-phosphate. We examined whether the down-regulation of NCDase was involved in the increase in ceramide and cell differentiation. ATRA was found to down-regulate mRNA, protein and the enzyme activity of NCDase. Interestingly, GATA-2 was also decreased with ATRA treatment, and experiments using its expression vector and siRNA and chromatin immunoprecipitation assay demonstrated GATA-2 acted as transcription-factor of NCDase gene expression. By establishing stable transfectants with decreased NCDase expression and activity, we clarified the significance of NCDase down-regulation for ATRA-induced neuronal differentiation. Those sub-clones showed both increased cellular ceramide and reduced cell growth as well as neuronal differentiation phenotypes. These results demonstrate that down-regulation of NCDase through ATRA-induced GATA-2 decrease plays an important role in induction of ceramide accumulation and neuronal differentiation in SH-SY5Y cells.

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by cAMP in HepG2 liver cells.

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a physiologic fibrinolysis inhibitor, is implicated in atherosclerosis. Cyclic adenosine monophosphate (cAMP) alters PAI-1 expression in several cells. Nevertheless, posttranscriptional regulation of PAI-1 has not been elucidated. To determine whether cAMP affects PAI-1 expression at posttranscriptional level, we determined promoter activity, mRNA levels, 3'-untranslated region (UTR) activity and protein levels of PAI-1 using HepG2 cells. cAMP decreased PAI-1 promoter activity at 24 h and mRNA expression at 4 h while it increased mRNA expression and accumulation of PAI-1 protein into media at 24 h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2 kb), and cAMP increased baseline luciferase activity of 3'-UTR of the 3.2 kb PAI-1 mRNA [3'-UTR (+1358-3176)] and 1 kb fragment of 3'-terminus of 3'-UTR of 3.2 kb mRNA [3'-UTR (+2177-3176)]. cAMP increased PAI-1 protein expression despite decrease in promoter activity, presumably by regulating PAI-1 expression at the posttranscriptional level and thereby affecting mRNA stability. The 53-nt fragment in 3'-UTR (+2591 to +2643 nt) was involved in posttranscriptional regulation by cAMP. Thus, cAMP can stabilize 3.2 kb PAI-1 mRNA mediated by specific effects on 3'-UTR, and these effects are associated with increased expression of PAI-1 protein.