Protein O-N-acetylglucosaminylation modulates promoter activities of cyclic AMP response element and activator protein 1 and enhances E-selectin expression on HuH-7 human hepatoma cells

High glucose accelerates O-N-acetylglucosaminylation (O-GlcNAcylation) of proteins and causes diabetic complications. In the present study, we found that treatment of HuH-7 human hepatoma cells with high glucose or the protein O-N-acetylglucosaminidase (O-GlcNAcase) inhibitor O-(2-acetoamide-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) increased the cell surface expression of E-selectin. A dual luciferase reporter assay indicated that high glucose and PUGNAc suppressed promoter activities of the cyclic AMP response element (CRE) and enhanced those of activator protein 1 (AP-1). Enhanced CRE promoter activities in HuH-7 cells treated with dibutyryl cAMP or co-transfected with a protein kinase A expression vector pFC-PKA that enhances the phosphorylation of CRE binding protein (CREB) were suppressed by PUGNAc. In contrast, PUGNAc further increased the enhanced AP-1 promoter activity in cells transfected with a mitogen-activated protein kinase kinase kinase expression vector pFC-MEKK that enhances c-Jun phosphorylation. Immuno-blotting using an anti-O-GlcNAc antibody revealed that high glucose and PUGNAc accelerated protein O-GlcNAcylation and that there were substantial differences in the O-GlcNAcylated proteins in the cytoplasmic and nuclear fractions. In addition, PUGNAc increased the nuclear import of O-GlcNAcylated CREB. These results suggest that protein O-GlcNAcylation modulates the promoter activities of E-selectin gene, suppression of CRE and enhancement of AP-1, and enhances E-selectin protein expression on hepatocytes.     

Release of GM-CSF and G-CSF by human arterial and venous smooth muscle cells: differential regulation by COX-2

In addition to their traditional contractile function, vascular smooth muscle cells can be stimulated under inflammatory conditions to release a range of potent biological mediators. Indeed, we and others have shown that human vascular smooth muscle release the colony stimulating factors (CSF) granulocyte macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF) as well as large amounts of prostaglandins following the induction of cyclo-oxygenase-2 (COX-2), when stimulated with cytokines. Here we demonstrate, for the first time, that co-induced COX-2 activity simultaneously suppresses GM-CSF release and potentiates G-CSF release by human vascular cells. Moreover, the differential regulation of GM-CSF and G-CSF release by COX-2 was mimicked by the prostacyclin (PGI(2)) mimetic, cicaprost. These observations suggest that PGI(2), released following the induction of COX-2, differentially regulates the release of GM-CSF (suppresses) and G-CSF (potentiates) from human vascular cells.     

Activation of cAMP-dependent signaling pathway induces mouse organic anion transporting polypeptide 2 expression

Rodent Oatp2 is a hepatic uptake transporter for such compounds as cardiac glycosides. In the present study, we found that fasting resulted in a 2-fold induction of Oatp2 expression in liver of mice. Because the cAMP-protein kinase A (PKA) signaling pathway is activated during fasting, the role of this pathway in Oatp2 induction during fasting was examined. In Hepa-1c1c7 cells, adenylyl cyclase activator forskolin as well as two cellular membrane-permeable cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, induced Oatp2 mRNA expression in a time- and dose-dependent manner. These three chemicals induced reporter gene activity in cells transfected with a luciferase reporter gene construct containing a 7.6-kilobase (kb) 5'-flanking region of mouse Oatp2. Transient transfection of cells with 5'-deletion constructs derived from the 7.6-kb Oatp2 promoter reporter gene construct, as well as 7.6-kb constructs in which a consensus cAMP response element (CRE) half-site CGTCA (-1808/-1804 bp) was mutated or deleted, confirms that this CRE site was required for the induction of luciferase activity by forskolin. Luciferase activity driven by the Oatp2 promoter containing this CRE site was induced in cells cotransfected with a plasmid encoding the protein kinase A catalytic subunit. Cotransfection of cells with a plasmid encoding the dominant-negative CRE binding protein (CREB) completely abolished the inducibility of the reporter gene activity by forskolin. In conclusion, induction of Oatp2 expression in liver of fasted mice may be caused by activation of the cAMP-dependent signaling pathway, with the CRE site (-1808/-1804) and CREB being the cis- and trans-acting factors mediating the induction, respectively.     

2-hydroxy-4'-methoxychalcone inhibits proliferation and inflammation of human aortic smooth muscle cells by increasing the expression of peroxisome proliferator-activated receptor gamma

Chalcone is a class of flavonoid compounds that are widely biosynthesized in plants. Epidemiological studies suggest that increased intake of flavonoids from fruits and vegetables reduces the risk of cardiovascular disease. However, the effect of chalcone on cardiovascular diseases has not been fully investigated. The aims of this study were to evaluate the antiatherosclerotic effect of 2-hydroxy-4'-methoxychalcone (AN07, a synthetic chalcone derivate) and to investigate its potential pharmacological mechanisms. Oxidized low-density lipoprotein (Ox-LDL) has been reported to stimulate proliferation of human aortic smooth muscle cells and that is one of the mechanisms resulting in atherosclerosis. In this study, we demonstrate that AN07 significantly inhibits the Ox-LDL-induced proliferation of human aortic smooth muscle cells. This effect is mediated via the inhibition of p44/42 mitogen-activated protein kinase and E-twenty six 1 phosphorylations. In the effect of anti-inflammation, AN07 decreases the Ox-LDL-stimulated upregulation of interleukin (IL) 1β and IL-6. In addition, AN07 acts synergistically with rosiglitazone and pioglitazone to inhibit the Ox-LDL-induced proliferation of human aortic smooth muscle cells and upregulation of cyclin D1, cyclin D3, IL-1β, and IL-6. These effects are a result of an increase in peroxisome proliferator-activated receptor gamma mRNA and protein expression stimulated by AN07 in human aortic smooth muscle cells. In conclusion, the chalcone derivate AN07 has versatile therapeutic potential against atherosclerosis by acting as peroxisome proliferator-activated receptor gamma inducer, p44/42 mitogen-activated protein kinase inhibitor, and cell cycle blocker.     

Stimulation of prostacyclin release from aortic smooth muscle cells by purine and pyrimidine nucleotides

ATP and ATP gamma S(10-100 microM) stimulated the release of prostacyclin (PGI2) from bovine aortic smooth muscle cells. This effect was reproduced by UTP, ITP and partially by GTP. ADP and ADP beta S, the P2X-selective agonist alpha, beta-methylene ATP (APCPP), AMP and adenosine were all inactive. This effect of ATP gamma S was not inhibited by Reactive Blue 2, an antagonist of P2Y receptors. The stimulation of PGI2 production in aortic smooth muscle cells by these nucleotides thus seems to involve receptors distinct from both P2X and P2Y subtypes, which are responsible for smooth muscle contraction and PGI2 release from endothelial cells, respectively.     

Upregulation of cyclooxygenase-2 by 4-nonylphenol is mediated through the cyclic amp response element activation pathway

The organic compound nonylphenol (NP) belongs to the family of alkylphenols and is a product of industrial synthesis formed during phenol alkylation. Nonylphenol is considered to be an endocrine disruptor due to weak ability to mimic estrogen and subsequently to disrupt the natural balance of hormones in a given organism. Since the endocrine and immune systems share portions of common signaling pathways, it is conceivable that NP may also affect immune system functions. However, the influence of NP on inflammation and macrophages responsiveness to NP is unclear. Thus, the effects of NP were investigated on cyclooxygenase (COX)-2 expression in cultured macrophages. NP induced COX-2 protein and gene expression in murine macrophage RAW264.7 cells and enhanced COX-2 promoter activity and prostaglandin E(2) production. Transfection of RAW264.7 cells with hCOX-2 or various deletion and mutation promoter constructs revealed that the cyclic AMP response element (CRE) was the predominant mediator responsive to NP-induced effects. Moreover, transfection with pCRE-Luc plasmid followed by immunoblotting demonstrated that NP activated CRE sites and CRE binding protein (CREB) phosphorylation. NP also increased nuclear CREB accumulation and CREB binding to the COX-2 promoter. Phosphatidylinositol 3 (PI3)-kinase, Akt, and the mitogen-activated protein kinases (MAP kinases) p38 and JNK were also significantly activated by NP. Our data demonstrate that NP induces COX-2 expression through the PI3-kinase/Akt/MAP kinases/CRE pathway. These findings provide insight into the signal transduction pathways involved in the inflammatory responses induced by NP in macrophages.     

Elevation of cyclic AMP by prostacyclin is accompanied by relaxation of bovine coronary arteries and contraction of rabbit aortic rings

The role of cyclic AMP (cAMP) in the control of vascular smooth muscle tension was examined by comparing the effects of prostacyclin (PGI2) on tension and cAMP levels in helical strips of bovine coronary arteries and in rabbit aortic rings, both denuded of endothelium. In bovine coronary arteries, PGI2 elevated cAMP levels and relaxed the muscles. The PGI2-induced cAMP elevation preceded the relaxation and both parameters were altered in a dose-dependent manner by increasing concentrations of PGI2 (0.3, 3 and 30 microM). These results are consistent with a role for cAMP as a mediator of vascular smooth muscle relaxation. Cyclic AMP levels were also elevated by PGI2 in a concentration- and time-dependent manner in rabbit aortic rings. However, in direct contrast to the results in the bovine coronary arteries, PGI2-induced elevation of cAMP in the aortic rings was accompanied by contraction rather than relaxation. Isoproterenol, a drug which is generally believed to relax smooth muscles by virtue of its ability to increase tissue levels of cAMP, relaxed PGI2-contracted aortic rings with no further elevation of cAMP beyond that caused by the PGI2 alone. These results demonstrate that cAMP elevation and relaxation of vascular smooth muscle are not always well correlated. It is possible that some form of functional compartmentalization of cAMP or cAMP-dependent protein kinase exists in these tissues.     

参麦注射液对培养大鼠主动脉平滑肌细胞增殖的影响和机理研究

目的 研究参麦注射液对血管平滑肌细胞增殖的影响和机理。方法 以培养的大鼠主动脉血管平滑肌细胞为模型，通过细胞计数及测量3H-胸腺嘧啶核苷掺入量观察细胞增殖情况，同时测定培养液中超氧化物歧化酶(SOD)活性、脂质过氧化物、前列环素(PGI2)的含量，比较分析加入不同剂量参麦注射液后上述指标的变化和相互关系。结果 不同剂量参麦注射液组的平滑肌细胞增殖及3H—胸腺嘧啶核苷掺入量均显著低于对照组，SOD活性及PGI2含量显著高于对照组，脂质过氧化程度显著降低。结论 参麦注射液对血管平滑肌细胞增殖具有抑制作用，并呈剂量依赖关系，其作用可能与增加SOD活性、降低脂质过氧化物水平，升高PGI2／TXA2有关。     

Transcriptional regulation of mu opioid receptor gene by cAMP pathway

The utility of morphine for the treatment of chronic pain is hindered by the development of tolerance. Fentanyl has been shown to be a potent analgesic with a lower propensity to produce tolerance and physical dependence in the clinical setting. Previous finding has shown that fentanyl induces mu opioid receptor gene expression in PC-12 cells (Brain Res 859:217-223, 2000). In this report, we aim to identify the molecular mechanism of mu-opioid receptor (MOR) gene regulation by fentanyl. We demonstrated that the 4.7-kilobase MOR promoter could be induced by fentanyl in PC-12 cells, and we defined a partial cAMP response element (CRE) located at -106/-111 in 5'-untranslated region of the MOR gene. In electrophoretic mobility shift assay, cAMP response element-binding protein (CREB) was found in the protein-DNA complex formed on the CRE box. CREB was phosphorylated after forskolin induction, and both CREB and CREB-binding protein (CBP) binding to the endogenous MOR promoter was increased by forskolin in chromatin immunoprecipitation assay. The functional role of CREB in the induction of MOR gene was further elucidated by an experiment in which a dominant-negative mutant CREB, CREB-S133A, abolished the forskolin-mediated MOR induction. Moreover, we found that this CRE box is conserved in mouse, rat, and human MOR gene, implying physiological relevance in different species. Collectively, this study demonstrated that fentanyl-triggered MOR gene induction was mediated by the sequential activation of CREB and the binding of CREB and CBP to MOR promoter, thus provides direct evidence for lower propensity of fentanyl to produce tolerance.     

Double blockade of cell cycle progression by coptisine in vascular smooth muscle cells

Coptisine, an isoquinoline alkaloid isolated from rhizome of Coptis japonica, inhibits proliferation of vascular smooth muscle cells (VSMCs). The aim of this study was to evaluate the action of coptisine, along with berberine (a structurally similar isoquinoline alkaloid), on progression of the cell cycle in VSMCs. Coptisine displayed antiproliferative action against VSMCs by blocking the cell cycle at G(1) and G(2)/M phases. The G(1) block was shown by inhibition of [(3)H]thymidine incorporation into VSMCs at coptisine concentrations higher than 15 microM. The mechanism underlying the G(1) arrest involved a decrease in cyclin D1 protein, although cyclin E, A, and B were not affected by coptisine treatment. The selective reduction in cyclin D1 protein was mainly attributable to accelerated proteolysis via proteasome-dependent pathway, since it was inhibited by a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norleucinal (MG132) and further the mRNA level of cyclin D1, protein synthesis, and mitogen-activated protein kinase (MAPK) activity remained unaltered. The mechanism underlying the G(2)/M arrest involved partial inhibition of tubulin polymerization, which was apparent at coptisine concentration of 3 microM. Berberine arrested the cell cycle at G(1) phase via a mechanism identical with coptisine, but did not cause block at G(2)/M phase. The results demonstrate that a small difference in the structure between isoquinoline alkaloids produces a big difference in activity, and that coptisine has a unique double action in arresting the cell cycle of VSMCs.