ApoA-I mimetics favorably impact cyclooxygenase 2 and bioactive lipids that may contribute to cardiometabolic syndrome in chronic treated HIV.

OBJECTIVE: We investigated whether apolipoprotein A-I (apoA-I) mimetic peptides 4F and 6F can be a novel therapeutic strategy to reduce blood and gut bioactive lipids, proinflammatory effects of endotoxin (LPS) and aberrant activation of cyclooxygenase 2 (COX-2) as instigators of increased risk for cardiometabolic disease in chronic treated HIV. METHODS: We used two humanized murine models of chronic treated HIV infection (n = 109 mice) and gut explants from HIV infected (n = 10) persons to determine whether Tg6F and 4F attenuate in vivo and ex vivo increased blood and gut bioactive lipids (measured by mass spectrometry) and intestinal protein levels of COX-2 (measured by immunoassays) in chronic treated HIV. RESULTS: In these models of HIV, when compared to HIV-1 infected mice on antiretroviral therapy (ART) alone, oral Tg6F in combination with ART attenuated increases in plasma and gut bioactive lipids (and particularly COX lipids) and intestinal COX-2. 4F and Tg6F also reduced ex vivo production of COX-2 protein and associated secretion of bioactive lipids in gut explants from HIV-1 infected persons treated with LPS. CONCLUSION: ApoA-I mimetics favorably impact the proinflammatory effects of LPS, COX-2 and production of bioactive lipids that collectively drive gut and systemic inflammation in chronic treated HIV. Given prior experimental evidence that the proinflammatory effects of LPS, COX-2 and gut dysfunction contribute to cardiometabolic syndrome in chronic HIV, apoA-I mimetic peptides may be a novel therapy to treat cardiometabolic syndrome in chronic HIV.

Remodeling of the HDL proteome with treatment response to abatacept or adalimumab in the AMPLE trial of patients with rheumatoid arthritis.

BACKGROUND AND AIMS: To evaluate changes in the high-density lipoprotein (HDL) proteome and HDL function in active rheumatoid arthritis (RA) patients initiating therapy with abatacept or adalimumab in the Abatacept Versus Adalimumab Comparison in Biologic-Naïve RA Subjects with Background Methotrexate (AMPLE) study. METHODS: Ultra high-pressure liquid chromatography (UHPLC) coupled with ion mobility mass spectrometry (LC-IM-MS) was used to analyze proteins associated with immunoaffinity-captured HDL from plasma of 30 patients with RA randomized to either abatacept (n = 15) or adalimumab (n = 15) therapy. Paraoxonase 1 (PON1) activity, HDL anti-oxidant capacity, cholesterol profiles, and homocysteine levels were also measured at baseline and following treatment. Repeated-measures analyses were performed using mixed-effect linear models to model the within-subject covariance over time. RESULTS: In models controlling for age, sex and treatment group, improvement in inflammation measured by decreases in CRP was associated with improvement in HDL function and changes in several HDL-associated proteins including significant decreases in lipopolysaccharide-binding protein, serum amyloid A-I (SAA-I) and inter-alpha-trypsin inhibitor heavy chain H4 (p values < 0.05). Improvement in disease activity was also associated with changes in multiple HDL-associated proteins. Adalimumab was associated with higher PON1 activity, HDL-associated serotransferrin, and HDL-associated immunoglobulin J chain, and lower HDL-associated SAA-I over time compared with abatacept. CONCLUSIONS: Improvement in inflammation associated with treatment of RA, using either abatacept or adalimumab in the AMPLE study, was associated with improvement in HDL function and significant alterations in the HDL proteome, including proteins involved in the immune response, proteinase inhibition, and lipid metabolism.

A short-term increase in dietary cholesterol and fat intake affects high-density lipoprotein composition in healthy subjects.

BACKGROUND AND AIMS: High-cholesterol and high-fat diets alter biochemical composition and anti-oxidant properties of high-density lipoproteins (HDL) in animals. Whether this occurs in humans is unknown. Therefore, we examined the effect of a short-term elevation in dietary cholesterol and fat intake on HDL composition in healthy subjects. METHODS AND RESULTS: In a randomized, crossover clinical trial, 14 healthy young volunteers followed a 14-day low-cholesterol/low-fat diet (LChF) and a 14-day isocaloric high-cholesterol/high-fat diet (HChF) in a random order. After each diet, we measured HDL concentrations of hydroxyeicosatetraenoic acids (HETE), hydroxyoctadecadienoic acids (HODE), and haptoglobin, as well as serum amyloid A (SAA) and paroxonase-1 activity (PON-1). HDL concentrations of 15-HETE (+254%, p = 0.002), 5-HETE (+116%, p = 0.004), 13-HODE (+102%, p = 0.049), and SAA levels (+75%, p = 0.007) were significantly higher after the HChF than after the LChF. Furthermore, haptoglobin was marginally increased (+32%, p = 0.091) while PON-1 activity was unaffected (-16%, p = 0.366) by the HChF. CONCLUSION: In healthy subjects, a short-term elevation in dietary cholesterol and fat intake increases HDL lipid hydroperoxide content (15-HETE, 5-HETE, 13-HODE) and SAA levels, which are key features of dysfunctional HDL. This is the first study showing that a physiologic manipulation of dietary cholesterol and fat intake affects HDL lipidome and proteome in healthy subjects independently of weight changes. CLINICAL TRIAL REGISTRATION: NCT02549144.

Deep sequencing in library selection projects: what insight does it bring?

High throughput sequencing is poised to change all aspects of the way antibodies and other binders are discovered and engineered. Millions of available sequence reads provide an unprecedented sampling depth able to guide the design and construction of effective, high quality naïve libraries containing tens of billions of unique molecules. Furthermore, during selections, high throughput sequencing enables quantitative tracing of enriched clones and position-specific guidance to amino acid variation under positive selection during antibody engineering. Successful application of the technologies relies on specific PCR reagent design, correct sequencing platform selection, and effective use of computational tools and statistical measures to remove error, identify antibodies, estimate diversity, and extract signatures of selection from the clone down to individual structural positions. Here we review these considerations and discuss some of the remaining challenges to the widespread adoption of the technology.

HDL lipid composition is profoundly altered in patients with type 2 diabetes and atherosclerotic vascular disease.

BACKGROUND AND AIMS: We have previously shown that the anti-inflammatory and anti-oxidant functions of HDL are impaired in T2D patients. In this study, we examined whether HDL from T2D patients contains elevated levels of oxidized fatty acids and whether those levels correlate with cardiovascular disease (CVD). METHODS AND RESULTS: HETEs and HODEs on HDL were determined by LC-MS/MS in 40 non-diabetic controls (ND), 40 T2D without CVD (D⁺CVD⁻) and 38 T2D with known history of CVD (D⁺CVD⁺). HDL oxidant index was evaluated by a cell-free assay using dichlorofluorescein. Twenty-six randomly selected subjects from the three groups underwent coronary calcium score evaluation (CAC). Major cardiovascular risk factors were similar among the groups. HETEs and HODEs content were significantly increased in HDL from D⁺CVD⁺ when compared to D⁺CVD⁻ and ND patients. HDL oxidant index was not different among the three groups; however, it was significantly higher in patients with CAC score >100 when compared to patients with CAC score <100. CONCLUSION: Patients with D⁺CVD⁻ and D⁺CVD⁺ are characterized by a severe, graded enrichment of oxidized fatty acids on HDL. In the present study, a loss of HDL function (as estimated by the HDL oxidant index) is observed only in patients with more advanced atherosclerosis.

PON3 is upregulated in cancer tissues and protects against mitochondrial superoxide-mediated cell death.

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.

Hand-off education and evaluation: piloting the observed simulated hand-off experience (OSHE).

AIM: The Observed Simulated Hand-off Experience (OSHE) was created to evaluate medical students' sign-out skills using a real-time assessment tool, the Hand-off CEX. SETTING: Thirty-two 4th year medical students participated as part of an elective course. PROGRAM DESCRIPTION: One week following an interactive workshop where students learned effective hand-off strategies, students participated in an experience in which they performed a hand-off of a mock patient using simulated history and physical examination data and a brief video. PROGRAM EVALUATION: Internal medicine residents served as standardized hand-off receivers and were trained on expectations. Students were provided feedback using a newly developed Hand-off CEX, based on the "Mini-CEX," which rates overall hand-off performance and its components on a 9-point Likert-type scale. Outcomes included performance ratings and pre- and post-student self-assessments of hand-off preparedness. Data were analyzed using Wilcoxon signed-rank tests and descriptive statistics. Resident receivers rated overall student performance with a mean score of 6.75 (range 4-9, maximum 9). Statistically significant improvement was observed in self-perceived preparedness for performing an effective hand-off (67% post- vs. 27% pre-reporting 'well-prepared,' p<0.009). DISCUSSION: This brief, standardized hand-off training exercise improved students' confidence and was rated highly by trained observers. Future work focuses on formal validation of the Hand-off CEX instrument. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11606-009-1170-y) contains supplementary material, which is available to authorized users.

DNA immunization using a non-viral promoter.

Most DNA vaccines rely on strong viral promoters to optimize levels of transgene expression. Some studies have demonstrated that the potency of viral promoters does not necessarily correlate with DNA vaccine efficacy in vivo. This has partly been attributed to downregulation of these promoters by cytokines such as interferon gamma induced by the CpG motives of these vaccines. In an attempt to avoid downregulation of viral promoters by IFN-gamma, we tested vaccine vectors driven by the MHC class II promoter. To enhance the activity of this promoter, another plasmid expressing the human MHC class II transactivator driven by a viral promoter, the native IFN-gamma inducible CIITA type IV promoter (PIV) or a synthetic promoter containing IFN-gamma inducible elements was co-inoculated. Our data show that a non-viral promoter such as the MHC class II promoter tested in this study can indeed be used in DNA vaccines.

Over- and ectopic expression of Wnt3 causes progressive loss of ameloblasts in postnatal mouse incisor teeth.

Intercellular signaling is essential for the development of teeth during embryogenesis and in maintenance of the continuously growing incisor teeth in postnatal rodents. WNT intercellular signaling molecules have been implicated in the regulation of tooth development, and the Wnt3 gene shows specific expression in the enamel knot at the cap stage. We demonstrate here that Wnt3 also is expressed in specific epithelial cell layers in postnatal incisor teeth. To begin to delineate the functions of Wnt3 in developing and postnatal teeth, we determined the effects of over- and ectopic expression of Wnt3 in the tooth epithelium of mice carrying a keratin 14-Wnt3 transgene. Expression of the transgene caused a progressive loss of ameloblasts from postnatal lower incisor teeth. Loss of ameloblasts may be due to defective proliferation or differentiation of ameloblast precursors, progressive apoptosis of ameloblasts, or loss of ameloblast stem cells.

Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) upregulates a spectrum of inflammatory cytokines and adhesion molecules different from those induced by classic inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide. Interestingly, Ox-PAPC also induces the expression of a set of proteins similar to those induced by TNF-alpha or lipopolysaccharide, which include the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8. To elucidate the molecular mechanisms of Ox-PAPC-induced gene expression and to determine whether Ox-PAPC and other inflammatory mediators such as TNF-alpha utilize common signaling pathways, we examined the transcriptional regulation of IL-8 by Ox-PAPC and TNF-alpha in human aortic endothelial cells. Both Ox-PAPC and TNF-alpha induced the expression of IL-8 mRNA in a dose-dependent fashion; however, the kinetics of IL-8 mRNA accumulation between the 2 ligands differed. Ox-PAPC-induced IL-8 mRNA was seen as early as 30 minutes, peaked between 4 and 8 hours, and decreased substantially by 24 hours. In contrast, TNF-alpha-induced IL-8 mRNA synthesis was elevated at 30 minutes, peaked at 2 hours, and reached basal/undetectable levels by 6 hours. Actinomycin D experiments suggested that both Ox-PAPC and TNF-alpha regulate the expression of IL-8 at the transcriptional level. Furthermore, the half-life of IL-8 mRNA for both ligands was similar (<30 minutes), suggesting that mRNA stability was not responsible for the differences in the kinetics of IL-8 accumulation between the 2 ligands. Transient transfection studies with reporter constructs containing 1.48 kb of the IL-8 promoter identified an Ox-PAPC-specific response region between -133 and -1481 bp of the IL-8 promoter. In contrast, TNF-alpha activation of the IL-8 promoter was mediated almost entirely through the nuclear factor-kappaB and activation protein-1 response elements present between -70 and -133 bp of the IL-8 promoter. Thus, although Ox-PAPC and TNF-alpha both induced IL-8 synthesis, our data suggest that the 2 ligands utilize different mechanisms in the regulation of IL-8 transcription.

Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein.

The oxidation of apolipoprotein B-containing lipoproteins and cell membrane lipids is believed to play an integral role in the development of fatty streak lesions, an initial step in atherogenesis. We have previously shown that two antioxidant-like enzymes, paraoxonase (PON)-1 and PON3, are high density lipoprotein-associated proteins capable of preventing the oxidative modification of low density lipoprotein (LDL) (Reddy, S. T., Wadleigh, D. J., Grijalva, V., Ng, C., Hama, S., Gangopadhyay, A., Shih, D. M., Lusis, A. J., Navab, M., and Fogelman, A. M. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 542-547). In the present study, we demonstrate that PON2 (i) is not associated with high density lipoprotein; (ii) has antioxidant properties; and (iii) prevents LDL lipid peroxidation, reverses the oxidation of mildly oxidized LDL (MM-LDL), and inhibits the ability of MM-LDL to induce monocyte chemotaxis. The PON2 protein was overexpressed in HeLa cells using the tetracycline-inducible ("Tet-On") system, and its antioxidant capacity was measured in a fluorometric assay. Cells that overexpressed PON2 showed significantly less intracellular oxidative stress following treatment with hydrogen peroxide or oxidized phospholipid. Moreover, cells that overexpressed PON2 were also less effective in oxidizing and modifying LDL and, in fact, were able to reverse the effects of preformed MM-LDL. Our results suggest that PON2 possesses antioxidant properties similar to those of PON1 and PON3. However, in contrast to PON1 and PON3, PON2 may exert its antioxidant functions at the cellular level, joining the host of intracellular antioxidant enzymes that protect cells from oxidative stress.

Modulation of omega-3/omega-6 polyunsaturated ratios with dietary fish oils in men with prostate cancer.

OBJECTIVES: The results of epidemiologic and animal studies support the role of a low-fat diet supplemented with omega-3 fatty acids contained in fish oil in preventing the development and progression of prostate cancer. As a first step in studying the role of a low-fat, fish oil-supplemented (LF/FOS) diet in a clinical setting, we conducted a prospective study in men with untreated prostate cancer to evaluate whether a 3-month dietary intervention affects the ratio of omega-3 to omega-6 fatty acids in plasma and gluteal fat. In addition, we evaluated the feasibility of studying cyclooxygenase-2 (COX-2) expression in serial prostate needle biopsy specimens before and after the diet. METHODS: Nine men with untreated prostate cancer consumed an LF/FOS diet for 3 months. Plasma, gluteal adipose tissue, and prostate needle biopsy specimens were obtained from each patient before and after the intervention. The fatty acid compositions of the plasma and gluteal adipose tissue were determined by gas-liquid chromatography, and the COX-2 expression in the prostatic tissue specimens was determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Short-term intervention with an LF/FOS diet caused a significant increase in the omega-3/omega-6 fatty acid ratio in plasma (P = 0.002) and gluteal adipose tissue (P = 0.002) in men with prostate cancer. The COX-2 expression in prostatic tissue was quantitated by RT-PCR in 7 of 9 patients, and COX-2 expression decreased in 4 of these 7 patients. CONCLUSIONS: A short-term dietary intervention in men with prostate cancer leads to a significant increase in the omega-3/omega-6 fatty acid ratios in plasma and adipose tissue. The potential for this diet to prevent the development and progression of prostate cancer by way of altered COX-2 expression and prostaglandin production in prostatic tissue requires further study.

A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids.

We have developed a novel and rapid cell-free assay of the ability of HDL to prevent the formation of or inactivate oxidized phospholipids. HDL was tested for its ability to inhibit the oxidation of LDL, or inhibit the oxidation of l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) by hydroperoxyoctadecadienoic acid (HPODE), or inactivate oxidized PAPC (Ox-PAPC). In each case the fluorescent signal generated in the presence of the test substances and the test HDL was determined. As little as 2.5 microg of normal human HDL cholesterol significantly inhibited the fluorescent signal generated by Ox-PAPC; results did not differ regardless of whether the HDL was prepared by gel electrophoresis, fast protein liquid chromatography, or dextran sulfate precipitation. HDL from each of 27 patients with coronary atherosclerosis failed to inhibit the fluorescent signal generated by a control LDL, whereas HDL from each of 31 matched normal subjects with the same levels of HDL cholesterol significantly inhibited the signal. Results from an established cell-based assay (Navab, M., S. Hama, J. Cooke, G. M. Anantharamaiah, M. Chaddha, L. Jin, G. Subbanagounder, K. F. Faull, S. T. Reddy, N. E. Miller, and A. M. Fogelman. 2000. J. Lipid Res. 41: 1481-1494) were identical. HDL from the patients also failed to inhibit the fluorescent signal generated from PAPC plus HPODE (10 of 10 patients) whereas HDL from matched controls (8 of 8 patients) significantly inhibited the fluorescent signal. We conclude that this new assay has the potential to allow widespread testing of the hypothesis that HDL that is dysfunctional in preventing the formation or inactivating oxidized phospholipids may play an important role in the development of atherosclerosis.

Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids.

Paraoxonase-1 (PON1) is a secreted protein associated primarily with high density lipoprotein (HDL) and participates in the prevention of low density lipoprotein (LDL) oxidation. Two other paraoxonase (PON) family members, namely, PON2 and PON3, have been identified. In this study, we report the cloning and characterization of the human PON3 gene from HepG2 cells. Tissue Northern analysis identifies an approximately 1.3-kb transcript for PON3 primarily in the liver. PON3-specific peptide antibodies detect an approximately 40-kDa protein associated with HDL and absent from LDL. Pretreatment of cultured human aortic endothelial cells with supernatants from HeLa Tet On cell lines overexpressing PON3 prevents the formation of mildly oxidized LDL and inactivates preformed mildly oxidized LDL. In contrast to PON1, PON3 is not active against the synthetic substrates paraoxon and phenylacetate. Furthermore, PON3 expression is not regulated in HepG2 cells by oxidized phospholipids and is not regulated in the livers of mice fed a high-fat atherogenic diet.

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1.

Apolipoprotein A-I (apoA-I) and an apoA-I peptide mimetic removed seeding molecules from human low density lipoprotein (LDL) and rendered the LDL resistant to oxidation by human artery wall cells. The apoA-I-associated seeding molecules included hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE). LDL from mice genetically susceptible to fatty streak lesion formation was highly susceptible to oxidation by artery wall cells and was rendered resistant to oxidation after incubation with apoA-I in vitro. Injection of apoA-I (but not apoA-II or murine serum albumin) into mice rendered their LDL resistant to oxidation within 3 h. Infusion of apoA-I into humans rendered their LDL resistant to oxidation within 6 h. We conclude that 1) oxidation of LDL by artery wall cells requires seeding molecules that include HPODE and HPETE; 2) LDL from mice genetically susceptible to atherogenesis is more readily oxidized by artery wall cells; and 3) normal HDL and its components can remove or inhibit the activity of lipids in freshly isolated LDL that are required for oxidation by human artery wall cells.

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3.

Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.

Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages.

Cyclooxygenase-2 (COX-2), the enzyme primarily responsible for induced prostaglandin synthesis, is an immediate early gene induced by endotoxin in macrophages. We investigated the cis-acting elements of the COX-2 5'-flanking sequence, the transcription factors and signaling pathways responsible for transcriptional activation of the COX-2 gene in endotoxin-treated murine RAW 264.7 macrophages. Luciferase reporter constructs with alterations in presumptive cis-acting transcriptional regulatory elements demonstrate that the cyclic AMP-response element and two nuclear factor interleukin-6 (CCAAT/enhancer-binding protein (C/EBP)) sites of the COX-2 promoter are required for optimal endotoxin-dependent induction. In contrast, the E-box and NF-kappaB sites are not required for endotoxin-dependent induction. Inhibition of endotoxin-induced NF-kappaB activation by expression of an inhibitor-kappaB alpha mutant does not block endotoxin-dependent COX-2 reporter activity. Overexpression of c-Jun, C/EBPbeta, and C/EBPdelta enhances induction of the COX-2 reporter, while overexpression of cyclic AMP-response element-binding protein or "dominant negative" C/EBPbeta represses COX-2 induction. In addition, endotoxin rapidly and transiently elicits c-Jun phosphorylation in RAW 264.7 macrophages. Cotransfection of the COX-2 reporter with dominant negative expression vectors shows that endotoxin-induced COX-2 gene expression requires signaling through a Ras-independent pathway involving the adapter protein ECSIT and the signaling kinases MEKK1 and JNK. In contrast, endotoxin-induced COX-2 reporter activity is not blocked by overexpression of dominant-negative forms of Raf-1, ERK1, or ERK2.

Transcriptional regulation of the cyclooxygenase-2 gene in activated mast cells.

Activation of mast cells by aggregation of their IgE receptors induces rapid and transient synthesis of cyclooxygenase-2 (COX-2). In this study we investigated (i) the cis-acting response elements and transcription factors active at the COX-2 promoter and (ii) the signal transduction pathways mediating COX-2 induction following aggregation of mast cell IgE receptors. Transient transfection assays with COX-2 promoter/luciferase constructs suggest that a consensus cyclic AMP response element is essential for induced COX-2 expression. Cotransfection studies with plasmids expressing c-Jun, dominant negative Ras, dominant negative c-Jun NH(2)-terminal kinase, and dominant negative MEKK1 demonstrate that activation of the Ras/MEKK1/c-Jun NH(2)-terminal kinase/c-Jun pathway is required for COX-2 promoter-mediated luciferase expression. Attenuation of COX-2 promoter activity by dominant negative constructs for Raf-1, ERK1, and ERK2 suggests that the Ras/Raf-1/extracellular signal-regulated kinase pathway is also necessary for COX-2 induction. Although mutating the two NF-IL6 sites individually did not affect COX-2 promoter activity, mutating both NF-IL6 sites substantially inhibits COX-2 promoter activity. Moreover, overexpression of wild type CCAAT/enhancer-binding protein-beta (C/EBPbeta) augments COX-2 promoter activity in activated mast cells and cotransfection of a dominant negative C/EBPbeta construct completely blocks COX-2 promoter/luciferase expression. Our data suggest that in activated mast cells, a Ras/MEKK1/c-Jun NH(2)-terminal kinase signal transduction pathway activating c-Jun, a Ras/Raf-1/extracellular signal-regulated kinase pathway, and activated C/EBPbeta facilitate COX-2 induction via the cyclic AMP response element and NF-IL6 sites of the COX-2 promoter.