Carla Cook

All-trans-retinoic acid induces manganese superoxide dismutase in human neuroblastoma through NF-κB

Retinoids are signaling molecules that are involved in proliferation, differentiation, and apoptosis during development. Retinoids exert their effects, in part, by binding to nuclear receptors, thereby altering gene expression. Clinical use of retinoids in the treatment of neuroblastoma is of interest due to their success in management of acute promyelocytic leukemia. Using the SK-N-SH human neuroblastoma cell line we investigated the effects of the differentiation agent all-trans-retinoic acid (ATRA) on the expression of manganese superoxide dismutase (MnSOD), an enzyme previously shown to enhance differentiation in vitro. Manganese superoxide dismutase mRNA, protein, and activity levels increased in a time-dependent manner upon treatment with ATRA. Nuclear levels of the NF-kappaB proteins p50 and p65 increased within 24 h of ATRA administration. This increase paralleled the degradation of the cytoplasmic inhibitor IkappaB-beta. Furthermore an increase in DNA binding to a NF-kappaB element occurred within a 342-bp enhancer (I2E) of the SOD2 gene with 10 microM ATRA treatment. Reporter analysis showed that ATRA-mediated I2E-dependent luciferase expression was attenuated upon mutation of the NF-kappaB element, suggesting a contribution of this transcription factor to retinoid-mediated upregulation of MnSOD. This study identifies SOD2 as a retinoid-responsive gene and demonstrates activation of the NF-kappaB pathway in response to ATRA treatment of SK-N-SH cells. These results suggest that signaling events involving NF-kappaB and SOD2 may contribute to the effects of retinoids used in cancer therapy.

Nicotinic acetylcholine receptor signaling in atherogenesis.

Smoking is a major risk factor for the development of atherosclerosis, stroke and myocardial infarction. Cigarette smoke consists of a complex mixture of about 4000 compounds. Out of these, polycyclic hydrocarbons, tobacco-specific nitrosamines, oxidizing agents and carbon monoxide have been implicated in the development of atherosclerosis. Recent studies have shown that nicotine (the addictive component of cigarettes) binds to high affinity cell-surface receptors and accelerates the atherogenic process. These receptors are called nicotinic acetylcholine receptors (nAChRs) and are expressed ubiquitously in almost all cells existing in the blood vessels. The present review summarizes the pro-atherogenic effects of nAChR ligands such as nicotine and tobacco nitrosamines. The contribution of different nAChR subunits in plaque growth, progression and neovascularization are discussed in detail. The signaling pathways underlying the actions of the nAChRs ligands in blood vessels are also described. Finally, the feasibility of nAChR ligands as therapeutic targets for atherosclerosis is summarized. We believe that the information presented in this review is relevant for atherosclerosis patients who are active smokers, exposed to environmental tobacco smoke or use nicotine patches or gums for smoking cessation.

Age and sex mediated changes in epicardial fat adipokines.

Aging, which is an independent risk factor for heart disease, alters body fat mass and its function. Epicardial fat plays an important physiological and pathophysiological role on cardiac structure and function. This study investigated if aging altered the abundance of epicardial (EF) and abdominal fat (AF) derived mediators in a sex dependent manner in female and male Fischer 344×Brown Norway hybrid (FBN) rats. EF and AF were obtained from 48 female and male, young (6 months), aged (26/30 months) and very aged (30/36 months) FBN rats. Adipose derived anti-inflammatory and pro-inflammatory mediators were measured using ELISA, adipokine array and real-time qPCR. No dramatic changes in circulating lipids other than a higher triglyceride and high density lipoprotein in aged females and a significantly increased circulating adiponectin (p<0.005) in aged rats were observed. Real time PCR results showed that compared to 6 months old female rats, the aged (26 months) and very aged (30 months) rats had significantly lower levels of EF genes: adiponectin (p<0.005), PPARγ (p<0.01, 0.005), IL-6 (p<0.01) and PAI-1 (p<0.01, 0.01), respectively, but not in AF. In contrast, the male rats exhibited an increase in IL-6 in EF (p<0.005) but a decrease in adiponectin and PPARγ in AF with aging. These changes might be attributed to differences in adipocyte make-up or macrophage infiltration. In conclusion, aging had a more profound impact on EF derived mediators in female rather than male rats, which might help explain the increased risk to cardiovascular disease seen in older women.

Oxidation Sensitive Nociception Involved in Endometriosis Associated Pain

Abstract Endometriosis is a disease characterized by the growth of endometrial tissue outside the uterus and is associated with chronic pelvic pain. Peritoneal fluid (PF) of women with endometriosis is a dynamic milieu and is rich in inflammatory markers, pain-inducing prostaglandins prostaglandin E2 and prostaglandin F2&agr;, and lipid peroxides; and the endometriotic tissue is innervated with nociceptors. Our clinical study showed that the abundance of oxidatively modified lipoproteins in the PF of women with endometriosis and the ability of antioxidant supplementation to alleviate endometriosis-associated pain. We hypothesized that oxidatively modified lipoproteins present in the PF are the major source of nociceptive molecules that play a key role in endometriosis-associated pain. In this study, PF obtained from women with endometriosis or control women were used for (1) the detection of lipoprotein-derived oxidation-sensitive pain molecules, (2) the ability of such molecules to induce nociception, and (3) the ability of antioxidants to suppress this nociception. LC–MS/MS showed the generation of eicosanoids by oxidized-lipoproteins to be similar to that seen in the PF. Oxidatively modified lipoproteins induced hypothermia (intracerebroventricular) in CD-1 mice and nociception in the Hargreaves paw withdrawal latency assay in Sprague-Dawley rats. Antioxidants, vitamin E and N-acetylcysteine, and the nonsteroidal anti-inflammatory drug indomethacin suppressed the pain-inducing ability of oxidatively modified lipoproteins. Treatment of human endometrial cells with oxidatively modified lipoproteins or PF from women with endometriosis showed upregulation of similar genes belonging to opioid and inflammatory pathways. Our finding that oxidatively modified lipoproteins can induce nociception has a broader impact not only on the treatment of endometriosis-associated pain but also on other diseases associated with chronic pain.

MicroRNA Regulation of Adipose Derived Stem Cells in Aging Rats

Background Perturbations in abdominal fat secreted adipokines play a key role in metabolic syndrome. This process is further altered during the aging process, probably due to alterations in the preadipocytes (aka. stromal vascular fraction cells-SVF cells or adipose derived stem cells-ASCs) composition and/or function. Since microRNAs regulate genes involved both in development and aging processes, we hypothesized that the impaired adipose function with aging is due to altered microRNA regulation of adipogenic pathways in SVF cells. Methodology and Principal Findings Alterations in mRNA and proteins associated with adipogenic differentiation (ERK5 and PPARg) but not osteogenic (RUNX2) pathways were observed in SVF cells isolated from visceral adipose tissue with aging (6 to 30 mo) in female Fischer 344 x Brown Norway Hybrid (FBN) rats. The impaired differentiation capacity with aging correlated with altered levels of miRNAs involved in adipocyte differentiation (miRNA-143) and osteogenic pathways (miRNA-204). Gain and loss of function studies using premir or antagomir-143 validated the age associated adipocyte dysfunction. Conclusions and Significance Our studies for the first time indicate a role for miRNA mediated regulation of SVF cells with aging. This discovery is important in the light of the findings that dysfunctional adipose derived stem cells contribute to age related chronic diseases.

Atherogenic ω-6 Lipids Modulate PPAR- EGR-1 Crosstalk in Vascular Cells

Atherogenic ω-6 lipids are physiological ligands of peroxisome proliferator-activated receptors (PPARs) and elicit pro- and antiatherogenic responses in vascular cells. The objective of this study was to investigate if ω-6 lipids modulated the early growth response-1 (Egr-1)/PPAR crosstalk thereby altering vascular function. Rat aortic smooth muscle cells (RASMCs) were exposed to ω-6 lipids, linoleic acid (LA), or its oxidized form, 13-HPODE (OxLA) in the presence or absence of a PPARα antagonist (MK886) or PPARγ antagonist (GW9662) or PPAR-specific siRNA. Our results demonstrate that ω-6 lipids, induced Egr-1 and monocyte chemotactic protein-1 (MCP-1) mRNA and protein levels at the acute phase (1–4 hrs) when PPARα was downregulated and at subacute phase (4–12 hrs) by modulating PPARγ, thus resulting in altered monocyte adhesion to RASMCs. We provide novel insights into the mechanism of action of ω-6 lipids on Egr-1/PPAR interactions in vascular cells and their potential in altering vascular function.

Aryl Hydrocarbon Receptor Ligands Inhibit IGF-II and Adipokine Stimulated Breast Cancer Cell Proliferation

Obesity increases human cancer risk and the risk for cancer recurrence. Adipocytes secrete paracrine factors termed adipokines that stimulate signaling in cancer cells that induce proliferation. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that plays roles in tumorigenesis, is regulated by exogenous lipophilic chemicals, and has been explored as a therapeutic target for cancer therapy. Whether exogenous AHR ligands modulate adipokine stimulated breast cancer cell proliferation has not been investigated. We provide evidence that adipocytes secrete insulin-like growth factor 2 (IGF-2) at levels that stimulate the proliferation of human estrogen receptor (ER) positive breast cancer cells. Using highly specific AHR ligands and AHR short interfering RNA (AHR-siRNA), we show that specific ligand-activated AHR inhibits adipocyte secretome and IGF-2-stimulated breast cancer cell proliferation. We also report that a highly specific AHR agonist significantly (P < 0.05) inhibits the expression of E2F1, CCND1 (known as Cyclin D1), MYB, SRC, JAK2, and JUND in breast cancer cells. Collectively, these data suggest that drugs that target the AHR may be useful for treating cancer in human obesity.

ω-6 lipids regulate PPAR turnover via reciprocal switch between PGC-1 alpha and ubiquitination

OBJECTIVE Dietary ω-6 lipids such as linoleic acid and its oxidized forms (13-HPODE OxLA) interact with peroxisome proliferator-activated receptors (PPARs) and elicit pro and anti-atherogenic effects in vascular cells. Ligand-dependent PPAR protein turnover is promoted by ubiquitination, but attenuated by binding to its co-activator, peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1α). The objective of our study was to investigate if the dual atherogenic effects of ω-6 lipids are due to its regulation of PPAR turnover. METHODS AND RESULTS In rat aortic smooth muscle cells (RASMCs), oxidized linoleic acid (OxLA) at 10-50 μM induced and stabilized PPARα protein at earlier time points (0-4 h) but suppressed it at 12 h. Conversely, it activated PPARγ protein turnover at a later time point (12 h). Pre-treatment with the proteasome inhibitor (MG132) prevented OxLA mediated loss of PPAR stability and transactivity. Co-immunoprecipitation studies indicated a ligand mediated time-dependent reciprocal exchange of PPAR interaction between ubiquitination and PGC-1α. This ω-6 lipid mediated time-dependent switch between PPAR degradation versus stability helped modulate the pro and anti-atherogenic effects of these dietary lipids. CONCLUSION Our findings provide insights into the dual pro and anti-atherogenic effects of dietary ω-6 lipids on vascular cells by the regulation of PPAR turnover.

The Aporphine Alkaloid Boldine Induces Adiponectin Expression and Regulation in 3T3-L1 Cells

Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor (PPAR)-gamma, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H(2)O(2)) (100 microM) or tumor necrosis factor-alpha (TNFalpha) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5-100 microM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARgamma, and C/EBPalpha to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H(2)O(2) or TNFalpha and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5-25 microM) having a larger inductive effect compared to higher concentrations (50-100 microM). Boldine treatment alone in the absence of H(2)O(2) or TNFalpha was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease.