Muyao Li

Down-regulation of Manganese-Superoxide Dismutase through Phosphorylation of FOXO3a by Akt in Explanted Vascular Smooth Muscle Cells from Old Rats

Manganese-superoxide dismutase (MnSOD) is one of the major cellular antioxidant defense systems. To study the effect of age on the regulation of MnSOD in the vasculature, we compared MnSOD expression and its transcriptional regulation in explanted vascular smooth muscle cells (VSMC) isolated from old (24 months old) versus young (6 months old) rats and grown in a normal (5 mm) or high (12.5 and 25 mm) glucose or tumor necrosis factor α (5 ng/ml) environment to induce oxidative stress. Both MnSOD protein and activity were reduced in VSMC from old compared with young animals. FOXO3a, a member of the family of Forkhead transcription factors, interacted with the promoter of the rat MnSOD gene at a specific binding site. Inhibition of FOXO3a transcription with small interfering RNA led to a reduction in MnSOD gene expression. VSMC from old rats had increased phosphorylated FOXO3a at Ser253, which paralleled the reduction of MnSOD protein. Treatment of VSMC with 5 nm insulin-like growth factor-1 induced phosphorylation of Akt and FOXO3a over time, repressing FOXO3a DNA binding and consequently MnSOD gene expression. Furthermore, Akt activity was selectively increased in VSMC from the old, supporting the hypothesis that increased age-related Akt activity might be responsible for the phosphorylation and inactivation of FOXO3a, which in turn down-regulates MnSOD transcription.

Age-Related Changes in Redox Signaling and VSMC Function

Epidemiological studies have shown that advancing age is associated with an increased prevalence of cardiovascular disease (CVD). Vascular smooth muscle cells (VSMC) comprise the major arterial cell population, and changes in VSMC behavior, function, and redox status with age contribute to alterations in vascular remodeling and cell signaling. Over two decades of work on aged animal models provide support for age-related changes in VSMC and/or arterial tissues. Enhanced production of reactive oxygen species (ROS) and insufficient removal by scavenging systems are hallmarks of vascular aging. VSMC proliferation and migration are core processes in vascular remodeling and influenced by growth factors and signaling networks. The intrinsic link between gene regulation and aging often relates directly to transcription factors and their regulatory actions. Modulation of growth factor signaling leads to up- or downregulation of transcription factors that control expression of genes associated with VSMC proliferation, inflammation, and ROS production. Four major signaling pathways related to the transcription factors, AP-1, NF-kappaB, FoxO, and Nrf2, will be reviewed. Knowledge of age-related changes in signaling pathways in VSMC that lead to alterations in cell behavior and function consistent with disease progression may help in efforts to attenuate age-related CVD, such as atherosclerosis.

AGE-RELATED DIFFERENCES IN INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR SIGNALING REGULATES AKT/FOXO3A AND ERK/FOS PATHWAYS IN VASCULAR SMOOTH MUSCLE CELLS

Advanced age is a major risk factor for atherosclerosis, but how aging per se influences pathogenesis is not clear. Insulin‐like growth factor‐1 receptor (IGF‐1R) promotes aortic vascular smooth muscle cell (VSMC) growth, migration, and extracellular matrix formation, but how IGF‐1R signaling changes with age in VSMC is not known. We previously found age‐related differences in the activation of Akt/FOXO3a and ERK1/2 pathways in VSMC, but the upstream signaling remains unclear. Using explanted VSMC from Fischer 344/Brown Norway F1 hybrid rats shown to display age‐related vascular pathology similar to humans, we compared IGF‐1R expression in early passages of VSMC and found a constitutive activation of IGF‐1R in VSMC from old compared to young rats, including IGF‐1R expression and its tyrosine kinase activity. The link between IGF‐1R activation and the Akt/FOXO3a and ERK pathways was confirmed through the induction of IGF‐1R with IGF‐1 in young cells and attenuation of IGF‐1R with an inhibitor in old cells. The effects of three kinase inhibitors: AG1024, LY294002, and TCN, were compared in VSMC from old rats to differentiate IGF‐1R from other upstream signaling that could also regulate the Akt/FOXO and ERK pathways. Genes for p27kip‐1, catalase and MnSOD, which play important roles in the control of cell cycle arrest and stress resistance, were found to be FOXO3a‐targets based on FOXO3a‐siRNA treatment. Furthermore, IGF‐1R signaling modulated these genes through activation of the Akt/FOXO3a pathway. Therefore, activation of IGF‐1R signaling influences VSMC function in old rats and may contribute to the increased risk for atherosclerosis. J. Cell. Physiol. 217: 377–387, 2008.

Soy biodiesel and petrodiesel emissions differ in size, chemical composition and stimulation of inflammatory responses in cells and animals.

Debate about the biological effects of biodiesel exhaust emissions exists due to variation in methods of exhaust generation and biological models used to assess responses. Because studies in cells do not necessarily reflect the integrated response of a whole animal, experiments were conducted in two human cell lines representing bronchial epithelial cells and macrophages and female mice using identical particle suspensions of raw exhaust generated by a Volkswagen light-duty diesel engine using petrodiesel (B0) and a biodiesel blend (B20: 20% soy biodiesel/80% B0 by volume). Tailpipe particle emissions measurement showed B0 generated two times more particle mass, larger ultrafine particle number distribution modes, and particles of more nonpolar organic composition than the B20 fuel. Biological assays (inflammatory mediators, oxidative stress biomarkers) demonstrated that particulate matter (PM) generated by combustion of the two fuels induced different responses in in vitro and in vivo models. Concentrations of inflammatory mediators (Interleukin-6, IL-6; Interferon-gamma-induced Protein 10, IP-10; Granulocyte-stimulating factor, G-CSF) in the medium of B20-treated cells and in bronchoalveolar lavage fluid of mice exposed to B20 were ∼20-30% higher than control or B0 PM, suggesting that addition of biodiesel to diesel fuels will reduce PM emissions but not necessarily adverse health outcomes.

Modulation of cell injury and survival by high glucose and advancing age.

Old age is associated with a higher prevalence of cardiovascular disease and diabetes mellitus. Vascular smooth muscle cells (VSMC) play a role in the pathogenesis of vascular diseases, often a complication of diabetes mellitus. We examined in explanted aortic VSMC from young vs. older rats glucose-related activation of nuclear factor kappaB (NF-kappaB), a transcription factor induced by many oxidants. Data demonstrate that old age is associated with enhanced NF-kappaB activity in unstimulated VSMC that is further increased after exposure to high glucose medium. Furthermore, VSMC from old animals exhibit increased levels of protein carbonyls, an indicator of oxidative stress, and less apoptosis in response to glucose than VSMC isolated from young animals. These changes are accompanied by increased expression of NF-kappaB-related genes, gamma-glutamylcysteine synthetase, inhibitor of apoptosis protein-1 (IAP-1), and inducible nitric oxide synthase (iNOS). Results suggest that high glucose, a putative oxidative stress, causes apoptosis in VSMC from young animals and is associated with greater induction of NF-kappaB in VSMC from older animals. Increases in IAP-1 and decreased apoptosis implicate NF-kappaB as a survival factor in VSMC.

High glucose concentrations induce oxidative damage to mitochondrial DNA in explanted vascular smooth muscle cells

Oxidative stress is considered to be one of the mechanisms leading to atherosclerosis. It occurs in response to injury or to altered metabolic state. Alterations in cell growth (proliferation or apoptosis) can also contribute to the pathogenesis of atherosclerosis and is influenced by oxidative stress. Smooth muscle cells (SMC) from aortic explants of JCR:LA-cp homozygous cp/cp corpulent rats who are genetically predisposed to develop atherosclerosis exhibit increased SMC proliferation, which can be attenuated by exercise and food restriction. This study was conducted to characterize the effects fo oxidative stress and high glucose media on cell growth and its relationship to mitochondrial DNA integrity and gene expression in explanted aortic SMC from corpulent and lean JCR:LA-cp rats. The results show that SMC from the cp/cp rat appear to be resistant to oxidant-induced cell death and that they accumulate mitochondrial DNA mutations, probably as a result of a reduction in apoptosis. These data suggest that susceptibility to age- and glucose-related atherosclerosis may be related to alterations in redox signaling.

Age‐related differences in MAP kinase activity in VSMC in response to glucose or TNF‐α

Aortic vascular smooth muscle cells (VSMC) were used to study the effect of age on responses to high glucose concentrations or the cytokine, tumor necrosis factor‐alpha (TNF‐α). Activator protein‐1 (AP‐1) binding to DNA increased more in VSMC from old versus young rats (P < 0.02) and was related to increased expression of its components, c‐Fos, Fra‐1, and JunD. The relationship to upstream signals, i.e., activities of mitogen‐activated protein kinases (MAPK), was studied using antibodies to total and phosphorylated forms of extracellular signal‐regulated kinases (ERK), c‐Jun N‐terminal kinases (JNK) and p38. High glucose and TNF‐α increased ERK phosphorylation more in old (P < 0.05); whereas only TNF‐α induced JNK activation in young (P < 0.04). PD98059, a MEK inhibitor, attenuated AP‐1 activation, lowered c‐Fos and Fra‐1 protein levels and reduced cell number and cells positive for proliferating cell nuclear antigen in old. We concluded that age differentially influenced activation of signaling pathways in VSMC exposed to high glucose or TNF‐α. This may contribute to the increased risk for vascular disease associated with aging and diabetes mellitus (DM). J. Cell. Physiol. 197: 418–425, 2003© 2003 Wiley‐Liss, Inc.

Molecular changes during arsenic-induced cell transformation†

Arsenic and its derivatives are naturally occurring metalloid compounds widely distributed in the environment. Arsenics are known to cause cancers of the skin, liver, lung, kidney, and bladder. Although numerous carcinogenic pathways have been proposed, the exact molecular mechanisms remain to be delineated. To further characterize the role of oxidative stress in arsenite‐induced cell transformation via the reactive oxygen species (ROS)‐mediated Ras/Erk pathway, here we demonstrated arsenite‐induced rat lung epithelial cell (LEC) transformation, epithelial–mesenchymal transition, stimulation of the extracellular signal‐regulated kinase signaling pathway, and enhancement of cell proliferation. However, there was no evidence of activation of the phosphoinositide 3‐kinase/protein kinase B pathway in arsenite‐induced transformed LECs. Since ROS is involved in arsenite‐induced LEC cell transformation, Redox‐status regulatory proteins (Cu/Zn SOD and thioredoxin) and arsenite‐induced LEC cell transformation were significantly inhibited by concurrent treatment with the antioxidants. Our experimental results clearly demonstrated that induction of p‐ERK and cell proliferation by arsenite is mediated via oxidative stress, since antioxidants can inhibit arsenite‐induced cell transformation. J. Cell. Physiol. 226: 3225–3232, 2011.

Identification and characterization of an Nrf2-mediated ARE upstream of the rat glutamate cysteine ligase catalytic subunit gene (GCLC).

The antioxidant response element (ARE) is an essential component of upstream regulatory sequences present on genes for most phase II detoxification enzymes, including the glutamate cysteine ligase catalytic subunit (GCLC). NF‐E2‐related factor 2 (Nrf2) is a principal transcription factor that binds to the ARE and plays a key role in cellular responses to stress via the Keap1‐Nrf2‐ARE pathway. However, the ARE that mediates human GCLC gene expression has not been found in the rat. Thus, how the ARE‐mediated Keap1‐Nrf2‐ARE pathway regulates glutathione homeostasis in the rat remains a puzzle. We have identified a putative ARE sequence ∼4 kb upstream in the rat GCLC. We further defined the rat GCLC‐ARE in the category with the most ARE characters, that is, this rat GCLC‐ARE is a sequence‐specific site that significantly enhances promoter activity in reporter genes. The rat GCLC‐ARE is an Nrf2‐mediated element to which binding has been demonstrated in nuclear extracts and induced by tert‐butylhydroquinone. Given the central role that rat models play in toxicology and pathology, this first discovery of the rat GCLC‐ARE enhancer similar to that found in the human gene has broad implications for the study of antioxidant defenses and their regulation in a number of different fields. J. Cell. Biochem. 107: 944–954, 2009.

Differential activation of the inflammasome in THP-1 cells exposed to chrysotile asbestos and Libby “six-mix” amphiboles and subsequent activation of BEAS-2B cells

Inflammatory responses of THP-1 cells (macrophage cell line) exposed to chrysotile asbestos (Chry) and Libby six-mix (LIB) and the subsequent impact on bronchial epithelial cells were determined. Direct treatment of THP-1 cells with Chry caused cell death, activation of caspase-1 and release of IL-1β, while the addition of caspase-1 inhibitor, Z-YVAD-FMK, reduced IL-1β, suggesting that Chry activated the caspase-1 mediated Nod-like receptor protein 3 (NLRP3) inflammasome; by comparison, LIB had less effects on all of these parameters. Expression of antioxidant enzymes, protein oxidation and nitration, and lipid peroxides in THP-1 cells treated with the two particles suggest that LIB generated more reactive oxygen species (ROS) than the same dose of Chry. Differences in fiber length and surface area suggest a possible role for particulate size in the differential activation of the inflammasome. BEAS-2B cells, representing the bronchial epithelium, treated with supernatants of medium from Chry- or LIB-treated THP-1 cells (conditioned medium) activated the MAPK cascade, increased phosphorylation of ERK and Cot (MAP3K8), increased AP-1 binding activity and induced IL-6 release. To verify that IL-1β from THP-1 cells was responsible for activation of BEAS-2B, conditioned medium with added IL-1Ra, an IL-1β antagonist, was applied to BEAS-2B. Results show that IL-1Ra attenuated effects of conditioned medium, supporting a role of IL-1β, as a secondary mediator, in the transduction of inflammatory signaling from the macrophage to epithelial cells. The effects of LIB-conditioned medium appeared to be less dependent on IL-1β. In conclusion, Chry and LIB induce differential inflammatory responses in THP-1 cells that subsequently lead to differential effects in epithelial cells.