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Survival signals induced by low concentrations of 27-hydroxycholesterol in human monocytic cells via ERK activation
[0316]
doi:10.1016/j.freeradbiomed.2012.08.488
Multi-tiered protective response of Nrf2 transactivational activity in endothelial cells in vitro
[0325]
M. Xue*, H. Momiji, G. Barker, D.A. Rand, N. Rabbani, P.J. Thornalley University of Warwick, UK
Survival signals induced by low concentrations of 27-hydroxycholesterol in human monocytic cells via ERK activation
Introduction: Nrf2 binds to promoter antioxidant response elements (AREs) and thereby regulates the expression of a battery of protective genes countering oxidative stress. Sulforaphane (SFN) is an exogenous activator of Nrf2 derived from Brassica vegetables. Recent studies suggest Nrf2 activation also counters dicarbonyl glycation (dicarbonyl stress), metabolic dysfunction in hyperglycaemia (metabolic stress) and increases protein turnover. The aim of this study was to investigate changes in ARE-linked gene expression linked to multiple pathways of the protective response induced by SFN and effect on functional markers in human aortal endothelial cells (HAECs) in vitro. Methods: HAECs in primary culture were incubated with and without 2 µM SFN. Cells were collected at 6, 12, 24, 48 and 72 h post-stimulation digital mRNA profiling performed by the Nanostring technique. Results: Treatment of HAECs with SFN produced changes in expression of pathway classified ARE-linked genes and functional markers: Oxidative stress mRNA of quinone reductase, γ-glutamylcysteine ligase, thioredoxin and thioredoxin reductase increased 3-fold and glutathione reductase and haem oxygenase increased 2-fold and 9-fold respectively in the initial 24 h and decreased thereafter. Dicarbonyl stress mRNA of aldoketo reductase isozymes AKR1B1, AKR1C1 and AKR1C3 increased 17%, 5-fold and 3-fold respectively, maximising at 24 h and decreasing slowly thereafter. Metabolic stress mRNA of glucose-6-phosphate dehydrogenase, transketolase and transaldolase increased 2-fold in the initial 24 h and decreased slowly thereafter. Proteasomal proteolysis and autophagy mRNA of proteasome subunits A1 and B5 were little changed whereas mRNA of autophagy adaptor p62 was increased 2-fold. Functional status markers After 72 h SFN treatment, mRNA of VCAM1, E-selectin and NF-kB system p65 was decreased 53%, 31% and 13%, respectively. mRNA of endothelial nitric oxide synthase and gap junction CD146 was unchanged. Conclusions: Activation of Nrf2 protects against multiple stresses in HAECs in vitro with concomitant decreased in markers of cellular inflammation.
B. Vurusaner*1, G. Poli2, H. Basaga1 1 Sabanci University, Turkey, 2University of Turin, Italy Oxysterols are oxidized derivatives of cholesterol, most likely contributing to the development of atherosclerosis. On the basis of the clear findings by previous workers about oxysterol-mediated induction of survival pathways aside that of death pathways, we aimed to identify the main genes and related products involved in the transduction of survival signals and elucidate the relevant molecular mechanisms in human macrophagic cells challenged with 27-hydroxycholesterol in the low micromolar range. Experiments performed in our laboratory demonstrated that 27-OH had a dual effect on the viability of macrophage lineage. In U937 cells, the flow cytometric analyses of Annexin V showed that 27-OH is antiapoptotic at low concentrations (5, 10, 25µM) while it promoted cell death at 100µM or higher concentrations. Since ERKs are implicated in the survival pathways, the role of ERKs in the effects induced by 27-OH was investigated. Exposure of cells to 27-OH (10µM) resulted in phosphorylation of ERK1/2 within 30 min, where ERK phosphorylation peaked at 6h, as monitored through the use of phospho-specific antibodies. To characterize this survival pathway, we analyzed the expression of proapoptotic and antiapoptotic Bcl-2 family members in which several of these proteins constitute ERK targets, including Bad and Bim. Our data clearly show that Bad was phosphorylated at serine 75 and this phosphorylation, leading to its inactivation, peaked at 12h. In contrast, the level of Bim did not apparently change between 3h and 48h treatment with 27-OH. Bcl2 protein levels were found to decrease within 3h and then return to basal levels at 24h. The results of ongoing experiments on the actual role of ERK pathway in modulating the activity of Bad and possibly that of other Bcl-2 family components, namely Mcl-1 and Bcl-xl, will be also presented and discussed. Keywords: proteins
27-hydroxycholesterol,
MEK/ERK,
Bcl-2
doi:10.1016/j.freeradbiomed.2012.08.489
Keywords: Nrf2, Oxidative stress, Endothelial cell, Vascular inflammation
S233
doi:10.1016/j.freeradbiomed.2012.08.488
Multi-tiered protective response of Nrf2 transactivational activity in endothelial cells in vitro
[0325]
M. Xue*, H. Momiji, G. Barker, D.A. Rand, N. Rabbani, P.J. Thornalley University of Warwick, UK
Survival signals induced by low concentrations of 27-hydroxycholesterol in human monocytic cells via ERK activation
Introduction: Nrf2 binds to promoter antioxidant response elements (AREs) and thereby regulates the expression of a battery of protective genes countering oxidative stress. Sulforaphane (SFN) is an exogenous activator of Nrf2 derived from Brassica vegetables. Recent studies suggest Nrf2 activation also counters dicarbonyl glycation (dicarbonyl stress), metabolic dysfunction in hyperglycaemia (metabolic stress) and increases protein turnover. The aim of this study was to investigate changes in ARE-linked gene expression linked to multiple pathways of the protective response induced by SFN and effect on functional markers in human aortal endothelial cells (HAECs) in vitro. Methods: HAECs in primary culture were incubated with and without 2 µM SFN. Cells were collected at 6, 12, 24, 48 and 72 h post-stimulation digital mRNA profiling performed by the Nanostring technique. Results: Treatment of HAECs with SFN produced changes in expression of pathway classified ARE-linked genes and functional markers: Oxidative stress mRNA of quinone reductase, γ-glutamylcysteine ligase, thioredoxin and thioredoxin reductase increased 3-fold and glutathione reductase and haem oxygenase increased 2-fold and 9-fold respectively in the initial 24 h and decreased thereafter. Dicarbonyl stress mRNA of aldoketo reductase isozymes AKR1B1, AKR1C1 and AKR1C3 increased 17%, 5-fold and 3-fold respectively, maximising at 24 h and decreasing slowly thereafter. Metabolic stress mRNA of glucose-6-phosphate dehydrogenase, transketolase and transaldolase increased 2-fold in the initial 24 h and decreased slowly thereafter. Proteasomal proteolysis and autophagy mRNA of proteasome subunits A1 and B5 were little changed whereas mRNA of autophagy adaptor p62 was increased 2-fold. Functional status markers After 72 h SFN treatment, mRNA of VCAM1, E-selectin and NF-kB system p65 was decreased 53%, 31% and 13%, respectively. mRNA of endothelial nitric oxide synthase and gap junction CD146 was unchanged. Conclusions: Activation of Nrf2 protects against multiple stresses in HAECs in vitro with concomitant decreased in markers of cellular inflammation.
B. Vurusaner*1, G. Poli2, H. Basaga1 1 Sabanci University, Turkey, 2University of Turin, Italy Oxysterols are oxidized derivatives of cholesterol, most likely contributing to the development of atherosclerosis. On the basis of the clear findings by previous workers about oxysterol-mediated induction of survival pathways aside that of death pathways, we aimed to identify the main genes and related products involved in the transduction of survival signals and elucidate the relevant molecular mechanisms in human macrophagic cells challenged with 27-hydroxycholesterol in the low micromolar range. Experiments performed in our laboratory demonstrated that 27-OH had a dual effect on the viability of macrophage lineage. In U937 cells, the flow cytometric analyses of Annexin V showed that 27-OH is antiapoptotic at low concentrations (5, 10, 25µM) while it promoted cell death at 100µM or higher concentrations. Since ERKs are implicated in the survival pathways, the role of ERKs in the effects induced by 27-OH was investigated. Exposure of cells to 27-OH (10µM) resulted in phosphorylation of ERK1/2 within 30 min, where ERK phosphorylation peaked at 6h, as monitored through the use of phospho-specific antibodies. To characterize this survival pathway, we analyzed the expression of proapoptotic and antiapoptotic Bcl-2 family members in which several of these proteins constitute ERK targets, including Bad and Bim. Our data clearly show that Bad was phosphorylated at serine 75 and this phosphorylation, leading to its inactivation, peaked at 12h. In contrast, the level of Bim did not apparently change between 3h and 48h treatment with 27-OH. Bcl2 protein levels were found to decrease within 3h and then return to basal levels at 24h. The results of ongoing experiments on the actual role of ERK pathway in modulating the activity of Bad and possibly that of other Bcl-2 family components, namely Mcl-1 and Bcl-xl, will be also presented and discussed. Keywords: proteins
27-hydroxycholesterol,
MEK/ERK,
Bcl-2
doi:10.1016/j.freeradbiomed.2012.08.489
Keywords: Nrf2, Oxidative stress, Endothelial cell, Vascular inflammation
S233