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Class A scavenger receptor upregulation in smooth muscle cells by oxidized LDL
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I1731 I
4-HYDROXYNONENAL INCREASES EXPRESSION OF yGLUTAMYLCYSTEINE SYNTHETASE GENES IN TYPE II CELLS. Rui-Ming Liua. Zea Borokb. and Henry Jay Formana. a Department of Molecular Pharmacology & Toxicology, b Department of Medicine, University of Southern California, Los Angeles, CA.
GADDlS3 SENSITIZES CELLS TO ER STRESS BY DOWNREGULATING BCL2 AND PERTURBING THE CELLULAR REDOX STATE. Karen D. McCullouoh, Jennifer L. Martindale, Lars-Oliver Klotz, Tak-Ye Aw and Nikki J. Holbrook. Laboratory of Biological Chemistry, National Institute on Aging, Baltimore MD 21224
Previous studies from this laboratory have shown that 4hydroxynonenal (4HNE), a lipid peroxidation product, induces expression of y-glutamylcysteine synthtase (GCS), the ratelimiting enzyme in de nova glutathione (GSH) synthesis, in L2 cells. originally derived from type II pneumocytes of adult rat lungs. This study demonstrated that 4HNE, at the same concentration, also increased the expression of GCS genes in primary-cultured rat lung type II cells (Type II). The enzyme activity, the protein content, and the mRNA content of both subunits of GCS were significantly increased when Type II cells were treated with 5 or 10 pM 4HNE, the same concentration used to treat L2 cells. GCS regulatory subunit showed a greater induction than GCS catalytic subunit upon 4HNE treatment, a phenomenon that has also been observed in L2 cells. The data suggested that L2 cells maintain the same responsiveness to oxidant challenge as freshly isolated Type II cells. This observation provides important evidence to support the utilization of L2 cells as mode1 for Type II cells in studies of GSH synthesis. Supported by NIEHS grant ES-OS5 I I.
I 174
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GADD153 (CHOP) is a stress responsive gene that is strongly induced by agents that perturb the endoplasmic reticulum (ER). It has been suggested that gadd153 participates in regulating cell death, though the mechanism for this is unknown, To investigate the relationship between gadd153 and cell death, Rat1 fibroblasts that constitutively overexpress gadd153 (A94 cells) were treated with ER stress agents. Long term survival of A94 cells was dramatically reduced relative to Rat1 cells, the parental cells from which the A94 line was derived. Flow cytometric analysis revealed that A94 cells undergo significant apoptosis with 24 hours treatment with ER stress agents, an effect not observed with Western blotting revealed that bcl2 levels were parental cells. substantially reduced in A94 cells relative to parental Rat1 cells. Furthermore, bcl2 promoter activity was inhibited in cells that overexpress gadd153. Coincident with inhibition of bc12 expression, a dramatic lowering of the GSH:GSSG ratio (>I0 fold reduction) was found in A94 cells relative to the parental Ratl. As a result, ROS production increased in A94 cells, but not in parental Rat1 cells, after ER stress. Replenishment of bcl2 expression in A94 cells resulted in a restoration of GSH to normal levels, prevented ROS production following ER stress, and protected the cells from ER stress-induced death. Thus, gadd153 sensitizes cells to death by decreasing bcl2 levels, depleting GSH stores and perturbing cellular redox states. These data describe for the first time a relationship between gadd153 and oxidative stress.
11751
OXIDATIVELY-FRAGMENTED PHOSPHOLIPIDS ARE AGONISTS OF THE RECEPTOR/TRANSCRIPTION FACTOR PPARy Thomas M. McIntvre. Sean Davies, G. Marathe, Stephen M. Prescott, and Guy A Zimmerman Department of Medicine, University of Utah, Salt Lake Ciry, UT 84112, USA Oxidation modifies low density lipoproteins to particles that promote chronic inflammation of the vessel wall by attracting and activating monocytes. MCP-1 has a major role in these atherogenic events, but how oxidized (ox) LDL induces expression of this essential chemokine is unknown. We show ox-fragmented phospholipids derived from oxLDL or ox synthetic phosphatidylcholine (PC) stimulate MCP-1 synthesis by monocytes. We synthesized aze1aoylPC (azPC), found in oxPC, and it also induced MCP-1 synthesis. PPARy is a transcription factor that binds and is activated by negatively charged lipid ligands. The negatively charged azPC, but not free azelaic acid, was a high affinity PPARy ligand and it drove reporter expression from a consensus PPAR-responsive element (PPRE). The MCP-1 gene contained a PPRE in the first intron, and established xenobiotic PPARr agonists induced monocyte MCP-1 secretion. The presence of PPARy is regulated in monocytes, and azPC induced MCP-1 only when PPARy was present. We have identified ox-fragmented PC as a new class of potent ligands for PAPRy that induces MCP-1 secretion from monocytes. OXPC is found in plaques, so certain OXPC are endogenous ligands for PPARy that promote monocyte influx.
OXYGEN
CLASS A SCAVENGER RECEPTOR UPREGULATION SMOOTH MUSCLE CELLS BY OXIDIZED LDL Michele Mietus-Snvder, Maya Gowri, and Robert Pitas Gladstone Institute of Cardiovascular Disease Research, University of California, San Francisco
IN
We previously demonstrated that oxidative stress caused by phorbol esters or reactive oxygen upregulates the class A scavenger receptor @R-A) in human smooth muscle cells (SMC). The increase in SR-A expression correlates with activation of the redox-sensitive transcription factors activating protein-l (AP1)/&n and CCAAT enhancer-binding proteit$ (CEBP/p). We now show that these same regulatory pathways are activated and SR-A expression is stimulated by coincubation of SMC with macrophages or with oxidized low-density lipoproteins (OxLDL) from THP-1 macrophage-conditioned medium. SR-A expression was also enhanced by LDL oxidized in vitro. Inhibition of calcium flux suppressed SR-A induction by OxLDL. Conversely, calcium ionophore greatly enhanced SR-A upregulation by either Ox LDL or other treatments previously shown to promote intracellular oxidative stress. The enhancement of SR-A expression by calcium ionophore was dependent upon concurrent upregulation of SMC cyclooxygenase-2 (COX-2) expression and activity. The synergistic increase in SR-A upregulation with calcium ionophore was blocked by treatment with the COX-2 inhibitors, NS-398 or resveratrol. Incubation of THP-1 cells with OxLDL also resulted in monocyte-macrophage differentiation and increased SR-A expression. These findings support a role for OxLDL in the redox regulation of macrophage differentiation and SR-A expression and suggest that increased vascular oxidative stress may contribute to both SMC and macrophage foam cell formation.
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563
I1731 I
4-HYDROXYNONENAL INCREASES EXPRESSION OF yGLUTAMYLCYSTEINE SYNTHETASE GENES IN TYPE II CELLS. Rui-Ming Liua. Zea Borokb. and Henry Jay Formana. a Department of Molecular Pharmacology & Toxicology, b Department of Medicine, University of Southern California, Los Angeles, CA.
GADDlS3 SENSITIZES CELLS TO ER STRESS BY DOWNREGULATING BCL2 AND PERTURBING THE CELLULAR REDOX STATE. Karen D. McCullouoh, Jennifer L. Martindale, Lars-Oliver Klotz, Tak-Ye Aw and Nikki J. Holbrook. Laboratory of Biological Chemistry, National Institute on Aging, Baltimore MD 21224
Previous studies from this laboratory have shown that 4hydroxynonenal (4HNE), a lipid peroxidation product, induces expression of y-glutamylcysteine synthtase (GCS), the ratelimiting enzyme in de nova glutathione (GSH) synthesis, in L2 cells. originally derived from type II pneumocytes of adult rat lungs. This study demonstrated that 4HNE, at the same concentration, also increased the expression of GCS genes in primary-cultured rat lung type II cells (Type II). The enzyme activity, the protein content, and the mRNA content of both subunits of GCS were significantly increased when Type II cells were treated with 5 or 10 pM 4HNE, the same concentration used to treat L2 cells. GCS regulatory subunit showed a greater induction than GCS catalytic subunit upon 4HNE treatment, a phenomenon that has also been observed in L2 cells. The data suggested that L2 cells maintain the same responsiveness to oxidant challenge as freshly isolated Type II cells. This observation provides important evidence to support the utilization of L2 cells as mode1 for Type II cells in studies of GSH synthesis. Supported by NIEHS grant ES-OS5 I I.
I 174
I
GADD153 (CHOP) is a stress responsive gene that is strongly induced by agents that perturb the endoplasmic reticulum (ER). It has been suggested that gadd153 participates in regulating cell death, though the mechanism for this is unknown, To investigate the relationship between gadd153 and cell death, Rat1 fibroblasts that constitutively overexpress gadd153 (A94 cells) were treated with ER stress agents. Long term survival of A94 cells was dramatically reduced relative to Rat1 cells, the parental cells from which the A94 line was derived. Flow cytometric analysis revealed that A94 cells undergo significant apoptosis with 24 hours treatment with ER stress agents, an effect not observed with Western blotting revealed that bcl2 levels were parental cells. substantially reduced in A94 cells relative to parental Rat1 cells. Furthermore, bcl2 promoter activity was inhibited in cells that overexpress gadd153. Coincident with inhibition of bc12 expression, a dramatic lowering of the GSH:GSSG ratio (>I0 fold reduction) was found in A94 cells relative to the parental Ratl. As a result, ROS production increased in A94 cells, but not in parental Rat1 cells, after ER stress. Replenishment of bcl2 expression in A94 cells resulted in a restoration of GSH to normal levels, prevented ROS production following ER stress, and protected the cells from ER stress-induced death. Thus, gadd153 sensitizes cells to death by decreasing bcl2 levels, depleting GSH stores and perturbing cellular redox states. These data describe for the first time a relationship between gadd153 and oxidative stress.
11751
OXIDATIVELY-FRAGMENTED PHOSPHOLIPIDS ARE AGONISTS OF THE RECEPTOR/TRANSCRIPTION FACTOR PPARy Thomas M. McIntvre. Sean Davies, G. Marathe, Stephen M. Prescott, and Guy A Zimmerman Department of Medicine, University of Utah, Salt Lake Ciry, UT 84112, USA Oxidation modifies low density lipoproteins to particles that promote chronic inflammation of the vessel wall by attracting and activating monocytes. MCP-1 has a major role in these atherogenic events, but how oxidized (ox) LDL induces expression of this essential chemokine is unknown. We show ox-fragmented phospholipids derived from oxLDL or ox synthetic phosphatidylcholine (PC) stimulate MCP-1 synthesis by monocytes. We synthesized aze1aoylPC (azPC), found in oxPC, and it also induced MCP-1 synthesis. PPARy is a transcription factor that binds and is activated by negatively charged lipid ligands. The negatively charged azPC, but not free azelaic acid, was a high affinity PPARy ligand and it drove reporter expression from a consensus PPAR-responsive element (PPRE). The MCP-1 gene contained a PPRE in the first intron, and established xenobiotic PPARr agonists induced monocyte MCP-1 secretion. The presence of PPARy is regulated in monocytes, and azPC induced MCP-1 only when PPARy was present. We have identified ox-fragmented PC as a new class of potent ligands for PAPRy that induces MCP-1 secretion from monocytes. OXPC is found in plaques, so certain OXPC are endogenous ligands for PPARy that promote monocyte influx.
OXYGEN
CLASS A SCAVENGER RECEPTOR UPREGULATION SMOOTH MUSCLE CELLS BY OXIDIZED LDL Michele Mietus-Snvder, Maya Gowri, and Robert Pitas Gladstone Institute of Cardiovascular Disease Research, University of California, San Francisco
IN
We previously demonstrated that oxidative stress caused by phorbol esters or reactive oxygen upregulates the class A scavenger receptor @R-A) in human smooth muscle cells (SMC). The increase in SR-A expression correlates with activation of the redox-sensitive transcription factors activating protein-l (AP1)/&n and CCAAT enhancer-binding proteit$ (CEBP/p). We now show that these same regulatory pathways are activated and SR-A expression is stimulated by coincubation of SMC with macrophages or with oxidized low-density lipoproteins (OxLDL) from THP-1 macrophage-conditioned medium. SR-A expression was also enhanced by LDL oxidized in vitro. Inhibition of calcium flux suppressed SR-A induction by OxLDL. Conversely, calcium ionophore greatly enhanced SR-A upregulation by either Ox LDL or other treatments previously shown to promote intracellular oxidative stress. The enhancement of SR-A expression by calcium ionophore was dependent upon concurrent upregulation of SMC cyclooxygenase-2 (COX-2) expression and activity. The synergistic increase in SR-A upregulation with calcium ionophore was blocked by treatment with the COX-2 inhibitors, NS-398 or resveratrol. Incubation of THP-1 cells with OxLDL also resulted in monocyte-macrophage differentiation and increased SR-A expression. These findings support a role for OxLDL in the redox regulation of macrophage differentiation and SR-A expression and suggest that increased vascular oxidative stress may contribute to both SMC and macrophage foam cell formation.
’ 9
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563