Fluid Shear Stress Attenuates Hydrogen Peroxide–Induced c-Jun NH 2 -Terminal Kinase Activation via a Glutathione Reductase–Mediated Mechanism Cellular Biology

—c-Jun NH 2 -terminal kinase (JNK) is activated by a number of cellular stimuli including reactive oxygen species (ROS). Previous studies have demonstrated that fluid shear stress (flow) inhibits cytokine-induced JNK activation in endothelial cells (ECs). In the present study, we show JNK activation by ROS in ECs and hypothesized that flow inhibits ROS-induced JNK activation in ECs via modulation of cellular protection systems against ROS. JNK was activated by 300 (cid:1) mol/L hydrogen peroxide (H 2 O 2 ) in bovine lung microvascular ECs (BLMVECs) with a peak at 60 minutes after stimulation (6.3 (cid:1) 1.2-fold increase). Preexposure of BLMVECs to physiological steady laminar flow (shear stress (cid:2) 12 dyne/cm 2 ) for 10 minutes significantly decreased H 2 O 2 -induced JNK activation. Thioredoxin and glutathione are cellular antioxidants that protect cells against ROS. Flow induced a significant increase in the ratio of reduced glutathione to oxidized glutathione consistent with a 1.6-fold increase in glutathione reductase (GR) activity. Preincubation of BLMVECs with the GR inhibitor, 1,3 bis-(2 chloroethyl)-1-nitrosourea, abolished the inhibitory effect of flow. In contrast, preincubation of BLMVECs with azelaic acid, a specific inhibitor for thioredoxin reductase, did not alter the effect of flow on H 2 O 2 -induced JNK activation. Overexpression of GR mimicked the effect of flow to inhibit JNK activation. These results suggest that flow activates GR, an important regulator of the intracellular redox state of glutathione, and exerts a protective mechanism against oxidative stress in endothelial cells. ( Circ Res . 2002;91:712-718.) study, we hypothesized that flow inhibits ROS-induced JNK activation in endothelial cells via modulation of these cellular antioxidant systems.

[1]  A. Farré,et al.  Heart Failure, Redox Alterations, and Endothelial Dysfunction , 2001, Hypertension.

[2]  J. Abe,et al.  Endothelial Atheroprotective and Anti‐inflammatory Mechanisms , 2001, Annals of the New York Academy of Sciences.

[3]  B. Berk,et al.  Fluid shear stress inhibits TNF-α activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[4]  W. Min,et al.  Laminar flow inhibits TNF-induced ASK1 activation by preventing dissociation of ASK1 from its inhibitor 14-3-3. , 2001, The Journal of clinical investigation.

[5]  D. Harrison,et al.  Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. , 2000, Circulation research.

[6]  D. Lefer,et al.  Oxidative stress and cardiac disease. , 2000, The American journal of medicine.

[7]  N. Dhalla,et al.  Role of oxidative stress in cardiovascular diseases , 2000, Journal of hypertension.

[8]  Qunhua Huang,et al.  Src and Cas Mediate JNK Activation but Not ERK1/2 and p38 Kinases by Reactive Oxygen Species* , 2000, The Journal of Biological Chemistry.

[9]  W. Zidek,et al.  Reactive oxygen species in essential hypertension and non-insulin-dependent diabetes mellitus. , 1999, American journal of hypertension.

[10]  Ze'ev Ronai,et al.  Role of redox potential and reactive oxygen species in stress signaling , 1999, Oncogene.

[11]  M. Gimbrone.  Vascular Endothelium , Hemodynamic Forces , and Atherogenesis , 1999 .

[12]  H. Jo,et al.  The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for γ‐glutamylcysteine synthetase and γ‐glutamyl transpeptidase , 1999 .

[13]  B. Berk,et al.  MAP kinases and vascular smooth muscle function. , 1998, Acta physiologica Scandinavica.

[14]  Kohei Miyazono,et al.  Mammalian thioredoxin is a direct inhibitor of apoptosis signal‐regulating kinase (ASK) 1 , 1998, The EMBO journal.

[15]  B. Berk,et al.  Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. , 1998, Arteriosclerosis, thrombosis, and vascular biology.

[16]  A. Zeiher,et al.  Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[17]  A. Holmgren,et al.  Selenium and the thioredoxin and glutaredoxin systems. , 1997, Biomedical and environmental sciences : BES.

[18]  M. Ishida,et al.  c-Src Is Required for Oxidative Stress-mediated Activation of Big Mitogen-activated Protein Kinase 1 (BMK1)* , 1997, The Journal of Biological Chemistry.

[19]  P. Angel,et al.  The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents , 1997, Molecular and cellular biology.

[20]  Minoru Takagi,et al.  Induction of Apoptosis by ASK1, a Mammalian MAPKKK That Activates SAPK/JNK and p38 Signaling Pathways , 1997, Science.

[21]  J. Abe,et al.  Big Mitogen-activated Protein Kinase 1 (BMK1) Is a Redox-sensitive Kinase* , 1996, The Journal of Biological Chemistry.

[22]  J. Woodgett,et al.  The stress-activated protein kinase subfamily of c-Jun kinases , 1994, Nature.

[23]  R. Davis,et al.  The mitogen-activated protein kinase signal transduction pathway. , 1993, The Journal of biological chemistry.

[24]  Nancy Y. Ip,et al.  ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF , 1991, Cell.

[25]  B. Halliwell.  Free radicals, reactive oxygen species and human disease: a critical evaluation with special reference to atherosclerosis. , 1989, British journal of experimental pathology.

[26]  J. Leeder,et al.  Use of a microplate reader in an assay of glutathione reductase using 5,5'-dithiobis(2-nitrobenzoic acid). , 1989, Analytical biochemistry.

[27]  D. Ku,et al.  Pulsatile Flow and Atherosclerosis in the Human Carotid Bifurcation: Positive Correlation between Plaque Location and Low and Oscillating Shear Stress , 1985, Arteriosclerosis.

[28]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[29]  W. Valentine,et al.  Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. , 1967, The Journal of laboratory and clinical medicine.

[30]  A. Holmgren,et al.  Antioxidant function of thioredoxin and glutaredoxin systems. , 2000, Antioxidants & redox signaling.

[31]  N. Hogg.  Free Radicals in Disease , 1998, Seminars in reproductive endocrinology.

[32]  J. McCord.  Oxygen-derived radicals: a link between reperfusion injury and inflammation. , 1987, Federation proceedings.

[33]  H. Aebi,et al.  Catalase in vitro. , 1984, Methods in enzymology.