TY - JOUR
T1 - Nicotinamide adenine dinucleotide phosphate reduced oxidase 5 (Nox5) regulation by angiotensin II and endothelin-1 is mediated via calcium/calmodulin-dependent, Rac-1-independent pathways in human endothelial cells
AU - Montezano, Augusto C.
AU - Burger, Dylan
AU - Paravicini, Tamara M.
AU - Chignalia, Andreia Z.
AU - Yusuf, Hiba
AU - Almasri, Mahmoud
AU - He, Ying
AU - Callera, Glaucia E.
AU - He, Gang
AU - Krause, Karl Heinz
AU - Lambeth, David
AU - Quinn, Mark T.
AU - Touyz, Rhian M.
PY - 2010/4/30
Y1 - 2010/4/30
N2 - Rationale: Although Nox5 (Nox2 homolog) has been identified in the vasculature, its regulation and functional significance remain unclear. Objectives: We sought to test whether vasoactive agents regulate Nox5 through Ca2+/ calmodulin-dependent processes and whether Ca 2+-sensitive Nox5, associated with Rac-1, generates superoxide (O2.-) and activates growth and inflammatory responses via mitogen-activated protein kinases in human endothelial cells (ECs). Methods and Results: Cultured ECs, exposed to angiotensin II (Ang II) and endothelin (ET)-1 in the absence and presence of diltiazem (Ca2+ channel blocker), calmidazolium (calmodulin inhibitor), and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with small interfering RNA. Ang II and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca2+ ([Ca2+]e). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca2+]e, but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated O 2.- production and inhibited phosphorylation of extracellular signal-regulated kinase (ERK)1/2, but not p38 MAPK (mitogen-activated protein kinase) or SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase). Nox5 small interfering RNA blunted Ang II-induced, but not ET-1-induced, upregulation of proliferating-cell nuclear antigen and vascular cell adhesion molecule-1, important in growth and inflammation. Conclusions: Human ECs possess functionally active Nox5, regulated by Ang II and ET-1 through Ca2+/calmodulin-dependent, Rac-1-independent mechanisms. Nox5 activation by Ang II and ET-1 induces ROS generation and ERK1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by Ang II but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by Ang II and ET-1. Such phenomena link Ca2+/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by Ang II and ET-1.
AB - Rationale: Although Nox5 (Nox2 homolog) has been identified in the vasculature, its regulation and functional significance remain unclear. Objectives: We sought to test whether vasoactive agents regulate Nox5 through Ca2+/ calmodulin-dependent processes and whether Ca 2+-sensitive Nox5, associated with Rac-1, generates superoxide (O2.-) and activates growth and inflammatory responses via mitogen-activated protein kinases in human endothelial cells (ECs). Methods and Results: Cultured ECs, exposed to angiotensin II (Ang II) and endothelin (ET)-1 in the absence and presence of diltiazem (Ca2+ channel blocker), calmidazolium (calmodulin inhibitor), and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with small interfering RNA. Ang II and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca2+ ([Ca2+]e). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca2+]e, but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated O 2.- production and inhibited phosphorylation of extracellular signal-regulated kinase (ERK)1/2, but not p38 MAPK (mitogen-activated protein kinase) or SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase). Nox5 small interfering RNA blunted Ang II-induced, but not ET-1-induced, upregulation of proliferating-cell nuclear antigen and vascular cell adhesion molecule-1, important in growth and inflammation. Conclusions: Human ECs possess functionally active Nox5, regulated by Ang II and ET-1 through Ca2+/calmodulin-dependent, Rac-1-independent mechanisms. Nox5 activation by Ang II and ET-1 induces ROS generation and ERK1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by Ang II but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by Ang II and ET-1. Such phenomena link Ca2+/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by Ang II and ET-1.
KW - ERK1/2
KW - Reactive oxygen species
KW - Vascular cells
KW - Vasoactive peptides
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U2 - 10.1161/CIRCRESAHA.109.216036
DO - 10.1161/CIRCRESAHA.109.216036
M3 - Article
C2 - 20339118
AN - SCOPUS:77952489013
SN - 0009-7330
VL - 106
SP - 1363
EP - 1373
JO - Circulation research
JF - Circulation research
IS - 8
ER -