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

Abstract

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 Ca(2+)/calmodulin-dependent processes and whether Ca(2+)-sensitive Nox5, associated with Rac-1, generates superoxide (O(2)(*-)) 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 (Ca(2+) 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 Ca(2+) ([Ca(2+)](e)). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca(2+)](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 Ca(2+)/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 Ca(2+)/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by Ang II and ET-1.

Figure 1

Nox4 and Nox5 protein expression in human endothelial cells stimulated with Ang II (B) and ET-1 (C) for 2–24 hours. Representative immunoblots demonstrate Nox4, Nox5 and β-actin expression in human endothelial cells in control (C) and stimulated conditions (2–24 hrs). Top immunoblot (A) shows Nox5 expression in HEK cells that stably overexpress Nox5 (positive control), human endothelial cells (HMEC), human ovarian tumor cell line (positive control) and rat vascular smooth muscle cells (negative control). Bar graphs are means ± SEM from 7 experiments. Results were normalized to β-actin. Control (C) was taken as 100% and data presented as the percentage change relative to control conditions. *p<0.05 vs C.

Figure 2

Diltiazem, calmidazolium and low [Ca²⁺]_e attenuate agonist-induced effects on Nox5 expression. Cells were exposed to diltiazem (L-type Ca²⁺ channel blocker) or calmidazolium (calmodulin inhibitor) for 30 minutes prior to agonist stimulation (24 hrs). In some experiments cells were grown in reduced Ca²⁺ culture media containing 50% of normal Ca²⁺ levels (Ca²⁺ 50%). Upper panels are representative immunoblots. Results were normalized to GAPDH. Control (C) was taken as 100% and data presented as the percentage change relative to control conditions. Data are means ± SEM from 5 experiments. *p<0.05 vs C.

Figure 3

Regulation of NADPH oxidase by Ca²⁺. NADPH oxidase is activated by Ang II (A) and ET-1 (B) as assessed by enhanced lucigenin (5 μmol/L) chemiluminescence. Reduced Ca²⁺ culture media (Ca²⁺ 50%) attenuated Ang II (C) and ET-1 (D)-induced activation of NADPH oxidase. *p<0.05 vs other groups

Figure 4

Regulation of NADPH oxidase activity and Nox5 by calmodulin. Ang II- and ET-1-induced activation of NADPH oxidase is inhibited by calmidazolium (fig 4A and 4B). Data are means ± SEM from 6 experiments. *p<0.05 vs other groups. Figure 4C. Ang II and ET-1 promote association between Nox5 and calmodulin. Nox5 was immunoprecipitated (IP) and then probed for calmodulin by western blotting (WB). Data are means ± SEM from 6 experiments. *p<0.05 vs control (Ctl). IGG, immunoglobulin G, and Nox5, negative and positive controls respectively.

Figure 5

Nox5 is important in ROS generation stimulated by Ang II and ET-1. siRNA knockdown of Nox5 decreases ROS production. Nox5, but not Nox4, was downregulated with siRNA. Significant reduction in expression of Nox5, but not Nox4, was evident after 48 hours as shown by western blot (A). RNA interference knockdown of Nox5 abrogated ROS generation induced by Ang II and ET-1 in ECs (B,C). Data are means ± SEM from 5 experiments. *p<0.05 vs control (C) and non-silenced (NS) conditions. Figure 5D, effects of EHT1864 on Ang II- and ET-1-induced activation of NADPH oxidase. Cells were exposed to the Rac1 inhibitor for 30 mins prior to Ang II and ET-1 stimulation. Data are means ± SEM from 5 experiments. *p<0.05 vs control (C).

Figure 6

siRNA knockdown of Nox5 decreases ERK 1/2 activation. Downregulation of Nox5 and inhibition of calmodulin were associated with significantly reduced activation of ERK1/2. Short-term (5 minutes) exposure to Ang II and ET-1 increased ERK 1/2 phosphorylation. In Nox5 siRNA-transfected cells (A) and cells exposed to calmidazolium (B), neither Ang II nor ET-1 increased ERK 1/2 activation. Data are means ± SEM from 5 experiments. *p<0.05 vs control (C) and non-silenced (NS) conditions.

Figure 7

siRNA knockdown of Nox5 decreases VCAM-1 and PCNA expression induced by Ang II, but not by ET-1. Downregulation of Nox5 was associated with significantly reduced expression of VCAM-1 (A) and PCNA (B) induced by Ang II. Nox5 siRNA did not affect ET-1-induced effects on VCAM-1 (C) and PCNA (B) expression. Data are means ± SEM from 5 experiments. *p<0.05 vs control (C) and non-silenced (NS) conditions.

Figure 8

Schematic demonstrating mechanisms of Nox5 regulation by Ang II and ET-1 in human endothelial cells. Binding of Ang II and ET-1 to their respective receptors (ATR, ETR), stimulates Ca²⁺ influx through L-type Ca²⁺ channels and activation of calmodulin, which interact with Ca²⁺/calmodulin-binding domains to activate Nox5. Activated Nox5 results in generation of superoxide (•O₂⁻) and phosphorylation (p) of ERK1/2. These processes are independent of Rac-1. Stimulation of ECs by Ang II and ET-1 induces upregulation of PCNA (proliferating cell nuclear antigen) and VACM-1 (vascular cell adhesion molecule-1) important in growth and inflammation respectively. Whereas Ang II stimulates these responses through Nox5/•O₂⁻/ERK1/2, ET-1 influences PCNA and VCAM-1 through Nox5-independent processes.