Acute tumor necrosis factor alpha signaling via NADPH oxidase in microvascular endothelial cells: role of p47phox phosphorylation and binding to TRAF4

Abstract

Tumor necrosis factor alpha (TNF-alpha) receptor-associated factors (TRAFs) play important roles in TNF-alpha signaling by interacting with downstream signaling molecules, e.g., mitogen-activated protein kinases (MAPKs). However, TNF-alpha also signals through reactive oxygen species (ROS)-dependent pathways. The interrelationship between these pathways is unclear; however, a recent study suggested that TRAF4 could bind to the NADPH oxidase subunit p47phox. Here, we investigated the potential interaction between p47phox phosphorylation and TRAF4 binding and their relative roles in acute TNF-alpha signaling. Exposure of human microvascular endothelial cells (HMEC-1) to TNF-alpha (100 U/ml; 1 to 60 min) induced rapid (within 5 min) p47phox phosphorylation. This was paralleled by a 2.7- +/- 0.5-fold increase in p47phox-TRAF4 association, membrane translocation of p47phox-TRAF4, a 2.3- +/- 0.4-fold increase in p47phox-p22phox complex formation, and a 3.2- +/- 0.2-fold increase in NADPH-dependent O2- production (all P < 0.05). TRAF4-p47phox binding was accompanied by a progressive increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38(MAPK) activation, which was inhibited by an O2- scavenger, tiron. TRAF4 predominantly bound the phosphorylated form of p47phox, in a protein kinase C-dependent process. Knockdown of TRAF4 expression using siRNA had no effect on p47phox phosphorylation or binding to p22phox but inhibited TNF-alpha-induced ERK1/2 activation. In coronary microvascular EC from p47phox-/- mice, TNF-alpha-induced NADPH oxidase activation, ERK1/2 activation, and cell surface intercellular adhesion molecule 1 (ICAM-1) expression were all inhibited. Thus, both p47phox phosphorylation and TRAF4 are required for acute TNF-alpha signaling. The increased binding between p47phox and TRAF4 that occurs after p47phox phosphorylation could serve to spatially confine ROS generation from NADPH oxidase and subsequent MAPK activation and cell surface ICAM-1 expression in EC.

FIG.1.

Time course of TNF-α-induced p47^phox phosphorylation, p47^phox-TRAF4 binding, p47^phox-p22^phox binding, and NADPH-dependent SOD-inhibitable O₂⁻ production by HMEC-1. (A) Representative immunoblots. p47^phox was immunoprecipitated (IP), and then parallel membranes were immunoblotted (IB) either with a phosphoserine-specific antibody (top panel), an anti-TRAF4 antibody (second panel), an anti-p22^phox antibody (third panel), or an anti-p47^phox antibody (fourth panel). (B) The first three panels show quantitative densitometric analyses of immunoblots. Results are expressed as mean arbitrary absorbance units ± standard deviations obtained from three separate experiments. *, P < 0.01 compared to unstimulated cells (i.e., time zero). The bottom panel shows acute TNF-α-induced NADPH-dependent, SOD-inhibitable chemiluminescence (CL) in an HMEC-1 cell homogenate. (C) Control experiment to confirm specific capture of TRAF4 after immunoprecipitation for p47^phox. CMEC from p47^phox−/− mice (KO cells) were transfected with an empty vector or with p47^phox cDNA. Immunoprecipitation was performed for p47^phox followed by immunoblotting for TRAF4.

FIG. 2.

Assessment of p47^phox phosphorylation in p47^phox-TRAF4 complexes by sequential immunoprecipitation of ³²P-labeled cells. (A) HMEC-1 were labeled with ³²P before being treated with TNF-α (100 U/ml, 15 min) in the presence or absence of Bis (10 μmol/liter). Cell lysates were first immunoprecipitated (IP) with an anti-TRAF4 antibody to recover p47^phox-TRAF4 complexes, disrupted with detergent, and then reimmunoprecipitated with an anti-p47^phox antibody as described in Materials and Methods. IB, immunoblotted. Phosphorylated p47^phox was detected by autoradiography (middle panel). The same membrane was immunoblotted with an antibody against p47^phox to assess the total amount of p47^phox (phosphorylated and nonphosphorylated) bound to TRAF4 (bottom panel). The initial TRAF4 immunoprecipitate was immunoblotted with an anti-TRAF4 antibody (top panel) to confirm equal precipitation of TRAF4. (B) Analysis of relative proportions of phosphorylated p47^phox and total p47^phox. Autoradiographic and immunoblotted bands were scanned densitometrically, and the backgrounds were subtracted. The ratio of phospho-p47^phox to total p47^phox was calculated by dividing density results of autoradiography by density results of immunoblotting; the results are expressed as means ± standard deviations for three separate cell cultures and labelings.*, P < 0.01 compared to control cells; †, P < 0.01 compared to cells stimulated with TNF-α alone.

FIG. 3.

Effect of TNF-α on association between p47^phox and TRAF4 in the cytoskeleton and membrane fractions of HMEC-1. (A) TRAF4 was immunoprecipitated (IP) from the Triton X-100-insoluble (cytoskeleton) fraction, and p47^phox was then detected by immunoblotting (IB) in cells stimulated with TNF-α (30 min) either in the presence or absence of the PKC inhibitor Bis or chelerythrine (Chel). Immunoblotting for TRAF4 confirmed equal precipitation. (B) Assessment of translocation and binding to p22^phox. p22^phox was immunoprecipitated from the cellular membrane fraction, and p47^phox was detected by immunoblotting. Protein extracted from U937 cells after stimulation with phorbol myristate acetate was used as a positive control. Similar results were obtained in two independent experiments.

FIG. 4.

Immunofluorescence confocal micrographs of HMEC-1 probed for p47^phox and TRAF4. (A) HMEC-1 cultured onto chamber slides were exposed to TNF-α or vehicle for 30 min. Slides were double labeled with a rabbit anti-p47^phox polyclonal antibody (left panels, red) and a goat anti-TRAF4 polyclonal antibody (middle panels, green). The yellow color in the merged images (right panels) indicates a colocalization of p47^phox and TRAF4. The white arrows indicate areas of cell surface membrane labeling. All panels are the same scale. (B) CMEC isolated from p47^phox−/− mice and labeled with anti-TRAF4 antibody.

FIG. 5.

Time course of TNF-α-induced MAPK activation in HMEC-1. (A) Representative immunoblots showing expression of phospho-ERK1/2, phospho-p38^MAPK, and phospho-JNK and corresponding total MAPKs after various periods of exposure to TNF-α. (B) Densitometric quantification of expression level of phospho-ERK1/2, phospho-p38^MAPK, and phospho-JNK normalized by respective total ERK1/2, p38^MAPK, and JNK expression. Results are means ± standard deviations from three independent experiments. *, P < 0.01 for TNF-α-stimulated versus unstimulated (time zero).

FIG. 6.

MAPK activation in wild-type and p47^phox−/− CMEC. (A) Representative immunoblots showing expression of phospho-ERK1/2, phospho-p38^MAPK, and phospho-JNK and corresponding total MAPKs with or without TNF-α stimulation (30 min). (B) Densitometric quantification of expression levels of phospho-ERK1/2, phospho-p38^MAPK, and phospho-JNK normalized by respective total ERK1/2, p38^MAPK, and JNK expression. Results are means ± standard deviations from three CMEC isolations (six mice in each group for each isolation) *, P < 0.01 comparing TNF-α-stimulated versus unstimulated in each group.

FIG. 7.

Effects of tiron and DPI on TNF-α-induced ERK1/2 activation in wild-type and p47^phox−/− CMEC. (A) NADPH-dependent O₂⁻ production by wild-type and p47^phox−/− CMEC homogenates measured by lucigenin chemiluminescence. Results are expressed as mean light units (MLU) per minute per milligram of protein. (B) Densitometric quantification of expression levels of phospho-ERK1/2 normalized by total ERK1/2 in wild-type and p47^phox−/− CMEC. Results are means ± standard deviations from three CMEC isolations (six mice from each group for each isolation). *, significantly higher (P < 0.01) value than control unstimulated value; †, significantly lower (P < 0.01) value than control unstimulated value. (C) Representative immunoblot showing ERK1/2 phosphorylation in wild-type and p47^phox−/− CMEC stimulated with TNF-α or unstimulated in the presence or absence of tiron or DPI.

FIG. 8.

Effect of siRNA-mediated knockdown of TRAF4 on acute TNFα-induced ERK1/2 activation and p47^phox phosphorylation in HMEC-1. (A) Immunoblotting for TRAF4 expression and ERK1/2 activation after anti-TRAF4 siRNA treatment. HEK293 cells, which do not express TRAF4 endogenously (6a), were used as a negative control for TRAF4 expression. (B) Assessment of serine phosphorylation of p47^phox after knockdown of TRAF4. The same blot was reprobed with an anti-p47^phox antibody, while a parallel membrane was immunoblotted (IB) for p22^phox. IP, immunoprecipitated.

FIG. 9.

Immunofluorescence confocal micrographs of wild-type and p47^phox−/− CMEC showing surface expression of ICAM-1. Nonpermeabilized CMEC cultured onto chamber slides were stimulated with TNF-α (100 U/ml, 60 min) or not stimulated in the presence or absence of tiron. Cells were then probed for the surface membrane expression of ICAM-1. Normal rabbit IgG (5 μg/ml) was used as a negative control (lower right panel). All panels are the same scale.