Recruitment of the adaptor protein Nck to PECAM-1 couples oxidative stress to canonical NF-κB signaling and inflammation

Abstract

Oxidative stress stimulates nuclear factor κB (NF-κB) activation and NF-κB-dependent proinflammatory gene expression in endothelial cells during several pathological conditions, including ischemia/reperfusion injury. We found that the Nck family of adaptor proteins linked tyrosine kinase signaling to oxidative stress-induced activation of NF-κB through the classic IκB kinase-dependent pathway. Depletion of Nck prevented oxidative stress induced by exogenous hydrogen peroxide or hypoxia/reoxygenation injury from activating NF-κB in endothelial cells, increasing the abundance of the proinflammatory molecules ICAM-1 (intracellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) and recruiting leukocytes. Nck depletion also attenuated endothelial cell expression of genes encoding proinflammatory factors but not those encoding antioxidants. Nck promoted oxidative stress-induced activation of NF-κB by coupling the tyrosine phosphorylation of PECAM-1 (platelet endothelial cell adhesion molecule-1) to the activation of p21-activated kinase, which mediates oxidative stress-induced NF-κB signaling. Consistent with this mechanism, treatment of mice subjected to ischemia/reperfusion injury in the cremaster muscle with a Nck inhibitory peptide blocked leukocyte adhesion and emigration and the accompanying vascular leak. Together, these data identify Nck as an important mediator of oxidative stress-induced inflammation and a potential therapeutic target for ischemia/reperfusion injury.

Figure 1. Oxidant stress requires Nck for canonical NF-κB activation in endothelial cells

(A) Phosphorylation of NF-κB in HAECs treated with increasing doses of H₂O₂ was determined by Western blotting. Phospho-NF-κB was normalized to total NF-κB and conveyed as a fold change compared to untreated conditions. (B) Phosphorylation of NF-κB in HAECs treated with H₂O₂ for the indicated times was determined as in (A). (C/D) IKK activity was determined in HAECs transfected with Nck siRNA and treated with H₂O₂. (E) HAECs were treated as in (C) and nuclear translocation of NF-κB activation was determined by immunofluorescence staining (fig. S2B). At least 100 cells per condition were scored for the presence or absence of nuclear NF-κB staining for each experiment. (F) Phosphorylation of NF-κB in HAECs treated as in (C) was determined by Western blotting. (G) Phosphorylation of NF-κB in HAECs treated with Nck siRNA and exposed to hypoxia followed by reoxygenation (Hyp./reox.) was determined by Western blotting. n=4–5 independent experiments in all panels. * p<0.05, ** p<0.01, *** p<0.001.

Figure 2. Depleting Nck expression blunts oxidant stress-induced endothelial activation and leukocyte recruitment

(A–C) The abundance of VCAM-1 (A, B) or ICAM-1 (A, C) in HAECs transfected with Nck1/2 siRNA and treated with H₂O₂ or TNFα was determined by Western blotting. (D–F) The abundance of VCAM-1 (D, E) or ICAM-1 (D, F) in HAECs transfected with Nck siRNA and exposed to hypoxia/reoxygenation was determined by Western blotting. (G/H) HAECs transfected with Nck1/2 siRNA were exposed to H₂O₂ (G) or hypoxia/reoxygenation injury (H). The adhesion of Cell Tracker Green-labeled THP-1 monocytes was determined in static adhesion assays (fig. S3) and conveyed as percent of monocytes adhering under each condition. (I/J) HAECs transfected with Nck1/2 siRNA were exposed to H₂O₂ (I) or hypoxia/reoxygenation (J). Changes in the expression of genes encoding proinflammatory and antioxidant factors were determined by qRT-PCR. Results show the fold change in gene expression compared to untreated conditions. n=5 independent experiments in all panels. * p<0.05, ** p<0.01, *** p<0.001.

Figure 3. Nck couples oxidant stress-induced tyrosine phosphorylation to activation of PAK2

(A) Total cellular tyrosine phosphorylation was determined in cells treated with H₂O₂ for the indicated times. Representative blots are shown. (B) Nck co-immunoprecipitation with tyrosine phosphorylated proteins was determined in cells treated with H₂O₂. (C) HAECs were treated as in (B) and Nck/phosphotyrosine interactions were analyzed in situ by proximity ligation assays. Average proximal ligations per cell are shown. (D) Cells were treated as in (B), and Nck recruitment to the membrane fraction was determined by Western blotting and normalized to the integral membrane protein α5 integrin. (E) HAECs were treated as in (B) and PAK2 co-immunoprecipitation with Nck1 and Nck2 was analyzed. (F) Activation of PAK2 was determined by Western blotting in cells pretreated with Nck-blocking peptide before treatment with H₂O₂. (G) Cells were treated as in (F), and PAK2 recruitment to the membrane fraction was determined by Western blotting and normalization to the integral membrane protein α5 integrin. n=5 independent experiments for each panel. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4. Oxidant stress-induced PECAM-1 phosphorylation stimulates interactions with NcK

(A/B) Nck was immunoprecipitated from HAECs treated with H₂O₂, and Nck-interacting tyrosine phosphorylated proteins were identified by Western blotting with the 4G10 antibody. Enhanced interaction with a 130kDa tyrosine phosphorylated protein was blunted by PECAM-1 siRNA. Representative Western blots are shown. (C–E) PECAM-1 was immunoprecipitated from HAECs treated with H₂O₂. Changes in PECAM-1 phosphorylation (C/D) and Nck coimmunoprecipitation (C/E) were analyzed by Western blotting and normalized to total PECAM-1 in the immunoprecipitates. Representative Western blots are shown. (F) HAECs were treated as in (A) and PECAM-1/Nck interactions were analyzed in situ by proximity ligation assays. Average proximal ligations per cell is shown. n=5 independent experiments for A–E and n=4 independent experiments for (F). * p<0.05, ** p<0.01.

Figure 5. PECAM-1 phosphorylation is required for its interaction with the Nck SH2 domain

(A) Pulldown of PECAM-1 with GST-tagged Nck1 SH2 domains following H₂O₂ treatment in HAECs was determined by Western blotting and normalized to PECAM-1 abundance in the cell lysates. The GST-tagged R308K mutant SH2 domain served as a negative control for phosphotyrosine-dependent interactions. Representative Western blots are shown. (B/C) HEK293 cells expressing wild-type PECAM-1 or the phosphorylation deficient PECAM-1 YYFF mutant were lysed and precipitation with either (B) the GST-Nck1 SH2 domain or (C) endogenous Nck1/2 was determined by Western blotting. Precipitated PECAM-1 was normalized to either total PECAM-1 in the lysates (A) or total Nck1/2 abundance in the immunoprecipitates (B). Representative Western blots are shown. n=5 independent experiments for each panel. ** p < 0.01, *** p < 0.001.

Figure 6. PECAM-1 is required for oxidant stress-induced proinflammatory responses

(A/B) HAECs transfected with PECAM-1 siRNA were treated with H₂O₂ for the indicated times and analyzed for phosphorylated NF-κB (A, B) and VCAM-1 abundance (A, C) by Western blotting. Representative Western blots are shown. n=5 independent experiments. *** p<0.001, **** p < 0.0001. (D) Model of oxidant stress-induced activation of NF-κB activation through PECAM-1-dependent recruitment of Nck.

Figure 7. The Nck blocking peptide blunts inflammation and permeability during ischemia/reperfusion injury in vivo

Mice were pretreated with vehicle, control peptide (Ctrl Pep) or the Nck blocking peptide (Nck-Pep) and subjected to sham or ischemia/reperfusion (I/R) injury. Intravital microscopy was performed on cremasteric postcapillary venules. (A) Leukocyte adhesion within the venules (conveyed as the number of leukocytes adhering to the vessel wall per mm²), (B) leukocyte emigration out of the vessel (number of emigrated cells per mm² of interstitum), and (C) leakage of FITC-albumin (as a measure of vessel permeability) were measured before ischemia (pre) and throughout reperfusion. (D) Representative images of FITC-albumin leakage at 60 min reperfusion are shown. n=4–8 mice per group. Statistical comparisons (Bonferroni, P≤0.0033):* comparing Vehicle I/R and Control Peptide-I/R to pretreatment values (p < 0.001), # comparing Vehicle-I/R and Control Peptide-I/R to all other groups (p < 0.001), & comparing Vehicle-I/R group to pretreatment (p < 0.001), ‡ comparing Control Peptide-I/R to pretreatment (p < 001), $ comparing Vehicle-I/R to Nck Peptide-I/R (p < 0.01). § comparing Control Peptide-I/R to Nck Peptide-I/R (p < 0.01).