Altered nitric oxide production mediates matrix-specific PAK2 and NF-κB activation by flow

Abstract

Shear stress generated by distinct blood flow patterns modulates endothelial cell phenotype to spatially restrict atherosclerotic plaque development. Signaling through p21-activated kinase (PAK) mediates several of the deleterious effects of shear stress, including enhanced NF-κB activation and proinflammatory gene expression. Whereas shear stress activates PAK in endothelial cells on a fibronectin matrix, basement membrane proteins limit shear-induced PAK activation and inflammation through a protein kinase A-dependent pathway; however, the mechanisms underlying this regulation were unknown. We show that basement membrane proteins limit membrane recruitment of PAK2, the dominant isoform in endothelial cells, by blocking its interaction with the adaptor protein Nck. This uncoupling response requires protein kinase A-dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain. Of importance, shear stress does not stimulate nitric oxide production in endothelial cells on fibronectin, resulting in enhanced PAK activation, NF-κB phosphorylation, ICAM-1 expression, and monocyte adhesion. These data demonstrate that differential flow-induced nitric oxide production regulates matrix-specific PAK signaling and describe a novel mechanism of nitric oxide-dependent NF-κB inhibition.

FIGURE 1:

Matrix controls Nck-dependent PAK2 membrane targeting through PAK2 Ser-20 phosphorylation. (A) BAECs on basement membrane proteins (BM) or fibronectin (FN) were sheared for 15 min, and PAK2 levels in cytosolic and membrane fractions were analyzed by Western blotting. Results are normalized to α5 integrin levels in the membrane fraction. Representative immunoblots are shown. n = 3. (B) Coimmunoprecipitation of Nck in PAK2 immunoprecipitates was determined by Western blotting and normalized to PAK2 levels in the pull downs. Representative immunoblots are shown. n = 3 (C) Endothelial cells on BM or FN were sheared for the indicated times, and Ser-20 phosphorylation was determined by immunoblotting. Values are means ± SE, n = 3. *p < 0.05 compared with static condition, ^#p < 0.05 comparing matrices. (D) HAECs plated on either BM or FN were exposed to oscillatory shear stress (OSS) for 18 h, and Ser-20 phosphorylation was determined as in C. Values are means ± SE, n = 4. (E) BAECs expressing a multiepitope (ME)-tagged PAK2 construct or a S20A mutant were plated on basement membrane proteins and sheared for 30 min. PAK2 phosphorylation on Ser-141 corresponding to PAK activation was then assessed by Western blotting. Representative blots and mean densitometry values for PAK Ser-141 phosphorylation are shown. n = 3. *p < 0.05.

FIGURE 2:

Shear stress–induced PAK2 Ser-20 phosphorylation is PKA dependent. (A) BAECs were treated with the PKA inhibitor PKI (20 μM) or H89 (5 μM) for 30 min before application of fluid shear stress (15 min), and Ser-20 phosphorylation was determined by immunoblotting. Values are means ± SE, n = 3. (B) Endothelial cells transfected with siRNA against PKA Cα were sheared for 15 min, and Ser-20 phosphorylation was determined. Values are means ± SE, n = 3. (C) Endothelial cells were treated with PKI (20 μM) in the absence or presence of the PAK–Nck blocking peptide (20 μg/ml), and shear stress-induced PAK2 activation was determined by Western blotting as previously described. n = 3.

FIGURE 3:

Basement membrane proteins promote flow-induced NO production. (A) BAECs on BM or FN were exposed to shear stress for 2 h, and NO species (NOx) were quantified using the Sievers NO analyzer via chemiluminescence and normalized to total protein levels in the lysates. n = 3. (B), Endothelial cells on BM or FN were subjected laminar shear stress (LSS) or oscillatory shear stress (OSS) for 18 h or maintained under static conditions. NOx levels were measured as described in A. n = 3. (C–F) Endothelial cells on either BM or FN were sheared for the indicated times, and eNOS phosphorylation on Ser-1179, Ser-635, and Thr-497 was determined by Western blotting. Results were normalized to GAPDH, and values shown are means ± SE. Representative images are shown in C. n = 3. *p < 0.05 compared with static, ^#p < 0.05 compared between matrices.

FIGURE 4:

Enhanced NO production on basement membrane proteins is PKA dependent. (A) BAECs on BM were treated with PKI (20 μM, 15 min) or H89 (5 μM, 30 min) and sheared for 2 h, and NOx levels in cell lysates were determined using the Sievers NO analyzer via chemiluminescence and normalized to total protein levels in the lysates. n = 3. *p < 0.05. (B) Endothelial cells were treated as in A and sheared for 30 min. eNOS phosphorylation on Ser-1179 was determined by Western blotting. Results were normalized to total eNOS, and values shown are means ± SE. n = 3. *p < 0.05.

FIGURE 5:

NO/PKG inhibitors blunt PAK2 Ser-20 phosphorylation and rescue PAK activation. (A) BAECs were left untreated (NT) or treated with CPTIO (200 μM, 30 min), ODQ (10 μM, 30 min), or KT5823 (2 μM, 1 h). Cells were sheared for 15 min, and Ser-20 phosphorylation was determined. n = 4–7. (B) Endothelial cells were treated as in A, and PAK2 Ser-141 phosphorylation was determined. n = 3. (C) Endothelial cells were transfected with eNOS siRNA, plated on BM, and exposed to shear stress for 15 min. Ser-20 phosphorylation was determined as previously described. n = 3. (D) Endothelial cells treated with KT5823 as in A were sheared for 15 min and PAK2 membrane translocation determined. Results are normalized to α5 integrin levels in the membrane fraction. Representative immunoblots are shown. n = 3. (E) Endothelial cells on BM were treated with KT5823 as in A and exposed to shear stress for 15 min. PKA activity was determined by affinity precipitation and normalized to total PKA in cell lysates. Values are means ± SE, n = 3. *p < 0.05.

FIGURE 6:

Inhibiting NO/PKG signaling rescues shear stress–induced NF-κB activation on basement membrane proteins. (A) BAECs were treated with CPTIO (200 μM, 30 min), ODQ (10 μM, 30 min), or KT5823 (2 μM, 1 h) and sheared for 30 min. NF-κB activation was determined by Western blotting cell lysates for p65 Ser-536 phosphorylation. Results were normalized to GAPDH, and values shown are means ± SE, n = 3. (B) Endothelial cells were transfected with eNOS siRNA, plated on basement membrane proteins, and exposed to shear stress for 15 min. p65 phosphorylation was determined as in A, n = 3. (C) Endothelial cells expressing ME-PAK2 or the mutant S20A ME-PAK2 were exposed to shear stress for 30 min, and NF-κB activation was determined as previously described. Representative Western blots are shown. n = 3. (D) Endothelial cells plated on fibronectin were treated with 8-CPT-cGMP (200 μM, 1 h), and shear stress–induced NF-κB activation was determined. Values are means ± SE, n = 3. *p < 0.05.

FIGURE 7:

Blocking the PAK–Nck interaction prevents inflammatory signaling after PKG inhibition. (A) BAECs plated on basement membrane proteins were treated with KT5823 (2 μM) in either the absence or presence of the membrane-permeable, Tat-tagged PAK–Nck blocking peptide (20 μg/ml). Cells were then exposed to shear stress for 30 min, and NF-κB activation was determined. n = 3. (B) HAECs treated as in A were exposed to shear stress for 3 h to induce proinflammatory gene expression. ICAM-1 mRNA expression was determined by qRT-PCR and normalized to B2M mRNA levels. n = 3. *p < 0.05. (C) HAECs treated as in A were exposed to shear stress for 5 h, and the adhesion of Cell Tracker Green–labeled THP-1 monocytes was determined in static adhesion assays. Representative images are shown. (D) Monocyte adhesion in C was quantified by lysing cells in 100 mM NaOH and quantifying fluorescence in a plate reader. Results are normalized to total monocyte number (bound plus unbound) and expressed as a fold change compared with untreated cells under static conditions. Values are means ± SE, n = 3. *p < 0.05.

FIGURE 8:

Model for PKG-mediated PAK2 Ser-20 phosphorylation and loss of PAK–Nck binding on basement membrane proteins.