Khandaker N. Anwar

Protein Kinase C-δ Regulates Thrombin-Induced ICAM-1 Gene Expression in Endothelial Cells via Activation of p38 Mitogen-Activated Protein Kinase

ABSTRACT The procoagulant thrombin promotes the adhesion of polymorphonuclear leukocytes to endothelial cells by a mechanism involving expression of intercellular adhesion molecule 1 (ICAM-1) via an NF-κB-dependent pathway. We now provide evidence that protein kinase C-δ (PKC-δ) and the p38 mitogen-activated protein (MAP) kinase pathway play a critical role in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. We observed the phosphorylation of PKC-δ and p38 MAP kinase within 1 min after thrombin challenge of human umbilical vein endothelial cells. Pretreatment of these cells with the PKC-δ inhibitor rottlerin prevented the thrombin-induced phosphorylation of p38 MAP kinase, suggesting that p38 MAP kinase signals downstream of PKC-δ. Inhibition of PKC-δ or p38 MAP kinase by pharmacological and genetic approaches markedly decreased the thrombin-induced NF-κB activity and resultant ICAM-1 expression. The effects of PKC-δ inhibition were secondary to inhibition of IKKβ activation and of subsequent NF-κB binding to the ICAM-1 promoter. The effects of p38 MAP kinase inhibition occurred downstream of IκBα degradation without affecting the DNA binding function of nuclear NF-κB. Thus, PKC-δ signals thrombin-induced ICAM-1 gene transcription by a dual mechanism involving activation of IKKβ, which mediates NF-κB binding to the ICAM-1 promoter, and p38 MAP kinase, which enhances transactivation potential of the bound NF-κB p65 (RelA).

Gαq and Gβγ Regulate PAR-1 Signaling of Thrombin-Induced NF-κB Activation and ICAM-1 Transcription in Endothelial Cells

As thrombin binding to the G protein-coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, Galpha(q), and the Gbetagamma dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits Galpha(q), or alpha-transducin (Galpha(t)), a scavenger of the Gbetagamma, markedly decreased NF-kappaB activity induced by PAR-1 activation. We determined the downstream signaling targets activated by Galpha(q) and Gbetagamma that mediate NF-kappaB activation. Expression of the kinase-defective protein kinase C (PKC)-delta mutant inhibited NF-kappaB activation induced by the constitutively active Galpha(q) mutant, but had no effect on NF-kappaB activity induced by Gbeta(1)gamma(2). In related experiments, NF-kappaB as well as ICAM-1 promoter activation induced by Gbeta(1)gamma(2) were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated Galpha(q). Cotransfection of the catalytically inactive Akt mutant inhibited the NF-kappaB activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of Galpha(q) and PKC-delta. These results support a model in which ligation of PAR-1 induces NF-kappaB activation and ICAM-1 transcription by the engagement of parallel Galphaq/PKC-delta and Gbetagamma/PI3-kinase pathways that converge at Akt.

RhoA/Rho-Associated Kinase Pathway Selectively Regulates Thrombin-Induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells via Activation of IκB Kinase β and Phosphorylation of RelA/p65

We investigated the involvement of the RhoA/Rho-associated kinase (ROCK) pathway in regulating ICAM-1 expression in endothelial cells by the procoagulant, thrombin. Exposure of HUVECs to C3 exoenzyme, a selective inhibitor of Rho, markedly reduced thrombin-induced ICAM-1 expression. Inhibition of ROCK, the downstream effector of Rho, also prevented thrombin-induced ICAM-1 expression. Blockade of thrombin-induced ICAM-1 expression was secondary to inhibition of NF-κB activity, the key regulator of ICAM-1 expression in endothelial cells. In parallel studies we observed that inhibition of the RhoA/ROCK pathway by the same pharmacological and genetic approaches failed to inhibit TNF-α-induced NF-κB activation and ICAM-1 expression. The effect of RhoA/ROCK inhibition on thrombin-induced NF-κB activation was secondary to inhibition of IκB kinase activation and subsequent IκBα degradation and nuclear uptake and the DNA binding of NF-κB. Inhibition of the RhoA/ROCK pathway also prevented phosphorylation of Ser536 within the transactivation domain 1 of NF-κB p65/RelA, a critical event conferring transcriptional competency to the bound NF-κB. Thus, the RhoA/ROCK pathway signals thrombin-induced ICAM-1 expression through the activation of IκB kinase, which promotes NF-κB binding to ICAM-1 promoter and phosphorylation of RelA/p65, thus mediating the transcriptional activation of bound NF-κB.

Tumor Necrosis Factor-α Induces Early-Onset Endothelial Adhesivity by Protein Kinase Cζ–Dependent Activation of Intercellular Adhesion Molecule-1

Abstract— We tested the hypothesis that TNF-&agr; induces early-onset endothelial adhesivity toward PMN by activating the constitutive endothelial cell surface ICAM-1, the &bgr;2-integrin (CD11/CD18) counter-receptor. Stimulation of human pulmonary artery endothelial cells with TNF-&agr; resulted in phosphorylation of ICAM-1 within 1 minute, a response that was sustained up to 15 minutes after TNF-&agr; challenge. We observed that TNF-&agr; induced 10-fold increase in PMN adhesion to endothelial cells in an ICAM-1–dependent manner and that this response paralleled the rapid time course of ICAM-1 phosphorylation. We also observed that the early-onset TNF-&agr;–induced endothelial adhesivity was protein synthesis–independent and associated with cell surface ICAM-1 clustering. Pretreatment of cells with the pan-PKC inhibitor, chelerythrine, prevented the activation of endothelial adhesivity. As PKC&zgr;, an atypical PKC isoform abundantly expressed in endothelial cells, is implicated in signaling TNF-&agr;–induced ICAM-1 gene transcription, we determined the possibility that PKC&zgr; was involved in mediating endothelial adhesivity through ICAM-1 expression. We observed that TNF-&agr; stimulation of endothelial cells induced PKC&zgr; activation and its association with ICAM-1. Inhibition of PKC&zgr; by pharmacological and genetic approaches prevented the TNF-&agr;–induced phosphorylation and the clustering of the cell surface ICAM-1 as well as activation of endothelial adhesivity. Thus, TNF-&agr; induces early-onset, protein synthesis–independent expression of endothelial adhesivity by PKC&zgr;-dependent phosphorylation of cell surface ICAM-1 that precedes the de novo ICAM-1 synthesis. The rapid ICAM-1 expression represents a novel mechanism for promoting the stable adhesion of PMN to endothelial cells that is needed to facilitate the early-onset transendothelial migration of PMN.

Decreased oxidant buffering impairs NF-κB activation and ICAM-1 transcription in endothelial cells

The DNA binding activity of the transcription factor, NF-kappaB, is regulated by the phosphorylation and degradation of its inhibitory protein, IkappaB, and post-translational modification involving redox reaction of a cysteine residue (Cys62) of NF-kappaB. We addressed the role of the redox state of endothelial cells in modulating TNFalpha-induced NF-kappaB activity. The effects of TNFalpha on DNA-binding activity of NF-kappaB and expression of mRNA encoding ICAM-1 (an NF-kappaB-activated gene) were studied in human pulmonary artery endothelial (HPAE) cells under basal conditions and after decreasing the intracellular glutathione (GSH) concentration. HPAE cells were treated with buthionine sulfoximine (BSO) (16 h), an inhibitor of GSH synthesis, which caused concentration-dependent decreases in GSH concentration. Stimulation of control cells with TNFalpha resulted in reactive oxygen species (ROS) generation and activation of NF-kappaB binding to the ICAM-1 promoter and ICAM-1 transcription. However, stimulation of GSH-depleted cells with TNFalpha resulted in ROS accumulation secondary to the decreased ROS buffering capacity, and marked impairment of NF-kappaB-binding activity and ICAM-1 mRNA expression. Exposure of BSO-treated cells to the reducing agent dithiothreitol (DTT) before TNFalpha treatment or supplementation of nuclear extract (isolated after TNFalpha challenge of BSO-treated cells) with DTT significantly augmented the effect of TNFalpha on NF-kappaB-binding activity and ICAM-1 mRNA expression. Thus the oxidative modification of NF-kappaB secondary to the loss of ROS buffering capacity may regulate NF-kappaB binding to ICAM-1 promoter, and thereby ICAM-1 transcription in endothelial cells.

Critical Role of Non-Muscle Myosin Light Chain Kinase in Thrombin-Induced Endothelial Cell Inflammation and Lung PMN Infiltration

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.