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K N Anwar

Publications and source records attributed to K N Anwar.

6 recordsLinked to original sources
PubMed2001

Decreased oxidant buffering impairs NF-kappaB 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.

Buthionine Sulfoximine↗
PubMed2001

Protein kinase C-delta regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase.

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-kappaB-dependent pathway. We now provide evidence that protein kinase C-delta (PKC-delta) 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-delta and p38 MAP kinase within 1 min after thrombin challenge of human umbilical vein endothelial cells. Pretreatment of these cells with the PKC-delta inhibitor rottlerin prevented the thrombin-induced phosphorylation of p38 MAP kinase, suggesting that p38 MAP kinase signals downstream of PKC-delta. Inhibition of PKC-delta or p38 MAP kinase by pharmacological and genetic approaches markedly decreased the thrombin-induced NF-kappaB activity and resultant ICAM-1 expression. The effects of PKC-delta inhibition were secondary to inhibition of IKKbeta activation and of subsequent NF-kappaB binding to the ICAM-1 promoter. The effects of p38 MAP kinase inhibition occurred downstream of IkappaBalpha degradation without affecting the DNA binding function of nuclear NF-kappaB. Thus, PKC-delta signals thrombin-induced ICAM-1 gene transcription by a dual mechanism involving activation of IKKbeta, which mediates NF-kappaB binding to the ICAM-1 promoter, and p38 MAP kinase, which enhances transactivation potential of the bound NF-kappaB p65 (RelA).

Cells, Cultured↗
PubMed2000

Protein kinase C-zeta mediates TNF-alpha-induced ICAM-1 gene transcription in endothelial cells.

We addressed the role of protein kinase C (PKC) isozymes in mediating tumor necrosis factor-alpha (TNF-alpha)-induced oxidant generation in endothelial cells, a requirement for nuclear factor-kappaB (NF-kappaB) activation and intercellular adhesion molecule-1 (ICAM-1) gene transcription. Depletion of the conventional (c) and novel (n) PKC isozymes following 24 h exposure of human pulmonary artery endothelial (HPAE) cells with the phorbol ester, phorbol 12-myristate 13-acetate (500 nM), failed to prevent TNF-alpha-induced oxidant generation. In contrast, inhibition of PKC-zeta synthesis by the antisense oligonucleotide prevented the oxidant generation following the TNF-alpha stimulation. We observed that PKC-zeta also induced the TNF-alpha-induced NF-kappaB binding to the ICAM-1 promoter and the resultant ICAM-1 gene transcription. We showed that expression of the dominant negative mutant of PKC-zeta prevented the TNF-alpha-induced ICAM-1 promoter activity, whereas overexpression of the wild-type PKC-zeta augmented the response. These data imply a critical role for the PKC-zeta isozyme in regulating TNF-alpha-induced oxidant generation and in signaling the activation of NF-kappaB and ICAM-1 transcription in endothelial cells.

Cells, Cultured↗
PubMed1999

Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion.

We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation.

Cell Line↗
PubMed1999

Protein kinase C-activated oxidant generation in endothelial cells signals intercellular adhesion molecule-1 gene transcription.

We tested the hypothesis that activation of protein kinase C (PKC) and generation of oxidants are critical sequential signals mediating tumor necrosis factor (TNF)-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Stimulation of human pulmonary artery endothelial (HPAE) cells with TNF-alpha (100 U/ml) induced the activation of PKC and, subsequently, generation of oxidants. Pretreatment with calphostin C, a specific PKC inhibitor, prevented oxidant generation after TNF-alpha stimulation, indicating that PKC activation mediated the production of oxidants in HPAE cells. In contrast, pretreatment of HPAE cells with N-acetylcysteine, an antioxidant and a precursor of glutathione, failed to prevent PKC activation, indicating that PKC activation was not secondary to the oxidant production. These findings suggest that oxidant generation in endothelial cells occurs downstream of PKC activation. However, both PKC activation and oxidant generation were necessary for ICAM-1 mRNA expression because the pretreatment of HPAE cells with either calphostin C or N-acetylcysteine inhibited the TNF-alpha-induced activation of NF-kappaB and prevented the activation of ICAM-1 promoter. Prolonged exposure of HPAE cells to the phorbol ester, phorbol-12-myristate-13-acetate, which is known to deplete all except atypical PKC isozymes, failed to prevent TNF-alpha-induced ICAM-1 mRNA expression. We conclude that TNF-alpha-induced oxidant generation secondary to the activation of a phorbol ester-insensitive PKC isozyme signals the activation NF-kappaB and ICAM-1 gene transcription.

Cells, Cultured↗