MKK3 and -6-dependent activation of p38alpha MAP kinase is required for cytoskeletal changes in pulmonary microvascular endothelial cells induced by ICAM-1 ligation

Previous studies demonstrated that neutrophil adherence induces ICAM-1-dependent cytoskeletal changes in TNF-alpha-treated pulmonary microvascular endothelial cells that are prevented by a pharmacological inhibitor of p38 MAP kinase. This study determined whether neutrophil adherence induces activation of p38 MAP kinase in endothelial cells, the subcellular localization of phosphorylated p38, which MAP kinase kinases lead to p38 activation, which p38 isoform is activated, and what the downstream targets may be. Confocal microscopy showed that neutrophil adhesion for 2 or 6 min induced an increase in phosphorylated p38 in endothelial cells that was punctate and concentrated in the central region of the endothelial cells. Studies using small interfering RNA (siRNA) to inhibit the protein expression of MAP kinase kinase 3 and 6, either singly or in combination, showed that both MAP kinase kinases were required for p38 phosphorylation. Studies using an antisense oligonucleotide to p38alpha demonstrated that inhibition of the protein expression of p38alpha 1) inhibited activation of p38 MAP kinase without affecting the protein expression of p38beta; 2) prevented phosphorylation of heat shock protein 27, an actin binding protein that may induce actin polymerization upon phosphorylation; 3) attenuated cytoskeletal changes; and 4) attenuated neutrophil migration to the EC borders. Thus MAP kinase kinase3- and 6-dependent activation of the alpha-isoform of p38 MAP kinase is required for the cytoskeletal changes induced by neutrophil adherence and influences subsequent neutrophil migration toward endothelial cell junctions.     

Ca2+ influx induced by protease-activated receptor-1 activates a feed-forward mechanism of TRPC1 expression via nuclear factor-kappaB activation in endothelial cells

Thrombin activation of protease-activated receptor-1 induces Ca(2+) influx through store-operated cation channel TRPC1 in endothelial cells. We examined the role of Ca(2+) influx induced by the depletion of Ca(2+) stores in signaling TRPC1 expression in endothelial cells. Both thrombin and a protease-activated receptor-1-specific agonist peptide induced TRPC1 expression in human umbilical vein endothelial cells, which was coupled to an augmented store-operated Ca(2+) influx and increase in endothelial permeability. To delineate the mechanisms of thrombin-induced TRPC1 expression, we transfected in endothelial cells TRPC1-promoter-luciferase (TRPC1-Pro-Luc) construct containing multiple nuclear factor-kappaB (NF-kappaB) binding sites. Co-expression of dominant negative IkappaBalpha mutant prevented the thrombin-induced increase in TRPC1 expression, indicating the key role of NF-kappaB activation in mediating the response. Using TRPC1 promoter-deletion mutant constructs, we showed that NF-kappaB binding sites located between -1623 and -871 in the TRPC1 5'-regulatory region were required for thrombin-induced TRPC1 expression. Electrophoretic mobility shift assay utilizing TRPC1 promoter-specific oligonucleotides identified that the DNA binding activities of NF-kappaB to NF-kappaB consensus sites were located in this domain. Supershift assays using NF-kappaB protein-specific antibodies demonstrated the binding of p65 homodimer to the TRPC1 promoter. Inhibition of store Ca(2+) depletion, buffering of intracellular Ca(2+), or down-regulation of protein kinase Calpha downstream of Ca(2+) influx all blocked thrombin-induced NF-kappaB activation and the resultant TRPC1 expression in endothelial cells. Thus, Ca(2+) influx via TRPC1 is a critical feed-forward pathway responsible for TRPC1 expression. The NF-kappaB-regulated TRPC1 expression may be an essential mechanism of vascular inflammation and, hence, a novel therapeutic target.     

c-Src-dependent tyrosine phosphorylation of IKKbeta is involved in tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression

The signaling pathway involved in tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression was further studied in human A549 epithelial cells. TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ICAM-1 promoter activity was inhibited by a protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or an Src-specific tyrosine kinase inhibitor (PP2). TNF-alpha- or TPA-induced IkappaBalpha kinase (IKK) activation was also blocked by these inhibitors, which slightly reversed TNF-alpha-induced but completely reversed TPA-induced IkappaBalpha degradation. c-Src and Lyn, two members of the Src kinase family, were abundantly expressed in A549 cells, and their activation by TNF-alpha or TPA was inhibited by the same inhibitors. Furthermore, the dominant-negative c-Src (KM) mutant inhibited induction of ICAM-1 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKC or wild-type c-Src plasmids induced ICAM-1 promoter activity, this effect being inhibited by the dominant-negative c-Src (KM) or IKKbeta (KM) mutant but not by the nuclear factor-kappaB-inducing kinase (NIK) (KA) mutant. The c-Src (KM) mutant failed to block induction of ICAM-1 promoter activity caused by overexpression of wild-type NIK. In co-immunoprecipitation and immunoblot experiments, IKK was found to be associated with c-Src and to be phosphorylated on tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop of IKKbeta, were identified as being important for NF-kappaB activation. Substitution of these residues with phenylalanines abolished ICAM-1 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways converge at IKKbeta and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate ICAM-1 expression.     

Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-kappaB DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-kappaB pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.     

p38 MAP kinase-dependent regulation of endothelial cell permeability

We have previously shown that thrombin induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction and have determined the important role of endothelial cell myosin light chain kinase (MLCK) in this process. In the present study we explored p38 MAP kinase as a potentially important enzyme in thrombin-mediated endothelial cell contractile response and permeability. Thrombin induces significant p38 MAP kinase activation in a time-dependent manner with maximal effect at 30 min, which correlates with increased phosphorylation of actin- and myosin-binding protein, caldesmon. Both SB-203580 and dominant negative p38 adenoviral vector significantly attenuated thrombin-induced declines in transendothelial electrical resistance. Consistent with these data SB-203580 decreased actin stress fiber formation produced by thrombin in endothelium. In addition, dominant negative p38 had no effect on thrombin-induced myosin light chain diphosphorylation. Thrombin-induced total and site-specific caldesmon phosphorylation (Ser789) as well as dissociation of caldesmon-myosin complex were attenuated by SB-203580 pretreatment. These results suggest the involvement of p38 MAP kinase activities and caldesmon phosphorylation in the MLCK-independent regulation of thrombin-induced endothelial cell permeability.     

Midazolam suppresses thrombin-induced heat shock protein 27 phosphorylation through inhibition of p38 mitogen-activated protein kinase in cardiac myocytes

It has been shown that anesthetics have effects of cardiac preconditioning. Heat shock proteins (HSPs) function as molecular chaperone. Among them, HSP27, a low-molecular-weight HSP, abundantly exist in heart. However, the relationship between anesthetics and HSP27 in heart is not yet clarified. We investigated whether thrombin induces or phosphorylates HSP27 in primary cultured mouse myocytes and the effect of midazolam on the thrombin-stimulated HSP27 phosphorylation and the mechanism behind it. Thrombin time dependently phosphorylated HSP27 at Ser-15 and Ser-85 while having no effect on the levels of HSP27. Midazolam markedly suppressed the thrombin-induced phosphorylation of HSP27 at both Ser-15 and Ser-85. Thrombin induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase without affecting stress-activated protein kinase/c-Jun N-terminal kinase. In addition, midazolam attenuated the phosphorylation of thrombin-induced p38 MAP kinase but not that of p44/p42 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the thrombin-induced phosphorylation of HSP27 at both Ser-15 and Ser-85. These results strongly suggest that thrombin induces the HSP27 phosphorylation at least through the p38 MAP kinase activation in cardiac myocytes and that midazolam inhibits the thrombin-induced HSP27 phosphorylation via suppression of p38 MAP kinase activation.     

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.     

Tumor necrosis factor alpha-induced activation of downstream NF-kappaB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway

TNF-alpha induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-alpha-induced ICAM-1 expression. TNF-alpha produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-alpha-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-alpha resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-alpha-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C(2) ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by TNF-alpha and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-alpha- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCalpha or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-alpha activates PC-PLC to induce activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.     

Akt regulates thrombin-induced HSP27 phosphorylation in aortic smooth muscle cells: function at a point downstream from p38 MAP kinase

We previously reported that p38 MAP kinase takes part in thrombin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether Akt is involved in the phosphorylation of HSP27 and the role of adenylyl cyclase-cAMP system. Thrombin time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) and Akt in aortic smooth muscle A10 cells. SB203580, a p38 MAP kinase inhibitor, significantly suppressed the thrombin-induced phosphorylation of Akt and the Akt inhibitor suppressed the phosphorylation of HSP27. Furthermore, the thrombin-induced phosphorylation of HSP27, p38 MAP kinase and Akt were decreased by dibutyryl-cAMP (DBcAMP). These results strongly suggest that Akt functions the thrombin-induced phosphorylation of HSP27 at a point downstream from p38 MAP kinase in aortic smooth muscle cells and the adenylyl cyclase-cAMP system is upstream regulator of the HSP27 phosphorylation in these cells.     

Thrombin stimulation of vascular adhesion molecule-1 in endothelial cells is mediated by protein kinase C (PKC)-delta-NF-kappa B and PKC-zeta-GATA signaling pathways

We recently demonstrated that thrombin induces the expression of vascular adhesion molecule-1 (VCAM-1) in endothelial cells by an NF-kappaB- and GATA-dependent mechanism. In the present study, we describe the signaling pathways that mediate this response. Thrombin stimulation of the VCAM-1 gene and promoter in human umbilical vein endothelial cells was inhibited by preincubation with the phosphatidylinositol 3-kinase inhibitor, LY294002, the protein kinase C (PKC)-delta inhibitor, rottlerin, a PKC-zeta peptide inhibitor, or by overexpression of dominant negative (DN)-PKC-zeta. In electrophoretic mobility shift assays, thrombin-mediated induction of NF-kappaB p65 binding to two NF-kappaB motifs in the upstream promoter region of VCAM-1 was blocked by LY294002 and rottlerin, whereas the inducible binding of GATA-2 to a tandem GATA motif was inhibited by LY294002 and the PKC-zeta peptide inhibitor. In co-transfection assays, thrombin stimulation of a minimal promoter containing multimerized VCAM-1 NF-kappaB sites was inhibited by DN-PKC-delta but not DN-PKC-zeta. In contrast, thrombin-mediated transactivation of a minimal promoter containing tandem VCAM-1 GATA motifs was inhibited by DN-PKC-zeta but not DN-PKC-delta. Finally, thrombin failed to induce VCAM-1 expression in vascular smooth muscle cells. Taken together, these data suggest that the endothelial cell-specific effect of thrombin on VCAM-1 expression involves the coordinate activity of PKC-delta-NF-kappaB and PKC-zeta-GATA signaling pathways.     

Thrombin induces nitric-oxide synthase via Galpha12/13-coupled protein kinase C-dependent I-kappaBalpha phosphorylation and JNK-mediated I-kappaBalpha degradation

An imbalance between thrombin and antithrombin III contributed to vascular hyporeactivity in sepsis, which can be attributed to excess NO production by inducible nitric-oxide synthase (iNOS). In view of the importance of the thrombin-activated coagulation pathway and excess NO as the culminating factors in vascular hyporeactivity, this study investigated the effects of thrombin on the induction of iNOS and NO production in macrophages. Thrombin induced iNOS protein in the Raw264.7 cells, which was inhibited by a thrombin inhibitor, LB30057. Thrombin increased NF-kappaB DNA binding, whose band was supershifted with anti-p65 and anti-p50 antibodies. Thrombin elicited the phosphorylation and degradation of I-kappaBalpha prior to the nuclear translocation of p65. The NF-kappaB-mediated iNOS induction was stimulated by the overexpression of activated mutants of Galpha(12/13) (Galpha(12/13)QL). Protein kinase C depletion inhibited I-kappaBalpha degradation, NF-kappaB activation, and iNOS induction by thrombin or the iNOS induction by Galpha(12/13)QL. JNK, p38 kinase, and ERK were all activated by thrombin. JNK inhibition by the stable transfection with a dominant negative mutant of JNK1 (JNK1(-)) completely suppressed the NF-kappaB-mediated iNOS induction by thrombin. Conversely, the inhibition of p38 kinase enhanced the expression of iNOS. In addition, JNK and p38 kinase oppositely controlled the NF-kappaB-mediated iNOS induction by Galpha(12/13)QL. Hence, iNOS induction by thrombin was regulated by the opposed functions of JNK and p38 kinase downstream of Galpha(12/13). In the JNK1(-) cells, thrombin did not increase either the NF-kappaB binding activity or I-kappaBalpha degradation despite I-kappaBalpha phosphorylation. These results demonstrated that thrombin induces iNOS in macrophages via Galpha(12) and Galpha(13), which leads to NF-kappaB activation involving the protein kinase C-dependent phosphorylation of I-kappaBalpha and the JNK-dependent degradation of phosphorylated I-kappaBalpha.