Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF-kappaB (kidney), expression of iNOS and COX-2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF-kappaB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF-kappaB to DNA and the degradation of IkappaBalpha, IkappaBbeta, and IkappaBvarepsilon. Selective inhibition of iNOS activity with L-NIL reduced the circulatory failure and liver injury, while selective inhibition of COX-2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-kappaB and thus prevention of the expression of NFkappaB-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.     

TNF-alpha-induced cyclooxygenase-2 expression in human lung epithelial cells: involvement of the phospholipase C-gamma 2, protein kinase C-alpha, tyrosine kinase, NF-kappa B-inducing kinase, and I-kappa B kinase 1/2 pathway

TNF-alpha induced a dose- and time-dependent increase in cyclooxygenase-2 (COX-2) expression and PGE2 formation in human NCI-H292 epithelial cells. Immunofluorescence staining demonstrated that COX-2 was expressed in cytosol and nuclear envelope. Tyrosine kinase inhibitors (genistein or herbimycin) or phosphoinositide-specific phospholipase C inhibitor (U73122) blocked TNF-alpha-induced COX-2 expression. TNF-alpha also stimulated phosphatidylinositol hydrolysis and protein kinase C (PKC) activity, and both were abolished by genistein or U73122. The PKC inhibitor, staurosporine, also inhibited TNF-alpha-induced response. The 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, also stimulated COX-2 expression, this effect being inhibited by genistein or herbimycin. NF-kappaB DNA-protein binding and COX-2 promoter activity were enhanced by TNF-alpha, and these effects were inhibited by genistein, U73122, staurosporine, or pyrolidine dithiocarbamate. TPA stimulated both NF-kappaB DNA-protein binding and COX-2 promoter activity, these effects being inhibited by genistein, herbimycin, or pyrolidine dithiocarbamate. The TNF-alpha-induced, but not the TPA-induced, COX-2 promoter activity was inhibited by phospholipase C-gamma2 mutants, and the COX-2 promoter activity induced by either agent was attenuated by dominant-negative mutants of PKC-alpha, NF-kappaB-inducing kinase, or I-kappaB (inhibitory protein that dissociates from NF-kappaB) kinase (IKK)1 or 2. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by staurosporine or herbimycin. These results suggest that, in NCI-H292 epithelial cells, TNF-alpha might activate phospholipase C-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and protein tyrosine kinase, resulting in the activation of NF-kappaB-inducing kinase and IKK1/2, and NF-kappaB in the COX-2 promoter, then initiation of COX-2 expression and PGE2 release.     

Sphingosine-1-phosphate induces COX-2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX-2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX-2 expression by S1P in VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced the expression of COX-2 mRNA and protein in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen-activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX-2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by a selective NF-kappaB inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappaB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P-stimulated activation of NF-kappaB promoter activity was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF-kappaB. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappaB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappaB pathways was essential for S1P-induced COX-2 gene expression. Understanding the mechanisms involved in S1P-induced COX-2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.     

Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.     

Complement factor B gene regulation: synergistic effects of TNF-alpha and IFN-gamma in macrophages

Complement factor B (Bf) plays an important role in activating the alternative complement pathway. The inflammatory cytokines, in particular TNF-alpha and IFN-gamma, are critical in the regulation of Bf gene expression in macrophages. In this study, we investigated the mechanisms of Bf gene regulation by TNF-alpha and IFN-gamma in murine macrophages. Northern analysis revealed that Bf mRNA expression was synergistically up-regulated by TNF-alpha and IFN-gamma in MH-S cells. Truncations of the 5' Bf promoter identified a region between -556 and -282 bp that mediated TNF-alpha responsiveness as well as the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. Site-directed mutagenesis of a NF-kappaB-binding element in this region (-433 to -423 bp) abrogated TNF-alpha responsiveness and decreased the synergistic effect of TNF-alpha and IFN-gamma on Bf expression. EMSAs revealed nuclear protein binding to this NF-kappaB cis-binding element on TNF-alpha stimulation. Supershift analysis revealed that both p50 and p65 proteins contribute to induction of Bf by TNF-alpha. An I-kappaB dominant negative mutant blocked Bf induction by TNF-alpha and reduced the synergistic induction by TNF-alpha and IFN-gamma. In addition, the proteasome inhibitor MG132, which blocks NF-kappaB induction, blocked TNF-alpha-induced Bf promoter activity and the synergistic induction of Bf promoter activity by TNF-alpha and IFN-gamma. LPS was found to induce Bf promoter activity through the same NF-kappaB cis-binding site. These findings suggest that a NF-kappaB cis-binding site between -433 and -423 bp is required for TNF-alpha responsiveness and for TNF-alpha- and IFN-gamma-stimulated synergistic responsiveness of the Bf gene.     

Iron activates NF-kappaB in Kupffer cells

Iron exacerbates various types of liver injury in which nuclear factor (NF)-kappaB-driven genes are implicated. This study tested a hypothesis that iron directly elicits the signaling required for activation of NF-kappaB and stimulation of tumor necrosis factor (TNF)-alpha gene expression in Kupffer cells. Addition of Fe2+ but not Fe3+ (approximately 5-50 microM) to cultured rat Kupffer cells increased TNF-alpha release and TNF-alpha promoter activity in a NF-kappaB-dependent manner. Cu+ but not Cu2+ stimulated TNF-alpha protein release and promoter activity but with less potency. Fe2+ caused a disappearance of the cytosolic inhibitor kappaBalpha, a concomitant increase in nuclear p65 protein, and increased DNA binding of p50/p50 and p65/p50 without affecting activator protein-1 binding. Addition of Fe2+ to the cells resulted in an increase in electron paramagnetic resonance-detectable.OH peaking at 15 min, preceding activation of NF-kappaB but coinciding with activation of inhibitor kappaB kinase (IKK) but not c-Jun NH2-terminal kinase. In conclusion, Fe2+ serves as a direct agonist to activate IKK, NF-kappaB, and TNF-alpha promoter activity and to induce the release of TNF-alpha protein by cultured Kupffer cells in a redox status-dependent manner. We propose that this finding offers a molecular basis for iron-mediated accentuation of TNF-alpha-dependent liver injury.