Estrogen receptor inhibits interleukin-6 gene expression by disruption of nuclear factor kappaB transactivation

The estrogen receptor (ER) suppresses interleukin-6 (IL-6) gene expression through interaction with nuclear factor kappaB (NF-kappaB) in a hormone-dependent manner. Classic ER binding to DNA is not required and the mechanism of repression is unclear. Previously reported studies suggest that the interference of NF-kappaB binding to DNA by ER may play an important role. An alternative model for repression would be the disruption of NF-kappaB transactivation. In the present study, gel shift assays were used to examine the binding of RelA and p50 dimers to the IL-6 promoter in the presence of ER. The effect of ER on NF-kappaB transactivation was studied independent of NF-kappaB binding to DNA using the mammalian one-hybrid system. ER had little effect on the binding of homodimers or heterodimers of RelA and p50 to the IL-6 promoter. In transfection experiments, both ERalpha and ERbeta inhibited NF-kappaB-mediated expression in a hormone dependent manner with repression also dependent upon dimerization of RelA with p50. Mutant ER that is unable to transactivate failed to repress NF-kappaB expression, but deletion of the N-terminal portion of the receptor had no effect. Taken together, these results suggest that the disruption of NF-kappaB-mediated transactivation plays a significant role in ER inhibition of IL-6 gene expression.     

Nuclear factor of activated T cells 2 transactivation in mast cells: a novel isoform-specific transactivation domain confers unique FcepsilonRI responsiveness

Murine nuclear factor of activated T cells (NFAT)2.alpha/beta differ by 42 and 28 unique amino-terminal amino acids and are differentially expressed. Both isoforms share conserved domains that regulate DNA-binding and subcellular localization. A genetic "one-hybrid" assay was used to define two distinct transactivation (TA) domains: in addition to a conserved TAD present in both isoforms, a second, novel TAD exists within the beta-specific amino terminus. Pharmacologic inhibitors G?6976 and rottlerin demonstrate that both conventional and novel protein kinase C (PKC) family members regulate endogenous mast cell NFAT activity, and NFAT2 TA. Overexpression of dominant active PKC (which has been implicated in immune receptor signaling) induces NFAT2.alpha/beta TA. Mutations within the smallest PKC-responsive transactivation domain demonstrate that the PKC effect is at least partially indirect. Significantly, the beta-specific domain confers greater ability to TA in response to treatment with phorbol 12-myristate 13-acetate/ionomycin or lipopolysaccharide, and unique sensitivity to FcepsilonRI signaling. Accordingly, overexpression of NFAT2.beta results in significantly greater NFAT- and interleukin-4 reporter activity than NFAT2.alpha. These results suggest that whereas NFAT2 isoforms may share redundant DNA-binding preferences, there are specialized functional consequences of their isoform-specific domains.     

Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H(2)O(2) formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-kappaB activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-kappaB signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.     

WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway

WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.     

Thrombin up-regulates tissue factor pathway inhibitor-2 synthesis through a cyclooxygenase-2-dependent, epidermal growth factor receptor-independent mechanism

The serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) inhibits the tissue factor-factor VIIa complex and thereby impairs factor Xa and subsequently thrombin generation. Here we show that thrombin itself up-regulates TFPI-2 mRNA and protein expression in human liver myofibroblasts, a cell type shown to express high levels of TFPI-2 (Neaud, V., Hisaka, T., Monvoisin, A., Bedin, C., Balabaud, C., Foster, D. C., Desmoulière, A., Kisiel, W., and Rosenbaum, J. (2000) J. Biol. Chem. 275, 35565-35569). This effect required thrombin catalytic activity, as shown by its abolition with hirudin. Although the thrombin effect could be mimicked by agonists of both protease-activated receptor (PAR)-1 and PAR-4, it was largely blocked by a PAR-1 blocking antibody. Transactivation of the epidermal growth factor (EGF) receptor has been reported as a common event in thrombin signaling. However, thrombin did not detectably transactivate the EGF receptor in liver myofibroblasts, and blocking the EGF receptor did not affect TFPI-2 induction. On the other hand, thrombin increased the expression of cyclooxygenase-2 (COX-2) mRNA via a MAPK-dependent pathway, and a specific COX-2 inhibitor abolished the effect of thrombin on TFPI-2 expression. Thus, thrombin, through PAR-1 signaling, up-regulates the synthesis of TFPI-2 via a MAPK/COX-2-dependent pathway. The up-regulation of TFPI-2 expression by thrombin could in turn down-regulate thrombin generation and contribute to limit blood coagulation.     

Human cytomegalovirus induced cyclooxygenase-2 in human retinal pigment epithelial cells augments viral replication through a prostaglandin pathway

Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kappaB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p<0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.