The signaling pathways that couple tumor necrosis factor-␣ (TNF␣) receptors to functional, especially inf lammatory, responses have remained elusive. We report here that TNF␣ induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNF␣ resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dosedependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNF␣ on endothelial cells leading to extracellular signal-regulated kinases and NF-B activation, whereas ceramide or sphingosine was not. Furthermore, N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNF␣-induced extracellular signal-regulated kinases and NF-B activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNF␣-induced endothelial cell activation.Tumor necrosis factor-␣ (TNF␣) was originally described for its antitumor activity, but is now recognized to be one of the most pleiotropic cytokines in mediating systemic inflammatory and immune responses (1, 2). A major site for these TNF␣ actions is the vascular endothelium, where TNF␣ triggers endothelial cells to secrete various cytokines and induces or enhances the expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 and E-selectin (3). The regulated expression of these adhesion molecules is essential for the recruitment of circulating blood cells to the endothelium during the inflammatory and immune responses (3-5).TNF␣ activity is exerted through binding two distinct membrane receptors, p55 (TNF␣-R1) and p75 (TNF␣-R2). Engagement of the TNF␣ receptors results in recruitment of two distinct classes of receptor-associated proteins, one the TRADD, FADD͞MORT1 and RIP family, and the other, the TRAF family (6-8). Both of these appear to couple TNF␣ receptors to downstream signaling cascades such as cysteine proteases and NF-B activation to regulate cell proliferation, differentiation, and programmed cell death (6). Recently, the lipid second messenger, ceramide, has also received attention in TNF␣ signaling (6, 9). TNF␣ stimulates the activation of sphingomyelinase, yielding ceramide that, in turn, can induce apoptosis and may play a role in apoptotic signaling in various cell types (6, 9). In addition, ceramide can be subsequently metabolized to sphingosine and sphingosine 1-phosphate (S1P), via ceramidase and sphingosine kinase (SKase) activation, respectively (10). These sphingomyelin metabolites were also proposed to play a variety of roles in regulation of cellular activities such as calcium mobilization, cell motility, and mitogenesis (9, 10). In this study, we demonstrate that TNF␣ promoted generation of ceramide that was...

Since its discovery in 1920, a great deal of effort has gone into investigating the physiological actions of vitamin D and the impact its deficiency has on human health. Despite this intense interest, there is still disagreement on what constitutes the lower boundary of adequacy and on the Recommended Dietary Allowance. There has also been a major push to elucidate the biochemistry of vitamin D, its metabolic pathways and the mechanisms that mediate its action. Originally thought to act by altering the expression of target genes, it was realized in the mid-1980s that some of the actions of vitamin D were too rapid to be accounted for by changes at the genomic level. These rapid non-genomic actions have attracted as much interest as the genomic actions and they have spawned additional questions in an already busy field. This mini-review attempts to summarise the in vitro and in vivo work that has been conducted to characterise the rapid non-genomic actions, the mechanisms that give rise to these properties and the roles that these play in the overall action of vitamin D at the cellular level. Understanding the effects of vitamin D at the cellular level should enable the design of elegant human studies to extract the full potential of vitamin D to benefit human health.

This review covers basic aspects of histone modification and the role of posttranslational histone modifications in the development of allergic diseases, including the immune mechanisms underlying this development. Together with DNA methylation, histone modifications (including histone acetylation, methylation, phosphorylation, ubiquitination, etc.) represent the classical epigenetic mechanisms. However, much less attention has been given to histone modifications than to DNA methylation in the context of allergy. A systematic review of the literature was undertaken to provide an unbiased and comprehensive update on the involvement of histone modifications in allergy and the mechanisms underlying this development. In addition to covering the growing interest in the contribution of histone modifications in regulating the development of allergic diseases, this review summarizes some of the evidence supporting this contribution. There are at least two levels at which the role of histone modifications is manifested. One is the regulation of cells that contribute to the allergic inflammation (T cells and macrophages) and those that participate in airway remodeling [(myo-) fibroblasts]. The other is the direct association between histone modifications and allergic phenotypes. Inhibitors of histone-modifying enzymes may potentially be used as anti-allergic drugs. Furthermore, epigenetic patterns may provide novel tools in the diagnosis of allergic disorders.

Although it is well appreciated that arachidonic acid, a second messenger molecule that is released by ligandstimulated phospholipase A 2 , stimulates a wide range of cell types, the mechanisms that mediate the actions of arachidonic acid are still poorly understood. We now report that arachidonic acid stimulated the appearance of dual-phosphorylated (active) p38 mitogen-activated protein kinase as detected by Western blotting in HeLa cells, HL60 cells, human neutrophils, and human umbilical vein endothelial cells but not Jurkat cells. An increase in p38 kinase activity caused by arachidonic acid was also observed. Further studies with neutrophils show that the stimulation of p38 dual phosphorylation by arachidonic acid was transient, peaking at 5 min, and was concentration-dependent. The effect of arachidonic acid was not affected by either nordihydroguaiaretic acid, an inhibitor of the 5-, 12-, and 15-lipoxygenases or by indomethacin, an inhibitor of cyclooxygenase. Arachidonic acid also stimulated the phosphorylation and/or activity of the extracellular signal-regulated protein kinase and of c-jun N-terminal kinase in a cell-typespecific manner. An examination of the mechanisms through which arachidonic acid stimulated the phosphorylation/activity of p38 and extracellular signal-regulated protein kinase in neutrophils revealed an involvement of protein kinase C. Thus, arachidonic acid stimulated the translocation of protein kinase C ␣, ␤I, and ␤II to a particulate fraction, and the effects of arachidonic acid on mitogen-activated protein kinase phosphorylation/activity were partially inhibited by GF109203X, an inhibitor of protein kinase C. This study is the first to demonstrate that a polyunsaturated fatty acid causes the dual phosphorylation and activation of p38.

Arachidonic acid (20:4(n-6)), which is released by cells responding to a wide range of stimuli, may play an important role in intracellular signaling. We now report that incubation of WB cells with 20:4(n-6) resulted in the appearance of several tyrosine-phosphorylated cytosolic proteins. Two of the phosphotyrosine-containing proteins, migrating in SDS-polyacrylamide gels of approximately 43 and 45 kDa, corresponded in mobility to phosphorylated species of the 42- and 44-kDa mitogen-activated protein kinase (MAPK) isoforms. Immunoblots of soluble fractions from unstimulated WB cells with anti-MAPK antibodies revealed the presence of the 42- and 44-kDa isoforms of MAPK. Upon incubation with 20:4(n-6), the mobility of both isoforms was retarded, consistent with their activation by phosphorylation. Chromatography of soluble fractions from these cells on Mono Q columns revealed early and late eluting peaks of myelin basic protein kinase activity, which contained the 42- and 44-kDa MAPK isoforms, respectively. Activation of MAPK was transient, peaking at 5 min, and was detectable at 5 microM 20:4(n-6). Further studies into the mechanisms by which MAPK was activated by 20:4(n-6) strongly suggested the involvement of protein kinase C (PKC). Not only did incubation of WB cells with 20:4(n-6) result in the translocation of PKC alpha, delta, and epsilon to a particulate fraction, it was found that the fatty acid failed to activate MAPK in cells pretreated for 26 h with phorbol 12-myristate 13-acetate, which depleted WB cells of PKC alpha, delta and epsilon. In addition, fatty acids of the n-3 series were effective activators of MAPK. The present study, to our knowledge, is the first to report that polyunsaturated fatty acids can cause the activation of MAPK.

Epidemiological studies have shown a dramatic increase in the incidence and the prevalence of allergic diseases over the last several decades. Environmental triggers including risk factors (e.g., pollution), the loss of rural living conditions (e.g., farming conditions), and nutritional status (e.g., maternal, breastfeeding) are considered major contributors to this increase. The influences of these environmental factors are thought to be mediated by epigenetic mechanisms which are heritable, reversible, and biologically relevant biochemical modifications of the chromatin carrying the genetic information without changing the nucleotide sequence of the genome. An important feature characterizing epigenetically-mediated processes is the existence of a time frame where the induced effects are the strongest and therefore most crucial. This period between conception, pregnancy, and the first years of life (e.g., first 1000 days) is considered the optimal time for environmental factors, such as nutrition, to exert their beneficial epigenetic effects. In the current review, we discussed the impact of the exposure to bacteria, viruses, parasites, fungal components, microbiome metabolites, and specific nutritional components (e.g., polyunsaturated fatty acids (PUFA), vitamins, plant- and animal-derived microRNAs, breast milk) on the epigenetic patterns related to allergic manifestations. We gave insight into the epigenetic signature of bioactive milk components and the effects of specific nutrition on neonatal T cell development. Several lines of evidence suggest that atypical metabolic reprogramming induced by extrinsic factors such as allergens, viruses, pollutants, diet, or microbiome might drive cellular metabolic dysfunctions and defective immune responses in allergic disease. Therefore, we described the current knowledge on the relationship between immunometabolism and allergy mediated by epigenetic mechanisms. The knowledge as presented will give insight into epigenetic changes and the potential of maternal and post-natal nutrition on the development of allergic disease.

The current study investigated the action of 1,25-dihydroxyvitamin D 3 (1,25D) at the genomic and signal transduction levels to induce rat cytochrome P450C24 (CYP24) gene expression. A rat CYP24 promoter containing two vitamin D response elements and an Ets-1 binding site was used to characterize the mechanism of actions for the 1,25D secosteroid hormone. The Ets-1 binding site was determined to function cooperatively with the most proximal vitamin D response element in a hormone-dependent fashion. Evidence was obtained for distinct roles of ERK1/ERK2 and ERK5 in the 1,25D-inductive actions. Specifically, 1,25D stimulated the activities of ERK1/ERK2 and ERK5 in a Ras-dependent manner. Promoter induction was inhibited by mitogenactivated protein (MAP) kinase inhibitors (PD98059 and U0126) and a dominant-negative Ras mutant (Ras17N). Induction of CYP24 by 1,25D was also inhibited by overexpression of dominant-negative mutants of ERK1 and MEK5 (ERK1K71R and MEK5(A)). The p38 and JNK MAP kinases were not required for the action of 1,25D. 9-cis retinoid X receptor ␣ (RXR␣) interacted with ERK2 but not ERK5 in intact cells, whereas Ets-1 interacted preferentially with ERK5. Increased phosphorylation of RXR␣ and Ets-1 was detected in response to 1,25D. Activated ERK2 and ERK5 specifically phosphorylated RXR␣ and Ets-1, respectively. Mutagenesis of Ets-1 (T38A) reduced CYP24 promoter activity to levels observed with the dominant-negative MEK5(A) and inhibited ERK5-directed phosphorylation. Mutated RXR␣ (S260A) inhibited 1,25D-induced CYP24 promoter activity and abolished phosphorylation by activated ERK2. The 1,25D-inductive action through ERK5 involved Ets-1 phosphorylation at threonine 38, whereas hormone stimulation of ERK1/ERK2 required RXR␣ phosphorylation on serine 260. The ERK1/ERK2 and ERK5 modules provide a novel mechanism for linking the rapid signal transduction and slower transcription actions of 1,25D to induce CYP24 gene expression.The hormonally active form of vitamin D 3 is 1,25-dihydroxyvitamin D 3 (1,25D). 1 This secosteroid hormone plays a central role in calcium homeostasis and bone metabolism and participates in a diverse range of cellular actions including inhibition of tumor cell growth, induction of cell differentiation, and modulation of the immune response (1-4). The transcriptional actions of 1,25D are mediated by the nuclear vitamin D receptor (VDR), which heterodimerizes with retinoid X receptor (RXR) and binds to specific vitamin D response element (VDRE) sites in the promoter region of vitamin D-responsive genes (5). Transactivation by the liganded VDR/RXR is dependent upon the binding of one or more coactivator complexes that permit bridging to the RNA polymerase II machinery (6, 7). In the unliganded state, some nuclear receptors including VDR (8) can bind a co-repressor that inhibits transactivation (9, 10), but this repressor dissociates upon ligand binding.Maintenance of cellular 1,25D levels is critical to regulation of the function of the hormone in which high levels of 1,25D are to...

No abstract