Stephen M. Prescott

Cyclooxygenase-2 and carcinogenesis.

Numerous investigations have shown that COX-2 is a participant in the pathway of colon carcinogenesis, especially when mutation of the APC tumor suppressor is the initiating event. Moreover, it seems that the amount of COX-2 is important, since there is a correlation between its level of expression and the size of the tumors and their propensity to invade underlying tissue [40]. Inhibiting COX-2 at an early stage blocks the development of malignant tumors, causes pre-malignant tumors to regress and may improve the outcome once the cancer is completely established. This set of findings seems to link very strongly with the traditional observation that chronic inflammation is a precursor to a variety of types of cancer. By this formulation, inflammatory stimuli increase COX-2 and the downstream events that it induces promote tumor formation. All of these finding suggest that existing NSAIDs will be useful for the prophylaxis of colon cancer and polyps and we eagerly await clinical investigations that will generate guidelines that suggest those individuals that are the most appropriate recipients for such therapy. Although this field has progressed rapidly in the last few years, many important questions remain.

Many actions of cyclooxygenase‐2 in cellular dynamics and in cancer

Cyclooxygenase‐2 (COX‐2) is the inducible isoform of cyclooxygenase, the enzyme that catalyzes the rate‐limiting step in prostaglandin synthesis from arachidonic acid. Various prostaglandins are produced in a cell type‐specific manner, and they elicit cellular functions via signaling through G‐protein coupled membrane receptors, and in some cases, through the nuclear receptor PPAR. COX‐2 utilization of arachidonic acid also perturbs the level of intracellular free arachidonic acid and subsequently affects cellular functions. In a number of cell and animal models, induction of COX‐2 has been shown to promote cell growth, inhibit apoptosis and enhance cell motility and adhesion. The mechanisms behind these multiple actions of COX‐2 are largely unknown. Compelling evidence from genetic and clinical studies indicates that COX‐2 upregulation is a key step in carcinogenesis. Overexpression of COX‐2 is sufficient to cause tumorigenesis in animal models and inhibition of the COX‐2 pathway results in reduction in tumor incidence and progression. Therefore, the potential for application of non‐steroidal anti‐inflammatory drugs as well as the recently developed COX‐2 specific inhibitors in cancer clinical practice has drawn tremendous attention in the past few years. Inhibition of COX‐2 promises to be an effective approach in the prevention and treatment of cancer, especially colorectal cancer. J. Cell. Physiol. 190: 279–286, 2002.

Mammalian diacylglycerol kinases, a family of lipid kinases with signaling functions.

Many intracellular signaling pathways are initiated by a simple reaction, the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) 1 (1), which results in a transient rise in the amounts of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The initial signaling has two predictable components: IP3 binds to intracellular receptors to initiate calcium release from intracellular stores (2), and DAG functions as an allosteric activator of protein kinase C (PKC) (3). Both the polar product, IP3, and the lipid messenger, DAG, are converted to inactive products to return the cell to the basal state; the pathways that regulate inositol phosphate levels were reviewed in the fourth minireview in this series (22). In addition to activating PKC, DAG participates in other cellular events. For example, it is a potent activator of the guanine nucleotide exchange factors vav (4) and Ras-GRP (5), indicating a potential role for DAG in regulating Ras and Rho family proteins. In addition to these signaling roles, DAG occupies a central position in the synthesis of major phospholipids (phosphatidylcholine and phosphatidylethanolamine (6)) and triacylglycerols. Thus, to maintain cellular homeostasis, intracellular diacylglycerol levels must be tightly regulated. This is illustrated by evidence that inappropriate accumulation of diacylglycerol contributes to cellular transformation. For example, cell lines that overexpress PLC g have a malignant phenotype (7). Also, cells transformed with one of several oncogenes have elevated DAG levels (8–11), and growth factors that are proto-oncogenes stimulate this pathway. Most of the evidence for this pathological effect centers on excessive and/or prolonged activation of PKC, which is a common feature of the transformed state, both in tumors and in cell cultures (12). PKC function was identified, in part, by virtue of being the target for phorbol esters; these tumor promoters function in the same way as DAG to activate PKC but persist because they are not metabolized (or at least this happens very slowly). Thus, these observations have led to the hypothesis that prolonged elevation of DAG functions as a tumor promoter, the equivalent of an endogenous phorbol ester. This review focuses on a family of enzymes, the diacylglycerol kinases (DGKs) that phosphorylate diacylglycerol to phosphatidic acid (PA) (Fig. 1), which also has signaling functions; it stimulates DNA synthesis (13, 14) and modulates the activity of several enzymes including phosphatidylinositol 5-kinases (PI-5-K) (reviewed by Rameh and Cantley (84), first article in this series), PAK1 (15), PKCz, and Ras-GAP (16). Although the bulk of the signaling “pool” of PA (it, too, is an intermediate in phospholipid synthesis) is thought to derive from the action of phospholipase D (16), DGKs likely contribute to it as well. Thus, DGKs catalyze a reaction that removes DAG and would terminate the PKC-mediated signal but yield a product, PA, that has other functions both in signaling and phospholipid synthesis. The net result on cellular events is, therefore, difficult to predict, but the potential outcomes all support the conclusion that DGKs occupy an interesting niche.

Sol Sherry Lecture in Thrombosis: Molecular Events in Acute Inflammation

The inflammatory response is characterized by a multistep molecular interaction between “signaling” cells, such as endothelial cells, and “responding” cells, such as neutrophils and monocytes. In the first step, selectins produced by signaling cells mediate the tethering of responding cells at sites of inflammation. Subsequently, an additional mediator expressed by signaling cells activates the tethered responding cells. Under pathological conditions, the same mechanism is invoked in inappropriate ways: (1) by prolonged presentation of selectins on the cell surface and (2) by the unregulated production of oxidized phospholipids that mimic the normal secondary signaling molecule, platelet-activating factor (PAF). The enzyme PAF acetylhydrolase (PAF-AH) inactivates PAF and oxidized phospholipids and constitutes an “off” switch that suppresses inflammation. Inhibition of normal PAF-AH function or inactivating mutations of the PAF-AH gene can lead to increased susceptibility to inflammatory disease. These studies have relevance to atherosclerosis and thrombosis, because inflammation is a central feature of both.

Celecoxib inhibits meningioma tumor growth in a mouse xenograft model

Treatments for recurrent meningiomas are limited. We previously demonstrated universal expression of COX‐2 in meningiomas and dose‐dependent growth inhibition in vitro with celecoxib, a COX‐2 inhibitor. We therefore tested the effects of celecoxib on meningioma growth in a mouse xenograft model.

The p38 MAPK Pathway Mediates Transcriptional Activation of the Plasma Platelet-activating Factor Acetylhydrolase Gene in Macrophages Stimulated with Lipopolysaccharide

Administration of lipopolysaccharide (LPS) to experimental animals results in the up-regulation of expression of the plasma form of platelet-activating factor acetylhydrolase (PAF AH) in tissue macrophages. To investigate the mechanism underlying induction of PAF AH by LPS we used murine RAW264.7 and human THP-1 macrophages as model systems. We found that the p38 mitogen-activated protein kinase (p38 MAPK) pathway mediates transcriptional activation of the PAF AH gene through the participation of nucleotides -68/-316 relative to the transcriptional initiation site. This promoter region spans two Sp1/Sp3 binding sites (SP-A and SP-B) and is necessary and sufficient for the observed effect. Disruption of these Sp binding sites significantly reduces promoter activity in LPS-stimulated cells. The ability of LPS to induce transcriptional activation of PAF AH is not due to enhanced Sp1/Sp3 binding to the promoter but involves enhanced transactivation function of Sp1 via p38 MAPK activation. These studies characterize the mechanism by which LPS modulates expression of PAF AH at the transcriptional level, and they have important implications for our understanding of responses that occur during the development of LPS-mediated inflammatory diseases.

Molecular basis for susceptibility of plasma platelet-activating factor acetylhydrolase to oxidative inactivation

Platelet‐activating factor acetylhydrolase (PAF‐AH) is a phospholipase A2 that inactivates potent lipid messengers, such as PAF and modified phospho‐lipids generated in settings of oxidant stress. The catalytic activity of PAF‐AH is sensitive to oxidants, a feature that may have pathological consequences. We report that peroxynitrite, an oxidant species generated after cellular activation, mediates oxidative inactivation of PAF‐AH. We found that peroxynitrite inactivated and derivatized the recombinant protein and obtained evidence supporting a role for a methionine and two tyrosine residues in this process. We employed inter‐species comparisons and site‐directed mutagenesis and identified a role for M‐117, and a smaller contribution of Y‐307 and Y‐335 as targets of oxidant attack using free and lipoprotein‐associated recombinant proteins. M‐117 is adjacent to W‐115 and L‐116, which are essential for association of PAF‐AH with LDL. Oxidation of LDL‐associated PAF‐AH partially dissociated the enzyme from the particles. Similarly, oxidation of the purified enzyme in the absence of lipoproteins prevented subsequent association with LDL. These results provide new insights into the molecular mechanisms that mediate inactivation of PAF‐AH in settings of oxidant stress and the consequences of oxidation on the ability of this enzyme to associate with LDL.—MacRitchie, A. N., Gardner, A. A., Prescott, S. M., Stafforini, D. M. Molecular basis for susceptibility of plasma platelet‐activating factor acetylhydrolase to oxidative inactivation. FASEB J. 21, 1164–1176 (2007)

IFN-ε Mediates TNF-α-Induced STAT1 Phosphorylation and Induction of Retinoic Acid-Inducible Gene-I in Human Cervical Cancer Cells

Retinoic acid inducible gene-I (RIG-I) plays important roles during innate immune responses to viral infections and as a transducer of cytokine signaling. The mechanisms of RIG-I up-regulation after cytokine stimulation are incompletely characterized. It was previously reported that IFN–γ induces the expression of RIG-I in endothelial cells. In this study, we characterized the mechanism of type I IFN-mediated up-regulation of RIG-I in HeLa cells and found that, in addition to type I IFN, TNF-α, a cytokine that regulates innate immune responses, induced expression of RIG-I. To investigate whether TNF-α- and type I IFN-mediated up-regulations of RIG-I were causally related, we studied the kinetics of these responses. Our results were consistent with a model in which TNF-α functioned upstream of type I IFNs. The ability of TNF-α to up-regulate RIG-I required protein synthesis, expression of functional type I IFNRs, and STAT1 signaling. We also found that IFN-ε was the only IFN isoform expressed constitutively in HeLa cells and that its expression was up-regulated in response to stimulation with TNF-α. The mechanism of up-regulation involved stabilization of IFN-ε mRNA in the absence of transcriptional activation. Silencing the expression of IFN-ε attenuated STAT1 expression and phosphorylation and inhibited RIG-I expression, providing additional support for the participation of IFN-ε upstream of STAT1. Our findings support a sequential mechanism whereby TNF-α leads to stabilization of IFN-ε mRNA, increased IFN-ε synthesis, engagement of type I IFNRs, increased STAT1 expression and phosphorylation, and up-regulation of RIG-I expression. These findings have implications for our understanding of the immune responses that follow cytokine stimulation.

Fish oil fix

Fish oil has anti-inflammatory properties, but for years the mechanism has remained obscure. That mechanism now begins to come to light—and aspirin may feed into the system by promoting the production of lipid mediators derived from the oil.

The cloning and developmental regulation of murine diacylglycerol kinase ζ

Diacylglycerol kinases (DGKs) regulate the key signaling intermediates diacylglycerol (DAG) and phosphatidic acid (PA). We isolated cDNA clones of mouse diacylglycerol kinase ζ (mDGKζ) and found that it shares 88% identity at the nucleic acid level and 95.5% identity at the amino acid level with human DGKζ (hDGKζ). Murine DGKζ protein rose gradually during embryonic development, and was abundant in newborn and adult brains. By RNA whole‐mount in situ hybridization, mDGKζ was shown to be expressed in spinal ganglia and limb buds at low level in E11.5 embryos and at higher level in E12.5 embryos. In E13.5 embryos, DGKζ mRNA was highly expressed in vibrissa follicles, in spinal ganglia, and in the interdigital regions of the developing limbs. Northern blotting showed that DGKζ expression was limited to specific anatomical regions of the brain. Thus, the expression of DGKζ is regulated temporally and spatially during mammalian development and correlates with the development of sensory neurons and regions undergoing apoptosis.