Catherine H. Liu

Edg-1, the G protein–coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation

Sphingolipid signaling pathways have been implicated in many critical cellular events. Sphingosine-1-phosphate (SPP), a sphingolipid metabolite found in high concentrations in platelets and blood, stimulates members of the endothelial differentiation gene (Edg) family of G protein-coupled receptors and triggers diverse effects, including cell growth, survival, migration, and morphogenesis. To determine the in vivo functions of the SPP/Edg signaling pathway, we disrupted the Edg1 gene in mice. Edg1(-/-) mice exhibited embryonic hemorrhage leading to intrauterine death between E12.5 and E14.5. Vasculogenesis and angiogenesis appeared normal in the mutant embryos. However, vascular maturation was incomplete due to a deficiency of vascular smooth muscle cells/pericytes. We also show that Edg-1 mediates an SPP-induced migration response that is defective in mutant cells due to an inability to activate the small GTPase, Rac. Our data reveal Edg-1 to be the first G protein-coupled receptor required for blood vessel formation and show that sphingolipid signaling is essential during mammalian development.

Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate.

Vascular endothelial cells undergo morphogenesis into capillary networks in response to angiogenic factors. We show here that sphingosine-1-phosphate (SPP), a platelet-derived bioactive lipid, activates the EDG-1 and -3 subtypes of G protein-coupled receptors on endothelial cells to regulate angiogenesis. SPP induces the Gi/mitogen-activated protein kinase/cell survival pathway and the small GTPase Rho- and Raccoupled adherens junction assembly. Both EDG-1-and EDG-3-regulated signaling pathways are required for endothelial cell morphogenesis into capillary-like networks. Indeed, SPP synergized with polypeptide angiogenic growth factors in the formation of mature neovessels in vivo. These data define SPP as a novel regulator of angiogenesis.

Overexpression of Cyclooxygenase-2 Is Sufficient to Induce Tumorigenesis in Transgenic Mice

The cyclooxygenase (COX)-2 gene encodes an inducible prostaglandin synthase enzyme that is overexpressed in adenocarcinomas and other tumors. Deletion of the murine Cox-2 gene inMin mice reduced the incidence of intestinal tumors, suggesting that it is required for tumorigenesis. However, it is not known if overexpression of Cox-2 is sufficient to induce tumorigenic transformation. We have derived transgenic mice that overexpress the human COX-2 gene in the mammary glands using the murine mammary tumor virus promoter. The human Cox-2 mRNA and protein are expressed in mammary glands of female transgenic mice and were strongly induced during pregnancy and lactation. Female virgin Cox-2 transgenic mice showed precocious lobuloalveolar differentiation and enhanced expression of the β-casein gene, which was inhibited by the Cox inhibitor indomethacin. Mammary gland involution was delayed in Cox-2 transgenic mice with a decrease in apoptotic index of mammary epithelial cells. Multiparous but not virgin females exhibited a greatly exaggerated incidence of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors. Cox-2-induced tumor tissue expressed reduced levels of the proapoptotic proteins Bax and Bcl-xL and an increase in the anti-apoptotic protein Bcl-2, suggesting that decreased apoptosis of mammary epithelial cells contributes to tumorigenesis. These data indicate that enhanced Cox-2 expression is sufficient to induce mammary gland tumorigenesis. Therefore, inhibition of Cox-2 may represent a mechanism-based chemopreventive approach for carcinogenesis.

Immunosuppressive and Anti-angiogenic Sphingosine 1-Phosphate Receptor-1 Agonists Induce Ubiquitinylation and Proteasomal Degradation of the Receptor

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and β-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and T-cell responses: what we do and don't know

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic growth factor and immune modulator. GM-CSF also has profound effects on the functional activities of various circulating leukocytes. It is produced by a variety of cell types including T cells, macrophages, endothelial cells and fibroblasts upon receiving immune stimuli. Although GM-CSF is produced locally, it can act in a paracrine fashion to recruit circulating neutrophils, monocytes and lymphocytes to enhance their functions in host defense. Recent intensive investigations are centered on the application of GM-CSF as an immune adjuvant for its ability to increase dendritic cell (DC) maturation and function as well as macrophage activity. It is used clinically to treat neutropenia in cancer patients undergoing chemotherapy, in AIDS patients during therapy, and in patients after bone marrow transplantation. Interestingly, the hematopoietic system of GM-CSF-deficient mice appears to be normal; the most significant changes are in some specific T cell responses. Although molecular cloning of GM-CSF was carried out using cDNA library of T cells and it is well known that the T cells produce GM-CSF after activation, there is a lack of systematic investigation of this cytokine in production by T cells and its effect on T cell function. In this article, we will focus mainly on the immunobiology of GM-CSF in T cells.

Cyclooxygenase-1 and -2 isoenzymes.

The cyclooxygenase isoenzymes (COX-1 and -2) catalyze the rate-limiting steps in prostanoid biosynthesis. COX-1 and -2 genes encode two isoenzymes with overlapping yet distinct expression patterns and functions. Physiologically, various extracellular stimuli such as growth factors, cytokines and tumor promoters regulate the expression of COX-1 and -2 genes at both transcriptional and post-transcriptional levels. COX-2 is overexpressed in rheumatoid arthritis, colorectal and breast cancer. Prostanoids produced by the COX pathway signal via plasma membrane-localized, G-protein-coupled receptors as well as via nuclear receptors. Currently, several COX-2-selective inhibitors are developed to control the anti-inflammatory and anti-neoplastic activities of the COX-2 isoenzyme. Inhibition of the COX isoenzyme activity and/or expression may be the basis of future generation of anti-inflammatory and anti-neoplastic drugs.

Sphingosine-1-phosphate: extracellular mediator or intracellular second messenger?

Sphingosine-1-phosphate (SPP), a polar sphingolipid metabolite, has received much attention recently as an extracellular mediator and an intracellular second messenger. It regulates a wide range of biological responses such as cell growth, death, differentiation, and migration. Recent identification of plasma membrane receptors and the cloning of SPP metabolizing enzymes have increased our understanding of the biology of SPP synthesis and action. However, controversy exists regarding the mode of action of this molecule. EDG-1 and related G-protein-coupled receptors were identified recently as plasma membrane receptors for SPP. In light of this recent discovery, many of the functions of SPP previously thought to be due to intracellular second messenger action should be reevaluated. In addition, signaling properties and functions of the three known receptors for SPP need to be fully delineated. The structures and the evolutionary conservation of SPP metabolizing enzymes from yeast to mammals support the hypothesis that SPP also plays a role as an intracellular second messenger. However, definitive assignment of the intracellular role of SPP awaits purification/molecular cloning of elusive intracellular receptors. Better knowledge of the molecular basis of SPP action is needed to assess the physiological and pathophysiological significance of this bioactive lipid mediator.

Lysophosphatidic Acid Stimulates the G-protein-coupled Receptor EDG-1 as a Low Affinity Agonist

EDG-1, an inducible G-protein-coupled receptor from vascular endothelial cells, is a high affinity receptor for sphingosine 1-phosphate (SPP) (Lee, M-J., van Brocklyn, J. R., Thangada, S., Liu, C. H., Hand, A. R., Menzeleev, R., Spiegel, S., and Hla, T. (1998) Science 279, 1552–1555). In this study, we show that lysophosphatidic acid (LPA), a platelet-derived bioactive lipid structurally related to SPP, is an agonist for EDG-1. LPA binds to EDG-1 receptor with an apparentK d of 2.3 μm. In addition, LPA binding to EDG-1 induces receptor phosphorylation, mitogen-activated protein kinase activation, as well as Rho-dependent morphogenesis and P-cadherin expression. These data suggest that LPA is a low-affinity agonist for EDG-1. Activation of the endothelial receptor EDG-1 by platelet-derived lipids LPA and SPP may be important in thrombosis and angiogenesis, conditions in which critical platelet-endothelial interactions occur.

HER-2/neu status is a determinant of mammary aromatase activity in vivo: evidence for a cyclooxygenase-2-dependent mechanism.

Cytochrome P450 aromatase (aromatase), a product of the CYP19 gene, catalyzes the synthesis of estrogens from androgens. Given the significance of estrogen synthesis in hormone-dependent breast carcinogenesis, it is important to elucidate the mechanisms that regulate CYP19 expression. The main objective of this study was to define the interrelationship between HER-2/neu, cyclooxygenase-2 (COX-2), and aromatase in mammary tissue. Mammary aromatase activity and prostaglandin E(2) (PGE(2)) levels were increased in mice with mammary-targeted expression of a COX-2 transgene. In vitro, overexpressing COX-2 caused both increased PGE(2) production and aromatase activity, effects that were suppressed by celecoxib, a selective COX-2 inhibitor. Previously, we found that overexpression of HER-2/neu was associated with increased levels of COX-2 in human breast cancers. Here, we show that overexpression of HER-2/neu is also associated with increased aromatase activity. These results suggested the possibility that COX-2 was the functional intermediate linking HER-2/neu and aromatase. Consistent with this idea, COX-2 deficiency led to a gene dose-dependent reduction in mammary aromatase activity in a HER-2/neu transgenic mouse model. Complementary in vitro studies showed that HER-2/neu-mediated induction of PGE(2) synthesis and aromatase activity were suppressed by inhibiting COX-2. Collectively, our data indicate that COX-2 is the functional intermediate linking HER-2/neu and aromatase and suggest that inhibitors of PGE(2) synthesis will suppress estrogen biosynthesis in breast tissue.

Serum withdrawal-induced post-transcriptional stabilization of cyclooxygenase-2 mRNA in MDA-MB-231 mammary carcinoma cells requires the activity of the p38 stress-activated protein kinase.

Overexpression of thecyclooxygenase-2 (COX-2) gene is observed in several neoplastic diseases. However, molecular mechanisms involved in the regulation of expression of COX-2 are not well understood. In this report, we describe a unique post-transcriptional regulatory mechanism of COX-2 mRNA stabilization in MDA-MB-231 cells, a highly metastatic cell line derived from a human mammary tumor. High levels of COX-2 mRNA, protein, and enzyme activity were induced by serum withdrawal, which were potently inhibited by the addition of serum or >100-kDa serum factor. Nuclear run-on analysis and actinomycin D chase experiments indicate that regulation is primarily at the level of post-transcriptional mRNA stability. Interestingly, SB203580, an inhibitor of the p38 stress-activated protein kinase (SAPK), and overexpression of the dominant-negative p38α construct potently inhibited the serum withdrawal-induced COX-2 mRNA levels. Indeed, the half-life of COX-2 mRNA decreased from 9 to 4.5 h after SB203580 treatment, suggesting that signal transduction by the p38 SAPK pathway is required for COX-2 mRNA stability.