Victoria A. Blaho

Obesity Is Associated with Inflammation and Elevated Aromatase Expression in the Mouse Mammary Gland

Elevated circulating estrogen levels are associated with increased risk of breast cancer in obese postmenopausal women. Following menopause, the biosynthesis of estrogens through CYP19 (aromatase)-mediated metabolism of androgen precursors occurs primarily in adipose tissue, and the resulting estrogens are then secreted into the systemic circulation. The potential links between obesity, inflammation, and aromatase expression are unknown. In both dietary and genetic models of obesity, we observed necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS) in the mammary glands and visceral fat. The presence of CLS was associated with activation of NF-κB and increased levels of proinflammatory mediators (TNF-α, IL-1β, Cox-2), which were paralleled by elevated levels of aromatase expression and activity in the mammary gland and visceral fat of obese mice. Analyses of the stromal-vascular and adipocyte fractions of the mammary gland suggested that macrophage-derived proinflammatory mediators induced aromatase and estrogen-dependent gene expression (PR, pS2) in adipocytes. Saturated fatty acids, which have been linked to obesity-related inflammation, stimulated NF-κB activity in macrophages leading to increased levels of TNF-α, IL-1β, and Cox-2, each of which contributed to the induction of aromatase in preadipocytes. The discovery of the obesity → inflammation → aromatase axis in the mammary gland and visceral fat and its association with CLS may provide insight into mechanisms underlying the increased risk of hormone receptor-positive breast cancer in obese postmenopausal women, the reduced efficacy of aromatase inhibitors in the treatment of breast cancer in these women, and their generally worse outcomes. The presence of CLS may be a biomarker of increased breast cancer risk or poor prognosis. Cancer Prev Res; 4(3); 329–46. ©2011 AACR.

An update on the biology of sphingosine 1-phosphate receptors.

Sphingosine 1-phosphate (S1P) is a membrane-derived lysophospholipid that acts primarily as an extracellular signaling molecule. Signals initiated by S1P are transduced by five G protein-coupled receptors, named S1P1–5. Cellular and temporal expression of the S1P receptors (S1PRs) determine their specific roles in various organ systems, but they are particularly critical for regulation of the cardiovascular, immune, and nervous systems, with the most well-known contributions of S1PR signaling being modulation of vascular barrier function, vascular tone, and regulation of lymphocyte trafficking. However, our knowledge of S1PR biology is rapidly increasing as they become attractive therapeutic targets in several diseases, such as chronic inflammatory pathologies, autoimmunity, and cancer. Understanding how the S1PRs regulate interactions between biological systems will allow for greater efficacy in this novel therapeutic strategy as well as characterization of complex physiological networks. Because of the rapidly expanding body of research, this review will focus on the most recent advances in S1PRs.

Engagement of S1P1-degradative mechanisms leads to vascular leak in mice

GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P₁) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.

Cell-surface residence of sphingosine 1-phosphate receptor 1 on lymphocytes determines lymphocyte egress kinetics

The sphingosine 1-phosphate receptor 1 (S1P1) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein–coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P1 internalization is necessary for this effect. We characterize a knockin mouse (S1p1rS5A/S5A) in which the C-terminal serine-rich S1P1 motif, which is important for S1P1 internalization but dispensable for S1P1 signaling, is mutated. T cells expressing the mutant S1P1 showed delayed S1P1 internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P1 agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P1 expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P1 on T cells is a primary determinant of lymphocyte egress kinetics in vivo.

HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation

HDL, the good cholesterol, biases the endothelial response to the lipid S1P to protect blood vessels. Maintaining vascular health with HDL Flow through blood vessels subjects endothelial cells to abnormal shear forces at specific locations that trigger inflammation, which contributes to atherosclerotic plaque formation. Galvani et al. found that vascular inflammation and atherosclerosis in mice were suppressed by the endothelial cell receptor S1P1, which is activated by S1P, a lipid mediator that is abundant in blood bound to different chaperone proteins. S1P suppressed inflammation in cultured endothelial cells when bound to the lipoprotein ApoM+HDL. Thus, S1P bound to different chaperones triggered distinct or “biased” signaling pathways, which may also contribute to the protective effect of HDL, commonly called “good cholesterol,” in atherosclerosis. The sphingosine 1-phosphate receptor 1 (S1P1) is abundant in endothelial cells, where it regulates vascular development and microvascular barrier function. In investigating the role of endothelial cell S1P1 in adult mice, we found that the endothelial S1P1 signal was enhanced in regions of the arterial vasculature experiencing inflammation. The abundance of proinflammatory adhesion proteins, such as ICAM-1, was enhanced in mice with endothelial cell–specific deletion of S1pr1 and suppressed in mice with endothelial cell–specific overexpression of S1pr1, suggesting a protective function of S1P1 in vascular disease. The chaperones ApoM+HDL (HDL) or albumin bind to sphingosine 1-phosphate (S1P) in the circulation; therefore, we tested the effects of S1P bound to each chaperone on S1P1 signaling in cultured human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to ApoM+HDL-S1P, but not to albumin-S1P, promoted the formation of a cell surface S1P1–β-arrestin 2 complex and attenuated the ability of the proinflammatory cytokine TNFα to activate NF-κB and increase ICAM-1 abundance. Although S1P bound to either chaperone induced MAPK activation, albumin-S1P triggered greater Gi activation and receptor endocytosis. Endothelial cell–specific deletion of S1pr1 in the hypercholesterolemic Apoe−/− mouse model of atherosclerosis enhanced atherosclerotic lesion formation in the descending aorta. We propose that the ability of ApoM+HDL to act as a biased agonist on S1P1 inhibits vascular inflammation, which may partially explain the cardiovascular protective functions of HDL.

Regulation of Mammalian Physiology, Development, and Disease by the Sphingosine 1-Phosphate and Lysophosphatidic Acid Receptors

The moniker lysophospholipid incorporates two broad families of membrane-derived lipids: the glycerophospholipids and the sphingolipids1. Primary representatives of these two arms are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), the majority of whose activities are mediated via multiple G-protein-coupled receptors with a high degree of specificity for either LPA or S1P 2,3. Unlike S1P, which is a single molecular species (2S-amino-1-(dihydrogen phosphate)-4E-octadecene-1,3R-diol), LPA (1-O-acyl-2-hydroxy-sn-glycero-3-phosphate) is actually a diverse group of molecules consisting of either a saturated (e.g. 16:0; 18:0) or unsaturated (e.g. 16:1; 18:1; 18:2; 20:4) fatty acid chain esterified at the sn-1 or sn-2 position of a glycerol backbone 4,5. This can also be modified to alkyl or alkenyl at the sn-1 position 6. Other bioactive members of the LP family include the LPA analogues cyclic phosphatidic acid (cPA), sphigosylphosphotidylcholine (SPC), lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), and lysophosphatidylinositol (LPI) 1,5,7. Numerous emerging reports indicate roles in mammalian biology for other, less well-characterized LP members, as well 8. In the past decade, our understanding of LP biology has expanded exponentially, fueled by the identification of LP receptors, generation and analysis of LPA and S1P-receptor knockout mice, and small molecule compounds functioning as receptor-specific agonists or antagonists 3,9,10. Specific receptors for LPA and S1P were cloned and characterized beginning only about 15 years ago. The description of the first LPAR was reported in 1996, and the orphan receptor EDG-1, which was cloned as an immediate-early gene from endothelial cells, was identified as the first S1P receptor in 1998 11-13. Since then, four more S1P receptors and at least five more LPA receptors have been identified 3. The two families have been best characterized in specific organ systems; namely, LPARs in the nervous system and cancer, and S1PR in vascular biology and immunology. That is not to say that their contributions in other fields are minor, only that they are incompletely understood.

Defective sphingosine 1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation

Sphingosine 1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. The sphingosine phosphate receptor 1 (S1P1) agonist FTY-720 (Gilenya) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring an S1pr1 gene encoding phosphorylation-deficient receptors (S1P1(S5A)) developed severe experimental autoimmune encephalomyelitis (EAE) due to autoimmunity mediated by interleukin 17 (IL-17)–producing helper T cells (TH17 cells) in the peripheral immune and nervous system. S1P1 directly activated the Jak-STAT3 signal-transduction pathway via IL-6. Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

HDL-bound sphingosine-1-phosphate restrains lymphopoiesis and neuroinflammation

Lipid mediators influence immunity in myriad ways. For example, circulating sphingosine-1-phosphate (S1P) is a key regulator of lymphocyte egress. Although the majority of plasma S1P is bound to apolipoprotein M (ApoM) in the high-density lipoprotein (HDL) particle, the immunological functions of the ApoM–S1P complex are unknown. Here we show that ApoM–S1P is dispensable for lymphocyte trafficking yet restrains lymphopoiesis by activating the S1P1 receptor on bone marrow lymphocyte progenitors. Mice that lacked ApoM (Apom−/−) had increased proliferation of Lin− Sca-1+ cKit+ haematopoietic progenitor cells (LSKs) and common lymphoid progenitors (CLPs) in bone marrow. Pharmacological activation or genetic overexpression of S1P1 suppressed LSK and CLP cell proliferation in vivo. ApoM was stably associated with bone marrow CLPs, which showed active S1P1 signalling in vivo. Moreover, ApoM-bound S1P, but not albumin-bound S1P, inhibited lymphopoiesis in vitro. Upon immune stimulation, Apom−/− mice developed more severe experimental autoimmune encephalomyelitis, characterized by increased lymphocytes in the central nervous system and breakdown of the blood–brain barrier. Thus, the ApoM–S1P–S1P1 signalling axis restrains the lymphocyte compartment and, subsequently, adaptive immune responses. Unique biological functions imparted by specific S1P chaperones could be exploited for novel therapeutic opportunities.

Sphingosine‐1‐phosphate receptor 1 signalling in T cells: trafficking and beyond

Sphingosine‐1‐phosphate (S1P) is a lipid second messenger that signals via five G protein‐coupled receptors (S1P1–5). S1P receptor (S1PR) signalling is associated with a wide variety of physiological processes including lymphocyte biology, their recirculation and determination of T‐cell phenotypes. The effect of FTY720 (Fingolimod, Gilenya™) to regulate lymphocyte egress and to ameliorate paralysis in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis led to the use of FTY720 as a first‐line oral agent for treatment of relapsing–remitting multiple sclerosis. However, a significant body of research suggests that S1P signalling may participate in diverse immune regulatory functions other than lymphocyte trafficking. This review article discusses the current knowledge of S1P signalling in the fate and function of T regulatory, T helper type 17 and memory T cells in health and disease.

An engineered S1P chaperone attenuates hypertension and ischemic injury.

Cardiovascular diseases could be treated by chaperones that deliver the lipid mediator S1P that promotes endothelial function. Targeting S1P1 on endothelial cells The lipid mediator sphingosine 1-phosphate (S1P) is ferried in the blood by different chaperone proteins, the identity of which determines the specific signaling pathway triggered by S1P binding to its receptor S1P1. When bound to the lipoprotein ApoM+HDL, S1P suppresses endothelial cell inflammation and atherosclerosis. However, globally increasing HDL abundance does not confer these benefits, and ApoM is unstable when not bound to HDL. Swendeman et al. generated a stable form of ApoM (ApoM-Fc) that bound to S1P and activated S1P1 receptors in a sustained manner in endothelial cells. ApoM-Fc–S1P treatment of mice reduced hypertension induced by angiotensin II and improved outcomes after experimentally induced myocardial infarction or stroke, without inducing the lymphopenia characteristic of S1P1 agonists. These results provide proof-of-concept evidence that developing a chaperone that targets S1P to S1P1 selectively on endothelial cells could be used to treat cardiovascular diseases. Endothelial dysfunction, a hallmark of vascular disease, is restored by plasma high-density lipoprotein (HDL). However, a generalized increase in HDL abundance is not beneficial, suggesting that specific HDL species mediate protective effects. Apolipoprotein M–containing HDL (ApoM+HDL), which carries the bioactive lipid sphingosine 1-phosphate (S1P), promotes endothelial function by activating G protein–coupled S1P receptors. Moreover, HDL-bound S1P is limiting in several inflammatory, metabolic, and vascular diseases. We report the development of a soluble carrier for S1P, ApoM-Fc, which activated S1P receptors in a sustained manner and promoted endothelial function. In contrast, ApoM-Fc did not modulate circulating lymphocyte numbers, suggesting that it specifically activated endothelial S1P receptors. ApoM-Fc administration reduced blood pressure in hypertensive mice, attenuated myocardial damage after ischemia/reperfusion injury, and reduced brain infarct volume in the middle cerebral artery occlusion model of stroke. Our proof-of-concept study suggests that selective and sustained targeting of endothelial S1P receptors by ApoM-Fc could be a viable therapeutic strategy in vascular diseases.