Lymphocytes require sphingosine-1-phosphate (S1P) receptor-1 to exit lymphoid organs, but the source(s) of extracellular S1P and whether S1P directly promotes egress are unknown. By using mice in which the two kinases that generate S1P were conditionally ablated, we find that plasma S1P is mainly hematopoietic in origin, with erythrocytes a major contributor, whereas lymph S1P is from a distinct radiation-resistant source. Lymphocyte egress from thymus and secondary lymphoid organs was markedly reduced in kinase-deficient mice. Restoration of S1P to plasma rescued egress to blood but not lymph, and the rescue required lymphocyte expression of S1P-receptor-1. Thus, separate sources provide S1P to plasma and lymph to help lymphocytes exit the low-S1P environment of lymphoid organs. Disruption of compartmentalized S1P signaling is a plausible mechanism by which S1P-receptor-1 agonists function as immunosuppressives.

Summary G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane signaling molecules and regulate a host of physiological and disease processes. To better understand the functions of GPCRs in vivo, we quantified transcript levels of 353 non-odorant GPCRs in 41 adult mouse tissues. Cluster analysis placed many GPCRs into anticipated anatomical and functional groups and predicted novel roles for less studied receptors. From one such prediction, we showed that the Gpr91 ligand succinate can regulate lipolysis in white adipose tissue suggesting that signaling by this citric acid cycle intermediate may regulate energy homeostasis. We also showed that pairwise analysis of GPCR expression across tissues may help predict drug side effects. This resource will aid studies to understand GPCR function in vivo and may assist in the identification of therapeutic targets.

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Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. Sphingosine-1-phosphate (S1P) can regulate endothelial barrier function, but the sources of the S1P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for the S1P receptor 1 (S1pr1). S1pr1 agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S1P levels being sufficient for S1pr1 activation in wild-type mice. However, an agonist for another endothelial cell G i -coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S1P. Our results suggest that the blood communicates with blood vessels via plasma S1P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models. IntroductionSphingosine-1-phosphate (S1P), a lipid phosphate produced in the course of sphingosine metabolism in all cell types (1), promotes endothelial cell spreading and barrier function in cell culture (2-5) and in vivo (6, 7). S1P can regulate cell behavior via 5 GPCRs, designated S1P receptor 1 (S1pr1) through S1pr5 (also known as S1P 1 -S1P 5 ) (1,4,8). Models of receptor-dependent roles for S1P in regulating endothelial barrier function have focused on S1P produced by the endothelial cells themselves, casting S1P as a downstream, autocrine/paracrine mediator of the barrier-protective effects of other agents such as activated protein C (9, 10) and angiopoietin (7). However, S1P is present at high concentrations in plasma (11), and the importance of this source of S1P in regulating vascular integrity has not been examined. In addition, GPCR-independent S1P signaling mechanisms and cell-autonomous metabolic effects of disrupting sphingosine conversion to S1P have been reported and may affect vascular integrity (1-5, 7, 12, 13). Central to understanding the physiological roles of S1P in regulating blood vessel function are identification of the sources of S1P that are important for barrier protection in vivo as well as determination of the importance of S1P from blood acting in trans on endothelial cells by receptor-dependent mechanisms ver-

The in vivo roles of the hundreds of mammalian G protein-coupled receptors (GPCRs) are incompletely understood. To explore these roles, we generated mice expressing the S1 subunit of pertussis toxin, a known inhibitor of G i/o signaling, under the control of the ROSA26 locus in a Cre recombinase-dependent manner (ROSA26 PTX ). Crossing ROSA26 PTX mice to mice expressing Cre in pancreatic β cells produced offspring with constitutive hyperinsulinemia, increased insulin secretion in response to glucose, and resistance to dietinduced hyperglycemia. This phenotype underscored the known importance of G i/o and hence of GPCRs for regulating insulin secretion. Accordingly, we quantified mRNA for each of the approximately 373 nonodorant GPCRs in mouse to identify receptors highly expressed in islets and examined the role of several. We report that 3-iodothyronamine, a thyroid hormone metabolite, could negatively and positively regulate insulin secretion via the G i -coupled α 2A -adrenergic receptor and the G s -coupled receptor Taar1, respectively, and proteaseactivated receptor-2 could negatively regulate insulin secretion and may contribute to physiological regulation of glucose metabolism. The ROSA26 PTX system used in this study represents a new genetic tool to achieve tissue-specific signaling pathway modulation in vivo that can be applied to investigate the role of G i/o -coupled GPCRs in multiple cell types and processes.

Summary We report an unexpected role for protease signaling in neural tube closure and formation of the central nervous system. Mouse embryos lacking protease-activated receptor 1 and 2 showed defective hindbrain and posterior neuropore closure and developed exencephaly and spina bifida, important human congenital anomalies. Par1 and Par2 were expressed in surface ectoderm, Par2 selectively along the line of closure. Ablation of Gi/z and Rac1 function in these Par2-expressing cells disrupted neural tube closure, further implicating G protein-coupled receptors and identifying a likely effector pathway. Cluster analysis of protease and Par2 expression patterns revealed a group of membrane-tethered proteases often co-expressed with Par2. Among these, matriptase activated Par2 with picomolar potency, and hepsin and prostasin activated matriptase. Together, our results suggest a role for protease-activated receptor signaling in neural tube closure and identify a local protease network that may trigger Par2 signaling and monitor and regulate epithelial integrity in this context.

Bone formation is exquisitely controlled in space and time. Heterotopic ossification (HO), the pathologic formation of extra-skeletal bone, occurs as a common complication of trauma or in genetic disorders and can be disabling and lethal. However, the underlying molecular mechanisms are largely unknown. Here we demonstrate that Gαs restricts bone formation to the skeleton by inhibiting Hedgehog (Hh) signaling in mesenchymal progenitor cells. In progressive osseous heteroplasia (POH), a human disease caused by null mutations in GNAS that encodes Gαs, HH signaling is upregulated in ectopic osteoblasts and progenitor cells. Ectopic Hh signaling is sufficient to induce HO, while Hh signaling inhibition blocks HO in animal models. As our previous work has shown that GNAS gain of function mutations upregulate WNT/β-Catenin signaling in fibrous dysplasia (FD), our findings identify Gαs as a critical regulator of osteoblast differentiation by maintaining a balance between two key signaling pathways: Wnt/β-catenin and Hh. HH signaling inhibitors developed for cancer therapy may be repurposed to treat HO and other diseases caused by GNAS inactivation.

The skeleton as an organ is widely distributed throughout the entire vertebrate body. Wnt signaling has emerged to play major roles in almost all aspects of skeletal development and homeostasis. Because abnormal Wnt signaling causes various human skeletal diseases, Wnt signaling has become a focal point of intensive studies in skeletal development and disease. As a result, promising effective therapeutic agents for bone diseases are being developed by targeting the Wnt signaling pathway. Understanding the functional mechanisms of Wnt signaling in skeletal biology and diseases highlights how basic and clinical studies can stimulate each other to push a quick and productive advancement of the entire field. Here we review the current understanding of Wnt signaling in critical aspects of skeletal biology such as bone development, remodeling, mechanotransduction, and fracture healing. We took special efforts to place fundamentally important discoveries in the context of human skeletal diseases.T he skeleton has many important functions related to human health. Aside from the classical functions of the skeleton in structural support and movement, the bone matrix forms a major reservoir of calcium and other inorganic ions, and bone cells are active regulators of calcium homeostasis. Recent data suggest that bone cells can secrete hormones (e.g., FGF23 and osteocalcin) and likely play a physiologically significant role in regulating phosphate and energy homeostasis. It has emerged that Wnt signaling plays a major role controlling multiple aspects of skeletal development and maintenance. Thus, understanding how the Wnt pathway controls skeletal growth and homeostasis has broad implications for human health and disease.Cartilage and bone define the skeleton and are produced by chondrocytes and osteoblasts, respectively. During embryonic development, bones are formed by two distinct processes: intramembranous and endochondral ossification (Fig. 1A). A number of cranial bones and the lateral portion of the clavicles are formed by intramembranous ossification. In this process, mesenchymal progenitor cells condense and differentiate directly into bone-forming osteoblasts. The majority of bones in our body are formed by endochondral ossification, during which mesenchymal progenitor cells condense and differentiate first into cartilage-forming chondrocytes to generate an avascular template of the future bone. Chondrocytes in these