Constitutive expression of the S1P1 receptor in adult tissues
Introduction
Sphingosine-1-phosphate (S1P), a bioactive lipid molecule, is produced from the metabolism of sphingomyelin. S1P acts extracellularly as a ligand via the specific G protein-coupled receptors and perhaps intracellularly as a second messenger in various cell types. Its levels are tightly regulated by a series of enzymes, for example, sphingosine kinase, S1P phosphatase and S1P lyase. S1P receptors, namely, S1P1/EDG-1, S1P2/EDG-5, S1P3/EDG-3, S1P4/EDG-6, and S1P5/EDG-8 are now recognized as high-affinity receptors for S1P [1]. Differential expression of the S1P receptors and the levels of S1P in various tissues are thought to regulate a number of important physiological and pathological processes in higher eukaryotes.
The functional roles of the S1P1 receptor were derived primarily from the studies on endothelial cells in vitro. The S1P1 cDNA was originally cloned from human umbilical vein endothelial cells as a phobol 12-myristate 13-acetate-inducible gene [2]. Activation of S1P1 by S1P in endothelial cells regulates cell survival, adherens junction assembly, migration, and morphogenesis [3]. S1P treatment in many cell types, including endothelial cell and vascular smooth muscle cell, resulted in the induction of DNA synthesis and cell proliferation in a Gi-dependent manner, in part, through S1P1 [4], [5]. S1P is also a potent modulator of cell migration. Both S1P1 and S1P3 receptors are required for focal contact formation and chemotaxis in endothelial cells in which S1P1 is more dedicated to Racmediated cortical actin assembly and lamellapodia formation and S1P3 functions in stress fiber formation and focal contact assembly via Rho pathway [6]. Interestingly recent studies showed that S1P2 inhibited cell migration via a signaling pathway dependent of the GTPase Rho, suggesting that differential expression of S1P receptors may regulate S1P-mediated cellular responses [7]. S1P1 and S1P3 have been shown to collaborate in the assembly and maintenance of adherens junctions via the Rho and Rac pathways in endothelial cells [3]. Indeed, S1P stimulation of the endothelial cell monolayer inhibited thrombin-induced transcellular permeability in vitro [8]. An agonist of S1P receptors, FTY720-P, which is an experimental agent undergoing clinical trials for the treatment of transplant rejection, also induced adherens junction assembly and inhibited vascular permeability in vivo [9]. These data suggest that S1P receptors are important for the physiological homeostasis of the vasculature.
In vivo studies showed that S1P synergized with angiogenic factors such as fibroblast growth factor-2 and vascular endothelial growth factor (VEGF) to induce angiogenesis and vascular maturation [3]. Moreover, S1P1 knockout mice died in utero between embryonic day 13.5 and E14.5 due to hemorrhage, which is caused by defective vascular maturation [10]. S1P1−/− mice showed a defect in the VSMC and pericyte coverage of nascent blood vessels. These data suggest that S1P1 is critical for endothelial cell function and vascular development. While a number of studies indicate the pivotal role of S1P1 during development, its function in the adult is yet to be defined.
Recent studies show that S1P1 is expressed in various cell types in vitro, such as hepatocytes, cardiomyocytes, and immune cells and that exogenous S1P modulate the function of these cells [11], [12], [13]. It is therefore likely that S1P1 may play a role not only in vasculature but also in other tissues. Although S1P1 transcripts have been detected in major mouse organs and various cell types, the distribution of S1P1 in various adult tissues has not been reported. Therefore, we examined the distribution of S1P1 in adult mouse tissues to gain insights into the potential functions of S1P1 in the adult organs. Here we show that S1P1 is widely expressed in vasculature as well as non-vascular cells in various adult tissues.
Section snippets
Animals
S1P1+/−LacZ mice were bred as 129Sv/C57BL6J hybrids and genotyped as previously described [10]. S1P1+/−LacZ and S1P1+/+ mice at ages of 4–5 months were sacrificed and tissues were fixed for LacZ staining (see the following text) and immunohistochemistry.
β-galactosidase (LacZ) staining of tissues
Tissues were dissected in cold phosphate buffered saline and fixed in 0.2% glutaraldehyde and 1.5% paraformaldehyde in PBS, pH 7.4 at room temperature for 90 min, then washed in PBS. Staining was performed at 37 °C or room temperature in 0.02%
Expression of S1P1 in the adult cardiovascular system
We examined the expression of S1P1 in adult mouse tissues (Fig. 1). Quantitative analysis of β-galactosidase activity in adult tissues showed that brain, lung and spleen were among the organs that expressed the highest levels of S1P1. Detectable levels were seen in heart, kidney and liver.
In the adult heart, S1P1 was more strongly expressed in the atrium than in ventricle (Fig. 1B, Fig. 2A and B). Cardiomyocytes were the primary cell types that expressed the S1P1 driven β-galactosidase
Discussion
S1P1 is known to be important in endothelial cell survival, migration, and morphogenesis [4]. Recently it has become apparent that S1P1 is expressed in variety of cell types and S1P stimulation induces diverse cellular responses [14]. During embryonic development, S1P1 is strongly expressed in forebrain and heart. In addition, intense expression of S1P1 is detected in dorsal aorta, intersomitic arteries, and capillaries. Very weak or no expression is detected in the anterior and posterior
Acknowledgements
We thank Ji-Hye Paik for help with mouse procedures. This work is supported by NIH grants HL70694 and HL67330.
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