Signaling mechanisms and molecular characteristics of G protein-coupled receptors for lysophosphatidic acid and sphingosine 1-phosphate

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P 1-3 receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P 1 and S1P 2 was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P 2 receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P 2 to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P 2 -mediated activation of the lipid phosphatase PTEN.Spingosine 1-phosphate (S1P), 2 a serum-borne bioactive lipid mediator, regulates an array of biological activities in various cell types (1-4). Most, if not all, of S1P-regulated functions are mediated by the S1P family of G-protein-coupled receptors (GPCRs) (5-7). There are five identified members of the S1P receptor family: S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 (old nomenclature: EDG-1, -5, -3, -6, and -8, respectively) (8). It was demonstrated that S1P receptor subtypes couple to different G ␣ polypeptides to regulate distinct signaling pathways (9 -11). The S1P receptor subtypes were expressed in distinct combinations in different cell types to produce an appropriate biological effect. For example, S1P 1 and S1P 3 are expressed in endothelial cells (ECs) (12). The signaling pathways regulated by the S1P 1 and S1P 3 receptors are required for the chemotaxis of endothelial cells, adherens junction assembly, endothelial morphogenesis, and angiogenic response in vitro and in vivo (7,(12)(13). However, the functional outcomes resulting from the concerted effects of the signaling pathways mediated by the distinct S1P receptor subtypes are currently unknown in a physiological environment.In contrast to S1P 1 -stimulating chemotaxis, S1P 2 -mediated signaling was shown to inhibit cell migration (14 -16). For example, embryonic fibroblasts isolated from S1P 2 null mice exhibited an enhanced chemotaxis toward S1P, serum, and platelet-derived growth factor; this enhancement was reversed by the reintroduction of S...

Section: Methodsmentioning

confidence: 99%

“…A low level of S1P 4 receptor was detected in young and intermediate ECs and is absent in senescent ECs. S1P 5 was not detected in ECs with different in vitro ages. The data are from a representative experiment, which was repeated six times with identical results.…”

Section: Volume 283 • Number 44 • October 31 2008mentioning

confidence: 99%

Up-regulating Sphingosine 1-Phosphate Receptor-2 Signaling Impairs Chemotactic, Wound-healing, and Morphogenetic Responses in Senescent Endothelial Cells

Estrada

Zeng

Lü

et al. 2008

“…One of the hallmarks of Rho activation is the rapid formation of actin stress fibers as a result of LPA or serum stimulation (6), a process that is dependent upon the activation of the G 12/13 family of heterotrimeric G proteins and the subsequent stimulation of RGLcontaining RhoGEFs (29). To explore whether PAK4 affects LPAinduced Rho activation, we treated control-transfected HA PAK4 445N and HA PAK4 350 M-transfected Swiss 3T3 fibroblasts with LPA and examined the cytoskeletal changes by staining polymerized actin.…”

Section: Fig 1 Identification Of Pak4 As a Pdz Rhogef Binding Proteinmentioning

confidence: 99%

Direct Interaction of p21-Activated Kinase 4 with PDZ-RhoGEF, a G Protein-linked Rho Guanine Exchange Factor

Barac

Basile

Vázquez‐Prado

et al. 2004

RhoGTPases regulate a wide variety of cellular processes, ranging from actin cytoskeleton remodeling to cell cycle progression and gene expression. Cell surface receptors act through a complex regulatory molecular network that includes guanine exchange factors (GEFs), GTPase activating proteins, and guanine dissociation inhibitors to achieve the coordinated activation and deactivation of Rho proteins, thereby controlling cell motility and ultimately cell fate. Here we found that a member of the RGL-containing family of Rho guanine exchange factors, PDZ RhoGEF, which, together with LARG and p115RhoGEF, links the G 12/13 family of heterotrimeric G proteins to Rho activation, binds through its C-terminal region to the serine-threonine kinase p21-activated kinase 4 (PAK4), an effector for Cdc42. This interaction results in the phosphorylation of PDZ Rho-GEF and abolishes its ability to mediate the accumulation of Rho-GTP by G␣ 13 . Moreover, when overexpressed, active PAK4 was able to dramatically decrease Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation. Together, these results provide evidence that PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs, thus providing a novel potential mechanism for cross-talk among Rho GTPases.

“…Sphingosine 1-phosphate, in one paradigm, binds to members of a family of G protein-coupled receptors (S1P [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] ) triggering diverse effects including proliferation, survival, migration, morphogenesis, adhesion molecule expression, and cytoskeletal changes (4 -8). S1P 1 /Edg 1 , the first of these receptors described (9, 10), couples to a G i pathway.…”

mentioning

confidence: 99%

“…The S1P 1 receptor is widely expressed in the adult, in particular in endothelial cells, the brain, and the heart, and also in the cells of the immune system (13,14). S1P 1 and S1P 4 receptors have been detected in T-lymphocytes (15)(16)(17).…”

mentioning

confidence: 99%

Expression of the Sphingosine 1-Phosphate Receptor, S1P1, on T-cells Controls Thymic Emigration

Allende

Dreier

Mandala

et al. 2004

411

381

S1P 1 is a widely distributed G protein-coupled receptor whose ligand, sphingosine 1-phosphate, is present in high concentrations in the blood. The sphingosine 1-phosphate receptor-signaling pathway is believed to have potent effects on cell trafficking in the immune system. To determine the precise role of the S1P 1 receptor on T-cells, we established a T-cell-specific S1P 1 knock-out mouse. The mutant mice showed a block in the egress of mature T-cells into the periphery. The expression of the S1P 1 receptor was up-regulated in mature thymocytes, and its deletion altered the chemotactic responses of thymocytes to sphingosine 1-phosphate. The results indicated that the expression of the S1P 1 receptor on T-cells controls their exit from the thymus and entry into the blood and, thus, has a central role in regulating the numbers of peripheral T-cells.Sphingolipids are important signaling molecules in a variety of biologic contexts (1-3). Sphingosine 1-phosphate, in one paradigm, binds to members of a family of G protein-coupled receptors (S1P 1-15 ) triggering diverse effects including proliferation, survival, migration, morphogenesis, adhesion molecule expression, and cytoskeletal changes (4 -8).S1P 1 /Edg 1 , the first of these receptors described (9, 10), couples to a G i pathway. During embryonic development, the S1P 1 receptor is highly expressed in the vascular system. Gene disruption in mice has demonstrated an essential function of the S1P 1 receptor in endothelial cells for the formation of a stable vascular network (11,12).The S1P 1 receptor is widely expressed in the adult, in particular in endothelial cells, the brain, and the heart, and also in the cells of the immune system (13, 14). S1P 1 and S1P 4 receptors have been detected in T-lymphocytes (15-17). Stimulation of receptor signaling has been found to mediate and regulate cell migration and suppress proliferation and cytokine production (17, 18). Recently, S1P 1 receptors have also been implicated in lymphocyte trafficking and homing as the result of studies using FTY720, a potent immunosuppressive agent. FTY720, a sphingosine analogue, is phosphorylated by sphingosine kinase type I and more efficiently by sphingosine kinase type II (19 -21) and functions as an agonist ligand for S1P 1 , S1P 3 , S1P 4 , and S1P 5 receptors (19,22). FTY720 causes lymphopenia through sequestration of circulating lymphocytes within lymph nodes and Peyer's patches (19,(22)(23)(24) and also blocks the egress of T-cells from the thymus (25, 26). However, it is not known at which S1P receptor and cell type FTY720 exerts its effects.To begin to address the physiologic function of the S1P 1 receptor and sphingosine 1-phosphate signaling in T-cells, we have generated a T-cell-specific mouse knock-out of the S1P 1 receptor using the Cre/loxP system. Our results with these mice reveal a crucial role for the S1P 1 receptor in the egress of mature T-cells from the thymus into the circulation. EXPERIMENTAL PROCEDURESGeneration of the S1P 1 loxP/loxP Lck-Cre Mice-We previously g...