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

Abstract

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...

Balance of S1P1and S1P2signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature

“…Recently, we showed that early-passaged ECs in vitro abundantly express S1P 1 receptors and barely express S1P 2 receptors. In contrast, S1P 2 receptors are markedly increased in senescent ECs (9). The upregulation of S1P 2 receptors was shown to mediate the impaired wound-healing and angiogenic responses in senescent ECs (9).…”

“…Thus cultured young and senescent ECs were used as an in vitro model to examine whether endothelial S1P 1 -and S1P 2 -mediated signaling cascades are able to concertedly regulate vascular barrier function in vivo. A minimal rise of TEER, an in vitro measurement of endothelial barrier function (9,17), in senescent ECs after S1P treatment is shown in Fig. 8A.…”

“…In contrast, S1P 2 receptors are markedly increased in senescent ECs (9). The upregulation of S1P 2 receptors was shown to mediate the impaired wound-healing and angiogenic responses in senescent ECs (9). Thus cultured young and senescent ECs were used as an in vitro model to examine whether endothelial S1P 1 -and S1P 2 -mediated signaling cascades are able to concertedly regulate vascular barrier function in vivo.…”

S1P2, the G Protein–Coupled Receptor for Sphingosine-1-Phosphate, Negatively Regulates Tumor Angiogenesis and Tumor Growth In vivo in Mice

“…We previously observed that endogenous S1P 2 mediated inhibition of Rac activity, cell migration, and capillary-like tube formation in an EC line and that an S1P 2 -selective antagonist enhanced in vivo angiogenesis in Matrigel plug assays (26). In addition, using both in vitro and in vivo models, several other groups have shown the antiangiogenic activity of S1P 2 receptor (27,28). However, it remains unknown whether and how S1P 2 is involved in tumor angiogenesis.…”

Roles of sphingosine-1-phosphate signaling in angiogenesis

“…Additionally, the overexpression of S1P2 exaggerates these S1P-induced responses in ECs. In senescent ECs, S1P2 is upregulated, which leads to inhibition of migration and tube formation in vitro [41] . Therefore, it is an intriguing possibility that S1P2 might play an inhibitory role in angiogenesis, which contrasts with S1P1.…”