Teresa Sanchez

Structural and functional characteristics of S1P receptors.

The sphingosine‐1‐phosphate (S1P) family of G protein‐coupled receptors (GPCR) regulates essential cellular processes such as proliferation, migration, cytoskeletal organization, adherens junction assembly, and morphogenesis. S1P, a product from the breakdown of sphingomyelin, binds to the five members of this receptor family, S1P1, S1P2, S1P3, S1P4, and S1P5, previously referred to as endothelial differentiation gene (EDG)‐1, ‐5, ‐3, ‐6, and ‐8. S1P receptors are widely expressed in different tissues, so it is not surprising that the S1P receptor family regulates many physiological processes, such as vascular maturation, cardiac development, lymphocyte trafficking, and vascular permeability. FTY720, a new S1P receptor agonist, is undergoing clinical trials as an immunosuppressor. Understanding the physiological role of these receptors and the basics of the ligand‐receptor interaction will potentially provide new therapies to control a variety of diseases.

Antagonism of Sphingosine-1-Phosphate Receptors by FTY720 Inhibits Angiogenesis and Tumor Vascularization

FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF- and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis.

Induction of Vascular Permeability by the Sphingosine-1-Phosphate Receptor–2 (S1P2R) and its Downstream Effectors ROCK and PTEN

Objectives—S1P acts via the S1PR family of G protein–coupled receptors to regulate a variety of physiological responses. Whereas S1P1R activates Gi- and PI-3-kinase–dependent signals to inhibit vascular permeability, the related S1P2R inhibits the PI-3-kinase pathway by coupling to the Rho-dependent activation of the PTEN phosphatase. However, cellular consequences of S1P2R signaling in the vascular cells are not well understood. Methods and Results—Selective signaling of the S1P2R was achieved by adenoviral-mediated expression in endothelial cells. Secondly, endogenously expressed S1P2R was blocked by the specific pharmacological antagonist JTE013. Activation of S1P2R in endothelial cells resulted in Rho-ROCK– and PTEN-dependent disruption of adherens junctions, stimulation of stress fibers, and increased paracellular permeability. JTE013 treatment of naive endothelial cells potentiated the S1P1R-dependent effects such as formation of cortical actin, blockade of stress fibers, stimulation of adherens junction assembly, and improved barrier integrity. This observation was extended to the in vivo model of vascular permeability in the rat lung: the S1P2R antagonist JTE013 significantly inhibited H2O2-induced permeability in the rat lung perfused model. Conclusions—S1P2R activation in endothelial cells increases vascular permeability. The balance of S1P1 and S1P2 receptors in the endothelium may determine the regulation of vascular permeability by S1P.

Essential role of sphingosine 1–phosphate receptor 2 in pathological angiogenesis of the mouse retina

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator that signals via the S1P family of G protein-coupled receptors (S1PR), regulates vascular maturation, permeability, and angiogenesis. In this study, we explored the role of S1P 2 receptor (S1P2R) in normal vascularization and hypoxia-triggered pathological angiogenesis of the mouse retina. S1P2R is strongly induced in ECs during hypoxic stress. When neonatal mice were subjected to ischemia-driven retinopathy, pathologic neovascularization in the vitreous chamber was suppressed in S1p2-/- mice concomitant with reduction in endothelial gaps and inflammatory cell infiltration. In addition, EC patterning and normal revascularization into the avascular zones of the retina were augmented. Reduced expression of the proinflammatory enzyme cyclooxygenase-2 (COX-2) and increased expression of eNOS were observed in the S1p2-/- mouse retina. S1P2R activation in ECs induced COX-2 expression and suppressed the expression of eNOS. These data identify the S1P2R-driven inflammatory process as an important molecular event in pathological retinal angiogenesis. We propose that antagonism of the S1P2R may be a novel therapeutic approach for the prevention and/or treatment of pathologic ocular neovascularization.

Serum withdrawal-induced post-transcriptional stabilization of cyclooxygenase-2 mRNA in MDA-MB-231 mammary carcinoma cells requires the activity of the p38 stress-activated protein kinase.

Overexpression of thecyclooxygenase-2 (COX-2) gene is observed in several neoplastic diseases. However, molecular mechanisms involved in the regulation of expression of COX-2 are not well understood. In this report, we describe a unique post-transcriptional regulatory mechanism of COX-2 mRNA stabilization in MDA-MB-231 cells, a highly metastatic cell line derived from a human mammary tumor. High levels of COX-2 mRNA, protein, and enzyme activity were induced by serum withdrawal, which were potently inhibited by the addition of serum or >100-kDa serum factor. Nuclear run-on analysis and actinomycin D chase experiments indicate that regulation is primarily at the level of post-transcriptional mRNA stability. Interestingly, SB203580, an inhibitor of the p38 stress-activated protein kinase (SAPK), and overexpression of the dominant-negative p38α construct potently inhibited the serum withdrawal-induced COX-2 mRNA levels. Indeed, the half-life of COX-2 mRNA decreased from 9 to 4.5 h after SB203580 treatment, suggesting that signal transduction by the p38 SAPK pathway is required for COX-2 mRNA stability.

Critical role of sphingosine-1-phosphate receptor 2 (S1PR2) in acute vascular inflammation

The endothelium, as the interface between blood and all tissues, plays a critical role in inflammation. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, highly abundant in plasma, that potently regulates endothelial responses through interaction with its receptors (S1PRs). Here, we studied the role of S1PR2 in the regulation of the proadhesion and proinflammatory phenotype of the endothelium. By using genetic approaches and a S1PR2-specific antagonist (JTE013), we found that S1PR2 plays a key role in the permeability and inflammatory responses of the vascular endothelium during endotoxemia. Experiments with bone marrow chimeras (S1pr2(+/+) → S1pr2(+/+), S1pr2(+/+) → S1pr2(-/-), and S1pr2(-/-) → S1pr2(+/+)) indicate the critical role of S1PR2 in the stromal compartment, in the regulation of vascular permeability and vascular inflammation. In vitro, JTE013 potently inhibited tumor necrosis factor α-induced endothelial inflammation. Finally, we provide detailed mechanisms on the downstream signaling of S1PR2 in vascular inflammation that include the activation of the stress-activated protein kinase pathway that, together with the Rho-kinase nuclear factor kappa B pathway (NF-kB), are required for S1PR2-mediated endothelial inflammatory responses. Taken together, our data indicate that S1PR2 is a key regulator of the proinflammatory phenotype of the endothelium and identify S1PR2 as a novel therapeutic target for vascular disorders.

S1P/S1P1 signaling stimulates cell migration and invasion in Wilms tumor.

Sphingosine-1-phosphate (S1P) is an important regulator of cellular functions via interaction with its receptors S1P(1-5). To date, nothing is known about the S1P receptor expression and the effects of S1P signaling in Wilms tumor. In this study, we found ubiquitous expression of S1P receptors in Wilms tumor specimens and cell lines. We demonstrated that S1P(1) acted as a promigratory modulator by employing S1P(1) antagonist VPC44116, S1P(1) siRNA and adenoviral transduction in Wilms tumor cells. Further, we clarified that S1P(1)-mediated migration occurred via Gi coupling and activation of PI3K and Rac1. In addition, S1P stimulated WiT49 cell invasion through S1P(1)/Gi signaling pathway. We consider that targeting S1P(1) may be a point of therapeutic intervention in Wilms tumor.

Regulation of Prostaglandin E2 Production by the Superoxide Radical and Nitric Oxide in Mouse Peritoneal Macrophages

The purpose of this study was to elucidate the role of NO and O-2 on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time-dependent production of nitric oxide (NO) and prostaglandin E2 (PGE2). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O-2), and by increased expression of COX-2. Our results provide evidence that O-2 may be involved in the pathways that result in arachidonate release and PGE2 formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions.

Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells.

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 S1P1-3 receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P1 and S1P2 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 S1P2 receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P2 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 S1P2-mediated activation of the lipid phosphatase PTEN.

Role of prostaglandin H synthase-2-mediated conversion of arachidonic acid in controlling 3T6 fibroblast growth

The specific role(s) of arachidonic acid (AA) and its metabolites in the signaling pathways that regulated fibroblast growth was studied. A Western blot analysis demonstrated that prostaglandin H synthase-2 (PGHS-2) was expressed by 3T6 fibroblast cultures in RPMI 1640 supplemented with fetal calf serum (10%). Dexamethasone, which inhibits AA release and PGHS-2 expression, significantly reduced cell proliferation. Ketoprofen, a dual cyclooxygenase inhibitor, and CGP-28238, a specific PGHS-2 inhibitor, reduced fibroblast proliferation in a dose-dependent manner. These drugs also reduced [3H]thymidine incorporation into the DNA of fibroblasts. These effects were correlated with a decrease in prostaglandin (PG) E2 levels in the cell medium. However, piroxicam at doses that selectively inhibit PGHS-1 did not have a significant effect on fibroblast proliferation. Finally, we showed that the antiproliferative effect of dexamethasone and PGHS-2 inhibitors was significantly antagonized when PGE2 was added to the culture medium. Our results suggest that PGHS-2 and prostaglandins such as PGE2 might play an important role in the regulation of 3T6 fibroblast growth stimulated by growth factors of serum.The specific role(s) of arachidonic acid (AA) and its metabolites in the signaling pathways that regulated fibroblast growth was studied. A Western blot analysis demonstrated that prostaglandin H synthase-2 (PGHS-2) was expressed by 3T6 fibroblast cultures in RPMI 1640 supplemented with fetal calf serum (10%). Dexamethasone, which inhibits AA release and PGHS-2 expression, significantly reduced cell proliferation. Ketoprofen, a dual cyclooxygenase inhibitor, and CGP-28238, a specific PGHS-2 inhibitor, reduced fibroblast proliferation in a dose-dependent manner. These drugs also reduced [3H]thymidine incorporation into the DNA of fibroblasts. These effects were correlated with a decrease in prostaglandin (PG) E2 levels in the cell medium. However, piroxicam at doses that selectively inhibit PGHS-1 did not have a significant effect on fibroblast proliferation. Finally, we showed that the antiproliferative effect of dexamethasone and PGHS-2 inhibitors was significantly antagonized when PGE2 was added to the culture medium. Our results suggest that PGHS-2 and prostaglandins such as PGE2 might play an important role in the regulation of 3T6 fibroblast growth stimulated by growth factors of serum.