In this report, sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid, is shown to activate tight-junction-associated protein Zonula Occludens-1 (ZO-1), which in turn plays a critical role in regulating endothelial chemotaxis and barrier integrity. After S1P stimulation, ZO-1 was redistributed to the lamellipodia and cell-cell junctions via the S1P1/G i /Akt/Rac pathway. Similarly, both endothelial barrier integrity and cell motility were significantly enhanced in S1P-treated cells through the G i /Akt/Rac pathway. Importantly, S1P-enhanced barrier integrity and cell migration were abrogated in ZO-1 knockdown cells, indicating ZO-1 is functionally indispensable for these processes. To investigate the underlying mechanisms, we demonstrated that cortactin plays a critical role in S1P-induced ZO-1 redistribution to the lamellipodia. In addition, S1P significantly induced the formation of endothelial tight junctions. ZO-1 and ␣-catenin polypeptides were colocalized in S1P-induced junctional structures; whereas, cortactin was not observed in these regions. Together, these results suggest that S1P induces the formation of two distinct ZO-1 complexes to regulate two different endothelial functions: ZO-1/cortactin complexes to regulate chemotactic response and ZO-1/␣-catenin complexes to regulate endothelial barrier integrity. The concerted operation of these two ZO-1 complexes may coordinate two important S1P-mediated functions, i.e. migration and barrier integrity, in vascular endothelial cells.Endothelial barrier integrity is an important physiological function of the endothelium in vivo. Dysregulated barrier integrity is implicated in a variety of pathological conditions, such as stroke, inflammation, various immune responses, etc. (1). To elucidate the function and regulation of endothelial barrier integrity, cultured brain microvascular endothelial cells have been widely employed as an in vitro model system to study the blood-brain barrier (BBB) 2 (2). Evidence from these studies indicates that BBB plays a critical role in regulating the homeostatic environment of the brain and the transportation of plasma constituents into brain. Furthermore, it has been shown that severely impaired blood-brain barrier integrity is attributed to the pathological states of various neurological disorders, such as multiple sclerosis (3, 4), Alzheimer disease (5, 6), and human immunodeficiency virus-1-associated encephalitis or dementia (7,8).Sphingosine 1-phosphate (S1P), a serum-borne bioactive lipid mediator secreted by activated platelets (9), enhances barrier formation in cultured pulmonary endothelial cells (ECs) (10). However, the molecular details for the formation and maintenance of endothelial barrier integrity are poorly understood. It was recently reported that the association of cortactin, an F-actin cross-linking polypeptide, and myosin light chain kinase is crucial in S1P-enhanced endothelial barrier integrity (11). Furthermore, it is well documented that tight junctions are important in regulating BBB formation...

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...

this process. One such cooperation between GPCR and TLR can be attributed to the sphingosine 1-phosphate (S1P) receptor family. The S1P receptors (S1P 1-5 ) are a family of GPCR with a high affinity for S1P, a serum-borne bioactive lipid associated with diverse biological activities such as inflammation and healing. In this study, we show that proinflammatory cytokine production, including IL-6 and IL-8, was increased with LPS and concomitant S1P stimulation. Furthermore, elevated cytokine production following LPS and S1P challenge in human gingival epithelial cells (HGEC) was significantly reduced when TLR4, S1P 1 or S1P 3 signaling was blocked. Our study also shows that S1P 1 and S1P 3 expression was induced by LPS in HGEC, and this elevated expression enhanced the influence of S1P in its cooperation with TLR4 to increase cytokine production. This cooperation between TLR4 and S1P 1 or S1P 3 demonstrates that TLR4 and GPCR can interact to enhance cytokine production in epithelial cells.

Background: Glia-driven neuroinflammation promotes neuron injury in glaucoma that is a chronic neurodegenerative disease of the optic nerve and a leading cause of irreversible blindness. Although therapeutic modulation of neuroinflammation is increasingly viewed as a logical strategy to avoid inflammatory neurotoxicity in glaucoma, current understanding of the molecular regulation of neuroinflammation is incomplete, and the molecular targets for immunomodulation remains unknown. Growing datasets pointed to nuclear factor-kappaB (NF-κB), a key transcriptional activator of inflammation, which was identified to be most affected in glaucomatous astroglia. Using a cell type-specific experimental approach, this study aimed to determine the value of astroglial NF-κB as a potential treatment target for immunomodulation in experimental mouse glaucoma. Methods: Neuroinflammatory and neurodegenerative outcomes of experimental glaucoma were comparatively analyzed in mice with or without cre/lox-based conditional deletion of astroglial IκKβ, which is the main activating kinase involved in IκB degradation through the canonical pathway of NF-κB activation. Glial responses and the inflammatory status of the retina and optic nerve were analyzed by cell morphology and cytokine profiling, and neuron structure and function were analyzed by counting retinal ganglion cell (RGC) axons and somas and recording pattern electroretinography (PERG) responses. Results: Analysis of glial inflammatory responses showed immunomodulatory outcomes of the conditional transgenic deletion of IκKβ in astroglia. Various pro-inflammatory cytokines known to be transcriptional targets for NF-κB exhibited decreased production in IκKβ-deleted astroglia, which included TNF-α that can induce RGC apoptosis and axon degeneration during glaucomatous neurodegeneration. Indeed, transgenic modulation of inflammatory responses by astroglial IκKβ deletion reduced neurodegeneration at different neuronal compartments, including both RGC axons and somas, and protected PERG responses.

Purpose Besides glia-driven neuroinflammation, growing evidence from analysis of human blood samples, isolated autoantibodies, and postmortem tissues also support systemic immune responses during neurodegeneration in glaucoma patients. To explore the T-cell–mediated component of systemic immunity, this study analyzed T lymphocytes in patients' blood. Methods Blood samples were collected from 32 patients with glaucoma and 21 nonglaucomatous controls, and mononuclear cells were isolated by Histopaque density gradient centrifugation. T-cell subset distribution was analyzed by multicolor flow cytometry after helper (Th) and cytotoxic fractions, and Th subpopulations, were stained with antibodies to CD4, CD8, or distinctive markers, such as IFN-γ (for Th1), IL-4 (for Th2), IL-17A (for Th17), and CD25/FoxP3 (for T regulatory cells [Tregs]). In addition, proliferative activity and cytokine secretion of T cells were analyzed after in vitro stimulation. Results Analysis of T-cell subset distribution detected a glaucoma-related shift. Despite similar frequencies of CD4+ or CD8+ T cells, or Th1, Th2, or Th17 subsets in glaucoma and control groups, glaucomatous samples exhibited a trend toward decreased frequency of CD4+ (or CD8+)/CD25+/FoxP3+ Tregs within the entire CD4+ (or CD8+) population ( P < 0.001). Furthermore, CD4+ T cells in glaucomatous samples presented a greater stimulation response (∼3-fold) as characterized by increased proliferation and proinflammatory cytokine secretion ( P < 0.05). Conclusions These findings suggest that the immunity activated in glaucoma may not be counterbalanced by an efficient immune suppression. More work is encouraged to determine whether shifted T-cell homeostasis may contribute to neurodegeneration in glaucoma, and/or whether T-cell subset imbalance may serve as a biomarker of autoimmune susceptibility.