Dual Roles of Tight Junction-associated Protein, Zonula Occludens-1, in Sphingosine 1-Phosphate-mediated Endothelial Chemotaxis and Barrier Integrity

Abstract

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...

Sphingosine 1-Phosphate 1 and TLR4 Mediate IFN-β Expression in Human Gingival Epithelial Cells

“…Subsequently, HGECs were transfected with 100 pmol of small interfering TLF4 (siTLR4; Dharmacon), siRNA to laminin (Dharmacon), 100 nM siIRF3 (Dharmacon), or 1 g of lentiviral vector carrying siRNA to S1P1 (28) using the electroporation technique (Amaxa). Briefly, when the cells reached 70% confluence, they were collected and then 1 ϫ 10 6 cells were incubated in 100 l of Human Keratinocyte Nucleofector Solution containing siRNA to IRF3, S1P1, TLR4, or laminin for 15 min at room temperature.…”

“…S1P1 signals through the G i/O proteins; however, S1P2 and S1P3 stimulate G q and G 12/13 (19), and S1P5 activates both G 12/13 and G i (20). S1P has been extensively studied for its healing and repair cell functions, such as cell migration (21) and tightening (22), but its role in inflammation is largely unknown particularly in epithelial cells. LPS-induced IFN-␤ expression (23) has been extensively studied in mammalian cells, including dendritic cells and macrophages (7); however, a detailed investigation of the regulation of IFN-␤ in epithelial cells is lacking.…”

Sphingosine-1-Phosphate Metabolism and Intestinal Tumorigenesis: Lipid Signaling Strikes Again

“…In fact, interactions between S1P, SK and various catenin family members have been described. [79][80][81][82][83] A recent study demonstrated that Sphk1, the major enzyme necessary for synthesizing S1P, is required for enlargement of Min mouse adenomas, since Min mice that do not express this enzyme develop only small adenomas, in contrast to the significant number of large adenomas that developed in Min mice with intact Sphk1 expression. 84 Importantly, both S1P and sphingosine levels were high in Min mouse polyps compared to normal adjacent tissues, indicating that sphingolipid metabolism is abnormal in the neoplastic tissue.…”

Junctional Protein Regulation by Sphingosine Kinase 2 Contributes to Blood–Brain Barrier Protection in Hypoxic Preconditioning-Induced Cerebral Ischemic Tolerance

“…Sphingosine-1-phosphate regulates vascular integrity through several mechanisms, including the modulation of junctional protein expression and localization (Adamson et al, 2010;Lee et al, 2006;Wang and Dudek, 2009). Only recently has this sphingolipid pathway been considered as a potential stroke therapeutic (Czech et al, 2009;Hasegawa et al, 2010;Wacker et al, 2009;Wei et al, 2011).…”