Abstract
Sphingosine 1-phosphate (S1P) generated by cells of innate immunity and the type 1 S1P G protein-coupled receptor (S1P(1)) on mobile T cells constitute a major system for control of lymphoid organ traffic and tissue migration of T cells. Now we show that T cell activation mediated by the T cell antigen receptor translocates plasma membrane S1P(1) to nuclear envelope membranes for association there with G(i/o), Erk (1/2), and other proteins that plasma membrane S1P(1) uses to signal T cell proliferation. However, nuclear S1P(1) and plasma membrane S1P(1) transduce opposite effects of S1P on T cell proliferation and relevant signaling as exemplified by respective decreases and increases in T cell nuclear concentrations of both phospho-Erk and active (phosphorylated) c-Jun. T cell antigen receptor-mediated activation of T cells therefore both eliminates migration responses to S1P by down-regulation of plasma membrane S1P(1) and translocates the S1P-S1P(1) axis into the nuclear domain where signals are directed to transcriptional control of immune functions other than migration.
Highlights
Endothelial cells and hepatocytes, the LPA-nuclear LPA1 axis stimulates prominent transcription of proinflammatory proteins, including type 2 cyclooxygenase (COX-2) and inducible nitric-oxide synthase
Activation of T cells through the T cell antigen receptor (TCR) mechanism used by antigen, which evokes a full program of functional immune responses, results in nuclearization of most plasma membrane S1P1 and assembly of a functional signaling complex in nuclear membranes
Membrane-enveloped nuclei from TCR-activated T cells showed S1P1 and Gi/o immunocytochemically and by Western blots, S1P1 was expressed at higher levels in nuclei from TCR-activated than from unstimulated T cells, and the level of association of S1P1 and Gi/o was greater in nuclei from TCR-activated than unstimulated T cells (Figs. 3 and 4)
Summary
C57BL/6 mouse splenic CD4 T cells were isolated at a purity of at least 96% by two cycles of magnetic bead immunoaffinity chromatography as reported previously [10]. Assays of the enzymatic markers acid phosphatase for lysosomes (Diagnostic Chemicals, Ltd., Oxford, CT) and 5Ј-nucleotidase (Diazyme Laboratories, San Diego, CA) for plasma membranes showed that routinely Ͻ5% of their total initial activities in intact cells were detected in the nuclei This level of nuclear purity was confirmed by demonstrating the absence of the plasma membrane marker annexin II [12] in Western blots of nuclear extracts, whereas annexin II was prominent in Western blots of the 700 ϫ g supernatants of homogenates containing membrane and cytoplasmic constituents. Replicate suspensions of purified nuclei from 0.5 to 1 ϫ 107 T cells and, in some studies, 0.25– 0.5 ϫ 107 intact T cells were resuspended in 200 l of phosphate-buffered saline with 10 units/ml DNase I, 0.1 g/100 ml fatty acid-free bovine serum albumin, and 1 M activated sodium orthovanadate phosphatase inhibitor; preincubated for 20 min at 37 °C; and incubated at 37 °C for 1 h without and with 10Ϫ9–10Ϫ5 M S1P followed by addition of 1⁄100 HALT protease inhibitor and cooling to 4 °C. Time course studies compared effects of S1P on transcription factors and other signaling proteins in intact cells and isolated nuclei after 1, 4, and 24 h at 37 °C
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