Sphingosine Kinase Type 1 Induces G12/13-mediated Stress Fiber Formation, yet Promotes Growth and Survival Independent of G Protein-coupled Receptors

Abstract

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of G␣ q , G␣ i , G␣ 12/13 , and G␤␥ subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G 12/13 -mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.Sphingosine 1-phosphate (S1P), 1 a sphingolipid metabolite found in organisms as diverse as plants, yeast, worms, flies, and mammals, has been linked to a wide spectrum of biological processes, among which cell growth, survival, and motility are prominent (1, 2). S1P is formed by sphingosine kinase (SphK), a highly conserved enzyme that is activated by numerous stimuli (1, 3).The most well known actions of S1P are mediated by binding to a family of specific G protein-coupled receptors (GPCRs). To date, five members, EDG-1/S1P 1 , EDG-5/S1P 2 , EDG-3/S1P 3 , EDG-6/S1P 4 , and EDG-8/S1P 5 , have been identified (1, 2, 4, 5). S1P receptors (S1PRs) are differentially expressed; coupled to a variety of G proteins; and regulate angiogenesis, vascular maturation, cardiac development, neuronal survival, and immunity (1, 2, 4). In particular, S1PRs have been shown to play critical roles in cell migration (6 -10). Activation of S1P 1 or S1P 3 by S1P in many cell types increases directional or chemotactic migration (6, 8, 10 -12), whereas binding to S1P 2 abolishes chemotaxis and membrane ruffling (13).Downstream of heterotrimeric G proteins, the S1PRs regulate tyrosine kinases such as focal adhesion kinase (FAK) and Src, which reside in focal adhesions, and the small GTPases of the Rho family that are important for cytoskeletal rearrangements (14). Whereas binding of S1P to S1P 1 mediates cortical ...

The immunomodulator FTY720 interferes with effector functions of human monocyte‐derived dendritic cells

“…This mechanism of action has been shown to be highly effective in suppressing allograft rejection and autoimmune diseases in a variety of animal models [4,5] and in initial clinical trials [6,7]. As a synthetic analog of the lysophospholipid sphingosine 1-phosphate (S1P), phosphorylated FTY720 (FTY720-P) targets and activates G protein-coupled S1P receptors (S1PR), which are well characterized for their prominent functions in regulating growth-related and cytoskeleton-dependent cellular activities [8][9][10]. The current concept of FTY720-induced lymphopenia is that FTY720 modulates lymphocyte trafficking in a dual manner: acceleration of migration into secondary lymphoid organs and blocking the egress into efferent lymphatics.…”

CXCL4‐induced monocyte survival, cytokine expression, and oxygen radical formation is regulated by sphingosine kinase 1

“…In addition to the effects of SphK1 overexpression, Olivera et al [28,29] demonstrated that administration of micromolar (but not nanomolar) concentrations of exogenous S1P suppresses apoptosis in a dose-dependent manner, and these effects were independent of S1P receptors. Similar results were published by Van Brocklyn et al…”

“…A few studies have suggested that suppression of apoptosis by S1P is mediated via its intracellular action, many others have argued in favor of the involvement of S1P membrane receptors, making this a controversial area (for review, see Hla et al [27]). In 1999 and 2003 Olivera et al [28,29] described that overexpression of SphK1 alone markedly reduces serum deprivation-induced apoptosis in HEK293 cells, Jurkat T cells, and NIH 3T3 fibroblasts. As shown here, stimulation with CXCL4 induces an increased SphK1 expression in monocytes and rescues these cells from apoptosis.…”

“…In addition to the effects of SphK1 overexpression, Olivera et al [28,29] demonstrated that administration of micromolar (but not nanomolar) concentrations of exogenous S1P suppresses apoptosis in a dose-dependent manner, and these effects were independent of S1P receptors. Similar results were published by Van Brocklyn et al [24], who could demonstrate that S1P at high concentrations acts not necessarily through binding to S1P receptors, but rather following cellular uptake of the phospho-lipid.…”

Cloning and characterization of rat sphingosine kinase 1 with an N-terminal extension

“…In addition, they differ in their physiological functions [16]. While SK1 stimulates growth and survival [17,18], SK2, which contains putative BH3 domain, inhibits cell growth and enhances apoptosis [19].…”