Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1؊/؊ tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1؊/؊ mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1؊/؊ mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.Sphingosine-1-phosphate (S1P) 1 is a signaling molecule that influences cellular functions including proliferation, survival, migration, adhesion molecule expression, and morphogenesis (1-4). S1P binds to members of the S1P receptor family (also known as EDG receptors) and, via G proteins, triggers multiple signaling pathways (5, 6). S1P has also been shown to function intracellularly mediating calcium homeostasis, cell growth, and suppression of apoptosis (7,8). In mammals, vascular development and lymphocyte trafficking are dependent on S1P receptor signaling (9 -13).Sphingosine kinase (SPHK) catalyzes the synthesis of S1P via the phosphorylation of sphingosine. SPHK activity is elevated by several stimuli, including platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor-␣, and phorbol ester, which trigger an increase in cellular S1P levels (14). Sphk genes have been identified in mammals (15-18), insects (19), plants (20), yeast (21), worm (22), and slime mold (23, 24). Mammals carry two known SphK genes, which in mice are encoded by Sphk1 and Sphk2. The two enzymes contain five highly conserved regions (C1-C5) and an ATP binding site within a conserved lipid kinase catalytic domain (15, 16). SPHK1 has a predominantly cytoplasm localization but can be induced to localize to the inner leaflet of the plasma membrane. Interestingly in endothelial cells SPHK1 is secreted and is capable of producing S1P extracellularly (25). Sphk1 shows a tissue distribution and developmental expression pattern different from Sphk2, although both enzymes are widely expressed (16,26).The importance of S1P receptor signaling in lymphocyte trafficking was first illuminated by the activities of FTY720, a potent immunosuppressive agent. FT...

Sphingosine-1-phosphate (S1P) elicits diverse cellular responses through a family of G-protein-coupled receptors. We have shown previously that genetic disruption of the S1P 1 receptor, the most widely expressed of the family, results in embryonic lethality because of its key role within endothelial cells in regulating the coverage of blood vessels by vascular smooth muscle cells. To understand the physiologic functions of the two other widely expressed S1P receptors, we generated S1P 2 and S1P 3 null mice. Neither the S1P 2 null mice nor the S1P 3 null mice exhibited significant embryonic lethality or obvious phenotypic abnormalities. To unmask possible overlapping or collaborative functions between the S1P 1 , S1P 2 , and S1P 3 receptors, we examined embryos with multiple S1P receptor mutations. We found that S1P 1 S1P 2 double null and S1P 1 S1P 2 S1P 3 triple null embryos displayed a substantially more severe vascular phenotype than did embryos with only S1P 1 deleted. We also found partial embryonic lethality and vascular abnormalities in S1P 2 S1P 3 double null embryos. Our results indicate that the S1P 1 , S1P 2 , and S1P 3 receptors have redundant or cooperative functions for the development of a stable and mature vascular system during embryonic development. Sphingosine-1 phosphate (S1P)1 is a sphingolipid metabolite that is present at high levels in the blood (1-3). Through the interaction with a family of five G-protein-coupled receptors (S1P 1-5 ), originally known as EDG receptors, sphingosine-1-phosphate triggers diverse cellular responses, including cytoskeletal changes, proliferation, and migration (1, 4 -8). The S1P 1 , S1P 2 , and S1P 3 receptors are widely expressed, including on embryonic endothelial cells (Table I) (9 -14). S1P 4 and S1P 5 receptor expression is more restricted and found on the cells of the immune and nervous systems (15, 16). The S1P 1 receptor couples selectively to the G i signaling pathway, whereas the S1P 2 and S1P 3 receptors both couple to the G i , G q , and G 12/13 pathways (2,(17)(18)(19)(20). In addition to these five S1P receptors, GPR3, GPR6, GPR12, and GPR63 have been characterized as G-protein-coupled receptors that interact with sphingosine-1-phosphate (21-23).The major physiological effects of S1P receptor signaling defined thus far have been localized to the immune and vascular systems. A global deletion of the S1P 1 receptor in mice results in lethality beginning at E12.5 due to severe hemorrhage as the result of deficient coverage of vessels by vascular smooth muscle cells, a process that occurs during the last stages of angiogenesis and is necessary for stabilizing the vascular system (14). Through analysis of endothelial cell-specific S1P 1 receptor knock-out mice, we have shown that the S1P 1 receptor functions within endothelial cells to regulate vascular smooth muscle cell coverage (24). The function of the S1P 1 receptor in the developing vasculature is also essential for proper limb development (25). Deletion of the S1P 1 receptor in T-cells has re...

Glycosphingolipids (GSLs) are believed to be integral for the dynamics of many cell membrane events, including cellular interactions, signaling, and trafficking. We have investigated their roles in development and differentiation by eliminating the major synthesis pathway of GSLs through targeted disruption of the Ugcg gene encoding glucosylceramide synthase. In the absence of GSL synthesis, embryogenesis proceeded well into gastrulation with differentiation into primitive germ layers and patterning of the embryo but was abruptly halted by a major apoptotic process. In vivo, embryonic stem cells deficient in GSL synthesis were again able to differentiate into endodermal, mesodermal, and ectodermal derivatives but were strikingly deficient in their ability to form well differentiated tissues. In vitro, however, hematopoietic and neuronal differentiation could be induced. The results demonstrate that the synthesis of GSL structures is essential for embryonic development and for the differentiation of some tissues and support the concept that GSLs are involved in crucial cell interactions mediating these processes.

To evaluate the role of nitric oxide synthase (nNOS) in the pathogenesis of diabetic neuropathy, we investigated nociception and nNOS expression in dorsal root ganglion (DRG) of rats with streptozocin-induced diabetes. Paw withdrawal threshold to noxious mechanical stimuli was decreased in both L-NAME-treated and diabetic rats. The number of NADPH-diaphorase positive neurons was significantly decreased in untreated diabetic compared with control rats. Decreased expression of nNOS protein was confirmed by immunoblotting. Insulin treatment completely prevented decreases in withdrawal threshold and nNOS expression. Cyclic GMP content paralleled nNOS expression in experimental animals. These results suggest that decreased nNOS-cGMP system in DRG may play a role in the pathogenesis of diabetic sensory neuropathy.

Streptozocin (STZ)-induced diabetic rats show hyperalgesia that is partially attributed to altered protein kinase C (PKC) activity. Both attenuated neuronal nitric oxide synthase (nNOS)-cGMP system and tetrodotoxin-resistant (TTX-R) Na channels in dorsal root ganglion neurons may be involved in diabetic hyperalgesia. We examined whether PKC␤ inhibition ameliorates diabetic hyperalgesia and, if so, whether the effect is obtained through action on neurons by testing nociceptive threshold in normal and STZ-induced diabetic rats treated with or without PKC␤-selective inhibitor LY333531 (LY) and by assessing the implication of LY in either nNOS-cGMP system or TTX-R Na channels of isolated dorsal root ganglion neurons. The decreased nociceptive threshold in diabetic rats was improved either after 4 weeks of LY treatment or with a single intradermal injection into the footpads. The treatment of LY for 6 weeks significantly decreased p-PKC␤ and ameliorated a decrease in cGMP content in dorsal root ganglia of diabetic rats. The latter effect was confirmed in ex vivo condition. The treatment with NO donor for 4 weeks also normalized both diabetic hyperalgesia and decreased cGMP content in dorsal root ganglions. The expressions of nNOS and TTX-R Na channels were not changed with LY treatment. These results suggest that LY is effective for treating diabetic hyperalgesia through ameliorating the decrease in the nNOS-cGMP system.

To examine which isoform of protein kinase C (PKC) may be associated with impaired nerve regeneration in diabetes, we compared neurite outgrowth of isolated dorsal root ganglion (DRG) neurons in streptozocin (STZ)-induced diabetic and control rats. Neurite outgrowth was significantly retarded in diabetic neurons. Rottlerin, a PKCdelta specific inhibitor, significantly retracted neurite outgrowth whereas Gö6976, an inhibitor specific for classical PKCs, had no effect, suggesting a significant role of PKCdelta in neurite outgrowth of DRG neurons. The expression of phosphorylated PKCdelta, but not total PKCdelta, in DRGs was decreased in diabetic rats. When this reduced expression was restored by overexpressing the PKCdelta in isolated DRG neurons, retardation of neurite outgrowth was significantly reversed in diabetic rats. These results suggest that a decrease in phosphorylated PKCdelta is at least in part responsible for impaired neurite outgrowth in diabetes, and that PKCdelta plays a significant role in the pathogenesis of diabetic neuropathy. This observation provides a useful clue for the treatment of diabetic neuropathy.

The regenerative ability of unmyelinated nerve fibers (UNFs) in diabetes and the effect of aldose reductase inhibitor (ARI) on it were ultrastructurally evaluated after sciatic nerve crush in control and untreated and tolrestat-treated streptozocin-induced diabetic rats. The density and number of UNFs were significantly increased in all groups at 5 weeks after the injury. The increase returned to the baseline level in control rats, but not in diabetic rats at 24 weeks. Although the axon size showed a marked decrease at 5 weeks and an incomplete recovery at 24 weeks in all groups, the recovery was significantly worse in diabetic than in control groups. Tolrestat did not have any effect on regeneration of UNFs in diabetes. These results suggest impaired regeneration of UNFs after nerve crush injury in diabetes and less therapeutic effect of ARI on it.

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