Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation

Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation.

Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage

Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved.

MAP kinase kinase 1 (MKK1) is essential for transmission of Trypanosoma brucei by Glossina morsitans

MAP kinase kinase 1 (MKK1) is encoded by a single copy gene in Trypanosoma brucei. It has been shown recently that MKK1 is not essential for bloodstream forms [14]. To investigate the requirement for MKK1 in other life-cycle stages we generated null mutants in procyclic forms of a fly-transmissible strain. These grew normally in culture and were able to establish midgut infections in tsetse at normal rates and intensities, but were incapable of colonising the salivary glands. Transformation of null mutants with an ectopic copy of MKK1 enabled parasites to complete the life cycle in tsetse and infect mice. This is the first example of a gene that is indispensable for transmission of T. brucei. It also raises the possibility that activating the MKK1 signalling cascade in vitro might trigger the differentiation and proliferation of life-cycle stages of T. brucei that are currently refractory to culture.

Thiazolidinedione-dependent activation of sphingosine kinase 1 causes an anti-fibrotic effect in renal mesangial cells

PPARγ agonists [thiazolidinediones (TZDs)] are known to exert anti-fibrotic effects in the kidney. In addition, we previously demonstrated that sphingosine kinase 1 (SK-1) and intracellular sphingosine-1-phosphate (S1P), by reducing the expression of connective tissue growth factor (CTGF), have a protective role in the fibrotic process.

The nuclear receptor LRH-1 critically regulates extra-adrenal glucocorticoid synthesis in the intestine

The nuclear receptor liver receptor homologue-1 (LRH-1, NR5A2) is a crucial transcriptional regulator of many metabolic pathways. In addition, LRH-1 is expressed in intestinal crypt cells where it regulates the epithelial cell renewal and contributes to tumorigenesis through the induction of cell cycle proteins. We have recently identified the intestinal epithelium as an important extra-adrenal source of immunoregulatory glucocorticoids. We show here that LRH-1 promotes the expression of the steroidogenic enzymes and the synthesis of corticosterone in murine intestinal epithelial cells in vitro. Interestingly, LRH-1 is also essential for intestinal glucocorticoid synthesis in vivo, as LRH-1 haplo-insufficiency strongly reduces the intestinal expression of steroidogenic enzymes and glucocorticoid synthesis upon immunological stress. These results demonstrate for the first time a novel role for LRH-1 in the regulation of intestinal glucocorticoid synthesis and propose LRH-1 as an important regulator of intestinal tissue integrity and immune homeostasis.

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin.rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin.rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

Immunomodulatory lipids in plants: plant fatty acid amides and the human endocannabinoid system

Since the discovery that endogenous lipid mediators show similar cannabimimetic effects as phytocannabinoids from CANNABIS SATIVA, our knowledge about the endocannabinoid system has rapidly expanded. Today, endocannabinoid action is known to be involved in various diseases, including inflammation and pain. As a consequence, the G-protein coupled cannabinoid receptors, endocannabinoid transport, as well as endocannabinoid metabolizing enzymes represent targets to block or enhance cannabinoid receptor-mediated signalling for therapeutic intervention. Based on the finding that certain endocannabinoid-like fatty acid N-alkylamides from purple coneflower ( ECHINACEA spp.) potently activate CB2 cannabinoid receptors we have focused our interest on plant fatty acid amides (FAAs) and their overall cannabinomodulatory effects. Certain FAAs are also able to partially inhibit the action of fatty acid amide hydrolase (FAAH), which controls the breakdown of endocannabinoids. Intriguingly, plants lack CB receptors and do not synthesize endocannabinoids, but express FAAH homologues capable of metabolizing plant endogenous N-acylethanolamines (NAEs). While the site of action of these NAEs in plants is unknown, endogenous NAEs and arachidonic acid glycerols in animals interact with distinct physiological lipid receptors, including cannabinoid receptors. There is increasing evidence that also plant FAAs other than NAEs can pharmacologically modulate the action of these endogenous lipid signals. The interference of plant FAAs with the animal endocannabinoid system could thus be a fortunate evolutionary cross point with yet unexplored therapeutic potential.

Sphingosine kinase 1 is pivotal for Fc epsilon RI-mediated mast cell signaling and functional responses in vitro and in vivo

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, triggered by FcepsilonRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin

Transitory binding between photoactivated rhodopsin (Rho* or Meta II) and the G protein transducin (Gt-GDP) is the first step in the visual signaling cascade. Light causes photoisomerization of the 11-cis-retinylidene chromophore in rhodopsin (Rho) to all-trans-retinylidene, which induces conformational changes that allow Gt-GDP to dock onto the Rho* surface. GDP then dissociates from Gt, leaving a transient nucleotide-empty Rho*-Gt(e) complex before GTP becomes bound, and Gt-GTP then dissociates from Rho*. Further biochemical advances are required before structural studies of the various Rho*-Gt complexes can be initiated. Here, we describe the isolation of n-dodecyl-beta-maltoside solubilized, stable, functionally active, Rho*-Gt(e), Rho(e)*-Gt(e), and 9-cis-retinal/11-cis-retinal regenerated Rho-Gt(e) complexes by sucrose gradient centrifugation. In these complexes, Rho* spectrally remained in its Meta II state, and Gt(e) retained its ability to interact with GTPgammaS. Removal of all-trans-retinylidene from Rho*-Gt(e) had no effect on the stability of the Rho(e)*-Gt(e) complex. Moreover, opsin in the Rho(e)*-Gt(e) complex with an empty nucleotide-binding pocket in Gt and an empty retinoid-binding pocket in Rho was regenerated up to 75% without complex dissociation. These results indicate that once Rho* couples with Gt, the chromophore plays a minor role in stabilizing this complex. Moreover, in complexes regenerated with 9-cis-retinal/11-cis-retinal, Rho retains a conformation similar to Rho* that is stabilized by Gt(e) apo-protein.

Do N-arachidonyl-glycine (NA-glycine) and 2-arachidonoyl glycerol (2-AG) share mode of action and the binding site on the β 2 subunit of GABA A receptors?

NA-glycine is an endogenous lipid molecule with analgesic properties, which is structurally similar to the endocannabinoids 2-AG and anandamide but does not interact with cannabinoid receptors. NA-glycine has been suggested to act at the G-protein coupled receptors GPR18 and GPR92. Recently, we have described that NA-glycine can also modulate recombinant α1β2γ2 GABAA receptors. Here we characterize in more detail this modulation and investigate the relationship of its binding site with that of the endocannabinoid 2-AG.

The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta

To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

Ramipril increases the protein level of skeletal muscle IRS-1 and alters protein tyrosine phosphatase activity in spontaneously hypertensive rats

To investigate mechanisms by which angiotensin converting enzyme (ACE)-inhibition increases insulin sensitivity, spontaneously hypertensive (SH) rats were treated with or without ramipril (1 mg/kg per day) for 12 weeks. Insulin binding and protein levels of insulin receptor substrate-1 (IRS-1), p85-subunit of phosphatidylinositol 3'-kinase (p85) and Src homology 2 domain-containing phosphatase-2 (SHP2) were then determined in hindlimb muscle and liver. Additionally, protein tyrosine phosphatase (PTPase) activities towards immobilized phosphorylated insulin receptor or phosphorylated IRS-1 of membrane (MF) and cytosolic fractions (CF) of these tissues were measured. Ramipril treatment increased IRS-1-protein content in muscle by 31+/-9% (P<0.05). No effects were observed on IRS-1 content in liver or on insulin binding or protein expression of p85 or SHP2 in both tissues. Ramipril treatment also increased dephosphorylation of insulin receptor by muscle CF (22.0+/-1.0%/60 min compared to 16.8+/-1.5%/60 min; P<0.05), and of IRS-1 by liver MF (37.2+/-1.7%/7.5 min compared to 33.8+/-1.7%/7.5 min; P<0.05) and CF (36.8+/-1.0%/7.5 min compared to 33.2+/-1.0%/7.5 min; P<0.05). We conclude that the observed effects of ACE-inhibition by ramipril on the protein expression of IRS-1 and on PTPase activity might contribute to its effect on insulin sensitivity.