Anja Lüth

Osteoclast-specific cathepsin K deletion stimulates S1P-dependent bone formation

Cathepsin K (CTSK) is secreted by osteoclasts to degrade collagen and other matrix proteins during bone resorption. Global deletion of Ctsk in mice decreases bone resorption, leading to osteopetrosis, but also increases the bone formation rate (BFR). To understand how Ctsk deletion increases the BFR, we generated osteoclast- and osteoblast-targeted Ctsk knockout mice using floxed Ctsk alleles. Targeted ablation of Ctsk in hematopoietic cells, or specifically in osteoclasts and cells of the monocyte-osteoclast lineage, resulted in increased bone volume and BFR as well as osteoclast and osteoblast numbers. In contrast, targeted deletion of Ctsk in osteoblasts had no effect on bone resorption or BFR, demonstrating that the increased BFR is osteoclast dependent. Deletion of Ctsk in osteoclasts increased their sphingosine kinase 1 (Sphk1) expression. Conditioned media from Ctsk-deficient osteoclasts, which contained elevated levels of sphingosine-1-phosphate (S1P), increased alkaline phosphatase and mineralized nodules in osteoblast cultures. An S1P1,3 receptor antagonist inhibited these responses. Osteoblasts derived from mice with Ctsk-deficient osteoclasts had an increased RANKL/OPG ratio, providing a positive feedback loop that increased the number of osteoclasts. Our data provide genetic evidence that deletion of CTSK in osteoclasts enhances bone formation in vivo by increasing the generation of osteoclast-derived S1P.

Acid sphingomyelinase–ceramide system mediates effects of antidepressant drugs

Major depression is a highly prevalent severe mood disorder that is treated with antidepressants. The molecular targets of antidepressants require definition. We investigated the role of the acid sphingomyelinase (Asm)-ceramide system as a target for antidepressants. Therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduced Asm activity and ceramide concentrations in the hippocampus, increased neuronal proliferation, maturation and survival and improved behavior in mouse models of stress-induced depression. Genetic Asm deficiency abrogated these effects. Mice overexpressing Asm, heterozygous for acid ceramidase, treated with blockers of ceramide metabolism or directly injected with C16 ceramide in the hippocampus had higher ceramide concentrations and lower rates of neuronal proliferation, maturation and survival compared with controls and showed depression-like behavior even in the absence of stress. The decrease of ceramide abundance achieved by antidepressant-mediated inhibition of Asm normalized these effects. Lowering ceramide abundance may thus be a central goal for the future development of antidepressants.

Acid Sphingomyelinase Inhibitors Normalize Pulmonary Ceramide and Inflammation in Cystic Fibrosis

Employing genetic mouse models we have recently shown that ceramide accumulation is critically involved in the pathogenesis of cystic fibrosis (CF) lung disease. Genetic or systemic inhibition of the acid sphingomyelinase (Asm) is not feasible for treatment of patients or might cause adverse effects. Thus, a manipulation of ceramide specifically in lungs of CF mice must be developed. We tested whether inhalation of different acid sphingomyelinase inhibitors does reduce Asm activity and ceramide accumulation in lungs of CF mice. The efficacy and specificity of the drugs was determined. Ceramide was determined by mass spectrometry, DAG-kinase assays, and fluorescence microscopy. We determined pulmonary and systemic Asm activity, neutral sphingomyelinase (Nsm), ceramide, cytokines, and infection susceptibility. Mass spectroscopy, DAG-kinase assays, and semiquantitative immune fluorescence microscopy revealed that a standard diet did not influence ceramide in bronchial respiratory epithelial cells, while a diet with Peptamen severely affected the concentration of sphingolipids in CF lungs. Inhalation of the Asm inhibitors amitriptyline, trimipramine, desipramine, chlorprothixene, fluoxetine, amlodipine, or sertraline restored normal ceramide concentrations in murine bronchial epithelial cells, reduced inflammation in the lung of CF mice and prevented infection with Pseudomonas aeruginosa. All drugs showed very similar efficacy. Inhalation of the drugs was without systemic effects and did not inhibit Nsm. These findings employing several structurally different Asm inhibitors identify Asm as primary target in the lung to reduce ceramide concentrations. Inhaling an Asm inhibitor may be a beneficial treatment for CF, with minimal adverse systemic effects.

Topical application of sphingosine-1-phosphate and FTY720 attenuate allergic contact dermatitis reaction through inhibition of dendritic cell migration.

Migration of Langerhans cells (LCs) from the skin to the lymph node is an essential step in the pathogenesis of allergic contact dermatitis (ACD). Therefore, inhibition of LC-migration could be a promising strategy to improve this skin disease. Effects of sphingosine-1-phosphate (S1P) and the immunomodulator FTY720 on LC trafficking is not well defined, yet. Thus, we investigated the action of topically administered S1P and FTY720 in a murine model of ACD. Most interestingly, FTY720 as well as S1P inhibited the inflammatory reaction in the elicitation phase of ACD. In the sensitization phase, FTY720, and S1P reduced the weight and cell count of the draining auricular lymph node, as well as immigrated dendritic cells provoked by repetitive topical administration of the hapten. Correspondingly, the density of LCs in the epidermis was higher in FTY720- and S1P-treated mice compared to vehicle treatment. A skin dendritic cell migration assay confirmed the significant inhibition of dendritic cell migration by FTY720 and S1P. These data supply conclusive evidence that the strategy of targeting the migratory response of LCs with locally acting S1P or FTY720 represents an emerging option in the treatment of allergic skin diseases like contact hypersensitivity and atopic dermatitis.

Involvement of the ABC-transporter ABCC1 and the sphingosine 1-phosphate receptor subtype S1P(3) in the cytoprotection of human fibroblasts by the glucocorticoid dexamethasone

Glucocorticoids (GC) represent the most commonly used drugs for the treatment of acute and chronic inflammatory skin diseases. However, the topical long-term therapy of GC is limited by the occurrence of skin atrophy. Most interestingly, although GC inhibit proliferation of human fibroblasts, they exert a pronounced anti-apoptopic action. In the present study, we further elucidated the molecular mechanism of the GC dexamethasone (Dex) to protect human fibroblasts from programmed cell death. Dex not only significantly alters the expression of the cytosolic isoenzyme sphingosine kinase 1 but also initiated an enhanced intracellular formation of the sphingolipid sphingosine 1-phosphate (S1P). Investigations using S1P3(−/−)-fibroblasts revealed that this S1P-receptor subtype is essential for the Dex-induced cytoprotection. Moreover, we demonstrate that the ATP-binding cassette (ABC)-transporter ABCC1 is upregulated by Dex and may represent a crucial carrier to transport S1P from the cytosol to the S1P3-receptor subtype.

Factor-Xa-induced mitogenesis and migration require sphingosine kinase activity and S1P formation in human vascular smooth muscle cells

AIMS Sphingosine-1-phosphate (S1P) is a cellular signalling lipid generated by sphingosine kinase-1 (SPHK1). The aim of the study was to investigate whether the activated coagulation factor-X (FXa) regulates SPHK1 transcription and the formation of S1P and subsequent mitogenesis and migration of human vascular smooth muscle cells (SMC). METHODS AND RESULTS FXa induced a time- (3-6 h) and concentration-dependent (3-30 nmol/L) increase of SPHK1 mRNA and protein expression in human aortic SMC, resulting in an increased synthesis of S1P. FXa-stimulated transcription of SPHK1 was mediated by the protease-activated receptor-1 (PAR-1) and PAR-2. In human carotid artery plaques, expression of SPHK1 was observed at SMC-rich sites and was co-localized with intraplaque FX/FXa content. FXa-induced SPHK1 transcription was attenuated by inhibitors of Rho kinase (Y27632) and by protein kinase C (PKC) isoforms (GF109203X). In addition, FXa rapidly induced the activation of the small GTPase Rho A. Inhibition of signalling pathways which regulate SPHK1 expression, inhibition of its activity or siRNA-mediated SPHK1 knockdown attenuated the mitogenic and chemotactic response of human SMC to FXa. CONCLUSION These data suggest that FXa induces SPHK1 expression and increases S1P formation independent of thrombin and that this involves the activation of Rho A and PKC signalling. In addition to its key function in coagulation, this direct effect of FXa on human SMC may increase cell proliferation and migration at sites of vessel injury and thereby contribute to the progression of vascular lesions.

Sphingosine Kinase-1 (SphK-1) Regulates Mycobacterium smegmatis Infection in Macrophages

Sphingosine kinase-1 is known to mediate Mycobacterium smegmatis induced inflammatory responses in macrophages, but its role in controlling infection has not been reported to date. We aimed to unravel the significance of SphK-1 in controlling M. smegmatis infection in RAW 264.7 macrophages. Our results demonstrated for the first time that selective inhibition of SphK-1 by either D, L threo dihydrosphingosine (DHS; a competitive inhibitor of Sphk-1) or Sphk-1 siRNA rendered RAW macrophages sensitive to M. smegmatis infection. This was due to the reduction in the expression of iNOs, p38, pp-38, late phagosomal marker, LAMP-2 and stabilization of the RelA (pp-65) subunit of NF-κB. This led to a reduction in the generation of NO and secretion of TNF-α in infected macrophages. Congruently, overexpression of SphK-1 conferred resistance in macrophages to infection which was due to enhancement in the generation of NO and expression of iNOs, pp38 and LAMP-2. In addition, our results also unraveled a novel regulation of p38MAPK by SphK-1 during M. smegmatis infection and generation of NO in macrophages. Enhanced NO generation and expression of iNOs in SphK-1++ infected macrophages demonstrated their M-1bright phenotype of these macrophages. These findings thus suggested a novel antimycobacterial role of SphK-1 in macrophages.

Dexamethasone protects human fibroblasts from apoptosis via an S1P3-receptor subtype dependent activation of PKB/Akt and BclXL

Topical used glucocorticoids (GC) represent an important class of steroid hormones for the treatment of a broad range of acute or chronic inflammatory diseases. Most interestingly, GC exert a pronounced anti-apoptotic effect in primary human fibroblasts whereas in variety of hematopoietic cells a pro-apoptotic effect is visible. Recently, it has been discovered that in human fibroblasts the GC dexamethasone (Dex) exerts its protection from programmed cell death via the formation of the lipid mediator sphingosine 1-phosphate (S1P) followed by an activation of the S1P(3)-receptor subtype. In the present study, the molecular mechanism of Dex to protect human fibroblasts from apoptosis was elucidated. Thereupon, Dex not only mediates its anti-apoptotic effect via activation of phosphoinositide 3-kinase (PI3K)/Akt signalling but also includes an involvement of the Bcl-2 family protein Bcl(XL). Most interestingly, the use of S1P(3)-knockout fibroblasts revealed that the S1P(3)-receptor subtype is crucial for activation of PKB/Akt as well as Bcl(XL) by Dex.

Phosphorylation of the immunomodulator FTY720 inhibits programmed cell death of fibroblasts via the S1P3 receptor subtype and Bcl-2 activation.

Background: FTY720, a synthetic compound produced by modification of a metabolite from Isaria sinclairii, is known as a unique immunosuppressive agent that exerts its activity by inhibiting lymphocyte egress from secondary lymphoid tissues. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate (FTY720-P), which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. Despite its homology to S1P, which exerts antiapoptotic actions in different cells, FTY720 has also been reported to be able to induce apoptosis in a variety of cells. Methods: Therefore, we investigated the action of both, FTY720 and its phosphorylated version FTY720-P, on apoptosis. Moreover, signalling pathways of apoptosis in response to FTY720 and FTY720-P were examined. Results and Conclusions: Although FTY720 acts apoptotic at micromolar concentrations in human fibroblasts the phosphorylated compound FTY720-P possesses a pronounced antiapoptotic effect counteracting FTY720-induced programmed cell death. Interestingly, none of the classical antiapoptotic pathways like MAP kinases, Akt or mTOR play a role in the protective role of FTY720-P. Most important, we identified that the S1P3 receptor subtype is involved in the antiapoptotic action of FTY720-P leading to an increased phosphorylation of Bcl-2 and changes in the mitochondrial membrane potential.

Novel methods for the quantification of (2E)-hexadecenal by liquid chromatography with detection by either ESI QTOF tandem mass spectrometry or fluorescence measurement.

Sphingosine-1-phosphate lyase (SPL) is the only known enzyme that irreversibly cleaves sphingosine-1-phosphate (S1P) into phosphoethanolamine and (2E)-hexadecenal during the final step of sphingolipid catabolism. Because S1P is involved in a wide range of physiological and diseased processes, determining the activity of the degrading enzyme is of great interest. Therefore, we developed two procedures based on liquid chromatography (LC) for analysing (2E)-hexadecenal, which is one of the two S1P degradation products. After separation, two different quantification methods were performed, tandem mass spectrometry (MS) and fluorescence detection. However, (2E)-hexadecenal as a long-chain aldehyde is not ionisable by electrospray ionisation (ESI) for MS quantification and has an insufficient number of corresponding double bonds for fluorescence detection. Therefore, we investigated 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) as a derivatisation reagent. DAIH transforms the aldehyde into an ionisable and fluorescent analogue for quantitative analysis. Our conditions were optimised to obtain the outstanding limit of detection (LOD) of 1 fmol per sample (30 μL) for LC-MS/MS and 0.75 pmol per sample (200 μL) for LC determination with fluorescence detection. We developed an extraction procedure to separate and concentrate (2E)-hexadecenal from biological samples for these measurements. To confirm our new methods, we analysed the (2E)-hexadecenal level of different cell lines and human plasma for the first time ever. Furthermore, we treated HT-29 cells with different concentrations of 4-deoxypyridoxine (DOP), which competitively inhibits pyridoxal-5-phosphate (P5P), an essential cofactor for SPL activity, and observed a significant decrease in (2E)-hexadecenal relative to the untreated cells.