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Dissertação de Mestrado em Biologia Celular e Molecular apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra. Maus hábitos alimentares e um estilo de vida sedentário contribuem para o aumento das taxas de obesidade em todo o mundo. A remodelação do tecido adiposo branco em resposta ao excesso crónico de energia resulta do aumento de tamanho dos adipócitos existentes (hipertrofia) e da formação de novos adipócitos através do recrutamento de células mesenquimais do estroma residentes no tecido adiposo. O aumento dos gastos energéticos pode ser conseguido através da activação da termogénese no tecido adiposo castanho e formação de adipócitos beges dentro de tecido adiposo branco (browning). Os mecanismos reguladores destes processos ainda estão muito por explorar. Sabe-se que prostaglandinas como PGI2 promovem o recrutamento e browning de células mesenquimais do estroma, e recentemente a enzima esfingosina cinase-1 (Sphk1) foi identificada como um importante regulador da activação termogénica do tecido adiposo castanho em resposta a baixas temperaturas. Sphk1 pertence à via de sinalização de esfingolípidos, e em obesidade os seus níveis encontram-se elevados, assim como o seu produto esfingosina-1-fosfato (S1P) e ceramida. Esta tese teve objectivo determinar o papel da Sphk1 no recrutamento e diferenciação de células mesenquimais do estroma, e também a sua função em adipócitos maduros. O silenciamento de Sphk1 através de siRNA em adipócitos castanhos reduziu significativamente a expressão de genes envolvidos na termogénese e resposta a estimulação por norepinefrina (NE), e reduziu também a taxa basal de lipólise, o que indica que a função de adipócitos castanhos é claramente afectada na ausência de Sphk1/S1P. Para além disso, o silenciamento de Sphk1 em adipócitos brancos e beges, induzidos por adição de cPGI2, reduziu significativamente a expressão de genes reguladores da adipogénese e lipólise, o que no entanto se traduziu num aumento da taxa de lipolise em adipócitos beges. Por outro lado, o silenciamento de Sphk1 em células progenitoras de adipócitos castanhos diminuiu significativamente a sua diferenciação, o que indica que Sphk1 é necessária para o processo de diferenciação de adipócitos castanhos. Mais ainda, o silenciamento de Sphk1 em células progenitoras do tecido adiposo branco reduziu significativamente a expressão de genes marcadores de adipogénese, em condições normais e na presença de cPGI2, o que indica que Sphk1 tem um papel semelhante na regulação da diferenciação de adipócitos brancos e beges. A expressão de genes mitocondriais também foi reduzida significativamente, o que pode comprometer a capacidade oxidativa dos adipócitos. Para além disso, o silenciamento de Sphk1 aumentou significativamente apoptose e morte celular de adipócitos durante o processo de diferenciação, medido por marcação das células com AnnexinV e detecção por citometria de fluxo. Esta técnica também permitiu detectar marcação de DNA com EdU, e revelou que há uma acumulação de células na fase S/G2/M e redução da proliferação celular na ausência de Sphk1. Ambos os resultados permitem explicar a redução na adipogénese observada nas experiencias anteriores. Por último, a adição de S1P a células progenitoras do tecido adiposo branco reduziu adipogénese e promoveu proliferação celular. Concluindo, estes resultados mostram que a sinalização de Sphk1/S1P tem um papel importante no recrutamento e diferenciação de células progenitoras de adipócitos, com especial relevância no desenvolvimento de adipócitos castanhos. Por isso é relevante continuar a explorar o papel dos esfingolípidos na regulação do tecido adiposo, dado o seu potencial para aplicações terapêuticas em doenças metabólicas. A high calorie diet and sedentary lifestyle have led to the spiralling increase in obesity rates around the world. Remodelling of white adipose tissue (WAT) in response to chronic energy excess is accomplished by enlargement of existing adipocytes (hypertrophy) and generation of new ones by recruitment of mesenchymal stromal cells (MSC) resident in adipose depot. Increasing energy expenditure can be accomplished by activation of brown adipose tissue (BAT) thermogenesis and formation of beige adipocytes in white depots (browning). The mechanisms regulating these processes are still fairly unknown. Prostaglandins have been shown to promote MSC recruitment and browning, in particular PGI2, and recently sphingosine kinase 1 (Sphk1) was identified as an important gene in BAT thermogenic activation in response to cold exposure. Sphk1 is an enzyme of the sphingolipid pathway, and has been found upregulated in obesity along with its product sphingosine-1-phosphate and ceramide. The aim of this study was to determine the role of Sphk1 in MSC recruitment and differentiation, and also the role in mature adipocyte function. siRNA-mediated knock down of Sphk1 in brown adipocytes significantly reduced expression of thermogenic genes and norepinephrine (NE) responsiveness, and also reduced basal lipolysis, indicating a clear impairment of brown adipocyte function in the absence of Sphk1/S1P signalling. In addition, Sphk1 knock down in white and cPGI2-induced beige adipocytes markedly reduced expression of general adipogenic markers and genes regulating lipolysis, which surprisingly translated into an increase in lipolysis in beige adipocytes. On the other hand, Sphk1 knock down in brown progenitors significantly reduced differentiation, which indicates that Sphk1 is required for normal brown adipocyte differentiation. Moreover, Sphk1 knock down in undifferentiated WAT MSCs significantly reduced expression of adipogenic genes, both in normal conditions and in the presence of cPGI2, indicating that Sphk1 regulates white and beige adipogenesis in a similar way. Genes involved in mitochondrial biogenesis were also significantly reduced, which may compromise the oxidative capacity of adipocytes. Furthermore, knock down of Sphk1 significantly increased apoptosis and cell death of differentiating adipocytes, measured by increased AnnexinV staining detected by flow cytometry. Additionally, EdU labelling allowed detection of cell accumulation in S/G2/M phase and reduction in cell proliferation. Both results could explain the observed reduction in adipogenesis. Finally, in line with its known effects, S1P added to differentiating WAT MSC reduced adipogenesis and promoted cell proliferation. Overall, these results show that Sphk1/S1P signalling plays an important role in recruitment and differentiation of adipocyte progenitors, with particular relevance for brown adipocyte development. Therefore, it is relevant to further explore the role of sphingolipids in regulation of adipose tissue, with potential for therapeutic application in metabolic diseases.

International audience; BACKGROUND: The accumulation of beta amyloid (Aβ) peptides, a hallmark of Alzheimer's disease (AD) is related to mechanisms leading to neurodegeneration. Among its pleiotropic cellular effects, Aβ accumulation has been associated with a deregulation of sphingolipid metabolism. Sphingosine 1-phosphate (S1P) derived from sphingosine is emerging as a critical lipid mediator regulating various biological activities including cell proliferation, survival, migration, inflammation, or angiogenesis. S1P tissue level is low and kept under control through equilibrium between its synthesis mostly governed by sphingosine kinase-1 (SphK1) and its degradation by sphingosine 1-phosphate lyase (SPL). We have previously reported that Aβ peptides were able to decrease the activity of SphK1 in cell culture models, an effect that could be blocked by the prosurvival IGF-1/IGF-1R signaling. RESULTS: Herein, we report for the first time the expression of both SphK1 and SPL by immunohistochemistry in frontal and entorhinal cortices from 56 human AD brains. Immunohistochemical analysis revealed a decreased expression of SphK1 and an increased expression of SPL both correlated to amyloid deposits in the entorhinal cortex. Otherwise, analysis of brain tissue extracts showed a decrease of SphK1 expression in AD brains whereas SPL expression was increased. The content of IGF-1R, an activator of SphK1, was found decreased in AD brains as well as S1P1, the major receptor for S1P. CONCLUSIONS: Collectively, these results highlight the importance of S1P in AD suggesting the existence of a global deregulation of S1P signaling in this disease from its synthesis by SphK1 and degradation by SPL to its signaling by the S1P1 receptor.

Purpose: Sphingolipids are important signaling molecules mediating cell survival, proliferation, cell cycle regulation and apoptosis. Ceramide is the most vital sphingolipid which induces growth arrest, senescence, and apoptosis. In this study, we aimed to determine the roles of sphingosine kinase- 1 (SK-1) and glucosylceramide synthase (GCS) genes in paclitaxel, doxorubicin, tamoxifen, cyclophosphamide and docetaxel induced apoptosis in human MCF-7 breast cancer cells. Methods: IC50 values (drug concentration inhibiting cell growth by 50%) of the anticancer agents were calculated using XTT cell proliferation assay. Changes in mitochondrial membrane potential (MMP) were determined using JC-1 assay kit. Changes in the mRNA levels of SK-1 and GCS genes were measured by using RT-PCR technique. Results: The results demonstrated significant decrease in cellular proliferation and increase in loss of MMP in a dose-dependent manner. Paclitaxel, doxorubicin, tamoxifen, cyclophosphamide and docetaxel application downregulated SK-1 expression while paclitaxel, tamoxifen, cyclophosphamide and docetaxel but not doxorubicin downregulated GCS comparing to untreated control cells. Conclusion: These results show for the first time that these agents induce apoptosis in MCF-7 cells by downregulating the antiapoptotic SK-1 and GCS genes that may result in accumulation of apoptotic ceramides. © 2011 Zerbinis Medical Publications., Turkish Association for Cancer Research and Control and by the Turkish Academy of Sciences

The use of endothelial progenitor cells in vascular therapies has been limited due to their low numbers present in the bone marrow and peripheral blood. The aim of this study was to investigate the effect of sphingosine kinase on the de-differentiation of mature human endothelial cells toward a progenitor phenotype. Methods: The lipid enzyme sphingosine kinase-1 was lentivirally over-expressed in human umbilical vein endothelial cells and cells were analyzed for progenitor phenotype and function. Results: Sphingosine kinase-1 mRNA expression was induced approximately 150-fold with a resultant 20-fold increase in sphingosine kinase-1 enzymatic activity. The mRNA expression of the progenitor cell markers CD34, CD133, and CD117 and transcription factor NANOG increased, while the endothelial cell markers analyzed were largely unchanged. The protein level of mature endothelial cell surface markers CD31, CD144, and von Willebrand factor significantly decreased compared to controls. In addition, functional assays provided further evidence for a de-differentiated phenotype with increased viability, reduced uptake of acetylated low-density lipoprotein and decreased tube formation in Matrigel in the cells over-expressing sphingosine kinase-1. Conclusions: These findings suggest that over-expression of sphingosine kinase-1 in human endothelial cells promotes, in part, their de-differentiation to a progenitor cell phenotype, and is thus a potential tool for the generation of a large population of vascular progenitor cells for therapeutic use Refereed/Peer-reviewed

Purpose: Sphingosine kinase-1 (SphK1) was shown in preclinical models and non-genitourinary cancers to be instrumental in cancer progression, adaptation to hypoxia and in tumour angiogenesis. No data were available in human prostate cancer. The present study was designed to assess SphK1 expression and activity in radical prostatectomy specimens and to research correlations with clinical features.Materials and methods: Transverse section of fresh tissue was obtained from 30 consecutive patients undergoing laparoscopic prostatectomy. SphK1 enzymatic activities of tumour and normal counterpart were determined. Relationships with PSA, Gleason sum, pathological stage, resection margin status and treatment failure were researched. SphK1 pattern ofexpression was then assessed on tissue microarray. Results: A significant 2-fold increase in SphK1 enzymatic activity(11.1 ± 8.4 versus 5.9 ± 3.2(P < 0.04)) was observed in cancer. The upper quartile of SphK1 activity was associated with higher PSA (16.7 versus 6.4 ng/ml, P = 0.04), higher tumor volumes (20.7 versus 9.8,P = 0.002), higher rates of positive margins (85.7% versus 28.6%, P = 0.01) and surgical failure(71.4% versus 9.5%, P = 0.003) than the lower three quartiles. Odds ratios (OR) for treatment failure showed a strong relationship with SphK1 activity (OR: 23.7, P = 0.001), positive resection margins (OR: 15.0, P = 0.007) and Gleason sum (P4 + 3, OR: 8.0, P = 0.003). Tissue microarrays showed discrete epithelial expression that varied with Gleason sum with significant relationship between SphK1 expression and higher Gleason sum.Conclusion: In complement to preclinical literature, the demonstrated relationships between SphK1-increased activity in cancer and relevant clinical features confirm a central role for SphK1 in prostate cancer that herald promising avenues in risk-assessment and treatment. Refereed/Peer-reviewed

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

The enzyme sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which is an important survival factor for endothelial cells (EC). Modest increases in intracellular SK1 activity in the EC are known to confer a survival advantage upon the cells. Here, we investigated the effects of more dramatic increases in intracellular SK1 in the EC. We found that these cells show reduced cell survival under conditions of stress, enhanced caspase-3 activity, cell cycle inhibition, and cell-cell junction disruption. We propose that alterations in the phosphorylation state of the enzyme may explain the differential effects on the phenotype with modest versus high levels of enforced expression of SK1. Our results suggest that SK1 activity is subject to control in the EC, and that this control may be lost in conditions involving vascular regression.

The adipose-derived plasma protein, adiponectin (APN), has various protective effects on cardiovascular diseases. In this study, we show that endogenous APN is required for full cyclooxygenase-2 (COX-2) induction by ischemia-reperfusion injury in the heart in vivo. In rat neonatal cardiac myocytes, APN-induced COX-2 expression was reduced by treatment with a sphingosine kinase-1 (SphK-1) inhibitor or siRNA targeting SphK-1. Treatment with a sphingosine-1-phosphate (S1P) receptor antagonist also diminished COX-2 expression in response to APN stimulation. These findings suggest that APN is a physiological regulator of COX-2 signaling in the heart and that this regulation occurs in part via a SphK-1–S1P receptor dependent mechanism in cardiac myocytes.

2017