Mice with loss of SK1 in hematopoietic cells exhibit splenomegaly with DSS-induced colitis.

<p>Bone marrow transplants were performed with WT and SK1^−/− mice. Following 6 weeks of rest, mice were administered either regular drinking water or water containing 5% DSS for 5 days. <b>A</b>) Change in body weight was assessed. Data represent mean ±SD, n≥6 for each treatment group; (significance at Day 5 =  *WT^WTBM, **WT^SK1BM, **SK1^WTBM); *p<0.05, **p<0.01, ***p<0.001 vs strain Day 0. <b>B</b>) Spleen weight and <b>C</b>) colon length were assessed following treatment. <b>D</b>) Pathology damage scores were determined by a pathologist in a blinded fashion with H&E sections of colon tissue. Data represent mean ±SD, n≥6 for each treatment group; *p<0.05, **p<0.01, ***p<0.001 vs strain untreated, #p<0.05 as compared to WT^WTBM DSS treated, %p<0.05 as compared to SK1^SK1BM DSS. For the X-axis: regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

Hematopoietic genotype determines circulating sphingolipid levels.

<p>Whole blood was collected and analyzed for sphingolipid content using ESI/MS/MS. <b>A and C</b>) Ceramide and <b>B and D</b>) S1P levels were normalized to volume. Data represent mean ±SD, n≥6 for each treatment group, *p<0.05, **p<0.01 as compared to both strains of mice with WT bone marrow. X-axis: regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

Extra-hematopoietic SK1 is necessary for COX2 expression in the colon epithelium.

<p>COX2 expression levels were examined by IHC <b>A–D</b>): untreated mice; <b>E–H</b>) DSS treated mice. Scale bars  = 20 µm. Regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

Distinct Roles for Hematopoietic and Extra-Hematopoietic Sphingosine Kinase-1 in Inflammatory Bowel Disease

<div><p>Sphingosine kinase 1 (SK1), one of two SK enzymes, is highly regulated and has been shown to act as a focal point for the action of many growth factors and cytokines. SK1 leads to generation of sphingosine-1-phosphate (S1P) and potentially the activation of S1P receptors to mediate biologic effects. Our previous studies implicated SK1/S1P in the regulation of inflammatory processes, specifically in inflammatory bowel disease (IBD). These studies were conducted using a total body knockout mouse for SK1 and were unable to determine the source of SK1/S1P (hematopoietic or extra-hematopoietic) involved in the inflammatory responses. Therefore, bone marrow transplants were performed with wild-type (WT) and SK1-/- mice and colitis induced with dextran sulfate sodium (DSS). Irrespective of the source of SK1/S1P, bone marrow or tissue, DSS induced colitis in all mice; however, mice lacking SK1 in both hematopoietic and extra-hematopoietic compartments exhibited decreased crypt damage. Systemic inflammation was assessed, and mice with WT bone marrow demonstrated significant neutrophilia in response to DSS. In the local inflammatory response, mice lacking SK1/S1P in either bone marrow or tissue exhibited decreased induction of cytokines and less activation of STAT3 (signal transducer and activator of transcription 3). Interestingly, we determined that extra-hematopoietic SK1 is necessary for the induction of cyclooxygenase 2 (COX2) in colon epithelium in response to DSS-induced colitis. Taken together our data suggest that hematopoietic-derived SK1/S1P regulates specific aspects of the systemic inflammatory response, while extra-hematopoietic SK1 in the colon epithelium is necessary for the autocrine induction of COX2 in DSS-induced colitis.</p></div

STAT3 phosphorylation requires dual sources of SK1/S1P in acute DSS-induced colitis.

<p>Phospho-STAT3 (Ser727) was examined by IHC. <b>A–D</b>) untreated mice; <b>E–H</b>) DSS treated mice. Scale bars  = 20 µm. Regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

Dual sources of SK1/S1P regulate IL-1β and IL-6 expression in DSS-treated mice.

<p>Expression levels of <b>A</b>) IL-1β, <b>B</b>) IL-6, and <b>C</b>) TNFα in colon tissues were determined using real time-RTPCR and normalized to β-actin. Data represent mean ±SEM, n≥3 for each treatment group; *p<0.05, **p<0.01, and ****p<0.001 vs strain untreated, #p<0.05 as compared to WT^WTBM DSS. X-axis: regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

Mice with WT bone marrow exhibit significant neutrophilia following DSS-induced colitis.

<p><b>A</b>) Whole blood was collected and analyzed for neutrophil counts. <b>B</b>) Neutrophil counts are normalized to total lymphocytes and expressed as neutrophil-lymphocyte ratio. Data represent mean ±SD, n≥6 for each treatment group; *p<0.05, ***p<0.001 and ****p<0.001 vs strain untreated, #p<0.05 as compared to WT^WTBM DSS treated, %p<0.05 as compared to SK1^SK1BM DSS. X-axis: regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

Palmitate and glucose prevent colocalization of CERT and Golgi apparatus in INS-1 cells.

<p>INS-1 cells grown on a glass coverslip were transfected with the plasmid CERT-GFP as described in experimental procedures. 24 h later, the cells were treated with or without palmitate in the presence of 5 mM or 30 mM glucose for 12 h. Cells were then fixed and immunostained with WGA texas red-conjugated, a specific marker for the Golgi apparatus. a) Representative confocal microscopy images are shown; all images were processed and printed identically. b) The co-localization between CERT and WGA has been quantified through the Image J software and reported as Pearson colocalization coefficient. *p<0.05 G30+palmitate cells vs G30. c) The percentage of cells with co-localization of CERT and WGA was determined. The data are means ± the SD. **p<0.01 for G30+palmitate compared with G30.</p

Palmitate and glucose regulate the use of Cer for the biosynthesis of complex sphingolipids in INS-1 cells.

<p>a) Cells were treated for 12 h (left panel) or 24 h (right panel) with 0.4 mM palmitate (P4) or without palmitate in the presence of 5 mM or 30 mM glucose. Cell viability was assessed by the MTT assay. Results are expressed as percentage of cell viability with respect to 5 mM glucose-treated cells (100%). Data are the mean ± S.D. of three independent experiments. *, p<0.05; ** p, <0.01. b) INS-1 cells were treated with 5 mM or with 30 mM glucose ±0.4 mM palmitate and harvested in lysis buffer for immunoblot analysis of GRP78 and GAPDH levels as described in experimental procedures. INS-1 cells were pretreated 30 min ±0.1 µM thapsigargin (Tg). Equal amounts of protein from homogenates were analyzed by immunoblotting with an anti-GRP78 antibody and an anti-GAPDH antibody. c) Cells were treated for 12 h ±0.4 mM palmitate in the presence of 5 mM or 30 mM glucose and then pulsed with 0.3 µCi/ml [C3-³H]sphingosine for 1 h. At the end of pulse, cells were harvested and submitted to lipid extraction and partitioning. The methanolized organic phase and the aqueous phase were analyzed by HPTLC and digital autoradiography of HPTLC (see experimental procedures). G5, 5 mM glucose; G5P4, 5 mM glucose+0.4 mM palmitate; G30, 30 mM glucose; G30P4, 30 mM glucose+0.4 mM palmitate. Data are the mean ± S.D. of at least three independent experiments. *p<0.05 for Ceramide and Sphingomyelin G5P4 or G30 compared with G5 and for GSLs G30P4 compared with G30; **p <0.01 for Cer and SM G30P4 compared with G30.</p

Palmitate and glucose regulate CERT expression and activation in INS-1 cells.

<p>a) INS-1 cells were harvested in lysis buffer as described in material and methods. Equal amounts of protein from homogenates were analyzed by immunoblotting with an anti-CERT antibody, an anti-phosphoserine and an anti-GAPDH antibody; b) the amount of CERT expressed was determined by densitometric quantitation and normalized for GAPDH **p<0.01 for G30+palmitate compared with G30; c) the amount of pCERT expressed was determined by densitometric quantitation and normalized for CERT **p<0.01 for G30+palmitate compared with G30; d) Relative expression of CERT assessed by Real-Time PCR. Results are expressed as fold-change relative to G5 *p<0.05 G30+palmitate cells vs G30. Values are mean ± SD of three independent experiments.</p