Additional file 1: of Inhibition of TRPM7 suppresses cell proliferation of colon adenocarcinoma in vitro and induces hypomagnesemia in vivo without affecting azoxymethane-induced early colon cancer in mice

Cell Cycle Analysis. Description of data: Waixenicin A results in cell growth arrest in HT-29 cells as determined by cell cycle analysis using flow cytometry. (DOCX 613 kb

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

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

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