Thrombospondin-1 Type 1 Repeats in a Model of Inflammatory Bowel Disease: Transcript Profile and Therapeutic Effects

Thrombospondin-1 (TSP-1) is a matricellular protein with regulatory functions in inflammation and cancer. The type 1 repeats (TSR) domains of TSP-1 have been shown to interact with a wide range of proteins that result in the anti-angiogenic and anti-tumor properties of TSP-1. To ascertain possible functions and evaluate potential therapeutic effects of TSRs in inflammatory bowel disease, we conducted clinical, histological and microarray analyses on a mouse model of induced colitis. We used dextran sulfate sodium (DSS) to induce colitis in wild-type (WT) mice for 7 days. Simultaneously, mice were injected with either saline or one form of TSP-1 derived recombinant proteins, containing either (1) the three type 1 repeats of the TSP-1 (3TSR), (2) the second type 1 repeat (TSR2), or (3) TSR2 with the RFK sequence (TSR2+RFK). Total RNA isolated from the mice colons were processed and hybridized to mouse arrays. Array data were validated by real-time qPCR and immunohistochemistry. Histological and disease indices reveal that the mice treated with the TSRs show different patterns of leukocytic infiltration and that 3TSR treatment was the most effective in decreasing inflammation in DSS-induced colitis. Transcriptional profiling revealed differentially expressed (DE) genes, with the 3TSR-treated mice showing the least deviation from the WT-water controls. In conclusion, this study shows that 3TSR treatment is effective in attenuating the inflammatory response to DSS injury. In addition, the transcriptomics work unveils novel genetic data that suggest beneficial application of the TSR domains in inflammatory bowel disease

Thrombospondin-1 in a Murine Model of Colorectal Carcinogenesis

Colorectal Cancer (CRC) is one of the late complications observed in patients suffering from inflammatory bowel diseases (IBD). Carcinogenesis is promoted by persistent chronic inflammation occurring in IBD. Understanding the mechanisms involved is essential in order to ameliorate inflammation and prevent CRC. Thrombospondin 1 (TSP-1) is a multidomain glycoprotein with important roles in angiogenesis. The effects of TSP-1 in colonic tumor formation and growth were analyzed in a model of inflammation-induced carcinogenesis. WT and TSP-1 deficient mice (TSP-1-/-) of the C57BL/6 strain received a single injection of azoxymethane (AOM) and multiple cycles of dextran sodium sulfate (DSS) to induce chronic inflammation-related cancers. Proliferation and angiogenesis were histologically analyzed in tumors. The intestinal transcriptome was also analyzed using a gene microarray approach. When the area containing tumors was compared with the entire colonic area of each mouse, the tumor burden was decreased in AOM/DSS-treated TSP-1-/- versus wild type (WT) mice. However, these lesions displayed more angiogenesis and proliferation rates when compared with the WT tumors. AOM-DSS treatment of TSP-1-/- mice resulted in significant deregulation of genes involved in transcription, canonical Wnt signaling, transport, defense response, regulation of epithelial cell proliferation and metabolism. Microarray analyses of these tumors showed down-regulation of 18 microRNAs in TSP-1-/- tumors. These results contribute new insights on the controversial role of TSP-1 in cancer and offer a better understanding of the genetics and pathogenesis of CRC

Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity.

Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP) was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T), BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl) phthalate (MEHP) as a major metabolite of another important phthalate di (2-ethylhexyl) phthalate (DEHP) inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein) and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29) growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities

The effects of BBP and MEHP on mRNA translation in HEK-293T cells.

<p>HEK-293T cells were grown to 80% confluence and transfected with luciferase reporter. Eight hours later, cells were treated with BBP or MEHP for 24 hrs at the concentrations indicated. Equal volume of vehicle (methanol) was used as the control. Cells were harvested by washing once with cold PBS, and the cell lysate was prepared with PBL buffer (Promega) for the luciferase activity measurement using dual luciferase assay kit (Promega). <b>(A)</b> Schematic diagram of the reporter construct used in this experiment and throughout the whole study. <b>(B)</b> The effect of BBP on cap-dependent and IRES-driven translation; luciferase activity is the readout of translational activity. <b>(C)</b> The effect of MEHP on cap-dependent and IRES-driven translation. The structure of MEHP was inserted in the chart. The experiments were repeated three times, and the means were shown with standard deviations.</p

The effects of BBP on cell proliferation.

<p>Both HEK-293T and HT-29 cells were cultured to 80% confluency and treated with BBP at a series of concentrations as indicated. After the treatment for 24 hrs, the cell viability was measured as described in the “Materials and methods”. Each concentration point was repeated three times, and the means were shown with standard deviations.</p

Working model of regulation of mRNA translation by phthalates.

<p>The dashed arrows indicate the pathway not studied in the paper.</p

BBP inhibits mRNA translation <i>in vitro</i>.

<p>Rabbit reticulocyte lysate system was used to measure the translation activity <i>in vitro</i> using luciferase mRNA as the reporter. BBP was added to the final concentrations as indicated. Equal volume of methanol only was used as the control. Treatment with each concentration was repeated three times and the average values were shown. The structure of BBP was inserted in the chart.</p

BBP modulates the expression and activity of eIFs in HEK-293T cells.

<p>HEK-293T cells were cultured to 80% confluency and treated with a series of concentrations of BBP for 24 hrs. The cells were harvested and washed once with cold PBS for the preparation of total cell lysate as described in the “materials and methods”. <b>(A)</b> Equal amounts of cell lysate (50μg) were analyzed by Western blot with antibodies against eIFs as indicated. <b>(B)</b> A diagram to show the purification of eIF4F complex by m⁷GTP-resin affinity chromatography. <b>(C)</b> Analysis of eIF4G and eIF4E in eIF4F complex by Western blot.</p