Smad4 pathways modulate induction of the chemokine Ccl20 and repress inflammation-induced carcinogenesis in mouse colon. Mus musculus

To understand the extent of Smad-mediated gene regulation in the colon, we isolated colon epithelium from Smad4ΔLrig1 and from Smad4+ control mice (either mice lacking a CreERT allele and treated with tamoxifen, or mice bearing a CreERT allele but treated with vehicle only) and analyzed the colonic epithelium by RNAseq. The ability of TGFβ1 and/or BMP2 to block TNF-mediated induction of Ccl20 from our study suggests that these Smad-mediated pathways may act as gatekeepers for induction of other inflammation-associated genes. To determine if Smad-mediated signaling blocks all or specific subsets of TNF-induced genes, we analyzed both colonocytes and mouse colonoid treated with or without TNF, TGFβ1, and BMP2 by RNA seq. Overall design: In total, three RNAseq experiments were performed and three biological replicates were used for each condition: 1. Colon epithelium from Smad4ΔLrig1 and from Smad4+ control mice was isolated. Total RNA was isolated from these tissues using RNeasy kit (Qiagen). Processing of RNA using a TruSeq Stranded mRNA sample prep kit was conducted according to the manufacturer’s instructions (Illumina, San Diego, CA). 32~37 million 51 base pair single-end reads were generated per sample. 2. Total RNA was isolated from colonocytes treated with or without TNF, TGFβ1, and BMP2 using RNeasy kit (Qiagen). Processing of RNA using a TruSeq Stranded mRNA sample prep kit was conducted according to the manufacturer’s instructions (Illumina, San Diego, CA). 26~50 million 75 base pair paired-end reads were generated per sample. 3. Total RNA was isolated from mouse colonoid treated with or without TNF, TGFβ1, and BMP2 using RNeasy kit (Qiagen). Processing of RNA using a TruSeq Stranded mRNA sample prep kit was conducted according to the manufacturer’s instructions (Illumina, San Diego, CA). 50~72 million 75 base pair paired-end reads were generated per sample.