Project description:This experiment sought to characterize the epithelial-specific transcriptional response in the bleomycin model of lung fibrosis, and the contribution of IRE1a and the TGFb-activating integrin avb6 to this response. "Ribotag" mice were crossed to ShhCre mice, both on the C57BL/6 background to generate experimental cohorts. Mice used for the experiment conditionally expressed HA-tagged ribosomal protein Rpl22 in the epithelium. Mice were exposed to intranasal bleomycin (3 U/kg) on day 1 and subsequently treated with the IRE1a kinase inhibitor KIRA8 (50 mg/kg daily), or the anti-integrin beta6 antibody 3G9 (10 mg/kg on day 1 and day 4). Treatment controls were either drug vehicle (3% ethanol, 7% Tween80, and 90% saline) or the inert antibody Axum8 in saline). Lungs were harvested on day 7 after bleomycin exposure, flash-frozen, and later homogenized under native conditions with cycloheximide. An aliquot of the “input” homogenate was removed and total RNA purified by Qiagen RNeasy Mini and is representative of the whole-lung transcriptome/translatome. The remaining homogenate was subjected to immunoprecipitation of HA-tagged polysomes, followed by RNA purification by Qiagen RNeasy Micro, and is representative of the lung epithelial transcriptome/translatome.
Project description:The uncovering of genes involved in susceptibility to the sporadic cancer types is a great challenge. It is well established that the way in which an individual deals with DNA damage is related to the chance to develop cancer. Mutagen sensitivity is a phenotype that reflects an individual’s susceptibility to the major sporadic cancer types, including colon, lung and head and neck cancer. A standard test for mutagen sensitivity is measuring the number of chromatid breaks in lymphocytes after exposure to bleomycin. The aim of the present study was to search for the pathways involved in mutagen sensitivity. Lymphoblastoid cell lines of seven individuals with a low mutagen sensitivity were compared with seven with a high score. RNA was isolated from cells exposed to bleomycin (4 hr) and unexposed cells. Micro-array analysis (19K) was used to compare gene expression of insensitive and sensitive cells. The profile of most altered genes after bleomycin exposure, analyzed in all fourteen cell lines, included genes involved in multiple processes, most prominent in cell proliferation and DNA repair. When comparing the insensitive and sensitive individuals other differentially expressed genes were found, that were involved in proliferation (e.g. BUB1) and signal transduction (e.g. DUSP4). This difference in expression profiles between mutagen sensitive and insensitive individuals justifies further studies aiming to elucidate the genes responsible for the development of sporadic cancers. Keywords: exposure to dna damaging agent, comparison of phenotypic response to dna damaging agent
Project description:Bleomycin-induced acute lung injury is characterized by mesenchymal cell activation, which leads to pulmonary fibrosis. They also have the potential to increase epithelial cells to regenerate alveolar epithelial cell integrity. We used microarrays to detail the change of global gene expression in lung mesenchymal cells in this process.
Project description:We used mass spectrometry to profile changes in metabolites, proteins, and phosphorylation in silica-exposed BEAS-2B epithelial cells.
Project description:To understand the cellular composition and transcriptional phenotype of different cell populations in the mouse bleomycin induced fibrosis model, we performed RNAseq analysis on leukocyte, endothelial, epithelial and stroma (other) cell populations isolated from mouse lung samples.
Project description:Pulmonary fibrosis is a devatating lung disease with limited effective treatment. Because fibrotic responses are considered to be regulated by complex cellular and molecular circuits, it is difficult to demonstrate a causal relationship of cells, molecules, and cell-cell interactions with diseases in vivo. To overcome this issue, we established bleomycin (BLM)-stimulated alveolosphere model as an ex vivo culture model. To clarify whether our ex vivo model recapitulates the cellular and molecular response to BLM-induced lung injury, we performed RNA-seq (Bulk RNA-seq) analysis of the BLM-stimulated alveolospheres generated by co-culturing murine lung epithelial cells and lung fibroblasts.
Project description:We report histone modifications in alveolar epithelial type 2 cells in homeostatic mice as well as CTGF-positive alveolar eptihelial cells in mice treated with Bleomycin.