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Col6a1-/- Mouse Lung

ABSTRACT: Collagen 6 (COL6) is known for its role in a spectrum of congenital muscular dystrophies, which are often accompanied by respiratory dysfunction. However, little is known regarding the function of COL6 in the lung. We confirmed the presence of COL6 throughout the basement membrane region of mouse lung tissue. We studied lung structure and organization in a previously described Col6a1-/- mouse, which do not produce detectable COL6 in the lung. The Col6a1-/- mouse displayed multiple histopathological alveolar and airway abnormalities. The airspaces of Col6a1-/- lungs appeared simplified, with larger (29%, p<0.01) and fewer (31%, p<0.001) alveoli. These airspace abnormalities included a reduction in IsolectinB4+ alveolar capillaries and Sftpc+ ATII cells. Alterations in lung function consistent with these histopathological changes were evident. Col6a1-/- mice also displayed multiple airway changes including increased branching (59%, p<0.001), increased mucosal thickness (34%, p<0.001) and increased epithelial cell density (13%, p<0.001). Comprehensive transcriptome analysis revealed loss of COL6 is associated with reductions in integrin-paxillin-PI3K signaling in vivo. In vitro, COL6 promoted steady-state phospho-paxillin levels and reduced cell density (16-28%, p<0.05) at confluence. Inhibition of PI3K, or its downstream effectors, resulted in increased cell density to a level similar to that seen on matrices lacking COL6.

ORGANISM(S): Mus musculus

PROVIDER: GSE135667 | GEO | 2019/08/10

REPOSITORIES: GEO

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Project description:Non-small cell lung cancer (NSCLC) is a major cause of cancer-associated mortality worldwide, and bone metastasis is the most prevalent event observed in patients with advanced NSCLC. However, the pathogenesis of bone metastases has not been fully elucidated. In the present study, differentially expressed genes (DEGs) were identified by gene expression microarray analysis of NSCLC tissue samples with or without bone metastases. Subsequently, collagen family collagen 6A1 (COL6A1) was chosen as the target gene through Ingenuity Pathway Analysis and reverse transcription-quantitative (RT-q)PCR validation of the top eight DEGs. COL6A1 was overexpressed or knocked down, and the proliferation and invasion of NSCLC cells was assessed using Cell Counting Kit-8, colony formation and Transwell invasion assays. Additionally, the osteogenic capacity of HOB and hES-MP 002.5 cells was assessed using RT-qPCR, western blotting, Alizarin Red staining and alkaline phosphatase staining. A total of 364 DEGs were identified in NSCLC tissues with bone metastases compared with NSCLC tissues without bone metastases, including 140 upregulated genes and 224 downregulated genes. Gene Ontology analysis indicated that the upregulated and downregulated genes were primarily enriched in ‘cellular process’, ‘metabolic process’ and ‘biological regulation’. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that the upregulated genes were primarily enriched in ‘cysteine and methionine metabolism’, ‘oxidative phosphorylation’ and the ‘ribosome’, while the downregulated genes were primarily enriched in ‘transcriptional mis-regulation in cancer’, ‘ribosome’ and ‘mitophagy in animals’. COL6A1 was highly expressed in NSCLC tissue samples with bone metastases. Functionally, COL6A1 overexpression induced the proliferation and invasion of HARA cells, and knockdown prevented the proliferation and invasion of HARA-B4 cells. Finally, it was demonstrated that HOB and hES-MP 002.5 cells exhibited osteogenic capacity, and overexpression of COL6A1 in HARA cells increased adhesion of these cells to the osteoblasts, whereas knockdown of COL6A1 in HARA-B4 cells reduced their adhesive ability. In conclusion, COL6A1 may serve as a potential diagnostic marker and therapeutic target for bone metastasis in NSCLC.

Project description:Background: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) with asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthma is unclear. Objective: To explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthma. Methods: Primary human bronchial epithelial cell (HBEC) air-liquid interface (ALI) cultures were stimulated with IL-6 and sIL-6R to establish an IL-6TS gene signature. Two separate RNA sequencing (RNA-seq) studies were performed: The “IL-6 vs T2 study” compared gene expression after stimulation with control medium, IL-6, IL-6/sIL-6R and IL-4/IL-13, while the “JAK1-inhibition study” addressed the effect of JAK1 inhibition on IL-6TS induced gene expression. The IL-6TS gene signature was used to stratify lung epithelial transcriptomic data obtained from asthmatics (n=103) in the U-BIOPRED cohorts by hierarchical clustering. Molecular phenotyping was based on the transcriptional profiling of epithelial brushings, pathway analysis and immunohistochemistry analysis of bronchial biopsies. Results: Activation of IL-6TS in HBEC ALI cultures reduced epithelial barrier function and induced a specific epithelial gene signature enriched in airway remodeling genes. The IL-6TS signature identified a subset (n=17) of IL-6TS High asthma patients with increased epithelial expression of IL-6TS inducible genes in absence of increased systemic levels of IL-6 and sIL-6R. The IL-6TS High subset had an increased exacerbation frequency (p=0.028), blood (>300/μl; p=0.0028) and sputum (>20%; p=0.007) eosinophilia, and submucosal infiltration of CD4 T cells, CD8 T cells (p<0.001) and macrophages (p=0.001). In bronchial brushings, TLR pathway genes were up-regulated while the expression of epithelial tight junction genes was reduced (all with q<0.05). Sputum sIL-6R levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, MMP3, IL-8 and IL-1β (all with q<0.001). Conclusions: Local lung epithelial IL-6TS activation in absence of type 2 airway inflammation defines a novel subset of asthmatics and may drive airway inflammation and epithelial dysfunction in these patients.

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Col6a1-/- Mouse Lung

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