Positional Cloning Reveals Strain-Dependent Expression of <em>Trim16</em> to Alter Susceptibility to Bleomycin-Induced Pulmonary Fibrosis in Mice

<div><p>Pulmonary fibrosis is a disease of significant morbidity, with no effective therapeutics and an as yet incompletely defined genetic basis. The chemotherapeutic agent bleomycin induces pulmonary fibrosis in susceptible C57BL/6J mice but not in mice of the C3H/HeJ strain, and this differential strain response has been used in prior studies to map bleomycin-induced pulmonary fibrosis susceptibility loci named <em>Blmpf1</em> and <em>Blmpf2</em>. In this study we isolated the quantitative trait gene underlying <em>Blmpf2</em> initially by histologically phenotyping the bleomycin-induced lung disease of sublines of congenic mice to reduce the linkage region to 13 genes. Of these genes, <em>Trim16</em> was identified to have strain-dependent expression in the lung, which we determined was due to sequence variation in the promoter. Over-expression of <em>Trim16</em> by plasmid injection increased pulmonary fibrosis, and bronchoalveolar lavage levels of both interleukin 12/23-p40 and neutrophils, in bleomycin treated B6.C3H-<em>Blmpf2</em> subcongenic mice compared to subcongenic mice treated with bleomycin only, which follows the C57BL/6J versus C3H/HeJ strain difference in these traits. In summary we demonstrate that genetic variation in <em>Trim16</em> leads to its strain-dependent expression, which alters susceptibility to bleomycin-induced pulmonary fibrosis in mice.</p> </div

B6 and C3H <i>Trim16</i> promoter sequence variation alters transcription.

<p>B6/C3H sequence differences in the putative promoter region of <i>Trim16</i> (* indicates novel to Sanger, MGI). Allele specific promoter sequence alters the expression of a luciferase reporter vector transfected into the RAW 264.7 macrophage cell line.</p

Pulmonary expression of reduced region <i>Blmpf2</i> genes.

<p>Real-time quantitative PCR of genes mapping to the reduced <i>Blmpf2</i> region prior to (day 0: non-treated) and following bleomycin treatment (day 42) in the lungs of B6 and C3H mice. Gene expression was normalized to the Ataxin10 reference gene and is presented relative to the level in untreated C3H mice. Mean ±SEM of 5 per group. * indicates a significant difference in expression in lungs of bleomycin-treated mice relative to untreated controls, p<0.05; ^# indicates a significant difference in expression by strain, p<0.05.</p

Bleomycin-induced lung phenotype of <i>Blmpf2</i> subcongenic mice.

<p>The mice were treated with bleomycin by mini-osmotic pump and euthanized 42 days later. The percentage of the lung with fibrosis was determined from image analysis of histological sections and the mean ± SEM of 10–19 bleomycin-treated mice for each subcongenic line, and for the parental strains, is given. * indicates a significant difference in fibrosis from B6 mice, p<0.05. Genotypes [C3H alleles (white box); B6 alleles (black box)] were determined with microsatellite and SNP markers; genotypes of line 6 are expanded at the bottom of the figure.</p