10 research outputs found
Altered bleomycin-induced lung fibrosis in osteopontin-deficient mice
tin is a multifunctional matricellular protein abundantly expressed during inflammation and repair. Osteopontin deficiency is associated with abnormal wound repair characterized by aberrant collagen fibril-logenesis in the heart and skin. Recent gene microarray studies found that osteopontin is abundantly expressed in both human and mouse lung fibrosis. Macrophages and T cells are known to be major sources of osteopontin. During lung fibrosis, however, osteopontin expression continues to increase when inflammation has receded, suggesting alternative sources of ostepontin during this response. In this study, we demonstrate immunoreactivity for osteopontin in lung epithelial and inflammatory cells in human usual interstitial pneumonitis and murine bleomycin-induced lung fibrosis. After treatment with bleo-mycin, osteopontin-null mice develop lung fibrosis characterized by dilated distal air spaces and reduced type I collagen expression compared with wild-type controls. There is also a significant decreas
Altered bleomycin-induced lung fibrosis in osteopontin-deficient mice
Osteopontin is a multifunctional matricellular protein abundantly expressed during inflammation and repair. Osteopontin deficiency is associated with abnormal wound repair characterized by aberrant collagen fibrillogenesis in the heart and skin. Recent gene microarray studies found that osteopontin is abundantly expressed in both human and mouse lung fibrosis. Macrophages and T cells are known to be major sources of osteopontin. During lung fibrosis, however, osteopontin expression continues to increase when inflammation has receded, suggesting alternative sources of ostepontin during this response. In this study, we demonstrate immunoreactivity for osteopontin in lung epithelial and inflammatory cells in human usual interstitial pneumonitis and murine bleomycin-induced lung fibrosis. After treatment with bleomycin, osteopontin-null mice develop lung fibrosis characterized by dilated distal air spaces and reduced type I collagen expression compared with wild-type controls. There is also a significant decrease in levels of active transforming growth factor-β1 and matrix metalloproteinase-2 in osteopontin null mice. Type III collagen expression and total collagenase activity are similar in both groups. These results demonstrate that osteopontin expression is associated with important fibrogenic signals in the lung and that the epithelium may be an important source of osteopontin during lung fibrosis
The 2lox Lsd1 variant shows decreased binding to known interactors.
<p>(A) Immunoblots of FLAG-based coimmunoprecipitations of NIH 3T3 cells transfected with the Lsd1 variants demonstrate that the 2lox protein exhibits greatly reduced complex formation with known binding partners CoREST and HDAC1. For the two single point mutants, the M448V mutation has a more profound effect on CoREST binding. *: immunoglobulin heavy chain. (B) The relative coimmunoprecipitation of the Lsd1 binding partner CoREST was quantitated using ImageJ software. Data represent the mean +/− SD for two independent experiments. (C) Purification of endogenous Lsd1 complexes from MEF lines confirmed a decrease in Lsd1-CoREST interaction in cells expressing the mutant Lsd1. All westerns are representative of results from experiments performed in triplicate. (D) The decrease in the endogenous interaction of Lsd1 and CoREST by the 2lox variant was quantitated, using ImageJ software. Data represent the mean +/− SD for three independent experiments. **: p<0.001; *: p<0.005, compared to binding by wild-type Lsd1.</p
Protein expression in the developmentally-defective hearts.
<p>The expression of proteins in 3 hypomorphic and 2 wild-type control E18.5 hearts was examined by immunoblotting. A decrease in Lsd1 and a major increase in phosphorylation of E-cadherin were noted in the <i>Aof2</i><sup>2lox/2lox</sup> hearts, whereas all other proteins examined showed no obvious changes. In the E-cadherin blot, the arrow indicates the correct E-cadherin band, and the identity of the higher band is unknown. The Ncam antibody recognizes the 140 kDa isoform of this protein. Relevant molecular weight markers are indicated, in kDa, to the right of each panel.</p
Immunohistochemistry of the hypomorphic hearts.
<p>(A) Staining with an antibody specific for the phosphorylation of E-cadherin is strongly increased in the <i>Aof2</i><sup>2lox/2lox</sup> hearts compared to the control. The arrows indicate the regions (grey for heart wall, black for septum) from which the higher magnification images (on right) originate. (B) β-catenin localization is altered, with more of the protein present at the plasma membrane in <i>Aof2<sup>2lox/2lox</sup></i> hearts. (C) Lsd1 staining shows slightly decreased signals in <i>Aof2<sup>2lox/2lox</sup></i> hearts. (D) Staining with a non-specific IgG control antibody confirms the specificity of the staining. To minimize background, no counterstain was used. All photomicrographs constitute representative fields; magnification factor is provided above or beside the photographs.</p
Lsd1 expression in the developing heart.
<p>Wild-type embryos at the indicated development stages (E8.5 to E13.5, and E18.5) were dissected and processed for immunohistochemistry. H&E staining was performed, along with staining with anti-Lsd1 to visual the Lsd1 expression pattern during heart development. Note that in all panels, the black bar represents 100 µm.</p
Figure 5
<p><b>Changes in gene expression in the hypomorphic hearts.</b> Expression of gene products, examined by qRT-PCR using RNA isolated from E18.5 embryonic hearts. Data represents mean +/− SD from 3 to 5 animals of each genotype, and was normalized such that the expression of the mRNA in wild-type animals was equal to 1 (*: p<0.05). (A) Lsd1 mRNA levels were decreased by approximately 50%, confirming the microarray data. Nkx2-5, a heart marker, Ncam, and β-catenin were not found to be significantly altered in the hypomorphic hearts. The expression of Tescalcin and Fblim1, two proteins with potential roles in heart development that were identified by microarray, was also analyzed. The increase in Tescalcin expression was recapitulated by the qRT-PCR, while no difference in Fblim1 expression between the wild-type and hypomorphic hearts was noted. CamK2β showed minor increases in expression. (B) Expression of Wnt targets in the wild-type and hypomorphic hearts. The Wnt signaling pathway target genes Wnt11, Lrp6, Kit, and Isl1 were examined for changes in mRNA levels in the hypomorphic hearts by qRT-PCR. No statistically significant changes were noted in the expression of any of these genes. (C) mRNA expression of epithelial and mesenchymal markers is identical between wild-type and 2lox/2lox hearts. Expression of the epithelial markers Pecam and VE-Cadherin, and the mesenchymal markers B4galt6 and Fn1 was examined by qRT-PCR using RNA isolated from E18.5 embryonic hearts. No statistically significant difference was noted in the expression of any of these mRNAs.</p
Mice homozygous for the floxed (“2lox”) allele show heart defects.
<p>(A) Genotyping of late stage embryos. Genomic DNA was isolated from 6 representative E18.5 embryos, and the <i>Aof2</i> allele analyzed by PCR genotyping. Both the wild-type (253 bp) and 2lox (392 bp) alleles can be observed, with two animals homozygous for the 2lox allele. The size, in base pairs, of DNA size standard markers is indicated on the left of the image. (B) Embryos homozygous for the 2lox allele survive to birth, but die shortly thereafter, as shown by the lack of homozygous pups at day P21. The data represents the percent of each genotype present at the indicated time point; for E13.5, this is the total of 3 litters, while E18.5 and P21 represent data from 6 pooled litters each. (C) Representative photomicrograph (H&E staining) illustrating a ventricular septal defect in an E18.5 <i>Aof2</i><sup>2lox/1lox</sup> pup at low magnification (upper right hand panel; 1.0x) and higher magnification (lower right hand panel; 3.0x) compared to a wild type control animal (upper and lower left hand panels). (D) Sequence comparison of the wild-type and 2lox <i>Aof2</i> alleles. Sequencing results in the regions with the two point mutations are presented for <i>Aof2<sup>wt</sup></i> and <i>Aof2<sup>2lox</sup></i> alleles. The corresponding base pair sequence is written above the respective graphs. The A to G mutations at positions 1379 and 1483 (positions based on mRNA sequence) are highlighted in red, and indicated by the arrows.</p
Statistical analysis of microarray data (significant probes only).
<p>Legend: ID, Affymetrix probe ID; logFC, log value of the Fold change in expression in the 2lox/2lox hearts; AveExpr, average expression in wild-type hearts;</p><p>P.value, unadjusted P-value; adj.P.Val, adjusted P-value.</p