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Title: Early alterations in extracellular matrix and transforming growth factor [beta] gene expression in mouse lung indicative of late radiation fibrosis

Abstract

Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The expression of late radiation injury can be found immediately after irradiation by measuring messenger RNA (mRNA) abundance. To determine if extracellular matrix mRNA and transforming growth factor beta abundance was affected acutely after irradiation, the authors measured mRNA levels of collagen I (CI), collagen III (CIII), collagen IV (CIV), fibronectin (FN), and transforming growth factor [beta] (TGF[beta][sub 1,2 3]) in mouse lungs on day 1 and day 14 after graded doses of radiation. C57BL/6 female mice were irradiated with a single dose to the thorax of 5 or 12.5 Gy. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabelled cDNA probes for CI, CIII, CIV, FN, TGF[beta][sub 1,2 3] and a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiographic data were quantified by video densitometry and results normalized to GAPDH. Changes in the expression of CI, CIII, CIV, FN and TGF[beta][sub 1,2 3] were observed as early as 1 day after exposure. Through 14 days, changes in mRNA up to 5-fold were seen for any one dose. Dose related changes as high as 10-fold were also evident. Themore » CI:CIII ratio increased gradually for the 5 Gy dose at 14 days postirradiation while the CI:CII ratio for the 12.5 Gy dose decreased by approximately 4-fold as compared to the control. These studies suggest that alterations in expression of extracellular matrix and TGF[beta] mRNA occur very early after radiation injury even at low doses and may play a role in the development of chronic fibrosis. 37 refs., 6 figs.« less

Authors:
; ; ;  [1]
  1. (Univ. of Rochester School of Medicine, NY (United States))
Publication Date:
OSTI Identifier:
6581835
Resource Type:
Journal Article
Resource Relation:
Journal Name: International Journal of Radiation Oncology, Biology and Physics; (United States); Journal Volume: 28:3
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; FIBROSIS; ETIOLOGY; LUNGS; IRRADIATION; MESSENGER-RNA; BIOSYNTHESIS; BIOLOGICAL RADIATION EFFECTS; MICE; ANIMALS; BIOLOGICAL EFFECTS; BODY; MAMMALS; NUCLEIC ACIDS; ORGANIC COMPOUNDS; ORGANS; PATHOLOGICAL CHANGES; RADIATION EFFECTS; RESPIRATORY SYSTEM; RNA; RODENTS; SYNTHESIS; VERTEBRATES 560152* -- Radiation Effects on Animals-- Animals

Citation Formats

Finkelstein, J.N., Johnston, C.J., Baggs, R., and Rubin, P. Early alterations in extracellular matrix and transforming growth factor [beta] gene expression in mouse lung indicative of late radiation fibrosis. United States: N. p., 1994. Web. doi:10.1016/0360-3016(94)90187-2.
Finkelstein, J.N., Johnston, C.J., Baggs, R., & Rubin, P. Early alterations in extracellular matrix and transforming growth factor [beta] gene expression in mouse lung indicative of late radiation fibrosis. United States. doi:10.1016/0360-3016(94)90187-2.
Finkelstein, J.N., Johnston, C.J., Baggs, R., and Rubin, P. 1994. "Early alterations in extracellular matrix and transforming growth factor [beta] gene expression in mouse lung indicative of late radiation fibrosis". United States. doi:10.1016/0360-3016(94)90187-2.
@article{osti_6581835,
title = {Early alterations in extracellular matrix and transforming growth factor [beta] gene expression in mouse lung indicative of late radiation fibrosis},
author = {Finkelstein, J.N. and Johnston, C.J. and Baggs, R. and Rubin, P.},
abstractNote = {Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The expression of late radiation injury can be found immediately after irradiation by measuring messenger RNA (mRNA) abundance. To determine if extracellular matrix mRNA and transforming growth factor beta abundance was affected acutely after irradiation, the authors measured mRNA levels of collagen I (CI), collagen III (CIII), collagen IV (CIV), fibronectin (FN), and transforming growth factor [beta] (TGF[beta][sub 1,2 3]) in mouse lungs on day 1 and day 14 after graded doses of radiation. C57BL/6 female mice were irradiated with a single dose to the thorax of 5 or 12.5 Gy. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabelled cDNA probes for CI, CIII, CIV, FN, TGF[beta][sub 1,2 3] and a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiographic data were quantified by video densitometry and results normalized to GAPDH. Changes in the expression of CI, CIII, CIV, FN and TGF[beta][sub 1,2 3] were observed as early as 1 day after exposure. Through 14 days, changes in mRNA up to 5-fold were seen for any one dose. Dose related changes as high as 10-fold were also evident. The CI:CIII ratio increased gradually for the 5 Gy dose at 14 days postirradiation while the CI:CII ratio for the 12.5 Gy dose decreased by approximately 4-fold as compared to the control. These studies suggest that alterations in expression of extracellular matrix and TGF[beta] mRNA occur very early after radiation injury even at low doses and may play a role in the development of chronic fibrosis. 37 refs., 6 figs.},
doi = {10.1016/0360-3016(94)90187-2},
journal = {International Journal of Radiation Oncology, Biology and Physics; (United States)},
number = ,
volume = 28:3,
place = {United States},
year = 1994,
month = 2
}
  • Idiopathic pulmonary fibrosis is an inexorably fatal disorder characterized by connective tissue deposition within the terminal air spaces resulting in loss of lung function and eventual respiratory failure. Previously, the authors demonstrated that foci of activated fibroblasts expressing high levels of fibronectin, procollagen, and smooth muscle actin and thus resembling those found in healing wounds are responsible for the connective tissue deposition and scarring in idiopathic pulmonary fibrosis. Using in situ hybridization and immunohistochemistry, they now demonstrate the presence of transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}), a potent profibrotic cytokine, in the foci containing these activated fibroblasts. These resultsmore » suggest that matrix-associated TGF-{beta}{sub 1} may serve as a stimulus for the persistent expression of connective tissue genes. One potential source of the TGF-{beta}{sub 1} is the alveolar macrophage, and they demonstrate the expression of abundant TGF-{beta}{sub 1} mRNA in alveolar macrophages in lung tissue from patients with idiopathic pulmonary fibrosis.« less
  • Purpose: We determined whether anti-transforming growth factor-{beta} (TGF-{beta}) intervention could halt the progression of established radiation-induced liver fibrosis (RILF). Methods and Materials: A replication-defective adenoviral vector expressing the extracellular portion of human T{beta}RII and the Fc portion of immunoglobulin G fusion protein (AdT{beta}RIIFc) was produced. The entire rat liver was exposed to 30 Gy irradiation to generate a RILF model (RILFM). Then, RILFM animals were treated with AdT{beta}RIIFc (1 x 10{sup 11} plaque-forming units [PFU] of T{beta}RII), control virus (1 x 10{sup 11} PFU of AdGFP), or saline. Delayed radiation liver injury was assessed by histology and immunohistochemistry. Chronic oxidativemore » stress damage, hepatic stellate cell activation, and hepatocyte regeneration were also analyzed. Results: In rats infected with AdT{beta}RIIFc, fibrosis was significantly improved compared with rats treated with AdGFP or saline, as assessed by histology, hydroxyproline content, and serum level of hyaluronic acid. Compared with AdGFP rats, AdT{beta}RIIFc-treated rats exhibited decreased oxidative stress damage and hepatic stellate cell activation and preserved liver function. Conclusions: Our results demonstrate that TGF-{beta} plays a critical role in the progression of liver fibrosis and suggest that anti-TGF-{beta} intervention is feasible and ameliorates established liver fibrosis. In addition, chronic oxidative stress may be involved in the progression of RILF.« less
  • Transforming growth factor {beta} (TGF{beta}) is a multifunctional polypeptide4 that regulates proliferation, differentiation, and other functions of many cell types. The pathway of TGF{beta} signal transduction in cells is unknown. The authors report here that an early effect of TGF{beta} is an enhancement of the expression of two genes encoding serum- and phorbol ester tumor promoter-regulated transcription factors: the junB gene and the c-jun proto-oncogene, respectively. This stimulation was observed in human lung adenocarcinoma A549 cells which were growth inhibited by TGF{beta}, AKR-2B mouse embryo fibroblasts which were growth stimulated by TGF{beta}, and K562 human erythroleukemia cells, which were notmore » appreciably affected in their growth by TFG{beta}. The increase in jun mRNA occurred with picomolar TGF{beta} concentrations within 1 h of TGF{beta} stimulation, reached a peak between 1 and 5 h in different cells, and declined gradually to base-fine levels. This mRNA response was followed by a large increase in the biosynthesis of the c-jun protein (AP-1), as shown by metabolic labeling and immunoprecipitation analysis. However, differential and cell type-specific regulation appeared to determine the timing and magnitude of the response of each jun gene in a given cell. In AKR-2B and NIH 3T3 cells, only junB was induced by TGF{beta}, evidently in a protein synthesis-independent fashion. The junB response to TGF{beta} was maintained in c-Ha-ras and neu oncogene-transformed cells. Thus, one of the earliest genomic responses to TGF{beta} may involve nuclear signal transduction and amplification by the junB and c-jun transcription factors in concert with c-fos, which is also induced. The differential activation of the jun genes may explain some of the pleiotropic effects of TGF{beta}.« less
  • Transin is a transformation-associated gene which is expressed constitutively in rat fibroblasts transformed by a variety of oncogenes and in malignant mouse skin carcinomas but not benign papillomas or normal skin. It has been demonstrated that, in nontransformed Rat-1 cells, transin RNA expression is modulated positively by epidermal growth factor (EGF) and negatively by transforming growth factor beta (TGF-BETA); other peptide growth factors were found to have no effect on transin expression. Results presented here indicate that both protein synthesis and continuous occupancy of the EGF receptor by EGF were required for sustained induction of transin RNA. Treatment with TGF-BETAmore » inhibited the ability of EGF to induce transin, whether assayed at the transcriptional level by nuclear run-on analysis or at the level of transin RNA accumulation by Northern (RNA) blot analysis of cellular RNA. TGF-BETA both blocked initial production of transin transcription by EGF and halted established production of transin transcripts during prolonged treatment. These results suggest that TGF-BETA acts at the transcriptional level to antagonize EGF-mediated induction of transin gene expression.« less
  • The present study was undertaken to elucidate the mechanism for the antifibrotic effect of interferon gamma (IFN-{gamma}) in the bleomycin (BL)-mouse model of lung fibrosis. The expression of transforming growth factor (TGF-{beta}) and procollagen I and III and their mRNAs was investigated in the BL-mouse model of lung fibrosis with and without IFN-{gamma} treatment by Northern and slot blot analyses. Temporal changes in the content of procollagen and TGF-{beta} mRNAs in the lungs of mice receiving saline or BL by intratracheal route, with and without IFN-{gamma} treatment by intramuscular route, were quantitated. The level of TGF-{beta} mRNA increased rapidly andmore » peaked at day 5, whereas the levels of mRNAs for procollagens {alpha}{sub 1}(I) and {alpha}{sub 1}(III) peaked at 10 days after BL instillation. The peak levels of these mRNAs in BL-treated animals were five- to seven-fold higher than those of the control. BL treatment also increased the hydroxyproline content significantly from 3 to 14 days as compared to the corresponding saline control groups. A maximal increase to 447 {mu}g/lung from 223 {mu}g/lung in saline control was obtained at 10 days after instillation. Daily treatment with IFN-{gamma} markedly reduced the BL-induced increases in the mRNA levels of TGF-{beta}, and procollagen {alpha}{sub 1}(I) and {alpha}{sub 1}(III) without any effect on the lung level of {beta}-actin mRNA. IFN-{gamma} treatment also caused significant reduction in the BL-induced increase in the lung hydroxyproline content from 417 to 283 {mu}g/lung at 7 days and from 447 to 264 {mu}g/lung at 10 days. It may be concluded from the findings of the present study that the cellular mechanisms for the antifibrotic effect of IFN-{gamma} in the BL-mouse model of lung fibrosis are to initially downregulate the BL-induced overexpression of TFG-{beta} mRNA, and subsequently procollagen mRNAs, leading to a decreased collagen content. 42 refs., 7 figs.« less