K-ras activation generates an inflammatory response in lung tumors.

Activating mutations in K-ras are one of the most common genetic alterations in human lung cancer. To dissect the role of K-ras activation in bronchial epithelial cells during lung tumorigenesis, we created a model of lung adenocarcinoma by generating a conditional mutant mouse with both Clara cell secretory protein (CC10)-Cre recombinase and the Lox-Stop-Lox K-ras(G12D) alleles. The activation of K-ras mutant allele in CC10 positive cells resulted in a progressive phenotype characterized by cellular atypia, adenoma and ultimately adenocarcinoma. Surprisingly, K-ras activation in the bronchiolar epithelium is associated with a robust inflammatory response characterized by an abundant infiltration of alveolar macrophages and neutrophils. These mice displayed early mortality in the setting of this pulmonary inflammatory response with a median survival of 8 weeks. Bronchoalveolar lavage fluid from these mutant mice contained the MIP-2, KC, MCP-1 and LIX chemokines that increased significantly with age. Cell lines derived from these tumors directly produced MIP-2, LIX and KC. This model demonstrates that K-ras activation in the lung induces the elaboration of inflammatory chemokines and provides an excellent means to further study the complex interactions between inflammatory cells, chemokines and tumor progression.

Fibroblast tropoelastin and alpha-smooth-muscle actin expression are repressed by particulate-activated macrophage-derived tumor necrosis factor-alpha in experimental silicosis.

Lung elastin synthesis is normally confined to periods of development, is maximal during alveolarization, and declines to low levels in mature lung. We have previously described an elastogenic response in the adult rat lung associated with experimental granulomatous disease induced by silica instillation. Reinitiated tropoelastin expression was identified throughout the lung in fibroblasts expressing alpha-smooth-muscle actin, whereas fibroblasts within the granulomatous lesions failed to express both tropoelastin and alpha-smooth- muscle actin (Mariani and colleagues, Am. J. Pathol. 1995;147:988-1000). We hypothesized that inflammatory cells within the granulomatous lesions produce factors that alter fibroblast phenotype. We found that macrophages accumulating within granulomatous lesions of silicotic rat lungs produce and secrete tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine previously appreciated as a repressor of tropoelastin gene expression. In experimental cell systems, macrophages activated by particulates, either in vivo or in vitro, conditioned medium with a tropoelastin-repressing activity. This activity repressed both tropoelastin and alpha-smooth-muscle actin expression in primary cultures of rat lung fibroblasts in a time- dependent, transient manner. The particulate-activated macrophage-conditioned medium was found to contain TNF-alpha, which was both necessary and sufficient to induce these changes in lung fibroblast gene expression. These data indicate that macrophage-derived factors can modulate lung fibroblast tropoelastin expression in the diseased lung. Furthermore, the findings extend the association between expression by lung fibroblasts of tropoelastin and alpha-smooth-muscle actin.

Collagenase-3 induction in rat lung fibroblasts requires the combined effects of tumor necrosis factor-alpha and 12-lipoxygenase metabolites: a model of macrophage-induced, fibroblast-driven extracellular matrix remodeling during inflammatory lung injury.

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-alpha (TNF-alpha) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-alpha and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Regulation of lung fibroblast tropoelastin expression by alveolar epithelial cells.

Epithelial-mesenchymal interactions are of critical importance during tissue morphogenesis and repair. Although the cellular and molecular aspects of many of these interactions are beginning to be understood, the ability of epithelial cells to regulate fibroblast interstitial matrix production has not been extensively studied. We report here that cultured alveolar epithelial cells are capable of modulating the expression of tropoelastin, the soluble precursor of the interstitial lung matrix component elastin, by lung fibroblasts. Phorbol ester-stimulated alveolar epithelial cells secrete a soluble factor that causes a time- and dose-dependent repression of lung fibroblast tropoelastin mRNA expression. This alveolar epithelial cell-mediated repressive activity is specific for tropoelastin, is effective on lung fibroblasts from multiple stages of development, and acts at the level of transcription. Partial characterization of the repressive activity indicates it is an acid-stable, pepsin-labile protein. Gel fractionation of alveolar epithelial cell conditioned medium revealed two peaks of activity with relative molecular masses of approximately 25 and 50 kDa. These data support a role for epithelial cells in the regulation of fibroblast interstitial matrix production.

Elastin in lung development.

Elastin is a critical component of the lung interstitium, providing the property of recoil to the vascular, conducting airway, and terminal airspace compartments of the lung. Elastic fibers, consisting of soluble tropoelastin monomers cross-linked on a preexisting scaffold of microfibrils, are produced primarily during late fetal and neonatal stages of development. The factors and molecular mechanisms regulating the cell type-specific and tightly temporally regulated expression of tropoelastin are currently under investigation. The onset and inductive phase of tropoelastin expression are characterized by increased transcription of the tropoelastin gene. Glucocorticoids accelerate this induction in fetal rats during the canalicular stage of lung development. Many additional factors regulate tropoelastin expression in cultured lung fibroblasts and vascular smooth muscle cells, but the in vivo roles of such mediators are still under investigation. Cell-cell interactions may also promote elastogenesis during lung development, as localization of tropoelastin mRNA in pseudo-glandular and canalicular lungs demonstrates a close spatial relationship between epithelium and adjacent elastogenic mesenchyme. Elastin metabolism is altered in several experimental models of bronchopulmonary dysplasia, characterized by abnormal lung morphological development, suggesting that normal elastin production and deposition is necessary for proper development of alveoli. Studies employing reverse genetics may prove useful in further defining the role of elastin in lung development.

Localization of type I procollagen gene expression in silica-induced granulomatous lung disease and implication of transforming growth factor-beta as a mediator of fibrosis.

We have used the silica-induced model of pulmonary injury in the rat to study the pattern of collagen expression in granulomatous lung inflammation. A single intratracheal instillation of silica into adult rats resulted in granulomatous inflammation leading to fibrosis and alveolar proteinosis. The development of disease in these animals was characterized over a 27-day period after treatment by means of histological, biochemical, and molecular analyses. Biochemical analyses indicated that significant increases in the weights of silicotic lungs were due to elevated amounts of DNA and total protein. Analysis of hydroxyproline content showed a 15-fold increase in this amino acid in silicotic lungs, confirming the development of a fibrotic reaction. In situ hybridization for type I procollagen mRNA displayed increased gene expression in the parenchyma, conducting airways, and vasculature of silicotic rats. Within the parenchyma, type I procollagen was expressed uniquely within granulomatous lesions. Immunohistochemistry indicated type I procollagen was being expressed by an alpha-smooth muscle actin-negative population of cells. Immunolocalization of extra-cellular transforming growth factor-beta showed coincident temporal and spatial overlap with type I procollagen expression, implicating this cytokine as a mediator of collagen gene expression in this model.

Increased elastin production in experimental granulomatous lung disease.

In the normal, healthy lung, elastin production is restricted to periods of development and growth. However, elastin expression in the adult lung has been observed in some forms of pulmonary injury, including pulmonary fibrosis. Here, we report that elastin production is significantly increased within precise interstitial compartments of the lung in an experimental model of granulomatous lung disease. An increase in the number and volume of elastic fibers within the alveolar walls was apparent on histological examination of Verhoeff-van Gieson-stained sections of silicotic rat lungs. Quantitation of mature elastin cross-links indicated that silicosis was accompanied by a 17-fold increase in lung elastin content when compared with values from saline-treated controls. In situ hybridization for tropoelastin mRNA revealed that elastin production was absent from granulomatous lesions yet was prominent at nonfibrotic alveolar septal tips, where a high density of elastic fibers is seen in the normal lung. Immunohistochemistry indicated tropoelastin was being expressed by alpha-smooth muscle actin-containing cells. Transforming growth factor-beta was immunolocalized to granulomatous regions of the silicotic lung but was absent from regions showing increased tropoelastin expression. These data indicate that the reinitiation of tropoelastin gene expression is associated with granulomatous lung disease, and this expression leads to the aberrant accumulation of mature elastin in the lung.

Ultraviolet radiation increases tropoelastin accumulation by a post-transcriptional mechanism in dermal fibroblasts.

Chronically sun-damaged human skin is characterized by dermal connective tissue damage that includes the massive accumulation of abnormal elastic fibers. The content of elastin, the major protein component of elastic fibers, is increased two- to sixfold in sun-damaged skin. The aim of this study was to determine the mechanism responsible for the increase in elastin levels after ultraviolet (UV) irradiation. Confluent cultures of normal dermal fibroblasts were irradiated with 4.5 mJ/cm2 of UVB; sham-treated cells served as the control group. The accumulation of tropoelastin was determined at 5 d after treatment by measuring the incorporation of 14C-proline into radiolabeled tropoelastin isolated from cell layers and media. UV irradiation increased radiolabeled tropoelastin accumulation approximately twofold without affecting DNA content, the total amount of radiolabeled protein, or tropoelastin secretion. Moreover, the steady-state levels of tropoelastin mRNA, as determined by slot blot hybridizations, were unaffected by UV treatment. However, the translation of tropoelastin mRNA was increased when total RNA from irradiated cells was used in cell-free translation experiments. These results suggest that altered translational efficiency may account for the increase in tropoelastin accumulation after UV irradiation. In support of this hypothesis, nucleotide sequences were derived from tropoelastin mRNA isolated from UV-irradiated and nonirradiated dermal fibroblasts. Almost a 12% substitution rate was observed in nucleotide sequences derived from the 3' untranslated region of tropoelastin mRNA from the UV-treated cells. In contrast, a coding domain of tropoelastin did not contain base-substitution mutations. These multiple base substitutions in a noncoding domain of tropoelastin mRNA may be responsible for the post-transcriptional increase in tropoelastin accumulation after UV irradiation.

The size heterogeneity of human lysyl oxidase mRNA is due to alternate polyadenylation site and not alternate exon usage.

We have isolated the entire gene coding for human lysyl oxidase. Coding and untranslated domains of human lysyl oxidase mRNA were found in 7 exons, distributed throughout approximately 14 kb of human genomic DNA. The appearance of exon sequences in lysyl oxidase mRNA in several human tissues was determined using a reverse transcriptase - PCR assay. In contrast to a previous report, this analysis has unambiguously shown that the size heterogeneity of lysyl oxidase mRNA was not due to alternate usage of any of the exons of the lysyl oxidase gene. Moreover, DNA sequence analysis of the entire 3.8 kb 3'-untranslated region (UTR) within exon 7 revealed multiple poly-adenylation sites which were shown to be differentially expressed in human skin fibroblasts. This differential usage of polyadenylation sites within the 3'-UTR explains the appearance of multiple lysyl oxidase mRNAs of different sizes.

Elastin in lung development and disease.

Elastic fibres are present in lung structures including alveoli, alveolar ducts, airways, vasculature and pleura. The rate of lung elastin synthesis is greatest during fetal and neonatal development, and is minimal in the healthy adult. We have determined that glucocorticoids up-regulate fetal lung tropoelastin expression while concomitantly accelerating terminal airspace maturation. Because there is minimal turnover of elastin in healthy adult lung, the elastin incorporated into the lung early in development supports lung function for the normal lifespan. However, in the adult lung, in pathological circumstances such as emphysema or pulmonary fibrosis there may be reactivation of elastin expression. We have found in silica-induced pulmonary fibrosis that expression of tropoelastin is primarily increased in the walls and the septal tips of the alveolus, with modest increases in other compartments which normally express tropoelastin during development. This finding suggests that the mesenchymal cell of the alveolar wall increases tropoelastin expression during fibrotic disorders. In emphysema and fibrosis, elastin is present in abnormal-appearing, probably non-functional, elastic fibres, suggesting that the adult lung cannot recapitulate the elastic fibre assembly mechanisms operative during normal lung growth.

A restriction fragment length polymorphism results in a nonconservative amino acid substitution encoded within the first exon of the human lysyl oxidase gene.

A cDNA covering most of the coding sequence for human lysyl oxidase was used to screen, by Southern blot analysis, genomic DNA from circulating lymphocytes obtained from unrelated, apparently normal individuals. A heritable restriction fragment length polymorphism (RFLP) within a PstI restriction site was detected in 36% of individuals screened (a total of 72 chromosomes were analyzed). The major allele was represented as a 1.7-kb PstI restriction fragment. The minor allele was detected as 1.4 and 0.3kb restriction fragments. Lambda phage-DNA recombinants were isolated from a human lung fibroblast genomic DNA library using the human lysyl oxidase cDNA clone. DNA sequence analysis of several selected phage recombinants revealed that 83% of the coding sequence of lysyl oxidase was localized in four separate exons. Analysis of the coding sequence within exon 1, the most 5' exon within the lysyl oxidase gene, revealed that the PstI RFLP was due to a G-->A transition resulting in a nonconservative arginine to glutamine substitution proximal to a propeptide cleavage domain encoded by exon 1 of the lysyl oxidase gene.

The complete derived amino acid sequence of human lysyl oxidase and assignment of the gene to chromosome 5 (extensive sequence homology with the murine ras recision gene).

Lysyl oxidase catalyzes the oxidation of lysine residues to alpha-aminoadipic-delta-semialdehyde. This is the first step in the covalent cross-linking of collagen and tropoelastin and results in the formation of insoluble collagen and elastic fibers in the extracellular matrix. We have characterized the complete nucleotide sequence of human lysyl oxidase (EC 1.4.3.13) and compared the derived amino acid sequence (417-amino acids) to rat lysyl oxidase and the mouse ras recision gene (rrg). 88% of amino acids and 83% of nucleotides were conserved between human and rat lysyl oxidase. The mouse ras recision gene demonstrated 89% conservation of amino acids with human lysyl oxidase. The sequence conservation was not evenly distributed along the molecule. The carboxy terminus of the protein, which contains the putative copper binding sites and is likely to be the catalytically active domain, was more highly conserved than the amino terminus. The 89% amino acid sequence similarity between the murine ras recision gene and human lysyl oxidase suggests that they are the same gene product. Therefore, in addition to cross linking of extracellular matrix proteins, lysyl oxidase may have a direct role in tumor suppression. Northern blot analysis of poly A+RNA from cultured skin fibroblasts revealed at least three-distinct transcripts, sized 4.8 kb, 3.8 kb and 2.0 kb. In addition, using a panel of human mouse cell hybrids, the lysyl oxidase gene was assigned to human chromosome 5.

Abnormalities in the biosynthesis of type III procollagen in cultured skin fibroblasts from two patients with multiple aneurysms.

We examined the synthesis of collagenous proteins by cultured skin fibroblasts taken from 14 patients with an abdominal aortic aneurysm and either an aneurysm at a second site (8 patients) or a first order relative with an abdominal aortic aneurysm (6 patients). Fibroblasts were labeled with [3H] proline and, following pepsin digestion of media proteins, the ratio of type I/III collagen was examined by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). With the exception of two patients, the ratio of type I/III collagen in the media of fibroblasts from aneurysm patients was similar to control values (6 controls). In two of the patients, the type I/III collagen ratio was greater than 3 standard deviations from the mean of both control ratios and those of other aneurysm patients. mRNA levels coding for type III procollagen, however, were normal in both patients. Patient #1 (ME) showed reduced type III procollagen on SDS-PAGE analysis of intracellular proteins. Intracellular and media type III procollagen levels were normal in patient #2 (HR), but media type III collagen was reduced by over 50% after digestion with a combination of trypsin and alpha-chymotrypsin for 5 minutes at 36 degrees C. Control type III collagen was only reduced after digestion at 39 degrees C. These data suggest an altered thermal stability of the type III collagen trimer synthesized by this patient, probably due to a mutation in the amino acid sequence. The data presented in this paper suggest that some forms of common abdominal aortic aneurysms may be caused by mutations in the gene coding for type III procollagen.