Transcriptional regulation of CCSP by interferon-gamma in vitro and in vivo.

Interferon gamma (IFN-gamma), a potent cytokine inducing a wide range of immunologic activities, is increased in the airway secondary to viral infection or during an inflammatory response. This increase in IFN-gamma concentration may alter the expression of specific airway epithelial cell genes that regulate adaptation of airway inflammatory responses. One protein induced by IFN-gamma is Clara cell secretory protein (CCSP), which may contribute to the attenuation of airway inflammation. This study was done to investigate the molecular mechanism by which IFN-gamma stimulates the expression of the CCSP gene in mouse transformed Clara cells and transgenic mice. Deletion mapping and linker-scanning mutations demonstrated that IFN-gamma-induced expression of CCSP was regulated, in part, at the level of transcription. In vitro and in vivo studies verified that the minimal IFN-gamma-responsive segment was localized to the proximal 166 bp of the 5'-flanking region. Additionally, IFN-gamma-induced expression of CCSP was mediated indirectly through an interferon regulatory factor-1-mediated increase in hepatocyte nuclear factor-3beta.

Brain development: integrins and the Reelin pathway.

Integrins link the extracellular matrix to the intracellular environment. They have been implicated in the Reelin pathway in cortical development. But new genetic studies have revealed that, while beta(1) integrin plays a role in formation of the basement membrane, it is not essential for neuronal migration.

Identification of reelin-induced sites of tyrosyl phosphorylation on disabled 1.

The study of mice with spontaneous and targeted mutations has uncovered a signaling pathway that controls neuronal positioning during mammalian brain development. Mice with disruptions in reelin, dab1, or both vldlr and apoER2 are ataxic, and they exhibit severe lamination defects within several brain structures. Reelin is a secreted extracellular protein that binds to the very low density lipoprotein receptor and the apolipoprotein E receptor 2 on the surface of neurons. Disabled-1 (Dab1), an intracellular adapter protein containing a PTB (phosphotyrosine binding) domain, is tyrosyl-phosphorylated during embryogenesis, but it accumulates in a hypophosphorylated form in mice lacking Reelin or both very low density lipoprotein receptor and apolipoprotein E receptor 2. Dab1 is rapidly phosphorylated when neurons isolated from embryonic brains are stimulated with Reelin, and several tyrosines have been implicated in this response. Mice with phenylalanine substitutions of all five tyrosines (Tyr(185), Tyr(198), Tyr(200), Tyr(220), and Tyr(232)) exhibit a reeler phenotype, implying that tyrosine phosphorylation is critical for Dab1 function. Here we report that, although Src can phosphorylate all five tyrosines in vitro, Tyr(198) and Tyr(220) represent the major sites of Reelin-induced Dab1 phosphorylation in embryonic neurons.

Physiological regulation of uteroglobin/CCSP expression.

Uteroglobin/CCSP is expressed specifically in the Clara cells. This allows the gene to be used as a marker to identify the elements regulating the physiologic and cell-specific expression of this gene. The regulation of UG/CCSP by IFN-gamma was shown to be at the level of the proximal promoter by the upregulation of HNF3 beta. This has allowed the determination of the factors responsible for the expression of UG/CCSP.

Transgenic mouse models for lung cancer.

Transgenic technology allows the ability to target regulatory genes to the lungs in a cell-specific fashion. Using this technology, we have generated a model to investigate the phenotypic consequences of targeting oncogenes to particular cell types in the lungs and are developing a second model for the regulated expression of oncogenes in the lung. The transgenic model involves the constitutive expression of simian virus 40 large T antigen in the Clara cells of mouse lungs. This model has been used to investigate changes in expression of cell cycle regulatory genes in the Clara cells during the transformation process, as well as the expression of the transcription factors regulating the expression of Clara cell differentiation markers. The second model we are developing investigates the regulated expression of the genes in the lungs. This system is based on the establishment of two types of transgenic lines. The regulator line consists of a chimeric transcriptional factor placed under the control of a lung-specific SPC (surfactant protein C) promoter. This chimeric regulator is composed of a transcription activation domain, the GAL4 DNA-binding domain, and a truncated progesterone receptor that is responsive to RU 486, but not to endogenous progesterone. The second transgenic mouse line contains the silent target transgene under the control of a minimal promoter with upstream activating sequences (UAS) that are recognized by the regulator transgene. Upon breeding these two lines, the resulting bitransgenic mice can then be induced to express the target transgene only with the administration of RU 486. Two generations of regulators have been evaluated on their ability to regulate the expression of a growth hormone reporter gene. This system demonstrated the inducible expression of the reporter genes in the distal airways of the lungs.

Investigating stem cells in the lung.

Disruption of the lung architecture by genetic events, environmental insults, or transformation can lead to respiratory diseases such as acute respiratory distress syndrome and lung cancer. Identification of the stem cells of the lung and the processes by which they regulate homeostasis may lead to better targets for treatment of these diseases. There are a number of approaches to study stem cell biology. Development of the lung and the major pulmonary cells of the bronchioles and alveolar regions of the lung is discussed in this review. Likewise, identifying the proteins that are critical for cell-specific expression and differentiation may identify approaches for manipulation of gene expression for use as therapy or treatment of lung diseases. Furthermore, strategies for studying stem cells in the lung are addressed by using the mouse as a model system. Gaining a more detailed understanding of the stem cells of the lung may provide new insight into the processes that govern lung biology and may lead to better treatments for lung diseases. Enthusiasm for the use of exogenous stem cells to replace tissue, organs, or other defective or deficient cells is boundless. Before stem cells can be used indiscriminately for these purposes, understanding tissue genetics and immunology is essential. Progress has been made in these areas for pulmonary disease. Attention to these models will be applicable to other organs and diseases.

Interferon-gamma regulation of Clara cell gene expression: in vivo and in vitro.

This report demonstrates that Clara cell 10-kDa protein (CC10) mRNA levels are regulated by interferon-gamma (IFN-gamma). An analysis of total lung RNA from mice given IFN-gamma intratracheally showed increased levels of CC10 mRNA compared to control animals but no significant increases in surfactant proteins B and C. These results were confirmed in a Clara cell line, mtCC1-2, generated from the lungs of a transgenic mouse expressing the SV40 large T antigen under the control of a Clara cell-specific promoter. Significant increases in mtCC1-2 CC10 mRNA levels were observed in a time- and a dose-dependent manner. The expression of transacting factors hepatocyte nuclear factors 3 alpha and 3 beta (HNF-3 alpha and HNF-3 beta) were also analyzed, and a transient increase in the expression of HNF-3 beta but not HNF-3 alpha was detected. Deoxyribonuclease I footprint analysis identified a signal transducer and activator of transcription (STAT) binding site (at nucleotides -293 to -284 of CC10) adjacent to two thyroid transcription factor-1 (TTF-1) binding sites, suggesting a potential interaction between STAT1 and TTF-1. This report reinforces the hypothesis that CC10 functions as an anti-inflammatory protein and that increases in CC10 protein may provide additional protection from inflammation and disease in the lung.

Cyclin-dependent kinase inhibitor expression in pulmonary Clara cells transformed with SV40 large T antigen in transgenic mice.

Expression of cell cycle regulatory genes in mouse lung was investigated in transgenic models for Clara cell transformation. Clara cells were transformed by generating transgenic mice in which the SV40 large T antigen was expressed under the control of the mouse Clara cell M(r) 10,000 protein promoter. The resulting lung tumors express the large T antigen in normal Clara cells and in tumors, and these tumors express reduced levels of CC10 mRNA. The expression of cell cycle regulatory protein, p53, and the cyclin-dependent kinase inhibitors was analyzed by Northern blot analysis and in situ hybridization throughout the progression of Clara cell transformation in the lung. Increases in specific cyclin-dependent kinase inhibitor steady-state mRNA levels were detected in p15, p18, p27, and p57 during tumor progression. The expression of p15, p57, and p21 mRNAs were verified by in situ hybridization. Using this approach, regulatory genes have been identified that may be involved in the regulation of Clara cell differentiation.

cis-acting elements involved in the regulation of mouse Clara cell-specific 10-kDa protein gene. In vitro and in vivo analysis.

Transient transfection and murine germ line gene transfer analysis was used to determine the regions of DNA necessary to confer the appropriate level and cell specificity of the expression of the gene coding for the murine Clara cell 10-kDa protein, mCC10. To identify the cis-acting elements involved in the regulation of mCC10 gene, different lengths of the 5'-flanking sequence were ligated to the bacterial chloramphenicol acetyltransferase gene for transient transfection to H441 cells (human lung adenocarcinoma cell line). The corresponding sequences were also fused to the human growth hormone gene and transferred to the murine genome for an in vivo analysis of mCC10 promoter activity. The results of the transient transfection analysis identified the region from -166 to -124 of the 5'-flanking region of the mCC10 gene as necessary for the expression of this gene in H441 cells. The transgenic mouse analysis confirmed that the 166 base pairs of 5'-flanking DNA was sufficient to confer cell-specific expression. However, the transgenic mouse analysis also showed that, to achieve the full quantitative level of transgene (human growth hormone) expression, regions between -803 and -166 base pairs of the 5'-flanking sequences are required for maximum expression of mCC10 gene promoter activity.

Cloning and characterization of the mouse Clara cell specific 10 kDa protein gene: comparison of the 5'-flanking region with the human rat and rabbit gene.

The mouse Clara Cell 10 kiloDalton (kDa) protein (mCC10) cDNA was used to isolate a recombinant phage containing the mCC10 gene sequence in a 14 kilobase (kb) insert from a mouse genomic library. A total of 7.7 kb of this clone was sequenced. The sequenced region included: 3.3 kb of 5'-flanking region, 4.2 kb intragenic sequence and 0.2 kb of DNA flanking the 3' end of the gene. Computer assisted sequence analysis identified potential cis acting response elements for the glucocorticoid receptor, hepatocyte nuclear factor (HNF3) and octamer (Oct1) binding protein. The presence of B1 murine repetitive sequence also has been identified in a similar position reported in rat CC10 5'-flanking sequence. As with the rat CC10, the mCC10 5'-flanking region also contains deletions of a 2.1 kb and a 0.3 kb sequence present in the rabbit uteroglobin gene, these regions are reported to contain a cluster of glucocorticoid/progesterone receptor binding sites and estrogen receptor binding sites, respectively.