Analysis of genomic regions involved in regulation of the rabbit surfactant protein A gene in transgenic mice

1999 ◽  
Vol 277 (2) ◽  
pp. L349-L361 ◽  
Author(s):  
Joseph L. Alcorn ◽  
Robert E. Hammer ◽  
Katherine R. Graves ◽  
Margaret E. Smith ◽  
Shanna D. Maika ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Fusion Gene ◽  
Surfactant Protein ◽  
Type Ii Cells ◽  
Type Ii ◽  
Regulation Of Expression ◽  
Camp Induction ◽  
Genomic Regions

The gene encoding surfactant protein (SP) A, a developmentally regulated pulmonary surfactant-associated protein, is expressed in a lung-specific manner, primarily in pulmonary type II cells. SP-A gene transcription in the rabbit fetal lung is increased by cAMP. To delineate the genomic regions involved in regulation of SP-A gene expression, lines of transgenic mice carrying fusion genes composed of various amounts of 5′-flanking DNA from the rabbit SP-A gene linked to the human growth hormone structural gene as a reporter were established. We found that as little as 378 bp of 5′-flanking DNA was sufficient to direct appropriate lung cell-selective and developmental regulation of transgene expression. The same region was also sufficient to mediate cAMP induction of transgene expression. Mutagenesis or deletion of either of two DNA elements, proximal binding element and a cAMP response element-like sequence, previously found to be crucial for cAMP induction of SP-A promoter activity in transfected type II cells, did not affect lung-selective or temporal regulation of expression of the transgene; however, overall levels of fusion gene expression were reduced compared with those of wild-type transgenes.

A human SP-C promoter fragment targets α1-proteinase inhibitor gene expression to lung alveolar type II cells in transgenic mice

1996 ◽  
Vol 5 (2) ◽  
pp. 139-143 ◽  
Author(s):  
Eric Degryse ◽  
Maria M. De Santi ◽  
Mireille Dietrich ◽  
Dalila Ali Hadji ◽  
Jean François Spetz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Proteinase Inhibitor ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Promoter Fragment ◽  
Inhibitor Gene

Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice

1989 ◽  
Vol 9 (12) ◽  
pp. 5473-5479
Author(s):  
C M Shanahan ◽  
N W Rigby ◽  
J D Murray ◽  
J T Marshall ◽  
C A Townrow ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Transgenic Mice ◽  
Mrna Expression ◽  
Transgene Expression ◽  
Fusion Gene ◽  
Developmental Expression ◽  
Regulation Of Expression ◽  
Tissue Specific ◽  
Transgenic Livestock ◽  
Main Regulator

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.

Glucocorticoid inhibition of SP-A gene expression in lung type II cells is mediated via the TTF-1-binding element

2004 ◽  
Vol 286 (4) ◽  
pp. L767-L776 ◽  
Author(s):  
Joseph L. Alcorn ◽  
Kazi N. Islam ◽  
Pampee P. Young ◽  
Carole R. Mendelson
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Fusion Gene ◽  
Primary Cultures ◽  
Protein A ◽  
Fetal Lung ◽  
Binding Activity ◽  
Type Ii Cells ◽  
Type Ii ◽  
Flanking Sequence

Induction of surfactant protein-A ( SP-A) gene expression in fetal lung type II cells by cAMP and IL-1 is mediated by increased binding of thyroid transcription factor-1 (TTF-1) and NF-κB proteins p50 and p65 to the TTF-1-binding element (TBE) at -183 bp. In type II cell transfections, dexamethasone (Dex) markedly inhibits cAMP-induced expression of rabbit SP-A:human growth hormone ( hGH) fusion genes containing as little as ∼300 bp of the SP-A 5′-flanking sequence. Dex inhibition is blocked by RU-486, suggesting a role of the glucocorticoid receptor (GR). The present study was undertaken to define the mechanisms for GR inhibition of SP-A expression. Cotransfection of primary cultures of type II cells with a GR expression vector abrogated cAMP induction of SP-A promoter activity while, at the same time, causing a 60-fold induction of cotransfected mouse mammary tumor virus ( MMTV) promoter. In lung cells transfected with a fusion gene containing three TBEs fused to the basal SP-A promoter, Dex prevented the stimulatory effect of IL-1 on TTF-1 induction of SP-A promoter activity, suggesting that the GR inhibits SP-A promoter activity through the TBE. In gel shift assays using nuclear extracts from human fetal type II cells cultured in the absence or presence of cAMP, Dex markedly reduced binding of nuclear proteins to the TBE and blocked the stimulatory effect of cAMP on TBE-binding activity. Our finding that Dex increased expression of the NF-κB inhibitory partner IκB-α suggests that the decrease in TBE-binding activity may be caused, in part, by GR inhibition of NF-κB interaction with this site.

Effects of RSV infection on pulmonary surfactant protein SP-A in cultured human type II cells: contrasting consequences on SP-A mRNA and protein

2005 ◽  
Vol 289 (6) ◽  
pp. L1113-L1122 ◽  
Author(s):  
Joseph L. Alcorn ◽  
James M. Stark ◽  
Constance L. Chiappetta ◽  
Gaye Jenkins ◽  
Giuseppe N. Colasurdo
Keyword(s):  
Gene Expression ◽  
Protein Gene ◽  
Surfactant Protein ◽  
Type Ii Cells ◽  
Protein Homeostasis ◽  
Mrna Levels ◽  
Type Ii ◽  
Human Type ◽  
Rsv Infection ◽  
Type Ii Cell

Respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory illness in infants and children. Surfactant proteins A (SP-A) and D (SP-D) play critical roles in lung defense against RSV infections. Alterations in surfactant protein homeostasis in the lung may result from changes in production, metabolism, or uptake of the protein within the lung. We hypothesized that RSV infection of the type II cell, the primary source of surfactant protein, may alter surfactant protein gene expression. Human type II cells grown in primary culture possess lamellar bodies (a type II cell-specific organelle) and the ability to express surfactant protein mRNA. These cells were infected with RSV (by morphology and antibody binding). Surfactant protein mRNA levels determined by quantitative RT-PCR indicated a marked increase in SP-A mRNA levels (3-fold) 24 h after RSV exposure, whereas SP-D mRNA levels were unaffected. In contrast to mRNA levels, total SP-A protein levels (determined by Western blot analysis) were decreased 40% after RSV infection. The percentage of secreted SP-A was 43% of the total SP-A in the RSV-infected cells, whereas the percentage of secreted SP-A was 61% of the total SP-A in the uninfected cells. These changes in SP-A transcript levels and protein secretion in cultured human cells were recapitulated in RSV-infected mouse lung. Our findings suggest that type II cells are potentially important targets of RSV lower respiratory infection and that alterations in surfactant protein gene expression and SP-A protein homeostasis in the lung may arise via direct effects of RSV.

A CRE-like element plays an essential role in cAMP regulation of human SP-A2 gene in alveolar type II cells

1996 ◽  
Vol 271 (2) ◽  
pp. L287-L299 ◽  
Author(s):  
P. P. Young ◽  
C. R. Mendelson
Keyword(s):  
Gene Expression ◽  
Lung Tissue ◽  
Fusion Gene ◽  
Fetal Lung ◽  
Inducible Expression ◽  
Nuclear Proteins ◽  
Type Ii Cells ◽  
Type Ii ◽  
Nuclear Extracts ◽  
Human Fetal Lung

The human has two genes encoding surfactant protein-A (SP-A), termed SP-A1 and SP-A2; the SP-A2 gene is more highly regulated by cAMP and during fetal development than is SP-A1. In this study, by use of primary cultures of human type II cells transfected with fusion genes containing various amounts of SP-A2 5'-flanking DNA linked to human growth hormone (hGH) structural gene, as reporter, we found that -296 bp of SP-A2 upstream sequence is sufficient to direct high basal and cAMP-inducible expression in type II cells, but not in other cell types. By use of competitive EMSA, we observed that nuclear proteins isolated from midtrimester human fetal lung tissue bind specifically to a cAMP response element (CRE)-like sequence, TGACCTTA, at -242 bp, which we have termed CRESP-A2. Binding activity of CRESP-A2 for nuclear proteins from human fetal lung tissue before culture was manifest as two complexes of different mobilities and equivalent intensity. By contrast, upon differentiation of the human fetal lung in culture in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), the higher mobility complex was decreased to undetectable levels. By UV cross-linking analysis, using nuclear extracts from midgestation human fetal lung before culture and radiolabeled CRESP-A2 as a probe, we observed binding of proteins of approximately 50, 36, and 30 kDa. When nuclear extracts from human fetal lung cultured in the presence of DBcAMP were analyzed, binding of only the 50- and 36-kDa proteins was apparent. On the other hand, when the canonical CRE (TGACGTCA) known to bind the transcription factor CREB (M(r) approximately 43,000) was used as a probe, binding of only a approximately 43-kDa protein was evident using nuclear extracts from human fetal lung before and after culture. In type II cells transfected with an SP-A2(-296):hGH fusion gene in which CRESP-A2 was mutated, there was a marked reduction of basal and cAMP-stimulated fusion gene expression. These findings indicate that CRESP-A2 serves an important role in mediating basal and cAMP-inducible expression of the human SP-A2 gene in type II cells, that the fetal lung nuclear proteins bound to CRESP-A2 differ from those bound to the canonical palindromic CRE, and that changes in the complex of nuclear proteins bound to CRESP-A2 accompany induction of SP-A gene expression.

scholarly journals Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.

1989 ◽  
Vol 9 (12) ◽  
pp. 5473-5479 ◽  
Author(s):  
C M Shanahan ◽  
N W Rigby ◽  
J D Murray ◽  
J T Marshall ◽  
C A Townrow ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Transgenic Mice ◽  
Mrna Expression ◽  
Transgene Expression ◽  
Fusion Gene ◽  
Developmental Expression ◽  
Regulation Of Expression ◽  
Tissue Specific ◽  
Transgenic Livestock ◽  
Main Regulator

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.

Influence of the cytoskeleton on surfactant protein gene expression in cultured rat alveolar type II cells

1998 ◽  
Vol 274 (1) ◽  
pp. L87-L96 ◽  
Author(s):  
John M. Shannon ◽  
Tianli Pan ◽  
Karen E. Edeen ◽  
Larry D. Nielsen
Keyword(s):  
Gene Expression ◽  
Protein Gene ◽  
Primary Cultures ◽  
Surfactant Protein ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Partial Recovery ◽  
Type Ii ◽  
Alveolar Type Ii Cells

We have investigated the role of the cytoskeleton in surfactant protein gene expression. Cytochalasin D (CD), colchicine (Col), or nocodazole (Noco) were tested on primary cultures of adult rat alveolar type II cells. Treatment with any of the drugs did not result in dramatic cell shape changes, but ultrastructural examination revealed that the cytoplasm of cells treated with CD was markedly disorganized; cells treated with Col did not exhibit such changes. Treatment with any of the three drugs resulted in a reduction in surfactant protein (SP) mRNAs. These decreases were not the result of cell toxicity, since overall protein synthesis was unimpaired by drug treatment. Washing the cells followed by an additional 2 days of culture resulted in a reaccumulation of SP mRNAs in CD-treated cells but not in Col-treated cells. Washing of Noco-treated cultures resulted in partial recovery. SP mRNA stability was estimated in the presence or absence of cytoskeleton-disrupting drugs. Disruption of either microfilaments or microtubules significantly affected the half-lives of mRNAs for SP-A, SP-B, and SP-C. These data support a role for the cytoskeleton in the maintenance of type II cell differentiation and suggest that the role of the cytoskeleton is at least in part to stabilize SP mRNAs.

Changes in surfactant protein gene expression in a neonatal rabbit model of hyperoxia-induced fibrosis

1997 ◽  
Vol 272 (4) ◽  
pp. L720-L730 ◽  
Author(s):  
C. T. D'Angio ◽  
J. N. Finkelstein ◽  
M. B. Lomonaco ◽  
A. Paxhia ◽  
S. A. Wright ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Pulmonary Inflammation ◽  
Rabbit Model ◽  
Surfactant Protein ◽  
Type Ii Cells ◽  
Type Ii ◽  
Surfactant System ◽  
Lung Injuries

Lung injuries, including bronchopulmonary dysplasia, alter the surfactant system. We developed a newborn rabbit model of acute, followed by chronic, hyperoxic injury to study surfactant protein (SP) gene expression. Initial litters were exposed to >95% O2 until 50% died (LD50; 7-11 days old). Subsequent litters were exposed to >95% O2 for 8 days, followed by 60% O2 until 22-36 days. Controls were exposed to room air. LD50 animals displayed acute pulmonary inflammation, edema, protein leak, and surfactant dysfunction. These changes resolved, and fibrosis developed by 22 days. Whole lung SP-A mRNA expression (measured by membrane hybridization) was twice control levels at 4 days of >95% O2, with specific elevations in terminal bronchioles and type II cells at 4 days and the LD50 by in situ hybridization. Whole lung SP-B and SP-C mRNA were unchanged from control throughout exposure. However, in situ hybridization showed elevations in SP-B and SP-C mRNA in type II cells in inflamed areas at the LD50. SP mRNA alterations resolved by 22-36 days. The surfactant system recovers from acute hyperoxic injury, despite continued 60% O2 exposure.

scholarly journals Heparin and Fibroblast Growth Factors Affect Surfactant Protein Gene Expression in Type II Cells

2006 ◽  
Vol 35 (5) ◽  
pp. 611-618 ◽  
Author(s):  
Kevin A. Leiner ◽  
Donna Newman ◽  
Cheng-Ming Li ◽  
Eric Walsh ◽  
Jody Khosla ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Protein Gene ◽  
Fibroblast Growth Factors ◽  
Surfactant Protein ◽  
Type Ii Cells ◽  
Type Ii ◽  
Fibroblast Growth

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

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