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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 18, 2003 as doi:10.1096/fj.03-0048fje. |
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,2
Departments of
* Pediatrics and
Pathology, Baylor College of Medicine, Houston, Texas, USA;
Department of Pediatrics, Ajou University, Suwon, Korea;
Children's Research Institute, Columbus, Ohio, USA; and
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
2Correspondence: Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: fdemayo{at}bcm.tmc.edu
SPECIFIC AIMS
The goal of the present study was to investigate the mechanisms of hyperoxia-induced repression of the mouse Clara cell secretory protein (CCSP) promoter. The first specific aim was to determine whether hyperoxia-induced repression of the CCSP gene is mediated at least in part at the level of transcription. The second specific aim was to identify the DNA–protein interactions that govern the transcriptional regulation of the CCSP gene during hyperoxia.
PRINCIPAL FINDINGS
1. Hyperoxia represses CCSP expression in part at the level of transcription and the localization of the hyperoxia-responsive region of the CCSP gene to the proximal -166 bp of the 5'-flanking region in vitro and in vivo
Mouse transformed Clara Cells, mtCC, were tranfsected with various lengths of the CCSP 5' flanking region fused to the luciferase reporter gene and exposed to normoxic or hyperoxic conditions. This analysis localized repression of CCSP transcription by hyperoxia to -166 bp of the CCSP proximal promoter in vitro. This transcriptional repression CCSP gene expression by hyperoxia was confirmed in vivo by exposing transgenic mice with a human growth hormone reporter gene under control of the -166 bp promoter and observing repression of transgene expression to the same level as that of the endogenous CCSP gene.
2. Hyperoxia caused altered binding patterns of three trans-acting factors to cis-elements in the proximal -166 bp region of the CCSP gene in response to hyperoxia exposure
The effect of hyperoxia on the DNA–protein interactions in the -166 bp proximal promoter region was investigated by EMSA and Supershift analysis using nuclear extract isolated from mtCC exposed to normoxic and hyperoxic conditions. This analysis showed that hyperoxia caused an increase in c-Jun and C/EBPß binding and a decrease in Nkx2.1 binding to the CCSP promoter region. Western analyses revealed that hyperoxia exposure induced an increase in the expression of the C/EBP-ß isoform LIP (liver-inhibiting protein), a transcriptional repressor.
3. c-Jun and the C/EBP-ß LIP isoform inhibit CCSP gene expressions in vitro.
Cotransfection of CCSP expression plasmids with C/EBP-ß LIP isoforms or c-Jun expression plasmids decreased the transcriptional activity of the proximal -166 bp CCSP promoter. These observations confirm that these factors may play a role in the repression of CCSP expression after hyperoxic exposure.
4. Hyperoxia causes cytoplasmic sequestration of the known CCSP activator Nkx2.1
The reduced DNA binding of Nkx2.1 shown by EMSA prompted further investigations to determine whether hyperoxia-induced a decrease in total cellular Nkx2.1 content or a decrease in nuclear Nkx2.1. Immunohistochemical analysis showed that under normoxic conditions, Nkx2.1 protein was localized within the nucleus of airway cells (Fig. 1
A). However, staining of lungs of mice exposed to hyperoxic conditions revealed a more diffuse cytoplasmic distribution of Nkx2.1 immunostaining in the bronchiolar epithelium, (Fig. 1B
). Western blot analysis of mtCC cultured in normoxic or hyperoxic conditions (Fig. 1C
) confirmed that hyperoxia did not alter the total cellular content of Nkx2.1 but altered the nuclear content of this protein. These results suggest that cytoplasmic sequestration of the Nkx2.1 protein occurs in response to hyperoxia exposure both in vivo and in vitro.
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CONCLUSIONS AND SIGNIFICANCE
The present study was undertaken to investigate the molecular mechanisms of hyperoxia-induced repression of the mouse Clara cell secretory protein (CCSP) gene. Previous investigations suggest that CCSP, a 16 kDa protein secreted in the distal airways by the Clara cells, may serve an important role in the lung’s defense against inflammation and oxidant injury. However, exposure to supplemental oxygen, a common therapeutic modality for lung disease, represses the expression of CCSP in the adult mouse lung. The proximal -166 bp region of the CCSP gene is known to contain a complex composite of cis-elements that includes consensus sequences for activating protein-1 (AP-1), hepatocyte nuclear factor-3 (HNF-3), CCAAT/enhancer binding protein (C/EBP), and thyroid transcription factor-1 (Nkx2.1). The specific nucleotide sequences for the binding of these transcription factors are in very close proximity; in fact, the specific nucleotide sequences for AP-1, HNF-3, and C/EBP binding overlap one another. This composite of multipart cis-elements suggests the potential for interactions and cooperation between the transcription factors relevant to CCSP gene regulation.
In the present study, we demonstrated that there are multiple mechanisms involving three different trans-acting factors that coordinate the hyperoxia-induced decrease in CCSP gene expression. The hyperoxia-responsive region of the CCSP gene was localized to the proximal -166 bp region of the CCSP gene in transgenic mice and transient transfections. EMSAs using nuclear extracts obtained from mouse transformed Clara cells (mtCC) revealed that hyperoxia exposure altered the binding patterns of AP-1, C/EBP, and Nkx2.1 proteins to the CCSP proximal promoter. These altered patterns included an increase in c-Jun and C/EBPß binding, with a decrease in Nkx2.1 binding. Cotransfection of a c-Jun or the C/EBP-ß isoform LIP decreased the expression of a 166 bp-CCSP reporter gene. Moreover, cytoplasmic sequestration of the trans-acting factor Nkx2.1, a known CCSP gene activator, was observed by immunohistochemistry and Western blot analyses.
Oxidant stress is known to induce the transcriptional activity of AP-1 family members that mediate the induction of other target genes in the lung. The transcriptional activation potential varies considerably among specific members of the AP-1 family and the expression of specific AP-1 family members varies considerably between body tissues. c-Jun expression is highly inducible by oxidant stress and is expressed at relatively high levels in the lung. Thus, the discovery of c-Jun as a major constituent of the AP-1 complex binding the CCSP promoter secondary to hyperoxia exposure suggests a potentially specific role for c-Jun in regulating lung specific proteins in response to oxidant stress. Further support is provided by the evidence of a dose-dependent response of the CCSP gene expression to c-Jun in mtCC.
C/EBP-ß is known to modulate liver specific genes, but its role in the regulation of lung specific proteins is unreported. C/EBP-ß protein is a transcriptionally active protein that belongs to a family of C/EBP proteins in which two other members, C/EBP-
and C/EBP-
, are known to interact proximally with the CCSP. The C/EBP family of proteins functions by forming homo- or heterodimers that can bind the same C/EBP binding sites. The C/EBP-ß intronless gene product can undergo specific proteolytic cleavage of full-length C/EBP-ß to produce four isoforms: the full-length isoform (38 kDa), a 35 kDa LAP (liver-enriched transcriptional activating protein) isoform, 21 kDa LIP isoform, and a 14 kDa isoform. Truncation of the amino terminus results in a progressive loss of the trans-activation domain of these proteins, leaving the 21 and 14 kDa isoforms unable to activate gene transcription. The 21 and 14 kDa isoforms are able to function as dominant negative inhibitors of C/EBP-mediated gene activity. Therefore, induction of the LIP C/EBP-ß isoform in hyperoxia exposure has the potential to reduce the transcriptional activation of the C/EBP proteins interacting with the CCSP gene.
Nkx2.1, a strong transcriptional activator, is a homeodomain-containing phosphoprotein belonging to the Nkx family. Nkx2.1 is expressed throughout the respiratory epithelium and is critical to lung development as well as the expression of lung cell-specific proteins. Nkx2.1 also functions synergistically with C/EBP- to drive high levels of CCSP expression by the proximal CCSP promoter. Cytoplasmic sequestration of Nkx2.1 has been reported in the regulation of surfactant protein B in A549 cells secondary to the exposure to phorbol esters. Therefore, cytoplasmic sequestration of Nkx2.1 in Clara cells may decrease CCSP gene expression by the loss of Nkx2.1 binding directly with the proximal promoter and the loss of synergistic activity of Nkx2.1 with C/EBP-.
These proposed mechanisms for the hyperoxia-induced decrease in CCSP gene expression are shown in Fig. 2
. Under room air conditions, the transcriptional complex initially is highly active, with binding of the known transcriptional activators Nkx2.1, the LAP isoforms of C/EBP-, and HNF-3 proteins. However, with hyperoxia exposure the transcriptionally active complex is altered by the loss of nuclear Nkx2.1 and the increased binding of the LIP isoforms of C/EBP-ß and c-Jun, resulting in a decreased trans-activation potential of the protein complex interacting with the CCSP proximal promoter.
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These novel observations suggest that the hyperoxia-induced decrease in CCSP gene expression is mediated at least in part at the level of transcription and that there are multiple mechanisms mediating this decrease. Moreover, these results suggest a complex mechanism for the gene-specific regulation of lung proteins in response to oxidant stress. The utilization of common trans-acting factors interacting with compound nucleotide sequences may represent a common physiological mechanism for control of multiple tissue-specific genes in response to a common environmental stress, such as hyperoxia exposure. The underlying molecular events that initiate these altered DNA–protein interactions and change in subcellular localization remain to be elicited. The effects of oxidant stress may be direct through oxidative modifications of specific trans-acting factors or more indirect through the modification of redox-sensitive intracellular signaling pathways and/or strategic intracellular trafficking proteins.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0048fje; doi: 10.1096/fj.03-0048fje 
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